Methods of ex-vivo expansion of hematopoietic cells using multivariant IL-3 hematopoiesis chimera proteins
Granted 20 Aug 2002 · 3 office actions
Assignee: G. D. Searle & Co.
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Attorney: Attorney · Log in to unlock
Inventors: Maire Helena Caparon, Barbara Kure Klein, Peter O. Olins, Alan Michael Easton +6 · Examiner: Gary L. Kunz · AU 1647 · TC 1600
Life of the application
12 dated eventsAbstract
The present invention relates to methods of ex-vivo expansion of hematopoietic cells by culturing hematopoietic cells in a growth medium comprising a chimera protein which comprises a variant of human interleukin-3 (hIL-3) which contains multiple amino acid substitutions and which may have portions of the native hIL-3 molecule deleted and a hematopoietic growth factor. The present invention also relates to the ex-vivo expansion of hematopoietic cells for gene therapy. Additionally, the present invention relates to the use of the expanded hematopoietic cells for treating patients having a hematopoietic disorder.
Description
46 parts›This is a continuation-in-part of U.S. Ser. No…
This is a continuation-in-part of U.S. Ser. No. 08/446,872, filed Jun. 6, 1995, pending, which was filed under 35 USC §371 from PCT/US95/01185, filed Feb. 4, 1995; which is a continuation-in-part of U.S. Ser. No. 08/192,325, filed Feb. 4, 1994, now U.S. Patent No. 6,057,133; which is a continuation-in-part of U.S. Ser. No. 08/411,795, filed Apr. 6, 1995, now U.S. Pat. No. 5,604,116; said Ser. No. 08/411,795 was filed under 35 USC §371 from PCT/US93/11197, filed Nov. 22, 1993; which is a continuation-in-part of U.S. Ser. No. 07/981,044, filed Nov. 24, 1992, now abandoned. The noted applications are incorporated herein by reference.
›FIELD OF THE INVENTION
The present invention relates to methods of ex-vivo expansion of hematopoietic cells by culturing hematopoietic cells in a medium which includes a chimera protein comprising a variant of human interleukin-3 (hIL-3) joined with or without a linker to a second colony stimulating factors, cytokines, lymphokines, interleukins, hematopoietic growth factors or IL-3 variants and the use of the expanded hematopoietic cells for treating patients having a hematopoietic disorder.
›BACKGROUND OF THE INVENTION · 1 of 3
Colony stimulating factors, cytokines, lymphokines, interleukins or hematopoietic growth factors (herein collectively referred to as “hematopoietic growth factors”) which stimulate the differentiation and/or proliferation of bone marrow cells have generated much interest because of their therapeutic potential for restoring depressed levels of hematopoietic stem cell-derived cells. Hematopoietic growth factors in both human and murine systems have been identified and distinguished according to their activities. For example, granulocyte-CSF (G-CSF) and macrophage-CSF (M-CSF) stimulate the in vitro formation of neutrophilic granulocyte and macrophage colonies, respectively while GM-CSF and interleukin-3 (IL-3) have broader activities and stimulate the formation of both macrophage, neutrophilic and eosinophilic granulocyte colonies. IL-3 also stimulates the formation of mast, megakaryocyte and pure and mixed erythroid colonies.
Because of its ability to stimulate the proliferation of a number of different cell types and to support the growth and proliferation of progenitor cells, IL-3 has potential for therapeutic use in restoring hematopoietic cells to normal amounts in those cases where the number of cells has been reduced due to diseases or to therapeutic treatments such as radiation and/or chemotherapy.
Interleukin-3 (IL-3) is a hematopoietic growth factor which has the property of being able to promote the survival, growth and differentiation of hematopoietic cells. Among the biological properties of IL-3 are the ability (a) to support the growth and differentiation of progenitor cells committed to all, or virtually all, blood cell lineages; (b) to interact with early multipotential stem cells; (c) to sustain the growth of pluripotent precursor cells; (d) to stimulate proliferation of chronic myelogenous leukemia (CML) cells; (e) to stimulate proliferation of mast cells, eosinophils and basophils; (f) to stimulate DNA synthesis by human acute myelogenous leukemia (AML) cells; (g) to prime cells for production of leukotrienes and histamines; (h) to induce leukocyte chemotaxis; and (i) to induce cell surface molecules needed for leukocyte adhesion.
Mature human interleukin-3 (hIL-3) consists of 133 amino acids. It has one disulfide bridge and two potential glycosylation sites (Yang, et al., CELL 47:3 (1986)).
Murine IL-3 (mIL-3) was first identified by Ihle, et al., J. IMMUNOL. 126:2184 (1981) as a factor which induced expression of a T cell associated enzyme, 20-hydroxysteroid dehydrogenase. The factor was purified to homogeneity and shown to regulate the growth and differentiation of numerous subclasses of early hematopoietic and lymphoid progenitor cells.
In 1984, cDNA clones coding for murine IL-3 were isolated (Fung, et al., NATURE 307:233 (1984) and Yokota, et al., PROC. NATL. ACAD. SCI. USA 81:1070 (1984)). The murine DNA sequence coded for a polypeptide of 166 amino acids including a putative signal peptide.
The gibbon IL-3 sequence was obtained using a gibbon cDNA expression library. The gibbon IL-3 sequence was then used as a probe against a human genomic library to obtain a human IL-3 sequence.
Gibbon and human genomic DNA homologues of the murine IL-3 sequence were disclosed by Yang, et al., CELL 47:3 (1986). The human sequence reported by Yang, et al. included a serine residue at position 8 of the mature protein sequence. Following this finding, others reported isolation of Pro 8 hIL-3 cDNAs having proline at position 8 of the protein sequence. Thus it appears that there may be two allelic forms of hIL-3.
Dorssers, et al., GENE 55:115 (1987), found a clone from a human cDNA library which hybridized with mIL-3. This hybridization was the result of the high degree of homology between the 3′ noncoding regions of mIL-3 and hIL-3. This cDNA coded for an hIL-3 (Pro 8 ) sequence.
U.S. Pat. Nos. 4,877,729 and 4,959,455 disclose human IL-3 and gibbon IL-3 cDNAs and the protein sequences for which they code. The hIL-3 disclosed has serine rather than proline at position 8 in the protein sequence.
Clark-Lewis, et al., SCIENCE 231:134 (1986) performed a functional analysis of murine IL-3 analogs synthesized with an automated peptide synthesizer. The authors concluded that the stable tertiary structure of the complete molecule was required for full activity. A study on the role of the disulfide bridges showed that replacement of all four cysteines by alanine gave a molecule with {fraction (1/500)}th the activity as the native molecule. Replacement of two of the four Cys residues by Ala(Cys 79 , Cys 140 →Ala 79 , Ala 140 ) resulted in an increased activity. The authors concluded that in murine IL-3 a single disulfide bridge is required between cysteines 17 and 80 to get biological activity that approximates physiological levels and that this structure probably stabilizes the tertiary structure of the protein to give a conformation that is optimal for function. (Clark-Lewis, et al., PROC. NATL. ACAD. SCI. USA 85:7897 (1988)).
International Patent Application (PCT) WO 88/00598 discloses gibbon- and human-like IL-3. The hIL-3 contains a Ser 8 →Pro 8 replacement. Suggestions are made to replace Cys by Ser, thereby breaking the disulfide bridge, and to replace one or more amino acids at the glycosylation sites.
EP-A-0275598 (WO 88/04691) illustrates that Ala 1 can be deleted while retaining biological activity. Some mutant hIL-3 sequences are provided, e.g., two double mutants, Ala 1 →Asp 1 , Trp 13 →Arg 13 (pGB/IL-302) and Ala 1 →Asp 1 , Met 3 →Thr 3 (pGB/IL-304) and one triple mutant Ala 1 →Asp 1 , Leu 9 →Pro 9 , Trp 13 →Arg 13 (pGB/IL-303).
WO 88/05469 describes how deglycosylation mutants can be obtained and suggests mutants of Arg 54 Arg 55 and Arg 108 Arg 109 Lys 110 might avoid proteolysis upon expression in Saccharomyces cerevisiae by KEX2 protease. No mutated proteins are disclosed. Glycosylation and the KEX2 protease activity are only important, in this context, upon expression in yeast.
WO 88/06161 mentions various mutants which theoretically may be conformationally and antigenically neutral. The only actually performed mutations are Met 2 →Ile 2 and Ile131→Leu131. It is not disclosed whether the contemplated neutralities were obtained for these two mutations.
›BACKGROUND OF THE INVENTION · 2 of 3
WO 91/00350 discloses nonglycosylated hIL-3 analog proteins, for example, hIL-3 (Pro 8 Asp 15 Asp 70 ), Met 3 rhuIL-3 (Pro 8 Asp 15 Asp 70 ); Thr 4 rhuIL-3 (Pro 8 Asp 15 Asp 70 ) and Thr 6 rhuIL-3 (Pro 8 Asp 15 Asp 70 ). It is said that these protein compositions do not exhibit certain adverse side effects associated with native hIL-3 such as urticaria resulting from infiltration of mast cells and lymphocytes into the dermis. The disclosed analog hIL-3 proteins may have N termini at Met 3 , Thr 4 , or Thr 6 .
WO 90/12874 discloses cysteine added variants (CAVs) of IL-3 which have at least one Cys residue substituted for a naturally occurring amino acid residue.
U.S. Pat. No. 4,810,643 discloses the DNA sequence encoding human G-CSF.
WO 91/02754 discloses a fusion protein composed of GM-CSF and IL-3 which has increased biological activity compared to GM-CSF or IL-3 alone. Also disclosed are nonglycosylated IL-3 and GM-CSF analog proteins as components of the fusion.
WO 92/04455 discloses fusion proteins composed of IL-3 fused to a lymphokine selected from the group consisting of IL-3, IL-6, IL-7, IL-9, IL-11, EPO and G-CSF.
WO 92/06006 relates to hematopoietic molecules comprising an early acting factor (IL-3 or GM-CSF) and a late acting factor (EPO, IL-5, G-CSF or M-CSF) and the in vivo use for treating hematopoietic disorders.
Hematopoietic growth factors, such as hIL-3, have been administered alone, co-administered with other hematopoietic growth factors, or in combination with bone marrow transplants subsequent to high dose chemotherapy to treat the neutropenia and thrombocytopenia which are often the result of such treatment. However the period of severe neutropenia and thrombocytopenia may not be totally eliminated. The myeloid lineage, which is comprised of monocytes (macrophages), granulocytes (including neutrophils) and megakaryocytes, is critical in preventing infections and bleeding which can be life-threatening. Neutropenia and thrombocytopenia may also be the result of disease, genetic disorders, drugs, toxins, radiation and many therapeutic treatments such as conventional oncology therapy.
Bone marrow transplants have been used to treat this patient population. However, several problems are associated with the use of bone marrow to reconstitute a compromised hematopoietic system including: 1) the number of stem cells in bone marrow or other is limited, 2) Graft Versus Host Disease, 3) graft rejection and 4) possible contamination with tumor cells. Stem cells make up a very small percentage of the nucleated cells in the bone marrow, spleen and peripheral blood. It is clear that a dose response exits such that a greater number of stem cells will enhance hematopoietic recovery. Therefore, the use of hematopoietic cells that have been expanded ex-vivo should enhance hematopoietic recovery and patient survival. Bone marrow from an allogeneic donor has been used to provide bone marrow for transplant. However, Graft Versus Host Disease and graft rejection limit bone marrow transplantation even in recipients with HLA-matched sibling donors. An alternative to allogenic bone marrow transplants is autologous bone marrow transplants. In autologous bone marrow transplants, some of the patient's own marrow is harvested prior to myeloablative therapy, e.g. high dose chemotherapy, and is transplanted back into the patient afterwards. Autologous transplants eliminate the risk of Graft Versus Host Disease and graft rejection. However, autologous bone marrow transplants still present problems in terms of the limited number of stems cells in the marrow and possible contamination with tumor cells.
The limited number of stem cells may be overcome by ex-vivo expansion of the stem cells. In addition, stem cells can be specifically isolated selected based on the presence of specific surface antigen such as CD34+ in order to decrease tumor cell contamination of the marrow graft.
The following patents contain further details on separating stem cells, CD34+ cells, culturing the cells with hematopoietic growth factors, the use of the cells for the treatment of patients with hematopoietic disorders and the use of hematopoietic factors for cell expansion and gene therapy.
U.S. Pat. No. 5,061,620 relates to compositions comprising human hematopoietic stem cells provided by separating the stem cells from dedicated cells.
U.S. Pat. No. 5,199,942 describes a method for autologous hematopoietic cell transplantation comprising: (1) obtaining hematopoietic progenitor cells from a patient; (2) ex-vivo expansion of cells with a growth factor selected from the group consisting of IL-3, flt3 ligand, c-kit ligand, GM-CSF, IL-1, GM-CSF/IL-3 fusion protein and combinations thereof; (3) administering cellular preparation to a patient.
U.S. Pat. No. 5,240,856 relates to a cell separator that includes apparatus for automatically controlling the cell separation process.
U.S. Pat. No. 5,409,813 describes methods of positive and negative selection of a cell population from a mixture of cell populations utilizing a magnetically stabilized fluidized bed.
U.S. Pat. No. 5,409,825 relates to a method of growing hematopoietic stem cells in a liquid culture medium using mast cell growth factor (MGF) and optionally at least one cytokine selected from the group consisting of IL-3, GM-CSF and IL-3/GM-CSF fusion protein.
U.S. Pat. No. 5,459,069 relates to devices for maintaining and growing human stem cells and/or hematopoietic cells in culture.
U.S. Pat. No. 5,541,103 describes peripheral blood progenitor cells obtained by enriching blood progenitors expressing the cd34 antigen and culture the cells in a growth medium consisting of IL-1, IL-3, IL-6, erythropoietin and stem cell growth factor.
U.S. Pat. No. 5,464,753 describes a method of purifying pluripotent hematopoietic stem cells expressing P-glycoprotein from a mixture of blood or bone marrow cells.
U.S. Pat. No. 5,547,687 relates to a method of enriching CD34 cells from a cell mixture by density centrifugation.
›BACKGROUND OF THE INVENTION · 3 of 3
U.S. Pat. No. 5,571,686 depicts the use of megapoietin (c-mpl ligand) for the in vitro expansion of stem cells as a source of platelets for transplantation and for increasing the storage life of platelets.
WO 91/16116 describes devices and methods for selectively isolating and separating target cells from a mixture of cells.
WO 91/18972 describes methods for in vitro culturing of bone marrow, by incubating suspension of bone marrow cells, using a hollow fiber bioreactor.
WO 92/18615 relates to a process for maintaining and expanding bone marrow cells, in a culture medium containing specific mixtures of cytokines, for use in transplants.
WO 93/08268 describes a method for selectively expanding stem cells, comprising the steps of (a) separating CD34+ stem cells from other cells and (b) incubating the separated cells in a selective medium, such that the stem cells are selectively expanded.
WO 93/18136 describes a process for in vitro support of mammalian cells derived from peripheral blood.
WO 93/18648 relates to a composition comprising human neutrophil precursor cells with a high content of myeloblasts and promyelocytes for treating genetic or acquired neutropenia.
WO 94/08039 describes a method of enrichment for human hematopoietic stem cells by selection for cells which express c-kit protein.
WO 94/11493 describes a stem cell population that are CD34+and small in size, which are isolated using a counterflow elutriation method.
WO 94/27698 relates to a method combining immunoaffinity separation and continuous flow centrifugal separation for the selective separation of a nucleated heterogeneous cell population from a heterogeneous cell mixture.
WO 94/25848 describes a cell separation apparatus for collection and manipulation of target cells.
The long term culturing of highly enriched CD34+ precursors of hematopoietic progenitor cells from human bone marrow in cultures containing IL-1α, IL-3, IL-6 or GM-CSF is discussed in Brandt et al., J. Clin. Invest. 86:932-941, 1990.
›SUMMARY OF THE INVENTION · 1 of 3
The present invention encompasses the use of chimera proteins, comprising a recombinant human interleukin-3 (hIL-3) variant or mutant proteins (muteins) joined with or without a linker to a second colony stimulating factor (CSF), cytokine, lymphokine, interleukin, hematopoietic growth factor (herein collectively referred to as “hematopoietic growth factors”) or IL-3 variant, for the ex-vivo expansion of hematopoietic cells. These hIL-3 muteins contain amino acid substitutions and may also have amino acid deletions at either/or both the N- and C-termini. This invention encompasses mixed function hematopoietic growth factors formed from covalently linked polypeptides, each of which may act through a different and specific cell receptor to initiate complementary biological activities.
Novel compounds of this invention are represented by the formulas
R 1 -L-R 2 , R 2 -L-R 1 , R 1 -R 2 , R 2 -R 1 , R 1 -L-R 1 and R 1 -R 1
where R1 is a hIL-3 variant which contains multiple amino acid substitutions and which may have portions of the hIL-3 molecule deleted, R2 is an IL-3, IL-3 variant or hematopoietic growth factor with a different but complementary activity. The R1 polypeptide is joined either directly or through a linker segment to the R2 polypeptide. Thus L represents a chemical bond or polypeptide segment to which both R1 and R2 are joined. Preferably, these mutant IL-3 polypeptides of the present invention contain four or more amino acids which differ from the amino acids found at the corresponding positions in the native hIL-3 polypeptide.
These chimera molecules may be characterized by having the usual activity of both of the peptides forming the chimera molecule or it may be further characterized by having a biological or physiological activity greater than simply the additive function of the presence of IL-3 or the second hematopoietic growth factor alone. The chimera molecule may also unexpectedly provide an enhanced effect on the activity or an activity different from that expected by the presence of IL-3 or the second hematopoietic growth factor or IL-3 variant. The chimera molecule may also have an improved activity profile which may include reduction of undesirable biological activities associated with native hIL-3.
The present invention also includes mutants of hIL-3 in which from 1 to 14 amino acids have been deleted from the N-terminus and/or from 1 to 15 amino acids have been deleted from the C-terminus, containing multiple amino acid substitutions, to which a second hematopoietic growth factor or IL-3 variant has been joined. Preferred chimera molecules of the present invention are composed of hIL-3 variants in which amino acids 1 to 14 have been deleted from the N-terminus, amino acids 126 to 133 have been deleted from the C-terminus, and contains from about four to about twenty-six amino acid substitutions in the polypeptide sequence joined to second hematopoietic growth factor or IL-3 variant.
The present invention includes methods for selective ex vivo expansion of stem cells, comprising the steps of; (a) culturing said stem cells with a selected growth medium comprising a chimera protein having the formula selected from the group consisting of:
R 1 -L-R 2 , R 2 -L-R 1 , R 1 -R 2 , R 2 -R 1 , R 1 -L-R 1 and R 1 -R 1
wherein R 1 is a human interleukin-3 mutant polypeptide of SEQ ID NO:1
wherein
Xaa at position 17 is Ser, Lys, Gly, Asp, Met, Gln, or Arg;
Xaa at position 18 is Asn, His, Leu, Ile, Phe, Arg, or Gln;
Xaa at position 19 is Met, Phe, Ile, Arg, Gly, Ala, or Cys;
Xaa at position 20 is Ile, Cys, Gln, Glu, Arg, Pro, or Ala;
Xaa at position 21 is Asp, Phe, Lys, Arg, Ala, Gly, Glu, Gln, Asn, Thr, Ser or Val;
Xaa at position 22 is Glu, Trp, Pro, Ser, Ala, His, Asp, Asn, Gln, Leu, Val or Gly;
Xaa at position 23 is Ile, Val, Ala, Leu, Gly, Trp, Lys, Phe, Ser, or Arg;
Xaa at position 24 is Ile, Gly, Val, Arg, Ser, Phe, or Leu;
Xaa at position 25 is Thr, His, Gly, Gln, Arg, Pro, or Ala;
Xaa at position 26 is His, Thr, Phe, Gly, Arg, Ala, or Trp;
Xaa at position 27 is Leu, Gly, Arg, Thr, Ser, or Ala;
Xaa at position 28 is Lys, Arg, Leu, Gln, Gly, Pro, Val or Trp;
Xaa at position 29 is Gln, Asn, Leu, Pro, Arg, or Val;
Xaa at position 30 is Pro, His, Thr, Gly, Asp, Gln, Ser, Leu, or Lys;
Xaa at position 31 is Pro, Asp, Gly, Ala, Arg, Leu, or Gln;
Xaa at position 32 is Leu, Val, Arg, Gln, Asn, Gly, Ala, or Glu;
Xaa at position 33 is Pro, Leu, Gln, Ala, Thr, or Glu;
Xaa at position 34 is Leu, Val, Gly, Ser, Lys, Glu, Gln, Thr, Arg, Ala, Phe, Ile or Met;
Xaa at position 35 is Leu, Ala, Gly, Asn, Pro, Gln, or Val;
Xaa at position 36 is Asp, Leu, or Val;
Xaa at position 37 is Phe, Ser, Pro, Trp, or Ile;
Xaa at position 38 is Asn, or Ala;
Xaa at position 40 is Leu, Trp, or Arg;
Xaa at position 41 is Asn, Cys, Arg, Leu, His, Met, or Pro;
Xaa at position 42 is Gly, Asp, Ser, Cys, Asn, Lys, Thr, Leu, Val, Glu, Phe, Tyr, Ile, Met or Ala;
Xaa at position 43 is Glu, Asn, Tyr, Leu, Phe, Asp, Ala, Cys, Gln, Arg, Thr, Gly or Ser;
Xaa at position 44 is Asp, Ser, Leu, Arg, Lys, Thr, Met, Trp, Glu, Asn, Gln, Ala or Pro;
Xaa at position 45 is Gln, Pro, Phe, Val, Met, Leu, Thr, Lys, Trp, Asp, Asn, Arg, Ser, Ala, Ile, Glu or His;
Xaa at position 46 is Asp, Phe, Ser, Thr, Cys, Glu, Asn, Gln, Lys, His, Ala, Tyr, Ile, Val or Gly;
Xaa at position 47 is Ile, Gly, Val, Ser, Arg, Pro, or His;
Xaa at position 48 is Leu, Ser, Cys, Arg, Ile, His, Phe, Glu, Lys, Thr, Ala, Met, Val or Asn;
Xaa at position 49 is Met, Arg, Ala, Gly, Pro, Asn, His, or Asp;
Xaa at position 50 is Glu, Leu, Thr, Asp, Tyr, Lys, Asn, Ser, Ala, Ile, Val, His, Phe, Met or Gln;
Xaa at position 51 is Asn, Arg, Met, Pro, Ser, Thr, or His;
Xaa at position 52 is Asn, His, Arg, Leu, Gly, Ser, or Thr;
Xaa at position 53 is Leu, Thr, Ala, Gly, Glu, Pro, Lys, Ser, or Met;
Xaa at position 54 is Arg, Asp, Ile, Ser, Val, Thr, Gln, Asn, Lys, His, Ala or Leu;
Xaa at position 55 is Arg, Thr, Val, Ser, Leu, or Gly;
Xaa at position 56 is Pro, Gly, Cys, Ser, Gln, Glu, Arg, His, Thr, Ala, Tyr, Phe, Leu, Val or Lys;
›SUMMARY OF THE INVENTION · 2 of 3
Xaa at position 57 is Asn or Gly;
Xaa at position 58 is Leu, Ser, Asp, Arg, Gln, Val, or Cys;
Xaa at position 59 is Glu, Tyr, His, Leu, Pro, or Arg;
Xaa at position 60 is Ala, Ser, Pro, Tyr, Asn, or Thr;
Xaa at position 61 is Phe, Asn, Glu, Pro, Lys, Arg, or Ser;
Xaa at position 62 is Asn, His, Val, Arg, Pro, Thr, Asp, or Ile;
Xaa at position 63 is Arg, Tyr, Trp, Lys, Ser, His, Pro, or Val;
Xaa at position 64 is Ala, Asn, Pro, Ser, or Lys;
Xaa at position 65 is Val, Thr, Pro, His, Leu, Phe, or Ser;
Xaa at position 66 is Lys, Ile, Arg, Val, Asn, Glu, or Ser;
Xaa at position 67 is Ser, Ala, Phe, Val, Gly, Asn, Ile, Pro, or His;
Xaa at position 68 is Leu, Val, Trp, Ser, Ile, Phe, Thr, or His;
Xaa at position 69 is Gln, Ala, Pro, Thr, Glu, Arg, Trp, Gly, or Leu;
Xaa at position 70 is Asn, Leu, Val, Trp, Pro, or Ala;
Xaa at position 71 is Ala, Met, Leu, Pro, Arg, Glu, Thr, Gln, Trp, or Asn;
Xaa at position 72 is Ser, Glu, Met, Ala, His, Asn, Arg, or Asp;
Xaa at position 73 is Ala, Glu, Asp, Leu, Ser, Gly, Thr, or Arg;
Xaa at position 74 is Ile, Met, Thr, Pro, Arg, Gly, Ala;
Xaa at position 75 is Glu, Lys, Gly, Asp, Pro, Trp, Arg, Ser, Gln, or Leu;
Xaa at position 76 is Ser, Val, Ala, Asn, Trp, Glu, Pro, Gly, or Asp;
Xaa at position 77 is Ile, Ser, Arg, Thr, or Leu;
Xaa at position 78 is Leu, Ala, Ser, Glu, Phe, Gly, or Arg;
Xaa at position 79 is Lys, Thr, Asn, Met, Arg, Ile, Gly, or Asp;
Xaa at position 80 is Asn, Trp, Val, Gly, Thr, Leu, Glu, or Arg;
Xaa at position 81 is Leu, Gln, Gly, Ala, Trp, Arg, Val, or Lys;
Xaa at position 82 is Leu, Gln, Lys, Trp, Arg, Asp, Glu, Asn, His, Thr, Ser, Ala, Tyr, Phe, Ile, Met or Val;
Xaa at position 83 is Pro, Ala, Thr, Trp, Arg, or Met;
Xaa at position 84 is Cys, Glu, Gly, Arg, Met, or Val;
Xaa at position 85 is Leu, Asn, Val, or Gln;
Xaa at position 86 is Pro, Cys, Arg, Ala, or Lys;
Xaa at position 87 is Leu, Ser, Trp, or Gly;
Xaa at position 88 is Ala, Lys, Arg, Val, or Trp;
Xaa at position 89 is Thr, Asp, Cys, Leu, Val, Glu, His, Asn, or Ser;
Xaa at position 90 is Ala, Pro, Ser, Thr, Gly, Asp, Ile, or Met;
Xaa at position 91 is Ala, Pro, Ser, Thr, Phe, Leu, Asp, or His;
Xaa at position 92 is Pro, Phe, Arg, Ser, Lys, His, Ala, Gly, Ile or Leu;
Xaa at position 93 is Thr, Asp, Ser, Asn, Pro, Ala, Leu, or Arg;
Xaa at position 94 is Arg, Ile, Ser, Glu, Leu, Val, Gln, Lys, His, Ala, or Pro;
Xaa at position 95 is His, Gln, Pro, Arg, Val, Leu, Gly, Thr, Asn, Lys, Ser, Ala, Trp, Phe, Ile, or Tyr;
Xaa at position 96 is Pro, Lys, Tyr, Gly, Ile, or Thr;
Xaa at position 97 is Ile, Val, Lys, Ala, or Asn;
Xaa at position 98 is His, Ile, Asn, Leu, Asp, Ala, Thr, Glu, Gln, Ser, Phe, Met, Val, Lys, Arg, Tyr or Pro;
Xaa at position 99 is Ile, Leu, Arg, Asp, Val, Pro, Gln, Gly, Ser, Phe, or His;
Xaa at position 100 is Lys, Tyr, Leu, His, Arg, Ile, Ser, Gln, or Pro;
Xaa at position 101 is Asp, Pro, Met, Lys, His, Thr, Val, Tyr, Glu, Asn, Ser, Ala, Gly, Ile, Leu, or Gln;
Xaa at position 102 is Gly, Leu, Glu, Lys, Ser, Tyr, or Pro;
Xaa at position 103 is Asp, or Ser;
Xaa at position 104 is Trp, Val, Cys, Tyr, Thr, Met, Pro, Leu, Gln, Lys, Ala, Phe, or Gly;
Xaa at position 105 is Asn, Pro, Ala, Phe, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp, or His;
Xaa at position 106 is Glu, Ser, Ala, Lys, Thr, Ile, Gly, or Pro;
Xaa at position 108 is Arg, Lys, Asp, Leu, Thr, Ile, Gln, His, Ser, Ala or Pro;
Xaa at position 109 is Arg, Thr, Pro, Glu, Tyr, Leu, Ser, or Gly;
Xaa at position 110 is Lys, Ala, Asn, Thr, Leu, Arg, Gln, His, Glu, Ser, or Trp;
Xaa at position 111 is Leu, Ile, Arg, Asp, or Met;
Xaa at position 112 is Thr, Val, Gln, Tyr, Glu, His, Ser, or Phe;
Xaa at position 113 is Phe, Ser, Cys, His, Gly, Trp, Tyr, Asp, Lys, Leu, Ile, Val or Asn;
Xaa at position 114 is Tyr, Cys, His, Ser, Trp, Arg, or Leu;
Xaa at position 115 is Leu, Asn, Val, Pro, Arg, Ala, His, Thr, Trp, or Met;
Xaa at position 116 is Lys, Leu, Pro, Thr, Met, Asp, Val, Glu, Arg, Trp, Ser, Asn, His, Ala, Tyr, Phe, Gln, or Ile;
Xaa at position 117 is Thr, Ser, Asn, Ile, Trp, Lys, or Pro;
Xaa at position 118 is Leu, Ser, Pro, Ala, Glu, Cys, Asp, or Tyr;
Xaa at position 119 is Glu, Ser, Lys, Pro, Leu, Thr, Tyr, or Arg;
Xaa at position 120 is Asn, Ala, Pro, Leu, His, Val, or Gln;
Xaa at position 121 is Ala, Ser, Ile, Asn, Pro, Lys, Asp, or Gly;
Xaa at position 122 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys;
Xaa at position 123 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu;
wherein from 1 to 14 amino acids can be deleted from the N-terminus and/or from 1 to 15 amino acids can be deleted from the C-terminus of said human interleukin-3 mutant polypeptide; and wherein from 4 to 44 of the amino acids designated by Xaa are different from the corresponding amino acids of native (1-133) human interleukin-3;
R 2 is a hematopoietic growth factor;
L is a linker capable of Linking R 1 to R 2 ; and said chimera protein can additionally be immediately preceded by (methionine −1 ), (alanine −1 ), or (methionine −2 , alanine −1 ); and
(b) harvesting said cultured stem cells.
Additionally, the present invention encompasses methods of ex-vivo expansion of stem cells comprising the steps of (a) separating stem cells from a mixed population of cells; (b) culturing said separated stem cells with a growth medium comprising a chimera protein; (c) harvesting said cultured cells.
The present invention includes methods for treatment of a patient having a hematopoietic disorder, comprising the steps of; (a) removing stem cells from said patient or a blood donor; (b) culturing said stem cells with a growth medium comprising a chimera protein; (c) harvesting said cultured cells; and (d) transplanting said cultured cells into said patient.
The present invention also includes methods for treatment of a patient having a hematopoietic disorder, comprising the steps of; (a) removing stem cells from said patient or a blood donor; (b) separating stem cells from a mixed population of cells; (c) culturing said separated stem cells with a growth medium comprising a chimera protein; (d) harvesting said cultured cells; and (e) transplanting said cultured cells into said patient.
›SUMMARY OF THE INVENTION · 3 of 3
It is also envisioned that a patient could be given a hematopoietic growth factor, preferably a early acting factor, prior to removing stem cells for ex-vivo expansion to increase the number of early progenitors. It is also envisioned that a portion of the stem cells removed from a patient could be frozen and transplanted with the expanded stem cells to provide more early progenitors.
It is envisioned that the present invention includes methods of human gene therapy, comprising the steps of; (a) removing stem cells from a patient or blood donor; (b) culturing said stem cells with a selected growth medium comprising a chimera protein; (c) introducing DNA into said cultured cells; (d) harvesting said transduced cells; and (e) transplanting said transduced cells into said patient.
It is also envisioned that the present invention includes methods of human gene therapy, comprising the steps of; (a) removing stem cells from a patient or blood donor; (b) separating said stem cells from a mixed population of cells; (c) culturing said separated stem cells with a selected growth medium comprising a chimera protein; (d) introducing DNA into said cultured cells; (e) harvesting said transduced cells; and (f) transplanting said transduced cells into said patient.
It is also intended that the present invention includes methods of ex vivo expansion of hematopoietic cells, methods of expanding hematopoietic cells for gene therapy and methods of treating a patient using the expanded cells using the chimeric proteins of the present invention with other hematopoietic growth factors. A non-exclusive list of other appropriate hematopoietic growth factors, colony stimulating factors, cytokines, lymphokines, hematopoietic growth factors and interleukins for simultaneous or serial co-administration with the polypeptides of the present invention includes GM-CSF, CSF-1, G-CSF, G-CSF Ser 17 , c-mpl ligand (MGDF or TPO), c-mpl receptor agonists disclosed in PCT/US96/15938, M-CSF, erythropoietin (EPO), IL-1, IL-4, IL-2, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, LIF, flt3 ligand, B-cell growth factor, B-cell differentiation factor and eosinophil differentiation factor, stem cell factor (SCF) also known as steel factor or c-kit ligand, multi-functional hematopoietic receptor agonists disclosed in PCT/US96/15774, or combinations thereof.
›BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is the human IL-3 gene for E. coli expression (pMON5873), encoding the polypeptide sequence of natural (wild type) human IL-3 (SEQ ID NO:49), plus an initiator methionine, as expressed in E. coli , with the amino acids numbered from the N-terminus of the natural hIL-3.
FIG. 2 shows the bioactivity, as measured in the methylcellulose assay, of the polypeptide chimera pMON3988.
FIG. 3 shows the bioactivity, as measured in the methylcellulose assay, of the polypeptide chimeras pMON3987 and pMON26430, pMON3995 and pMON26415.
FIG. 4 shows the bioactivity, as measured in the methylcellulose assay, of the polypeptide chimera pMON26425.
FIG. 5 shows the bioactivity, as measured in the methylcellulose assay, of the polypeptide chimeras pMON26406 and pMON26433.
FIG. 6 shows the bioactivity, as measured in the methylcellulose assay, of the polypeptide chimeras pMON26431 and pMON26427.
›DETAILED DESCRIPTION OF THE INVENTION · 1 of 6
The present invention encompasses methods of ex-vivo expansion of hematopoietic cells using a chimera protein comprising a recombinant human interleukin-3 (hIL-3) variants or mutant proteins (muteins) joined with or without a linker to a second IL-3 mutein, IL-3 or a second factor including but not limited to colony stimulating factors, cytokines, lymphokines, interleukins, hematopoietic growth factors or IL-3 variants. This invention encompasses the ex-vivo expansion use of these mixed function hematopoietic growth factors (chimera protein) formed from covalently linked polypeptides, each of which may act through a different and specific cell receptor to initiate complementary biological activities.
Hematopoiesis requires a complex series of cellular events in which stem cells generate continuously into large populations of maturing cells in all major lineages. There are currently at least 20 known regulators with hematopoietic proliferative activity. Most of these proliferative regulators can stimulate one or another type of colony formation in vitro, the precise pattern of colony formation stimulated by each regulator is quite distinctive. No two regulators stimulate exactly the same pattern of colony formation, as evaluated by colony numbers or, more importantly, by the lineage and maturation pattern of the cells making up the developing colonies. Proliferative responses can most readily be analyzed in simplified in vitro culture systems. Three quite different parameters can be distinguished: alteration in colony size, alteration in colony numbers and cell lineage. Two or more factors may act on the progenitor cell, inducing the formation of larger number of progeny thereby increasing the colony size. Two or more factors may allow increased number of progenitor cells to proliferate either because distinct subsets of progenitors cells exist that respond exclusively to one factor or because some progenitors require stimulation by two or more factors before being able to respond. Activation of additional receptors on a cell by the use of two or more factors is likely to enhance the mitotic signal because of coalescence of initially differing signal pathways into a common final pathway reaching the nucleus (Metcalf, Nature 339:27, 1989). Other mechanisms could explain synergy. For example, if one signaling pathway is limited by an intermediate activation of an additional signaling pathway by a second factor may result in a superadditive response. In some cases, activation of one receptor type can induce a enhanced expression of other receptors (Metcalf, Blood 82(12):3515-3523 1993). Two or more factors may result in a different pattern of cell lineages then from a single factor. The use of chimera molecules may have the potential clinical advantage resulting from a proliferative response that is not possible by any single factor.
Hematopoietic and other growth factors can be grouped in to two distinct families of related receptors: (1) tyrosine kinase receptors, including those for epidermal growth factor, M-CSF (Sherr, 1990) and SCF (Yarden et al., EMBO J 6:3341, 1987): and (2) hematopoietic receptors, not containing a tyrosine kinase domain, but exhibiting obvious homology in their extracellular domain (Bazan, Proc. Natl. Acad. Sci. U.S.A. 87(18):6934-8 1990). Included in this later group are erythropoietin (EPO) (D'Andrea et al., Cell 57:277 1989), GM-CSF (Gearing et al., EMBO J 8:3667 1989), IL-3 (Kitamura et al., Cell 66:1165 1991), G-CSF (Fukunaga et al., J. Biol. Chem. 265(23):14008-15 1990), IL-4 (Harada et al., 1990), IL-5 (Takaki et al., EMBO J 9:4367 1990), IL-6 (Yamasaki et al., Science 241:825 1988), IL-7 (Goodwin et al., Cell 60(6):941-51 1990), LIF (Gearing et al., EMBO J 10:2839 1991) and IL-2 (Cosman et al., 1987). Most of the later group of receptors exists in high-affinity form as a heterodimers. After ligand binding, the specific α-chains become associated with at least one other receptor chain (β-chain, γ-chain). Many of these factors share a common receptor subunit. The α-chains for GM-CSF, IL-3 and IL-5 share the same β-chain (Kitamura et al., Cell 66:1165 1991, Takaki et al., EMBO. J. 10(10):2833-8 1991) and receptor complexes for IL-6, LIF and IL-11 share a common β-chain (gp130) (Taga et al., Cell 58(3):573-81 1989; Gearing et al., EMBO J 10:2839 1992). The receptor complexes of IL-2, IL-4 and IL-7 share a common γ-chain (Kondo et al., Science 262:1874 1993; Russell et al., Science 262:1880 1993; Noguchi et al., Science 262:1877 1993).
The ex-vivo expansion methods of the present invention use chimera proteins of the formula selected from the group consisting of
R 1 -L-R 2 , R 2 -L-R 1 , R 1 -R 2 , R 2 -R 1 , R 1 -L-R 1 and R 1 -R 1
where R1 is a hIL-3 variant which contains multiple amino acid substitutions and which may have portions of the hIL-3 molecule deleted as is disclosed in WO 94/12638, R2 is a hematopoietic growth factor with a different but complementary activity. By complementary activity is meant activity which enhances or changes the response to another cell modulator. The R1 polypeptide is joined either directly or through a linker segment to the R2 polypeptide. The term “directly” defines chimeras in which the polypeptides are joined without a peptide linker. Thus L represents a chemical bound or polypeptide segment to which both R1 and R2 are joined in frame, most commonly L is a linear peptide to which R1 and R2 are bound by amide bonds linking the carboxy terminus of R1 to the amino terminus of L and carboxy terminus of L to the amino terminus of R2. By “joined in frame” is meant that there is no translation termination or disruption between the reading frames of the DNA sequence encoding R1 and R2. A non-exclusive list of other growth factors, colony stimulating factors, cytokines, lymphokines, interleukins, and hematopoietic growth factors within the definition of R2, which can be joined to a hIL-3 variant of the present invention include GM-CSF, CSF-1, G-CSF, G-CSF Ser17, c-mpl ligand (MGDF or TPO), M-CSF, erythropoietin (EPO), IL-1, IL-4, IL-2, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, LIF, flt3 ligand, human growth hormone, B-cell growth factor, B-cell differentiation factor, eosinophil differentiation factor and stem cell factor (SCF) also known as steel factor or c-kit ligand. Additionally, this invention encompasses the use of modified R2 molecules or mutated or modified DNA sequences encoding these R2 molecules. The present invention also includes chimera molecules in which R2 is a hIL-3 variant which means an IL-3 in which has amino acid substitutions and which may have portions of the hIL-3 molecule deleted such as what is disclosed in WO 94/12638 and WO 94/12639 as well as other variants known in the art.
›DETAILED DESCRIPTION OF THE INVENTION · 2 of 6
As used herein human interleukin-3 corresponds to the amino acid sequence (1-133) as depicted in FIG. 1 and (15-125) hIL-3 corresponds to the 15 to 125 amino acid sequence of the hIL-3 polypeptide. Naturally occurring variants of hIL-3 polypeptide amino acids are also included in the present invention (for example, the allele in which proline rather than serine is at position 8 in the hIL-3 polypeptide sequence) as are variant hIL-3 molecules which are modified post-translationally (e.g. glycosylation).
“Mutant amino acid sequence,” “mutant protein” or “mutant polypeptide” refers to a polypeptide having an amino acid sequence which varies from a native sequence or is encoded by a nucleotide sequence intentionally made variant from a native sequence. “Mutant protein,” “variant protein” or “mutein” means a protein comprising a mutant amino acid sequence and includes polypeptides which differ from the amino acid sequence of native hIL-3 due to amino acid deletions, substitutions, or both. “Native sequence” refers to an amino acid or nucleic acid sequence which is identical to a wild-type or native form of a gene or protein.
Human IL-3 can be characterized by its ability to stimulate colony formation by human hematopoietic progenitor cells. The colonies formed include erythroid, granulocyte, megakaryocyte, granulocytic macrophages and mixtures thereof. Human IL-3 has demonstrated an ability to restore bone marrow function and peripheral blood cell populations to therapeutically beneficial levels in studies performed initially in primates and subsequently in humans (Gillio, A. P., et al. J. Clin. Invest. 85: 1560 (1990); Ganser, A., et al. Blood 76: 666 (1990); Falk, S., et al. Hematopathology 95: 355 (1991). Additional activities of hIL-3 include the ability to stimulate leukocyte migration and chemotaxis; the ability to prime human leukocytes to produce high levels of inflammatory mediators like leukotrienes and histamine; the ability to induce cell surface expression of molecules needed for leukocyte adhesion; and the ability to trigger dermal inflammatory responses and fever. Other IL-3-like properties are the interaction with early multipotential stem cells, the sustaining of the growth of pluripotent precursor cells, the ability to stimulate chronic myelogenous leukemia (CML) cell proliferation, the stimulation of proliferation of mast cells, the ability to support the growth of various factor-dependent cell lines, and the ability to trigger immature bone marrow cell progenitors. Other biological properties of IL-3 have been disclosed in the art. Many or all of these biological activities of hIL-3 involve signal transduction and high affinity receptor binding.
Biological activity of hIL-3 and hIL-3 chimera proteins of the present invention is determined by DNA synthesis by human acute myelogenous leukemia cells (AML). The factor-dependent cell line AML 193 was adapted for use in testing biological activity. The biological activity of hIL-3 and hIL-3 chimera proteins of the present invention is also determined by counting the colony forming units in a bone marrow assay.
Other in vitro cell based assays may also be useful to determine the activity of the chimera molecules depending on the hematopoietic growth factors that comprise the chimera. The following are examples of other useful assays.
TF-1 proliferation assay: The TF-1 cell line was derived from bone marrow of a patient with erythroleukemia (Kitamura et al., J. Cell Physiol. 140:323-334, 1989). TF-1 cells respond to IL-3, GM-CSF, EPO and IL-5.
32D proliferation assay: 32D is a murine IL-3 dependent cell line which does not respond to human IL-3 but does respond to human G-CSF which is not species restricted. T1165 proliferation assay: T1165 cells are a IL-6 dependent murine cell line (Nordan et al., Science 233:566, 1986) which respond to IL-6 and IL-11.
Human Plasma Clot meg-CSF Assay: Used to assay megakaryocyte colony formation activity (Mazur et al., Blood 57:277-286 1981).
Compounds of this invention are preferably made by genetic engineering techniques now standard in the art U.S. Pat. No. 4,935,233 and Sambrook et al., “Molecular Cloning. A Laboratory Manual”, Cold Spring Harbor Laboratory, 1989. One method of creating the preferred hIL-3 (15-125) mutant genes is cassette mutagenesis (Wells, et al. Gene, 34:315-323, 1985) in which a portion of the coding sequence of hIL-3 in a plasmid is replaced with synthetic oligonucleotides that encode the desired amino acid substitutions in a portion of the gene between two restriction sites. In a similar manner amino acid substitutions could be made in the full-length hIL-3 gene, or genes encoding variants of hIL-3 in which from 1 to 14 amino acids have been deleted from the N-terminus and/or from 1 to 15 amino acids have been deleted from the C-terminus. When properly assembled these oligonucleotides would encode hIL-3 variants with the desired amino acid substitutions and/or deletions from the N-terminus and/or C-terminus. These and other mutations could be created by those skilled in the art by other mutagenesis methods including; oligonucleotide-directed mutagenesis (Zoller and Smith Nucleic Acid Research, 10:6487-6500, 1982; Zoller and Smith Methods in Enzymology, 100:468-500, 1983; Zoller and Smith DNA, 3: 479, 1984 Smith M. Ann. Rev. Genet., 19:423-462, 1985; Kunkel Proc. Natl. Acad. Sci. USA, 82: 488-492, 1985, Taylor, et al. Nucl. Acids Res., 13:8764-8785 (1985), Deng and Nickoloff, Anal - Biochem 200:81-88, 1992) or polymerase chain reaction (PCR) techniques (Saiki, Science 230:1350-1354, 1985).
Additional details about recombinant techniques for construction of DNA sequences that encode the chimera proteins, plasmid DNA vectors for use in the expression of these novel chimera molecules, methods for producing the chimera molecules in bacterial cells, mammalian cells, or insect cells and the in-vitro and in-vivo activity of the chimera proteins can be found in WO 95/21254. It is understood that the chimera molecules of the present invention, used for the ex-vivo expansion of hematopoietic cells, can be made by other methods known to those skilled in the art.
›DETAILED DESCRIPTION OF THE INVENTION · 3 of 6
Hematopoietic cells that have been expanded ex-vivo using the chimera molecules of the present invention may be useful in the treatment of diseases characterized by a decreased levels of either myeloid, erythroid, lymphoid, or megakaryocyte cells of the hematopoietic system or combinations thereof. In addition, they may be used to activate mature myeloid and/or lymphoid cells. Among conditions susceptible to treatment with hematopoietic cells that have been expanded ex-vivo using the chimera proteins of the present invention is leukopenia, a reduction in the number of circulating leukocytes (white cells) in the peripheral blood. Leukopenia may be induced by exposure to certain viruses or to radiation. It is often a side effect of various forms of cancer therapy, e.g., exposure to chemotherapeutic drugs, radiation and of infection or hemorrhage. Therapeutic treatment of leukopenia with these chimera molecules of the present invention may avoid undesirable side effects caused by treatment with presently available drugs.
Hematopoietic cells that have been expanded ex-vivo using the chimera molecules of the present invention may be useful in the treatment of neutropenia and, for example, in the treatment of such conditions as aplastic anemia, cyclic neutropenia, idiopathic neutropenia, Chediak-Higashi syndrome, systemic lupus erythematosus (SLE), leukemia, myelodysplastic syndrome and myelofibrosis.
Hematopoietic cells that have been expanded ex-vivo using the chimera molecule of the present invention may be useful in the treatment or prevention of thrombocytopenia. Currently the only therapy for thrombocytopenia is platelet transfusions which are costly and carry the significant risks of infection (HIV, HBV) and alloimunization. Treatment involving the transplantation of the hematopoietic cells that have been expanded ex-vivo using chimera proteins of the present invention into a patient, may alleviate or diminish the need for platelet transfusions. Severe thrombocytopenia may result from genetic defects such as Fanconi's Anemia, Wiscott-Aldrich, or May-Hegglin syndromes. Acquired thrombocytopenia may result from auto- or allo-antibodies as in Immune Thrombocytopenia Purpura, Systemic Lupus Erythromatosis, hemolytic anemia, or fetal maternal incompatibility. In addition, splenomegaly, disseminated intravascular coagulation, thrombotic thrombocytopenic purpura, infection or prosthetic heart valves may result in thrombocytopenia. Severe thrombocytopenia may also result from chemotherapy and/or radiation therapy or cancer. Thrombocytopenia may also result from marrow invasion by carcinoma, lymphoma, leukemia or fibrosis.
One aspect of the present invention provides a novel hematopoietic factors for selective ex-vivo expansion of stem cells. The term “stem cell” refers to the totipiotent hematopoietic stem cells as well as early precursors and progenitor cells which can be isolated from bone marrow, spleen or peripheral blood. The term “expanding” refers to the differentiation and proliferation of the cells. The present invention provides a method for selective ex-vivo expansion of stem cells, comprising the steps of; (a) separating stem cells from a mixed population of cells, (b) culturing said separated stem cells with a selected media which contains a chimera protein(s) and (c) harvesting said cultured stems cells.
Stem cells as well as committed progenitor cells destined to become neutrophils, erythrocytes, platelets. etc., may be distinguished from most other cells by the presence or absence of particular progenitor marker antigens, such as CD34, that are present on the surface of these cells and/or by morphological characteristics. The phenotype for a highly enriched human stem cell fraction is reported as CD34+, Thy-1+ and lin-, but it is to be understood that the present invention is not limited to the expansion of this stem cell population. The CD34+ enriched human stem cell fraction can be separated by a number of reported methods, including affinity columns or beads, magnetic beads or flow cytometry using antibodies directed to surface antigens such as the CD34+. Further, physical separation methods such as counterflow elutriation may be used to enrich hematopoietic progenitors. The CD34+ progenitors are heterogeneous, and may be divided into several subpopulations characterized by the presence or absence of coexpression of different lineage associated cell surface associated molecules. The most immature progenitor cells do not express any known lineage-associated markers, such as HLA-DR or CD38, but they may express CD90(thy-1). Other surface antigens such as CD33, CD38, CD41, CD71, HLA-DR or c-kit can also be used to selectively isolate hematopoietic progenitors. The separated cells can be incubated in selected medium in a culture flask, sterile bag or in hollow fibers. Various hematopoietic growth factors may be utilized in order to selectively expand cells. Representative factors that have been utilized for ex-vivo expansion of bone marrow include, c-kit ligand, IL-3, G-CSF, GM-CSF, IL-1, IL-6, IL-11, flt-3 ligand or combinations thereof. The proliferation of the stem cells can be monitored by enumerating the number of stem cells and other cells, by standard techniques (e.g. hemacytometer, CFU, LTCIC) or by flow cytometry prior and subsequent to incubation.
Several methods for ex-vivo expansion of stem cells have been reported utilizing a number of selection methods and expansion using various hematopoietic growth factors including c-kit ligand (Brandt et al., Blood 83:1507-1514 (1994), McKenna et al., Blood 86:3413-3420 (1995), IL-3 (Brandt et al., Blood 83:1507-1514 (1994), Sato et al., Blood 82:3600-3609 (1993), G-CSF (Sato et al., Blood 82:3600-3609 (1993), GM-CSF (Sato et al., Blood 82:3600-3609 (1993), IL-1 (Muench et al., Blood 81:3463-3473 (1993), IL-6 (Sato et al., Blood 82:3600-3609 (1993), IL-11 (Lemoli et al., Exp. Hem. 21:1668-1672 (1993), Sato et al., Blood 82:3600-3609 (1993), flt-3 ligand (McKenna et al., Blood 86:3413-3420 (1995) and/or combinations thereof (Brandt et al., Blood 83:1507-1514 (1994), Haylock et al., Blood 80:1405-1412 (1992), Koller et al., Biotechnology 11:358-363 (1993), (Lemoli et al., Exp. Hem. 21:1668-1672 (1993), McKenna et al., Blood 86:3413-3420 (1995), Muench et al., Blood 81:3463-3473 (1993), Patchen et al., Biotherapy 7:13-26 (1994), Sato et al., Blood 82:3600-3609 (1993), Smith et al., Exp. Hem. 21:870-877 (1993), Steen et al., Stem Cells 12:214-224 (1994), Tsujino et al., Exp. Hem. 21:1379-1386 (1993). Among the individual hematopoietic growth factors, hIL-3 has been shown to be one of the most potent in expanding peripheral blood CD34+ cells (Sato et al., Blood 82:3600-3609 (1993), Kobayashi et al., Blood 73:1836-1841 (1989). However, no single factor has been shown to be as effective as the combination of multiple factors. The present invention provides methods for ex vivo expansion that utilize molecules that are more effective than IL-3 alone.
›DETAILED DESCRIPTION OF THE INVENTION · 4 of 6
Another projected clinical use of growth factors has been in the in vitro activation of hematopoietic progenitors and stem cells for gene therapy. Due to the long life-span of hematopoietic progenitor cells and the distribution of their daughter cells throughout the entire body, hematopoietic progenitor cells are good candidates for ex vivo gene transfection. In order to have the gene of interest incorporated into the genome of the hematopoietic progenitor or stem cell one needs to stimulate cell division and DNA replication. Hematopoietic stem cells cycle at a very low frequency which means that growth factors may be useful to promote gene transduction and thereby enhance the clinical prospects for gene therapy. Potential applications of gene therapy (review Crystal, Science 270:404-410 (1995) include; 1) the treatment of many congenital metabolic disorders and immunodifiencies (Kay and Woo, Trends Genet. 10:253-257 (1994), 2) neurological disorders (Freedmann, Trends Genet. 10:210-214 (1994), 3) cancer (Culver and Blaese, Trends Genet. 10:174-178 (1994) and 4) infectious diseases (Gilboa and Smith, Trends Genet. 10:139-144 (1994). Due to the long life-span of hematopoietic progenitor cells and the distribution of their daughter cells throughout the entire body, hematopoietic progenitor cells are good candidates for ex vivo gene transfection include the treatment of many congenital metabolic disorders and immunodifiencies (Kay and Woo, Trends Genet. 10:253-257 (1994) neurological disorders (Freedmann, Trends Genet. 10:210-214 (1994), cancer (Culver and Blaese, Trends Genet. 10:174-178 (1994) and infectious diseases (Gilboa and Smith, Trends Genet. 10:139-144 (1994).
There are a variety of methods, known to those with skill in the art, for introducing genetic material into a host cell. A number of vectors, both viral and non-viral have been developed for transferring therapeutic genes into primary cells. Viral based vectors include; 1) replication-deficient recombinant retrovirus (Boris-Lawrie and Temin, Curr. Opin. Genet. Dev. 3:102-109 (1993), Boris-Lawrie and Temin, Annal. New York Acad. Sci. 716:59-71 (1994), Miller, Current Top. Microbiol. Immunol. 158:1-24 (1992) and replication-deficient recombinant adenovirus (Berkner, BioTechniques 6:616-629 (1988), Berkner, Current Top. Microbiol. Immunol. 158:39-66 (1992), Brody and Crystal, Annal. New York Acad. Sci. 716:90-103 (1994). Non-viral based vectors include protein/DNA complexes (Cristiano et al., PNAS USA. 90:2122-2126 (1993), Curiel et al., PNAS USA 88:8850-8854 (1991), Curiel, Annal. New York Acad. Sci. 716:36-58 (1994), electroporation and liposome mediated delivery such as cationic liposomes (Farhood et al., Annal. New York Acad. Sci. 716:23-35 (1994).
The present invention provides an improvement to the existing methods of expanding hematopoietic cells, which new genetic material has been introduced, in that it provides methods utilizing chimera proteins that have improved biological activity, including an activity not seen by any single colony stimulation factor and/or physical properties.
Many drugs may cause bone marrow suppression or hematopoietic deficiencies. Examples of such drugs are AZT, DDI, alkylating agents and anti-metabolites used in chemotherapy, antibiotics such as chloramphenicol, penicillin, gancyclovir, daunomycin and sulfa drugs, phenothiazones, tranquilizers such as meprobamate, analgesics such as aminopyrine and dipyrone, anti convulsants such as phenytoin or carbamazepine, antithyroids such as propylthiouracil and methimazole and diuretics. Hematopoietic cells that have been expanded ex-vivo using the chimera molecules of the present invention may be useful in preventing or treating the bone marrow suppression or hematopoietic deficiencies which often occur in patients treated with these drugs.
Hematopoietic deficiencies may also occur as a result of viral, microbial or parasitic infections and as a result of treatment for renal disease or renal failure, e.g., dialysis. Hematopoietic cells that have been expanded ex-vivo using the chimera molecules of the present invention may be useful in treating such hematopoietic deficiency.
Various immunodeficiencies e.g., in T and/or B lymphocytes, or immune disorders, e.g., rheumatoid arthritis, may also be beneficially affected by treatment with hematopoietic cells that have been expanded ex-vivo using the chimera molecules of the present invention. Immunodeficiencies may be the result of viral infections e.g. HTLVI, HTLVII, HTLVIII, severe exposure to radiation, cancer therapy or the result of other medical treatment. The chimera molecules of the present invention may also be employed, alone or in combination with other hematopoietic growth factors, in the treatment of other blood cell deficiencies, including thrombocytopenia (platelet deficiency), or anemia. Other uses for these novel polypeptides are in the treatment of patients recovering from bone marrow transplants.
As indicated above, the therapeutic method may also include co-administration with other human factors. A non-exclusive list of other appropriate hematopoietic growth factors, colony stimulating factors, cytokines, lymphokines, hematopoietic growth factors and interleukins for simultaneous or serial co-administration with the polypeptides of the present invention includes GM-CSF, CSF-1, G-CSF, G-CSF Ser7, c-mpl ligand (MGDF or TPO), c-mpl receptor agonists disclosed in PCT/US96/15938, M-CSF, erythropoietin (EPO), IL-1, IL-4, IL-2, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, LIF, flt3 ligand, B-cell growth factor, B-cell differentiation factor and eosinophil differentiation factor, stem cell factor (SCF) also known as steel factor or c-kit ligand, multi-functional hematopoietic receptor agonists disclosed in PCT/US96/15774, or combinations thereof.
The treatment of hematopoietic deficiency may include removing hematopoietic cell from a patient, culturing the cell in a medium containing the chimera molecules to differentiate and proliferate the cells and returning the cultured cells to the patient following a medical treatment. In addition, hematopoietic cell can be removed from a blood donor, cultured and given to a patient suffering from a hematopoietic disorder.
›DETAILED DESCRIPTION OF THE INVENTION · 5 of 6
The present invention is directed to methods of ex-vivo expansion of hematopoietic cells by culturing the cells with a chimeric proteins(s) of the formula:
R 1 -L-R 2 , R 2 -L-R 1 , R 1 -R 2 , R 2 -R 1 , R 1 -L-R 1 and R 1 -R 1
wherein R 1 is a human interleukin-3 mutant polypeptide of the Formula:
(SEQ ID NO:1)
Ala Pro Met Thr Gln Thr Thr Ser Leu Lys Thr Ser
1 5 10
Trp Val Asn Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
15 20
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
25 30 35
Xaa Xaa Asn Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
40 45
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
50 55 60
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
65 70
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
75 80
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
85 90 95
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Phe Xaa
100 105
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
110 115 120
Xaa Xaa Xaa Gln Gln Thr Thr Leu Ser Leu Ala Ile
125 130
Phe
wherein
Xaa at position 17 is Ser, Lys, Gly, Asp, Met, Gln, or Arg;
Xaa at position 18 is Asn, His, Leu, Ile, Phe, Arg, or Gln;
Xaa at position 19 is Met, Phe, Ile, Arg, Gly, Ala, or Cys;
Xaa at position 20 is Ile, Cys, Gln, Glu, Arg, Pro, or Ala;
Xaa at position 21 is Asp, Phe, Lys, Arg, Ala, Gly, Glu, Gln, Asn, Thr, Ser or Val;
Xaa at position 22 is Glu, Trp, Pro, Ser, Ala, His, Asp, Asn, Gln, Leu, Val or Gly;
Xaa at position 23 is Ile, Val, Ala, Leu, Gly, Trp, Lys, Phe, Ser, or Arg;
Xaa at position 24 is Ile, Gly, Val, Arg, Ser, Phe, or Leu;
Xaa at position 25 is Thr, His, Gly, Gln, Arg, Pro, or Ala;
Xaa at position 26 is His, Thr, Phe, Gly, Arg, Ala, or Trp;
Xaa at position 27 is Leu, Gly, Arg, Thr, Ser, or Ala;
Xaa at position 28 is Lys, Arg, Leu, Gln, Gly, Pro, Val or Trp;
Xaa at position 29 is Gln, Asn, Leu, Pro, Arg, or Val;
Xaa at position 30 is Pro, His, Thr, Gly, Asp, Gln, Ser, Leu, or Lys;
Xaa at position 31 is Pro, Asp, Gly, Ala, Arg, Leu, or Gln;
Xaa at position 32 is Leu, Val, Arg, Gln, Asn, Gly, Ala, or Glu;
Xaa at position 33 is Pro, Leu, Gln, Ala, Thr, or Glu;
Xaa at position 34 is Leu, Val, Gly, Ser, Lys, Glu, Gln, Thr, Arg, Ala, Phe, Ile or Met;
Xaa at position 35 is Leu, Ala, Gly, Asn, Pro, Gln, or Val;
Xaa at position 36 is Asp, Leu, or Val;
Xaa at position 37 is Phe, Ser, Pro, Trp, or Ile;
Xaa at position 38 is Asn, or Ala;
Xaa at position 40 is Leu, Trp, or Arg;
Xaa at position 41 is Asn, Cys, Arg, Leu, His, Met, or Pro;
Xaa at position 42 is Gly, Asp, Ser, Cys, Asn, Lys, Thr, Leu, Val, Glu, Phe, Tyr, Ile, Met or Ala;
Xaa at position 43 is Glu, Asn, Tyr, Leu, Phe, Asp, Ala, Cys, Gln, Arg, Thr, Gly or Ser;
Xaa at position 44 is Asp, Ser, Leu, Arg, Lys, Thr, Met, Trp, Glu, Asn, Gln, Ala or Pro;
Xaa at position 45 is Gln, Pro, Phe, Val, Met, Leu, Thr, Lys, Trp, Asp, Asn, Arg, Ser, Ala, Ile, Glu or His;
Xaa at position 46 is Asp, Phe, Ser, Thr, Cys, Glu, Asn, Gln, Lys, His, Ala, Tyr, Ile, Val or Gly;
Xaa at position 47 is Ile, Gly, Val, Ser, Arg, Pro, or His;
Xaa at position 48 is Leu, Ser, Cys, Arg, Ile, His, Phe, Glu, Lys, Thr, Ala, Met, Val or Asn;
Xaa at position 49 is Met, Arg, Ala, Gly, Pro, Asn, His, or Asp;
Xaa at position 50 is Glu, Leu, Thr, Asp, Tyr, Lys, Asn, Ser, Ala, Ile, Val, His, Phe, Met or Gln;
Xaa at position 51 is Asn, Arg, Met, Pro, Ser, Thr, or His;
Xaa at position 52 is Asn, His, Arg, Leu, Gly, Ser, or Thr;
Xaa at position 53 is Leu, Thr, Ala, Gly, Glu, Pro, Lys, Ser, or Met;
Xaa at position 54 is Arg, Asp, Ile, Ser, Val, Thr, Gln, Asn, Lys, His, Ala or Leu;
Xaa at position 55 is Arg, Thr, Val, Ser, Leu, or Gly;
Xaa at position 56 is Pro, Gly, Cys, Ser, Gln, Glu, Arg, His, Thr, Ala, Tyr, Phe, Leu, Val or Lys;
Xaa at position 57 is Asn or Gly;
Xaa at position 58 is Leu, Ser, Asp, Arg, Gln, Val, or Cys;
Xaa at position 59 is Glu, Tyr, His, Leu, Pro, or Arg;
Xaa at position 60 is Ala, Ser, Pro, Tyr, Asn, or Thr;
Xaa at position 61 is Phe, Asn, Glu, Pro, Lys, Arg, or Ser;
Xaa at position 62 is Asn, His, Val, Arg, Pro, Thr, Asp, or Ile;
Xaa at position 63 is Arg, Tyr, Trp, Lys, Ser, His, Pro, or Val;
Xaa at position 64 is Ala, Asn, Pro, Ser, or Lys;
Xaa at position 65 is Val, Thr, Pro, His, Leu, Phe, or Ser;
Xaa at position 66 is Lys, Ile, Arg, Val, Asn, Glu, or Ser;
Xaa at position 67 is Ser, Ala, Phe, Val, Gly, Asn, Ile, Pro, or His;
Xaa at position 68 is Leu, Val, Trp, Ser, Ile, Phe, Thr, or His;
Xaa at position 69 is Gln, Ala, Pro, Thr, Glu, Arg, Trp, Gly, or Leu;
Xaa at position 70 is Asn, Leu, Val, Trp, Pro, or Ala;
Xaa at position 71 is Ala, Met, Leu, Pro, Arg, Glu, Thr, Gln, Trp, or Asn;
Xaa at position 72 is Ser, Glu, Met, Ala, His, Asn, Arg, or Asp;
Xaa at position 73 is Ala, Glu, Asp, Leu, Ser, Gly, Thr, or Arg;
Xaa at position 74 is Ile, Met, Thr, Pro, Arg, Gly, Ala;
Xaa at position 75 is Glu, Lys, Gly, Asp, Pro, Trp, Arg, Ser, Gln, or Leu;
Xaa at position 76 is Ser, Val, Ala, Asn, Trp, Glu, Pro, Gly, or Asp;
Xaa at position 77 is Ile, Ser, Arg, Thr, or Leu;
Xaa at position 78 is Leu, Ala, Ser, Glu, Phe, Gly, or Arg;
Xaa at position 79 is Lys, Thr, Asn, Met, Arg, Ile, Gly, or Asp;
Xaa at position 80 is Asn, Trp, Val, Gly, Thr, Leu, Glu, or Arg;
Xaa at position 81 is Leu, Gln, Gly, Ala, Trp, Arg, Val, or Lys;
Xaa at position 82 is Leu, Gln, Lys, Trp, Arg, Asp, Glu, Asn, His, Thr, Ser, Ala, Tyr, Phe, Ile, Met or Val;
Xaa at position 83 is Pro, Ala, Thr, Trp, Arg, or Met;
Xaa at position 84 is Cys, Glu, Gly, Arg, Met, or Val;
Xaa at position 85 is Leu, Asn, Val, or Gln;
Xaa at position 86 is Pro, Cys, Arg, Ala, or Lys;
Xaa at position 87 is Leu, Ser, Trp, or Gly;
Xaa at position 88 is Ala, Lys, Arg, Val, or Trp;
Xaa at position 89 is Thr, Asp, Cys, Leu, Val, Glu, His, Asn, or Ser;
Xaa at position 90 is Ala, Pro, Ser, Thr, Gly, Asp, Ile, or Met;
Xaa at position 91 is Ala, Pro, Ser, Thr, Phe, Leu, Asp, or His;
Xaa at position 92 is Pro, Phe, Arg, Ser, Lys, His, Ala, Gly, Ile or Leu;
›DETAILED DESCRIPTION OF THE INVENTION · 6 of 6
Xaa at position 93 is Thr, Asp, Ser, Asn, Pro, Ala, Leu, or Arg;
Xaa at position 94 is Arg, Ile, Ser, Glu, Leu, Val, Gln, Lys, His, Ala, or Pro;
Xaa at position 95 is His, Gln, Pro, Arg, Val, Leu, Gly, Thr, Asn, Lys, Ser, Ala, Trp, Phe, Ile, or Tyr;
Xaa at position 96 is Pro, Lys, Tyr, Gly, Ile, or Thr;
Xaa at position 97 is Ile, Val, Lys, Ala, or Asn;
Xaa at position 98 is His, Ile, Asn, Leu, Asp, Ala, Thr, Glu, Gln, Ser, Phe, Met, Val, Lys, Arg, Tyr or Pro;
Xaa at position 99 is Ile, Leu, Arg, Asp, Val, Pro, Gln, Gly, Ser, Phe, or His;
Xaa at position 100 is Lys, Tyr, Leu, His, Arg, Ile, Ser, Gln, or Pro;
Xaa at position 101 is Asp, Pro, Met, Lys, His, Thr, Val, Tyr, Glu, Asn, Ser, Ala, Gly, Ile, Leu, or Gln;
Xaa at position 102 is Gly, Leu, Glu, Lys, Ser, Tyr, or Pro;
Xaa at position 103 is Asp, or Ser;
Xaa at position 104 is Trp, Val, Cys, Tyr, Thr, Met, Pro, Leu, Gln, Lys, Ala, Phe, or Gly;
Xaa at position 105 is Asn, Pro, Ala, Phe, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp, or His;
Xaa at position 106 is Glu, Ser, Ala, Lys, Thr, Ile, Gly, or Pro;
Xaa at position 108 is Arg, Lys, Asp, Leu, Thr, Ile, Gln, His, Ser, Ala or Pro;
Xaa at position 109 is Arg, Thr, Pro, Glu, Tyr, Leu, Ser, or Gly;
Xaa at position 110 is Lys, Ala, Asn, Thr, Leu, Arg, Gln, His, Glu, Ser, or Trp;
Xaa at position 111 is Leu, Ile, Arg, Asp, or Met;
Xaa at position 112 is Thr, Val, Gln, Tyr, Glu, His, Ser, or Phe;
Xaa at position 113 is Phe, Ser, Cys, His, Gly, Trp, Tyr, Asp, Lys, Leu, Ile, Val or Asn;
Xaa at position 114 is Tyr, Cys, His, Ser, Trp, Arg, or Leu;
Xaa at position 115 is Leu, Asn, Val, Pro, Arg, Ala, His, Thr, Trp, or Met;
Xaa at position 116 is Lys, Leu, Pro, Thr, Met, Asp, Val, Glu, Arg, Trp, Ser, Asn, His, Ala, Tyr, Phe, Gln, or Ile;
Xaa at position 117 is Thr, Ser, Asn, Ile, Trp, Lys, or Pro;
Xaa at position 118 is Leu, Ser, Pro, Ala, Glu, Cys, Asp, or Tyr;
Xaa at position 119 is Glu, Ser, Lys, Pro, Leu, Thr, Tyr, or Arg;
Xaa at position 120 is Asn, Ala, Pro, Leu, His, Val, or Gln;
Xaa at position 121 is Ala, Ser, Ile, Asn, Pro, Lys, Asp, or Gly;
Xaa at position 122 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys;
Xaa at position 123 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu;
wherein from 1 to 14 amino acids can be deleted from the N-terminus and/or from 1 to 15 amino acids can be deleted from the C-terminus; and wherein from 4 to 44 of the amino acids designated by Xaa are different from the corresponding amino acids of native (1-133) human interleukin-3;
R 2 is a hematopoietic growth factor;
L is a linker capable of linking R 1 and R 2 ; and said chimera protein can additionally be immediately preceded by (methionine −1 ), (alanine −1 ), or (methionine −2 , alanine −1 ).
In a preferred embodiment, R 2 is a hematopoietic growth factor selected from the group consisting of GM-CSF, CSF-1, G-CSF, G-CSF Ser 7 , c-mpl ligand (MGDF or TPO), M-CSF, erythropoietin (EPO), IL-1, IL-4, IL-2, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, LIF, flt3 ligand, human growth hormone, B-cell growth factor, B-cell differentiation factor, eosinophil differentiation factor and stem cell factor (SCF).
The dosage regimen involved in ex-vivo expansion of hematopoietic cells and methods for treating the above-described conditions will be determined by the attending physician considering various factors which modify the action of drugs, e.g. the condition, body weight, sex and diet of the patient, the severity of any infection, time of administration and other clinical factors. Generally, a dosage regimen may be in the range of 1 ng to 100 ng of non-glycosylated IL-3 chimeric protein per mL of culture medium. This dosage regimen is referenced to a standard level of biological activity which recognizes that native IL-3 generally possesses an EC 50 at or about 10 picoMolar to 100 picoMolar in the AML proliferation assay described herein. Therefore, dosages would be adjusted relative to the activity of a given chimera protein vs. the activity of native (reference) IL-3 and it would not be unreasonable to note that dosage regimens may include doses as low as 0.1 ng and as high as 1 milligram per mL of culture medium. In addition, there may exist specific circumstances where dosages of chimera protein would be adjusted higher or lower. When administered with the chimera proteins of the present invention, other hematopoietic growth factors are used in the range of 1 ng to 100 ng per mL of culture medium. The other hematopoietic growth factors could be used as low as 1 pg/mL and as high as 1 mg/mL depending on the chimeria protein used, the various combination of hematopoietic growth factors used and the nature of the expanded hematopoietic cell population that is desired. Other factors that could effect the dosage of the chimera proteins and other hematopoietic growth factors include; co-administration with chemotherapeutic drugs and/or radiation; the use of glycosylated proteins; and various patient-related issues mentioned earlier in this section.
The following examples will illustrate the invention in greater detail although it will be understood that the invention is not limited to these specific examples.
›Examples31
›EXAMPLE 1 · 1 of 2
Determination of the In Vitro Activity of Chimera Proteins
The protein concentration of the chimera protein can be determined using a sandwich ELISA based on an affinity purified polyclonal antibody. Alternatively the protein concentration can be determined by amino acid composition. The bioactivity of the chimera molecule can be determined in a number of in vitro assays compared with native IL-3, the IL-3 variant or G-CSF alone or together. One such assay is the AML-193 cell proliferation assay. AML-193 cells respond to IL-3 and G-CSF which allows for the combined bioactivity of the IL-3 variant/G-CSF chimera to be determined. In addition other factor dependent cell lines, such as M-NFS-60 (ATCC. CRL 1838) or 32D which are murine IL-3 dependent cell line, may be used. The activity of IL-3 is species specific whereas G-CSF is not, therefor the bioactivity of the G-CSF component of the IL-3 variant/G-CSF chimera can be determined independently. The methylcellulose assay can be used to determine the effect of the IL-3 variant/G-CSF chimera protein on the expansion of the hematopoietic progenitor cells and the pattern of the different types of hematopoietic colonies in vitro. The methylcellulose assay can provide an estimate of precursor frequency since one measures the frequency of progenitors per 100,000 input cells. Long term, stromal dependent cultures have been used to delineate primitive hematopoietic progenitors and stem cells. This assay can be used to determine whether the chimera molecule stimulates the expansion of very primitive progenitors and/or stem cells. In addition, limiting dilution cultures can be performed which will indicate the frequency of primitive progenitors stimulated by the chimera molecule.
Determination of Bioactivity of Chimera Molecules in AML Proliferation Assay
The AML assay is useful for determining the activity of chimera molecules that respond to hIL-3, G-CSF and The factor-dependent cell line AML 193 was obtained from the American Type Culture Collection (ATCC, Rockville, Md.). This cell line, established from a patient with acute myelogenous leukemia, is a growth factor dependent cell line which displayed enhanced growth in GM-CSF supplemented medium (Lange, B., et al., Blood 70:192, 1987; Valtieri, M., et al., J. Immunol. 138:4042, 1987). The ability of AML 193 cells to proliferate in the presence of human IL-3 has also been documented. (Santoli, D., et al., J. Immunology 139:348, 1987). A cell line variant was used, AML 193 1.3, which was adapted for long term growth in IL-3 by washing out the growth factors and starving the cytokine dependent AML 193 cells for growth factors for 24 hours. The cells are then replated at 1×10 5 cells/well in a 24 well plate in media containing 100 U/mL IL-3. It took approximately 2 months for the cells to grow rapidly in IL-3. These cells are maintained as AML 193 1.3 thereafter by supplementing tissue culture medium (see below) with human IL-3.
AML 193 1.3 cells are washed 6 times in cold Hanks balanced salt solution (HBSS, Gibco, Grand Island, N.Y.) by centrifuging cell suspensions at 250×g for 10 minutes followed by decantation of the supernatant. Pelleted cells are resuspended in HBSS and the procedure is repeated until six wash cycles are completed. Cells washed six times by this procedure are resuspended in tissue culture medium at a density ranging from 2×10 5 to 5×10 5 viable cells/mL. This medium is prepared by supplementing Iscove's modified Dulbecco's Medium (IMDM, Hazelton, Lenexa, Kans.) with albumin, transferrin, lipids and 2-mercaptoethanol. Bovine albumin (Boehringer-Mannheim, Indianapolis, Ind.) is added at 500 μg/mL; human transferrin (Boehringer-Mannheim, Indianapolis, Ind.) is added at 100 μg/mL; soybean lipid (Boehringer-Mannheim, Indianapolis, Ind.) is added at 50 μg/mL; and 2-mercaptoethanol (Sigma, St. Louis, Mo.) is added at 5×10-5 M.
Serial dilutions of human interleukin-3 or chimera protein (hIL-3 mutein) are made in triplicate series in tissue culture medium supplemented as stated above in 96 well Costar 3596 tissue culture plates. Each well contained 50 μl of medium containing interleukin-3 or chimera protein once serial dilutions are completed. Control wells contained tissue culture medium alone (negative control). AML 193 1.3 cell suspensions prepared as above are added to each well by pipetting 50 μl (2.5×10 4 cells) into each well. Tissue culture plates are incubated at 37° C. with 5% CO 2 in humidified air for 3 days. On day 3, 0.5 μCi 3 H-thymidine (2 Ci/mM, New England Nuclear, Boston, Mass.) is added in 50 μl of tissue culture medium. Cultures are incubated at 37° C. with 5% CO 2 in humidified air for 18-24 hours. Cellular DNA is harvested onto glass filter mats (Pharmacia LKB, Gaithersburg, Md.) using a TOTED cell harvester (TOMTEC, Orange, Conn.) which utilized a water wash cycle followed by a 70% ethanol wash cycle. Filter mats are allowed to air dry and then placed into sample bags to which scintillation fluid (Scintiverse II, Fisher Scientific, St. Louis, Mo. or BetaPlate Scintillation Fluid, Pharmacia LKB, Gaithersburg, Md.) is added. Beta emissions of samples from individual tissue culture wells are counted in a LKB Betaplate model 1205 scintillation counter (Pharmacia LKB, Gaithersburg, Md.) and data is expressed as counts per minute of 3 H-thymidine incorporated into cells from each tissue culture well. Activity of each human interleukin-3 preparation or chimera protein preparation is quantitated by measuring cell proliferation ( 3 H-thymidine incorporation) induced by graded concentrations of interleukin-3 or chimera protein. Typically, concentration ranges from 0.05 pM-10 5 pM are quantitated in these assays. Activity is determined by measuring the dose of interleukin-3 or chimera molecule which provides 50% of maximal proliferation (EC 50 =0.5×(maximum average counts per minute of 3 H-thymidine incorporated per well among triplicate cultures of all concentrations of interleukin-3 tested—background proliferation measured by 3 H-thymidine incorporation observed in triplicate cultures lacking interleukin-3). This EC 50 value is also equivalent to 1 unit of bioactivity. Every assay is performed with native interleukin-3 as a reference standard so that relative activity levels could be assigned.
›EXAMPLE 1 · 2 of 2
Typically, the protein chimeras were tested in a concentration range of 2000 pM to 0.06 pM titrated in serial 2 fold dilutions. Biological activity of the chimera molecules was compared to the following standards as described below.
Protein chimeras comprised in part of G-CSF, pMON3987, pMON3995, pMON3997, pMON26406, pMON26433, pMON26415, pMON26416, and pMON26430, were compared to the dose response curve of equal molar concentrations of hG-CSF and pMON13288 or pMON13416.
Protein chimeras comprised in part of GM-CSF, pMON3989 and pMON3998 were compared to the dose response curve of equal molar concentrations of hGM-CSF and pMON13288. Protein chimeras comprised of dimers of hIL-3 variants, pMON3988, pMON26425, pMON26427, pMON26420, pMON26429 and pMON26431 were compared to the dose response curve of pMON13288 or pMON13416.
Activity for each sample was determined by the concentration which gave 50% of the maximal response by fitting a four-parameter logistic model to the data. It was observed that the upper plateau (maximal response) for the sample and the standard with which it was compared did not differ. Therefore relative potency calculation for each sample was determined from EC50 estimations for the sample and the standard as indicated above. Relative potency (EC50 of standard divided by EC50 of sample) reported in Table 3 is the mean of at least two independent assays unless indicated. AML 193.1.3 cells proliferate in response to hIL-3, hGM-CSF and hG-CSF.
›EXAMPLE 2
Determination of Bioactivity of Chimera Molecules in Methylcellulose Assay
This assays the ability of hematopoietic growth factors to stimulate normal bone marrow cells to produce different types of hematopoietic colonies in vitro (Bradley et al., Aust. Exp. Biol. Med. Sci. 44:287-300 1966; Pluznik et al., J Cell Comp Physiol 66:319-324 1965).
Methods
Approximately 30 mL of fresh, normal, healthy bone marrow aspirate are obtained from individuals. Under sterile conditions samples are diluted 1:5 with a 1×PBS (#14040.059 Life Technologies, Gaithersburg, Md.) solution in a 50 mL conical tube (#25339-50 Corning, Corning Md.). Ficoll (Histopaque 1077 Sigma H-8889) is layered under the diluted sample and centrifuged, 300×g for 30 min. The mononuclear cell band is removed and washed two times in 1×PBS and once with 1% BSA PBS (CellPro Co., Bothel, Wash.). Mononuclear cells are counted and CD34+ cells are selected using the Ceprate LC (CD34) Kit (CellPro Co., Bothel, Wash.) column. This fractionation is performed since all stem and progenitor cells within the bone marrow display CD34 surface antigen.
Cultures are set up in triplicate with a final volume of 1.0 mL in a 35×10 mm petri dish (Nunc#174926). Culture medium is purchased from Terry Fox Labs. (HCC-4230 medium (Terry Fox Labs, Vancouver, B. C., Canada) and erythropoietin (Amgen, Thousands Oaks, Calif.) is added to the culture media. 3,000-10,000 CD34+ cells are added per dish. Native IL-3 and chimera molecules are added to give final concentrations ranging from 0.001 nM 10 nM. Native IL-3 and chimera molecules are supplied in house. G-CSF (Neupogen) is from Amgen.
Cultures are resuspended using a 3 cc syringe and 1.0 mL is dispensed per dish. Control (baseline response) cultures received no hematopoietic growth factors . Positive control cultures received conditioned media (PHA stimulated human cells; Terry Fox Lab. H2400). Cultures are incubated at 37° C., 5% CO 2 in humidified air. Hematopoietic colonies which are defined as greater than 50 cells are counted on the day of peak response (days 10-11) using a Nikon inverted phase microscope with a 40× objective combination. Groups of cells containing fewer than 50 cells are referred to as clusters. Alternatively colonies can be identified by spreading the colonies on a slide and stained or they can be picked, resuspended and spun onto cytospin slides for staining.
›EXAMPLE 3
Determination of Bioactivity of Chimera Molecules in Human Cord Blood Hematopoietic Growth Factor Assay
Bone marrow cells are traditionally used for in vitro assays of hematopoietic growth factor activity. However, human bone marrow is not always available, and there is considerable variability between donors. Umbilical cord blood is comparable to bone marrow as a source of hematopoietic stem cells and progenitors (Broxmeyer et al., Proc. Natl. Acad. Sci. USA, 89:4109-4113 1992; Mayani et al., Blood 81:3252-3258 1993). In contrast to bone marrow, cord blood is more readily available on a regular basis. There is also a potential to reduce assay variability by pooling cells obtained fresh from several donors, or to create a bank of cryopreserved cells for this purpose. By modifying the culture conditions, and/or analyzing for lineage specific markers, it should be possible to assay specifically for granulocyte/macrophage colonies (CFU-GM), for megakaryocyte CSF activity, or for high proliferative potential colony forming cell (HPP-CFC) activity.
Methods
Mononuclear cells (MNC) are isolated from cord blood within 24 hr. of collection, using a standard density gradient (1.077 g/mL Histopaque). Cord blood MNC have been further enriched for stem cells and progenitors by several procedures, including immunomagnetic selection for CD14−, CD34+ cells; panning for SBA−, CD34+ fraction using coated flasks from Applied Immune Science (Santa Clara, Calif.); and CD34+ selection using a CellPro (Bothell, Wash.) avidin column. Either freshly isolated or cryopreserved CD34+ cell enriched fractions are used for the assay. Duplicate cultures for each serial dilution of sample (concentration range from 1 pM to 1204 pM) are prepared with 1×10 4 cells in 1 ml of 0.9% methylcellulose containing medium without additional growth factors (Methocult H4230 from Stem Cell Technologies, Vancouver, BC.). In some experiments, Methocult H4330 containing erythropoietin (EPO) was used instead of Methocult H4230, or Stem Cell Factor (SCF), 50 ng/mL (Biosource International, Camarillo, Calif.) was added. After culturing for 7-9 days, colonies containing >30 cells are counted. In order to rule out subjective bias in scoring, assays are scored blind.
›EXAMPLE 4
Determination of Bioactivity of Chimera Molecules in Megakaryocyte Proliferation Assay
Methods
1. Bone Marrow Proliferation Assay
a. CD34+ Cell Purification
Between 15-20 mL bone marrow aspirates were obtained from normal allogeneic marrow donors after informed consent. Cells were diluted 1:3 in phosphate buffered saline (PBS, Gibco-BRL), 30 mL were layered over 15 mL Histopaque-1077 (Sigma) and centrifuged for 30 minutes at 300 RCF. The mononuclear interface layer was collected and washed in PBS. CD34+ cells were enriched from the mononuclear cell preparation using an affinity column per manufacturers instructions (CellPro, Inc, Bothell Wash.). After enrichment, the purity of CD34+ cells was 70% on average as determined by using flow cytometric analysis using anti CD34 monoclonal antibody conjugated to fluorescein and anti CD38 conjugated to phycoerythrin (Becton Dickinson, San Jose Calif.).
Cells were resuspended at 40,000 cells/mL in X-Vivo 10 media (Bio-Whittaker, Walkersville, Md.) and 1 mL was plated in 12-well tissue culture plates (Costar). The growth factor rhIL-3 was added at 100 ng/mL (pMON5873) was added to some wells. hIL3 variant, pMON13288, was used at 10 ng/mL or 100 ng/mL. Conditioned media from BHK cells transfected with plasmid encoding c-mpl ligand were tested by addition of 100 μl of supernatant added to 1 mL cultures (approximately a 10% dilution). Cells were incubated at 37° C. for 8-14 days at 5% CO 2 in a 37° C. humidified incubator.
b. Cell Harvest and Analysis
At the end of the culture period a total cell count was obtained for each condition. For fluorescence analysis and ploidy determination cells were washed in megakaryocyte buffer (MK buffer, 13.6 mM Sodium Citrate, 1 mM Theophylline, 2.2 μm PGE1, 11 mM Glucose, 3% w/v BSA, in PBS, pH 7.4,) (Tomer et al., Blood 70(6):1735-1742, 1987) resuspended in 500 μl of MK buffer containing anti-CD41a FITC antibody (1:200, AMAC, Westbrook, Me.) and washed in MK buffer. For DNA analysis cells were permeablized in MK buffer containing 0.5% Tween 20 (Fisher, Fair Lawn N.J.)for 20 min. on ice followed by fixation in 0.5% Tween-20 and 1% paraformaldehyde (Fisher Chemical) for 30 minutes followed by incubation in Propidium Iodide (Calbiochem , La Jolla Calif.) (50 μg/mL) with RNA-ase (400 U/mL) in 55% v/v MK buffer (200 mOsm) for 1-2 hours on ice. Cells were analyzed on a FACScan or Vantage flow cytometer (Becton Dickinson, San Jose, Calif.). Green fluorescence (CD41a-FITC) was collected along with linear and log signals for red fluorescence (PI) to determine DNA ploidy. All cells were collected to determine the percent of cells that were CD41+. Data analysis was performed using software by LYSIS (Becton Dickinson, San Jose, Calif.). Percent of cells expressing the CD41 antigen was obtained from flow cytometry analysis(Percent). Absolute (Abs) number of CD41+ cells/mL was calculated by: (Abs)=(Cell Count)*(Percent)/100.
2. Megakaryocyte Fibrin Clot Assay
CD34+ enriched population were isolated as described above. Cells were suspended at 25,000 cells/mL with/without cytokine(s) in a media consisting of a base Iscoves IMDM media supplemented with 0.3% BSA, 0.4 mg/mL apo-transferrin, 6.67 μM FeCl 2 , 25 μg/mL CaCl 2 , 25 μg/mL L-asparagine, 500 μg/mL E-amino-n-caproic acid and Penicillin/Streptomycin. Prior to plating into 35 mm plates, thrombin was added (0.25 Units/mL) to initiate clot formation. Cells were incubated at 37° C. for 13 days at 5% CO 2 in a 37° C. humidified incubator.
At the end of the culture period plates were fixed with Methanol:Acetone (1:3), air dried and stored at −200° C. until staining. A peroxidase immunocytochemistry staining procedure was used (Zymed, Histostain-SP. San Francisco, Calif.) using a cocktail of primary monoclonal antibodies consisting of anti CD41a, CD42 and CD61. Colonies were counted after staining and classified as negative, CFU-MK (small colonies, 1-2 foci and less that approx. 25 cells), BFU-MK (large, multi-foci colonies with >25 cells) or mixed colonies (mixture of both positive and negative cells.
›EXAMPLE 5
Ex Vivo Expansion of CD34+ Cells from Peripheral Blood Using Chimera Molecules pMON13056 and pMON13148+/−SCF
Flow Cytometry Evaluation
The percentage of CD34+ cells in the thawed peripheral blood cell population was determined by flow cytometry. Cells were removed from the selected cell population and placed into two centrifuge tube and washed once in 9/1% albumin Phosphate buffer (PAB). Twenty microliters of anti-CD34 monoclonal antibody (8G12-FITC) or mouse monoclonal antibody IgG-FITC control was added to the tube. The tubes were incubated for 15 minutes on ice. The cells were washed once with PAB and resuspended in approximately 0.5 mL PAB. Propidium iodide (2 ug/mL) was added to each tube just prior to the analysis on the FACSort or FACScan. Selected cells that contain greater than 80% CD34+ cells were used to initiate the cultures.
On day 12, cultures were harvested and evaluated with CD41A-FITC (a megakaryocyte marker), CD15-FITC and CD11b-PE (early to late neutrophil marker) and CD34 by flow cytometry, using the same processes of preparation and analysis as described above.
Colony Assay Evaluation
Colony assay evaluation was performed on day 0 with 500-1000 selected CD34+ cells per dish and again on day 12 of culture with 5,000-10,000 cultured cells per dish. The cells were added to a colony assay culture tube containing 3 mL of Terry Fox Iscove's based methylcellulose and the following growth factors: 20 ng/mL SCF, 10 U/mL EPO, 300 U/mL GM-CSF, 300 U/mL G-CSF, 30 U/mL IL3 and 40 ng/mL IL6. Two 35 mm tissue culture dishes containing 1 mL were set up. All dishes were incubated at 37° C., 5% carbon dioxide, 5% oxygen and high humidity for 13-15 days. The dishes were scored for myeloid (CFU-GM), erythroid (BFU-E) or mixed myeloid and erythroid colonies (CFU-mix) using a Nikon SMZU stereoscope.
Cell Morphology Evaluation
On day 12 of culture cells were analyzed for cell morphology after Wright-Giemsa staining. Cultured cells were cytocentrifuged onto slides at 1000 rpm for 4 minutes. Each slide contained approximately 10000-20000 cells. Slides were allowed to air dry before staining with 0.5 mL Wright-Giemsa for 1 minutes and 0.5 mL tap water for 1-2 minutes. Slides were cover-slipped and evaluated using a Microstar light microscope. A differential cell count of neutrophils, megakaryocytes and other blood cells was performed.
Results
CD34+ Selection
Studies were performed on CD34+ cells selected using the Isolex™ 300 magnetic Cell Separator from apheresis products from normal donors mobilized with G-CSF. The selected cells were stored in X-VIVO 10 +12.5%HSA containing 10% DMSO in liquid nitrogen until required. Cultures were initiated as described in the methods section.
Proliferation Index of Cultures At Day 12
The proliferation index of cultures was calculated by diving the cell concentration at day 5-7 by 5×10 4 and then multiplying it by the cell concentration at day 12 divided by 1×10 5 . A summary of the proliferation index obtained from these CD34+ cell cultures is shown in Table 4.
Flow Cytometry Evaluation of Neutrophil Precursors
The percentage of neutrophil precursors in the CD34+ cell cultures at day 12 was assessed by flow cytometry using the CD15 marker for early to late neutrophil precursors and the CD1b marker found on late neutrophil precursors determined is shown in Table 4.
Flow Cytometry Evaluation of Megakaryocytes
The percentage of Mks in the CD34+ cell cultures was assessed by flow cytometry using the CD41a marker for megakaryocytes. The percentage of Mks observed in the CD34+ cell cultures is shown in Table 4.
Flow Cytometry Evaluation of CD34+ Cells
The percentage of CD34+ cells present in the cultures at day 12 was determined by flow cytometry. The percentage of CD34+ cells still remaining in the cultures at day 12 ranged from 0.103-19.3%, with no significant difference or patterns observed with the different growth factor combinations.
Total Number of Megakaryocytes Generated in Culture
The total number of megakaryocytes present in each culture is calculated by multiplying the total number of cells at day 12 by the percentage of CD15+ cells and is shown in Table 4.
Colony Forming Unit Granulocyte-Macrophage (CFU-GM) Index
CFU-GM index is calculate by dividing the total number of GM-colonies obtained at day 12 by the number of GM-colonies obtained at day 0. A CFU-GM index of 1 indicates that the number of colonies at day 12 is equivalent to the number of colonies at the start of the culture. A summary of the CFU-GM index for these cultures is shown in Table 4.
Colony Forming Unit (CFU) Index
CFU index is calculated by dividing the total number of colonies (CFU-GM, BFU-E and mixed) obtained at day 12 by the total number of colonies obtained at day 0. A CFU index of 1 indicates that the number of colonies at day 12 is equivalent to the number of colonies at the start of the culture. A summary of the CFU index for these cultures is shown in Table 4.
›EXAMPLE 5 · 1 of 25
Ex Vivo Expansion of CD34+ Cells from Bone Marrow Using pMON13056 vs. Native IL-3+/−G-CSF
Cells were cultured as in Example 4 except CD34+ cells were isolated from normal bone marrow. Native IL-3, IL-3 variant (pMON13288) and G-CSF were used at 50 ng/mL and pMON13056 was used at 100 ng/ml of culture medium. Starting cell number for each treatment was 20×10E4. The total cell expansion is shown in Table 5.
Amino acids are shown herein by standard one letter or three letter abbreviations as follows:
Further details known to those skilled in the art may be found in T. Maniatis, et al., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory (1982) and references cited therein, incorporated herein by reference; and in J. Sambrook, et al., Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory (1989) and references cited therein, incorporated herein by reference.
Additional details on the IL-3 variants of the present invention may be found in co-pending U.S. patent application Ser. No. 08/411,795 (WO 94/12638) which is hereby incorporated by reference in its entirety as if written herein.
Additional details on how to make the chimera protein can be found in WO 95/21254, WO 92/04455 and WO 91/02754.
Additional details about the lymphokine and the variants thereof can be found in U.S. Pat. Nos. 4,810,643, and 5,218,092 and E.P. Application 02174004.
All references, patents or applications cited herein are incorporated by reference in their entirety as if written herein.
Various other examples will be apparent to the person skilled in the art after reading the present disclosure without departing from the spirit and scope of the invention. It is intended that all such other examples be included within the scope of the appended claims.
Ala Pro Met Thr Gln Thr Thr Ser Leu Lys Thr Ser Trp Val Asn Cys
1 5 10 15
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
20 25 30
Xaa Xaa Xaa Xaa Xaa Xaa Asn Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
35 40 45
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
50 55 60
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
65 70 75 80
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
85 90 95
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Phe Xaa Xaa Xaa Xaa Xaa
100 105 110
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gln Gln Thr Thr Leu
115 120 125
Ser Leu Ala Ile Phe
130
Ala Pro Met Thr Gln Thr Thr Ser Leu Lys Thr Ser Trp Val Asn Cys
1 5 10 15
Xaa Xaa Xaa Ile Xaa Glu Xaa Xaa Xaa Xaa Leu Lys Xaa Xaa Xaa Xaa
20 25 30
Xaa Xaa Xaa Asp Xaa Xaa Asn Leu Asn Xaa Glu Xaa Xaa Xaa Ile Leu
35 40 45
Met Xaa Xaa Asn Leu Xaa Xaa Xaa Asn Leu Glu Xaa Phe Xaa Xaa Xaa
50 55 60
Xaa Xaa Xaa Xaa Xaa Asn Xaa Xaa Xaa Ile Glu Xaa Xaa Leu Xaa Xaa
65 70 75 80
Leu Xaa Xaa Cys Xaa Pro Xaa Xaa Thr Ala Xaa Pro Xaa Arg Xaa Xaa
85 90 95
Xaa Xaa Xaa Xaa Xaa Gly Asp Xaa Xaa Xaa Phe Xaa Xaa Lys Leu Xaa
100 105 110
Phe Xaa Xaa Xaa Xaa Leu Glu Xaa Xaa Xaa Xaa Gln Gln Thr Thr Leu
115 120 125
Ser Leu Ala Ile Phe
130
Ala Pro Met Thr Gln Thr Thr Ser Leu Lys Thr Ser Trp Val Asn Cys
1 5 10 15
Xaa Xaa Met Ile Asp Glu Xaa Ile Xaa Xaa Leu Lys Xaa Xaa Pro Xaa
20 25 30
Pro Xaa Xaa Asp Phe Xaa Asn Leu Asn Xaa Glu Asp Xaa Xaa Ile Leu
35 40 45
Met Xaa Xaa Asn Leu Arg Xaa Xaa Asn Leu Glu Ala Phe Xaa Arg Xaa
50 55 60
Xaa Lys Xaa Xaa Xaa Asn Ala Ser Ala Ile Glu Xaa Xaa Leu Xaa Xaa
65 70 75 80
Leu Xaa Pro Cys Leu Pro Xaa Xaa Thr Ala Xaa Pro Xaa Arg Xaa Pro
85 90 95
Ile Xaa Xaa Xaa Xaa Gly Asp Trp Xaa Glu Phe Xaa Xaa Lys Leu Xaa
100 105 110
Phe Tyr Leu Xaa Xaa Leu Glu Xaa Xaa Xaa Xaa Gln Gln Thr Thr Leu
115 120 125
Ser Leu Ala Ile Phe
130
Asn Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
1 5 10 15
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Xaa Xaa Xaa Xaa Xaa Xaa Xaa
20 25 30
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
35 40 45
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
50 55 60
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
65 70 75 80
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Phe Xaa Xaa Xaa
85 90 95
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gln Gln
100 105 110
Asn Cys Xaa Xaa Xaa Ile Xaa Glu Xaa Xaa Xaa Xaa Leu Lys Xaa Xaa
1 5 10 15
Xaa Xaa Xaa Xaa Xaa Asp Xaa Xaa Asn Leu Asn Xaa Glu Xaa Xaa Xaa
20 25 30
Ile Leu Met Xaa Xaa Asn Leu Xaa Xaa Xaa Asn Leu Glu Xaa Phe Xaa
35 40 45
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Xaa Xaa Xaa Ile Glu Xaa Xaa Leu
50 55 60
Xaa Xaa Leu Xaa Xaa Cys Xaa Pro Xaa Xaa Thr Ala Xaa Pro Xaa Arg
65 70 75 80
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Asp Xaa Xaa Xaa Phe Xaa Xaa Lys
85 90 95
Leu Xaa Phe Xaa Xaa Xaa Xaa Leu Glu Xaa Xaa Xaa Xaa Gln Gln
100 105 110
Asn Cys Xaa Xaa Met Ile Asp Glu Xaa Ile Xaa Xaa Leu Lys Xaa Xaa
1 5 10 15
Pro Xaa Pro Xaa Xaa Asp Phe Xaa Asn Leu Asn Xaa Glu Asp Xaa Xaa
20 25 30
Ile Leu Met Xaa Xaa Asn Leu Arg Xaa Xaa Asn Leu Glu Ala Phe Xaa
35 40 45
Arg Xaa Xaa Lys Xaa Xaa Xaa Asn Ala Ser Ala Ile Glu Xaa Xaa Leu
50 55 60
Xaa Xaa Leu Xaa Pro Cys Leu Pro Xaa Xaa Thr Ala Xaa Pro Xaa Arg
65 70 75 80
Xaa Pro Ile Xaa Xaa Xaa Xaa Gly Asp Trp Xaa Glu Phe Xaa Xaa Lys
85 90 95
Leu Xaa Phe Tyr Leu Xaa Xaa Leu Glu Xaa Xaa Xaa Xaa Gln Gln
100 105 110
Ala Pro Met Thr Gln Thr Thr Ser Leu Lys Thr Ser Trp Val Asn Cys
1 5 10 15
Ser Xaa Xaa Xaa Asp Glu Xaa Ile Xaa His Leu Lys Xaa Pro Pro Xaa
20 25 30
Pro Xaa Leu Asp Xaa Xaa Asn Leu Asn Xaa Glu Asp Xaa Xaa Ile Leu
35 40 45
Xaa Xaa Xaa Asn Leu Arg Xaa Xaa Asn Leu Xaa Xaa Phe Xaa Xaa Ala
50 55 60
Xaa Lys Xaa Leu Xaa Asn Ala Ser Xaa Ile Glu Xaa Ile Leu Xaa Asn
65 70 75 80
Leu Xaa Pro Cys Xaa Pro Xaa Xaa Thr Ala Xaa Pro Xaa Arg Xaa Pro
85 90 95
Ile Xaa Ile Xaa Xaa Gly Asp Trp Xaa Glu Phe Arg Xaa Lys Leu Xaa
100 105 110
Phe Tyr Leu Xaa Xaa Leu Glu Xaa Ala Gln Xaa Gln Gln Thr Thr Leu
115 120 125
Ser Leu Ala Ile Phe
130
Asn Cys Ser Xaa Xaa Xaa Asp Glu Xaa Ile Xaa His Leu Lys Xaa Pro
1 5 10 15
Pro Xaa Pro Xaa Leu Asp Xaa Xaa Asn Leu Asn Xaa Glu Asp Xaa Xaa
20 25 30
Ile Leu Xaa Xaa Xaa Asn Leu Arg Xaa Xaa Asn Leu Xaa Xaa Phe Xaa
35 40 45
Xaa Ala Xaa Lys Xaa Leu Xaa Asn Ala Ser Xaa Ile Glu Xaa Ile Leu
50 55 60
Xaa Asn Xaa Xaa Pro Cys Xaa Pro Xaa Xaa Thr Ala Xaa Pro Xaa Arg
›EXAMPLE 5 · 2 of 25
65 70 75 80
Xaa Pro Ile Xaa Ile Xaa Xaa Gly Asp Trp Xaa Glu Phe Arg Xaa Lys
85 90 95
Leu Xaa Phe Tyr Leu Xaa Xaa Leu Glu Xaa Ala Gln Xaa Gln Gln
100 105 110
111 amino acids
amino acid
linear
peptide
9
Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys Arg Pro
1 5 10 15
Pro Ala Pro Leu Leu Asp Pro Asn Asn Leu Asn Ala Glu Asp Val Asp
20 25 30
Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn
35 40 45
Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser Ile Leu
50 55 60
Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg
65 70 75 80
His Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys
85 90 95
Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln
100 105 110
111 amino acids
amino acid
linear
peptide
10
Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys Arg Pro
1 5 10 15
Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn Ser Glu Asp Met Asp
20 25 30
Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn
35 40 45
Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser Ile Leu
50 55 60
Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg
65 70 75 80
His Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys
85 90 95
Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln
100 105 110
111 amino acids
amino acid
linear
peptide
11
Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys Val Pro
1 5 10 15
Pro Ala Pro Leu Leu Asp Ser Asn Asn Leu Asn Ser Glu Asp Met Asp
20 25 30
Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn
35 40 45
Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser Ile Leu
50 55 60
Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg
65 70 75 80
His Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys
85 90 95
Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln
100 105 110
111 amino acids
amino acid
linear
peptide
12
Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln Pro
1 5 10 15
Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp
20 25 30
Ile Leu Met Glu Arg Asn Leu Arg Leu Pro Asn Leu Leu Ala Phe Val
35 40 45
Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Ala Ile Glu Ser Ile Leu
50 55 60
Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg
65 70 75 80
His Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys
85 90 95
Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln
100 105 110
111 amino acids
amino acid
linear
peptide
13
Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln Pro
1 5 10 15
Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp
20 25 30
Ile Leu Met Glu Arg Asn Leu Arg Leu Pro Asn Leu Glu Ser Phe Val
35 40 45
Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Ala Ile Glu Ser Ile Leu
50 55 60
Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg
65 70 75 80
His Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys
85 90 95
Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln
100 105 110
111 amino acids
amino acid
linear
peptide
14
Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln Pro
1 5 10 15
Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp
20 25 30
Ile Leu Met Glu Arg Asn Leu Arg Thr Pro Asn Leu Leu Ala Phe Val
35 40 45
Arg Ala Val Lys His Leu Glu Asn Ala Ser Ala Ile Glu Ser Ile Leu
50 55 60
Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg
65 70 75 80
His Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys
85 90 95
Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln
100 105 110
111 amino acids
amino acid
linear
peptide
15
Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln Pro
1 5 10 15
Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp
20 25 30
Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn
35 40 45
Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Gly Ile Glu Ala Ile Leu
50 55 60
Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg
65 70 75 80
His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Arg Lys
85 90 95
Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln
100 105 110
111 amino acids
amino acid
linear
peptide
16
Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln Pro
1 5 10 15
Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp
20 25 30
Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn
35 40 45
Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Gly Ile Glu Ala Ile Leu
50 55 60
Arg Asn Leu Val Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg
65 70 75 80
His Pro Ile Thr Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Arg Lys
85 90 95
Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln
100 105 110
111 amino acids
amino acid
linear
peptide
17
Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln Pro
1 5 10 15
Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp
20 25 30
Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn
35 40 45
Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser Ile Leu
50 55 60
Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg
65 70 75 80
His Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Glu Lys
85 90 95
Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln Gln
100 105 110
111 amino acids
amino acid
linear
peptide
18
Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln Pro
1 5 10 15
Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp
20 25 30
Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn
35 40 45
Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser Ile Leu
50 55 60
Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg
65 70 75 80
His Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Glu Lys
85 90 95
Leu Thr Phe Tyr Leu Val Ser Leu Glu His Ala Gln Glu Gln Gln
100 105 110
111 amino acids
amino acid
linear
peptide
19
Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln Pro
1 5 10 15
Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp
20 25 30
Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn
35 40 45
Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Gly Ile Glu Ala Ile Leu
50 55 60
Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg
65 70 75 80
His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu Lys
›EXAMPLE 5 · 3 of 25
85 90 95
Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln Gln
100 105 110
111 amino acids
amino acid
linear
peptide
20
Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln Pro
1 5 10 15
Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp
20 25 30
Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn
35 40 45
Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Gly Ile Glu Ala Ile Leu
50 55 60
Arg Asn Leu Val Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg
65 70 75 80
His Pro Ile Thr Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu Lys
85 90 95
Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln Gln
100 105 110
111 amino acids
amino acid
linear
peptide
21
Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln Pro
1 5 10 15
Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp
20 25 30
Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn
35 40 45
Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Gly Ile Glu Ala Ile Leu
50 55 60
Arg Asn Leu Val Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg
65 70 75 80
His Pro Ile Thr Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu Lys
85 90 95
Leu Thr Phe Tyr Leu Val Ser Leu Glu His Ala Gln Glu Gln Gln
100 105 110
111 amino acids
amino acid
linear
peptide
22
Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys Arg Pro
1 5 10 15
Pro Ala Pro Leu Leu Asp Pro Asn Asn Leu Asn Ala Glu Asp Val Asp
20 25 30
Ile Leu Met Glu Arg Asn Leu Arg Leu Pro Asn Leu Glu Ser Phe Val
35 40 45
Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Ala Ile Glu Ser Ile Leu
50 55 60
Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg
65 70 75 80
His Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys
85 90 95
Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln
100 105 110
111 amino acids
amino acid
linear
peptide
23
Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys Arg Pro
1 5 10 15
Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn Ser Glu Asp Met Asp
20 25 30
Ile Leu Met Glu Arg Asn Leu Arg Thr Pro Asn Leu Leu Ala Phe Val
35 40 45
Arg Ala Val Lys His Leu Glu Asn Ala Ser Ala Ile Glu Ser Ile Leu
50 55 60
Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg
65 70 75 80
His Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys
85 90 95
Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln
100 105 110
111 amino acids
amino acid
linear
peptide
24
Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys Val Pro
1 5 10 15
Pro Ala Pro Leu Leu Asp Ser Asn Asn Leu Asn Ser Glu Asp Met Asp
20 25 30
Ile Leu Met Glu Arg Asn Leu Arg Leu Pro Asn Leu Leu Ala Phe Val
35 40 45
Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Ala Ile Glu Ser Ile Leu
50 55 60
Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg
65 70 75 80
His Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys
85 90 95
Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln
100 105 110
113 amino acids
amino acid
linear
peptide
25
Met Ala Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys
1 5 10 15
Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp
20 25 30
Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala
35 40 45
Phe Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Gly Ile Glu Ala
50 55 60
Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro
65 70 75 80
Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg
85 90 95
Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln
100 105 110
Gln
113 amino acids
amino acid
linear
peptide
26
Met Ala Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys
1 5 10 15
Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp
20 25 30
Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala
35 40 45
Phe Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Gly Ile Glu Ala
50 55 60
Ile Leu Arg Asn Leu Val Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro
65 70 75 80
Ser Arg His Pro Ile Thr Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg
85 90 95
Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln
100 105 110
Gln
113 amino acids
amino acid
linear
peptide
27
Met Ala Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys
1 5 10 15
Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp
20 25 30
Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala
35 40 45
Phe Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Gly Ile Glu Ala
50 55 60
Ile Leu Arg Asn Leu Val Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro
65 70 75 80
Ser Arg His Pro Ile Thr Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg
85 90 95
Glu Lys Leu Thr Phe Tyr Leu Val Ser Leu Glu His Ala Gln Glu Gln
100 105 110
Gln
113 amino acids
amino acid
linear
peptide
28
Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys
1 5 10 15
Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn Leu Asn Ala Glu Asp
20 25 30
Val Asp Ile Leu Met Glu Arg Asn Leu Arg Leu Pro Asn Leu Glu Ser
35 40 45
Phe Val Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Ala Ile Glu Ser
50 55 60
Ile Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro
65 70 75 80
Thr Arg His Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg
85 90 95
Arg Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln
100 105 110
Gln
113 amino acids
amino acid
linear
peptide
29
Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys
1 5 10 15
Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn Ser Glu Asp
20 25 30
Met Asp Ile Leu Met Glu Arg Asn Leu Arg Thr Pro Asn Leu Leu Ala
35 40 45
Phe Val Arg Ala Val Lys His Leu Glu Asn Ala Ser Ala Ile Glu Ser
50 55 60
Ile Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro
65 70 75 80
Thr Arg His Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg
85 90 95
Arg Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln
100 105 110
Gln
113 amino acids
amino acid
linear
peptide
30
Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys
1 5 10 15
Val Pro Pro Ala Pro Leu Leu Asp Ser Asn Asn Leu Asn Ser Glu Asp
20 25 30
Met Asp Ile Leu Met Glu Arg Asn Leu Arg Leu Pro Asn Leu Leu Ala
35 40 45
Phe Val Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Ala Ile Glu Ser
50 55 60
Ile Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro
65 70 75 80
Thr Arg His Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg
85 90 95
Arg Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln
›EXAMPLE 5 · 4 of 25
100 105 110
Gln
113 amino acids
amino acid
linear
peptide
31
Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys
1 5 10 15
Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn Leu Asn Ala Glu Asp
20 25 30
Val Asp Ile Leu Met Glu Arg Asn Leu Arg Leu Pro Asn Leu Glu Ser
35 40 45
Phe Val Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Gly Ile Glu Ala
50 55 60
Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro
65 70 75 80
Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg
85 90 95
Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln
100 105 110
Gln
113 amino acids
amino acid
linear
peptide
32
Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys
1 5 10 15
Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn Ser Glu Asp
20 25 30
Met Asp Ile Leu Met Glu Arg Asn Leu Arg Thr Pro Asn Leu Leu Ala
35 40 45
Phe Val Arg Ala Val Lys His Leu Glu Asn Ala Ser Gly Ile Glu Ala
50 55 60
Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro
65 70 75 80
Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg
85 90 95
Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln
100 105 110
Gln
113 amino acids
amino acid
linear
peptide
33
Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys
1 5 10 15
Val Pro Pro Ala Pro Leu Leu Asp Ser Asn Asn Leu Asn Ser Glu Asp
20 25 30
Met Asp Ile Leu Met Glu Arg Asn Leu Arg Leu Pro Asn Leu Leu Ala
35 40 45
Phe Val Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Gly Ile Glu Ala
50 55 60
Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro
65 70 75 80
Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg
85 90 95
Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln
100 105 110
Gln
113 amino acids
amino acid
linear
peptide
34
Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys
1 5 10 15
Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn Leu Asn Ala Glu Asp
20 25 30
Val Asp Ile Leu Met Glu Arg Asn Leu Arg Leu Pro Asn Leu Glu Ser
35 40 45
Phe Val Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Gly Ile Glu Ala
50 55 60
Ile Leu Arg Asn Leu Val Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro
65 70 75 80
Ser Arg His Pro Ile Thr Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg
85 90 95
Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln
100 105 110
Gln
113 amino acids
amino acid
linear
peptide
35
Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys
1 5 10 15
Val Pro Pro Ala Pro Leu Leu Asp Ser Asn Asn Leu Asn Ser Glu Asp
20 25 30
Met Asp Ile Leu Met Glu Arg Asn Leu Arg Leu Pro Asn Leu Leu Ala
35 40 45
Phe Val Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Gly Ile Glu Ala
50 55 60
Ile Leu Arg Asn Leu Val Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro
65 70 75 80
Ser Arg His Pro Ile Thr Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg
85 90 95
Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln
100 105 110
Gln
113 amino acids
amino acid
linear
peptide
36
Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys
1 5 10 15
Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn Ser Glu Asp
20 25 30
Met Asp Ile Leu Met Glu Arg Asn Leu Arg Thr Pro Asn Leu Leu Ala
35 40 45
Phe Val Arg Ala Val Lys His Leu Glu Asn Ala Ser Gly Ile Glu Ala
50 55 60
Ile Leu Arg Asn Leu Val Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro
65 70 75 80
Ser Arg His Pro Ile Thr Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg
85 90 95
Glu Lys Leu Thr Phe Tyr Leu Val Ser Leu Glu His Ala Gln Glu Gln
100 105 110
Gln
113 amino acids
amino acid
linear
peptide
37
Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys
1 5 10 15
Val Pro Pro Ala Pro Leu Leu Asp Ser Asn Asn Leu Asn Ser Glu Asp
20 25 30
Met Asp Ile Leu Met Glu Arg Asn Leu Arg Leu Pro Asn Leu Leu Ala
35 40 45
Phe Val Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Gly Ile Glu Ala
50 55 60
Ile Leu Arg Asn Leu Val Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro
65 70 75 80
Ser Arg His Pro Ile Thr Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg
85 90 95
Glu Lys Leu Thr Phe Tyr Leu Val Ser Leu Glu His Ala Gln Glu Gln
100 105 110
Gln
113 amino acids
amino acid
linear
peptide
38
Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys
1 5 10 15
Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn Ser Glu Asp
20 25 30
Met Asp Ile Leu Met Glu Arg Asn Leu Arg Thr Pro Asn Leu Leu Ala
35 40 45
Phe Val Arg Ala Val Lys His Leu Glu Asn Ala Ser Gly Ile Glu Ala
50 55 60
Ile Leu Arg Asn Leu Val Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro
65 70 75 80
Ser Arg His Pro Ile Thr Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg
85 90 95
Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln
100 105 110
Gln
113 amino acids
amino acid
linear
peptide
39
Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys
1 5 10 15
Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn Leu Asn Ala Glu Asp
20 25 30
Val Asp Ile Leu Met Glu Arg Asn Leu Arg Leu Pro Asn Leu Glu Ser
35 40 45
Phe Val Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Gly Ile Glu Ala
50 55 60
Ile Leu Arg Asn Leu Val Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro
65 70 75 80
Ser Arg His Pro Ile Thr Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg
85 90 95
Glu Lys Leu Thr Phe Tyr Leu Val Ser Leu Glu His Ala Gln Glu Gln
100 105 110
Gln
113 amino acids
amino acid
linear
peptide
40
Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys
1 5 10 15
Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn Leu Asn Ala Glu Asp
20 25 30
Val Asp Ile Leu Met Asp Arg Asn Leu Arg Leu Ser Asn Leu Glu Ser
35 40 45
Phe Val Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Gly Ile Glu Ala
50 55 60
Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro
65 70 75 80
Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg
85 90 95
Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln
100 105 110
Gln
113 amino acids
amino acid
linear
peptide
41
Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ala Ile His His Leu Lys
1 5 10 15
Arg Pro Pro Ala Pro Ser Leu Asp Pro Asn Asn Leu Asn Asp Glu Asp
20 25 30
Met Ser Ile Leu Met Glu Arg Asn Leu Arg Leu Pro Asn Leu Glu Ser
35 40 45
Phe Val Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Gly Ile Glu Ala
50 55 60
Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro
65 70 75 80
Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg
85 90 95
Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln
›EXAMPLE 5 · 5 of 25
100 105 110
Gln
113 amino acids
amino acid
linear
peptide
42
Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys
1 5 10 15
Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn Leu Asn Asp Glu Asp
20 25 30
Met Ser Ile Leu Met Glu Arg Asn Leu Arg Leu Pro Asn Leu Glu Ser
35 40 45
Phe Val Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Gly Ile Glu Ala
50 55 60
Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro
65 70 75 80
Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg
85 90 95
Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln
100 105 110
Gln
113 amino acids
amino acid
linear
peptide
43
Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys
1 5 10 15
Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn Leu Asn Ala Glu Asp
20 25 30
Val Asp Ile Leu Met Asp Arg Asn Leu Arg Leu Pro Asn Leu Glu Ser
35 40 45
Phe Val Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Gly Ile Glu Ala
50 55 60
Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro
65 70 75 80
Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg
85 90 95
Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln
100 105 110
Gln
113 amino acids
amino acid
linear
peptide
44
Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys
1 5 10 15
Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn Leu Asn Asp Glu Asp
20 25 30
Val Ser Ile Leu Met Glu Arg Asn Leu Arg Leu Pro Asn Leu Glu Ser
35 40 45
Phe Val Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Gly Ile Glu Ala
50 55 60
Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro
65 70 75 80
Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg
85 90 95
Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln
100 105 110
Gln
113 amino acids
amino acid
linear
peptide
45
Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys
1 5 10 15
Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn Leu Asn Asp Glu Asp
20 25 30
Met Ser Ile Leu Met Glu Arg Asn Leu Arg Leu Pro Asn Leu Glu Ser
35 40 45
Phe Val Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Gly Ile Glu Ala
50 55 60
Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro
65 70 75 80
Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg
85 90 95
Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln
100 105 110
Gln
125 amino acids
amino acid
linear
peptide
46
Met Ala Tyr Pro Glu Thr Asp Tyr Lys Asp Asp Asp Asp Lys Asn Cys
1 5 10 15
Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys Arg Pro Pro Ala
20 25 30
Pro Leu Leu Asp Pro Asn Asn Leu Asn Ala Glu Asp Val Asp Ile Leu
35 40 45
Met Glu Arg Asn Leu Arg Leu Pro Asn Leu Glu Ser Phe Val Arg Ala
50 55 60
Val Lys Asn Leu Glu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn
65 70 75 80
Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His Pro
85 90 95
Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu Lys Leu Thr
100 105 110
Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln Gln
115 120 125
125 amino acids
amino acid
linear
peptide
47
Met Ala Tyr Pro Glu Thr Asp Tyr Lys Asp Asp Asp Asp Lys Asn Cys
1 5 10 15
Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys Arg Pro Pro Asn
20 25 30
Pro Leu Leu Asp Pro Asn Asn Leu Asn Ser Glu Asp Met Asp Ile Leu
35 40 45
Met Glu Arg Asn Leu Arg Thr Pro Asn Leu Leu Ala Phe Val Arg Ala
50 55 60
Val Lys His Leu Glu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn
65 70 75 80
Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His Pro
85 90 95
Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu Lys Leu Thr
100 105 110
Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln Gln
115 120 125
113 amino acids
amino acid
linear
peptide
48
Met Ala Asn Cys Ser Ile Met Ile Asp Glu Leu Ile His His Leu Lys
1 5 10 15
Ile Pro Pro Asn Pro Ser Leu Asp Ser Ala Asn Leu Asn Ser Glu Asp
20 25 30
Val Ser Ile Leu Met Glu Arg Asn Leu Arg Thr Pro Asn Leu Leu Ala
35 40 45
Phe Val Arg Ala Val Lys His Leu Glu Asn Ala Ser Gly Ile Glu Ala
50 55 60
Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro
65 70 75 80
Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg
85 90 95
Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln
100 105 110
Gln
134 amino acids
amino acid
linear
peptide
49
Met Ala Pro Met Thr Gln Thr Thr Ser Leu Lys Thr Ser Trp Val Asn
1 5 10 15
Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln Pro Pro
20 25 30
Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp Ile
35 40 45
Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn Arg
50 55 60
Ala Val Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser Ile Leu Lys
65 70 75 80
Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His
85 90 95
Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu
100 105 110
Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln Thr Thr
115 120 125
Leu Ser Leu Ala Ile Phe
130
36 amino acids
amino acid
linear
peptide
50
Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Glu Gly Gly Gly
1 5 10 15
Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser
20 25 30
Gly Gly Gly Ser
35
24 amino acids
amino acid
linear
peptide
51
Ile Ser Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn Pro Ser Pro Pro
1 5 10 15
Ser Lys Glu Ser His Lys Ser Pro
20
28 amino acids
amino acid
linear
peptide
52
Ile Glu Gly Arg Ile Ser Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn
1 5 10 15
Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro
20 25
906 base pairs
nucleic acid
double
linear
DNA (genomic)
53
ATGGCTAACT GCTCTATAAT GATCGATGAA ATTATACATC ACTTAAAGAG ACCACCTAAC 60
CCTTTGCTGG ACCCGAACAA CCTCAATTCT GAAGACATGG ATATCCTGAT GGAACGAAAC 120
CTTCGAACTC CAAACCTGCT CGCATTCGTA AGGGCTGTCA AGCACTTAGA AAATGCATCA 180
GGTATTGAGG CAATTCTTCG TAATCTCCAA CCATGTCTGC CCTCTGCCAC GGCCGCACCC 240
TCTCGACATC CAATCATCAT CAAGGCAGGT GACTGGCAAG AATTCCGGGA AAAACTGACG 300
TTCTATCTGG TTACCCTTGA GCAAGCGCAG GAACAACAGT ACGTAATCGA GGGAAGGATT 360
TCCCCGGGTG GTGGTTCTGG CGGCGGCTCC AACATGGCTA CACCATTAGG CCCTGCCAGC 420
TCCCTGCCCC AGAGCTTCCT GCTCAAGTGC TTAGAGCAAG TGAGGAAGAT CCAGGGCGAT 480
GGCGCAGCGC TCCAGGAGAA GCTGTGTGCC ACCTACAAGC TGTGCCACCC CGAGGAGCTG 540
GTGCTGCTCG GACACTCTCT GGGCATCCCC TGGGCTCCCC TGAGCTCCTG CCCCAGCCAG 600
GCCCTGCAGC TGGCAGGCTG CTTGAGCCAA CTCCATAGCG GCCTTTTCCT CTACCAGGGG 660
CTCCTGCAGG CCCTGGAAGG GATATCCCCC GAGTTGGGTC CCACCTTGGA CACACTGCAG 720
CTGGACGTCG CCGACTTTGC CACCACCATC TAACTGGGAA TGGCCCCTGC CCTGCAGCCC 780
›EXAMPLE 5 · 6 of 25
ACCCAGGGTG CCATGCCGGC CTTCGCCTCT GCTTTCCAGC GCCGGGCAGG AGGGGTCCTG 840
GTTGCTAGCC ATCTGCAGAG CTTCCTGGAG GTGTCGTACC GCGTTCTACG CCACCTTGCG 900
CAGCCC 906
732 base pairs
nucleic acid
double
linear
DNA (genomic)
54
ATGGCTAACT GCTCTATAAT GATCGATGAA ATTATACATC ACTTAAAGAG ACCACCTAAC 60
CCTTTGCTGG ACCCGAACAA CCTCAATTCT GAAGACATGG ATATCCTGAT GGAACGAAAC 120
CTTCGAACTC CAAACCTGCT CGCATTCGTA AGGGCTGTCA AGCACTTAGA AAATGCATCA 180
GGTATTGAGG CAATTCTTCG TAATCTCCAA CCATGTCTGC CCTCTGCCAC GGCCGCACCC 240
TCTCGACATC CAATCATCAT CAAGGCAGGT GACTGGCAAG AATTCCGGGA AAAACTGACG 300
TTCTATCTGG TTACCCTTGA GCAAGCGCAG GAACAACAGT ACGTAATCGA GGGAAGGATT 360
TCCCCGGGTG GTGGTTCTGG CGGCGGCTCC AACATGGCTA ACTGCTCTAT AATGATCGAT 420
GAAATTATAC ATCACTTAAA GAGACCACCT AACCCTTTGC TGGACCCGAA CAACCTCAAT 480
TCTGAAGACA TGGATATCCT GATGGAACGA AACCTTCGAA CTCCAAACCT GCTCGCATTC 540
GTAAGGGCTG TCAAGCACTT AGAAAATGCA TCAGGTATTG AGGCAATTCT TCGTAATCTC 600
CAACCATGTC TGCCCTCTGC CACGGCCGCA CCCTCTCGAC ATCCAATCAT CATCAAGGCA 660
GGTGACTGGC AAGAATTCCG GGAAAAACTG ACGTTCTATC TGGTTACCCT TGAGCAAGCG 720
CAGGAACAAC AG 732
777 base pairs
nucleic acid
double
linear
DNA (genomic)
55
ATGGCTAACT GCTCTATAAT GATCGATGAA ATTATACATC ACTTAAAGAG ACCACCTAAC 60
CCTTTGCTGG ACCCGAACAA CCTCAATTCT GAAGACATGG ATATCCTGAT GGAACGAAAC 120
CTTCGAACTC CAAACCTGCT CGCATTCGTA AGGGCTGTCA AGCACTTAGA AAATGCATCA 180
GGTATTGAGG CAATTCTTCG TAATCTCCAA CCATGTCTGC CCTCTGCCAC GGCCGCACCC 240
TCTCGACATC CAATCATCAT CAAGGCAGGT GACTGGCAAG AATTCCGGGA AAAACTGACG 300
TTCTATCTGG TTACCCTTGA GCAAGCGCAG GAACAACAGT ACGTAATCGA GGGAAGGATT 360
TCCCCGGGTG GTGGTTCTGG CGGCGGCTCC AACATGGCAC CGGCTCGTTC CCCGTCCCCG 420
TCTACCCAGC CGTGGGAACA CGTGAATGCC ATCCAGGAGG CCCGGCGTCT CCTGAACCTG 480
AGTAGAGACA CTGCTGCTGA GATGAATGAA ACAGTAGAAG TGATATCAGA AATGTTTGAC 540
CTCCAGGAGC CGACTTGCCT ACAGACCCGC CTGGAGCTGT ACAAGCAGGG CCTGCGGGGC 600
AGCCTCACCA AGCTCAAGGG CCCCTTGACC ATGATGGCCA GCCACTACAA GCAGCACTGC 660
CCTCCAACCC CGGAAACTTC CTGTGCAACC CAGATTATCA CCTTTGAAAG TTTCAAAGAG 720
AACCTGAAGG ACTTCCTGCT TGTCATCCCC TTTGACTGCT GGGAGCCAGT CCAGGAG 777
921 base pairs
nucleic acid
double
linear
DNA (genomic)
56
ATGGCTAACT GCTCTATAAT GATCGATGAA ATTATACATC ACTTAAAGAG ACCACCTAAC 60
CCTTTGCTGG ACCCGAACAA CCTCAATTCT GAAGACATGG ATATCCTGAT GGAACGAAAC 120
CTTCGAACTC CAAACCTGCT CGCATTCGTA AGGGCTGTCA AGCACTTAGA AAATGCATCA 180
GGTATTGAGG CAATTCTTCG TAATCTCCAA CCATGTCTGC CCTCTGCCAC GGCCGCACCC 240
TCTCGACATC CAATCATCAT CAAGGCAGGT GACTGGCAAG AATTCCGGGA AAAACTGACG 300
TTCTATCTGG TTACCCTTGA GCAAGCGCAG GAACAACAGT ACGTAATCGA GGGAAGGATT 360
TCCCCGGGTG GTGGTTCTGG CGGCGGCTCC AACATGGCTA CACCATTGGG CCCTGCCAGC 420
TCCCTGCCCC AGAGCTTCCT GCTCAAGTCT TTAGAGCAAG TGAGGAAGAT CCAGGGCGAT 480
GGCGCAGCGC TCCAGGAGAA GCTGTGTGCC ACCTACAAGC TGTGCCACCC CGAGGAGCTG 540
GTGCTGCTCG GACACTCTCT GGGCATCCCC TGGGCTCCCC TGAGCTCCTG CCCCAGCCAG 600
GCCCTGCAGC TGGCAGGCTG CTTGAGCCAA CTCCATAGCG GCCTTTTCCT CTACCAGGGG 660
CTCCTGCAGG CCCTGGAAGG GATATCCCCC GAGTTGGGTC CCACCTTGGA CACACTGCAG 720
CTGGACGTCG CCGACTTTGC CACCACCATC TGGCAGCAGA TGGAAGAACT GGGAATGGCC 780
CCTGCCCTGC AGCCCACCCA GGGTGCCATG CCGGCCTTCG CCTCTGCTTT CCAGCGCCGG 840
GCAGGAGGGG TCCTGGTTGC TAGCCATCTG CAGAGCTTCC TGGAGGTGTC GTACCGCGTT 900
CTACGCCACC TTGCGCAGCC C 921
951 base pairs
nucleic acid
double
linear
DNA (genomic)
57
ATGGCTAACT GCTCTATAAT GATCGATGAA ATTATACATC ACTTAAAGAG ACCACCTAAC 60
CCTTTGCTGG ACCCGAACAA CCTCAATTCT GAAGACATGG ATATCCTGAT GGAACGAAAC 120
CTTCGAACTC CAAACCTGCT CGCATTCGTA AGGGCTGTCA AGCACTTAGA AAATGCATCA 180
GGTATTGAGG CAATTCTTCG TAATCTCCAA CCATGTCTGC CCTCTGCCAC GGCCGCACCC 240
TCTCGACATC CAATCATCAT CAAGGCAGGT GACTGGCAAG AATTCCGGGA AAAACTGACG 300
TTCTATCTGG TTACCCTTGA GCAAGCGCAG GAACAACAGT ACGTAATCGA GGGAAGGATT 360
TCCCCGGGTG GTGGTTCTGG CGGCGGCTCC AACATGGCTC CAGTACCACC AGGTGAAGAT 420
TCCAAAGATG TGGCCGCCCC ACACAGACAG CCACTCACCT CTTCAGAACG AATTGACAAA 480
CAAATTCGGT ACATCCTCGA CGGGATATCA GCCCTGAGAA AGGAGACATG TAACAAGAGT 540
AACATGTGTG AAAGCAGCAA AGAGGCGCTA GCAGAAAACA ACCTGAACCT TCCAAAGATG 600
GCTGAAAAAG ATGGATGCTT CCAATCCGGA TTCAATGAGG AGACTTGCCT GGTGAAAATC 660
ATCACTGGTC TTTTGGAGTT TGAGGTATAC CTCGAGTACC TCCAGAACAG ATTTGAGAGT 720
AGTGAGGAAC AAGCCAGAGC TGTGCAGATG TCGACAAAAG TCCTGATCCA GTTCCTGCAG 780
AAAAAGGCAA AGAATCTAGA TGCAATAACC ACCCCTGACC CAACCACAAA TGCATCCCTG 840
CTGACGAAGC TGCAGGCACA GAACCAGTGG CTGCAGGACA TGACAACTCA TCTCATTCTG 900
CGCAGCTTTA AGGAGTTCCT GCAGTCCAGC CTGAGGGCTC TTCGGCAAAT G 951
732 base pairs
nucleic acid
double
linear
DNA (genomic)
58
ATGGCTAACT GCTCTATAAT GATCGATGAA ATTATACATC ACTTAAAGAG ACCACCTAAC 60
CCTTTGCTGG ACCCGAACAA CCTCAATTCT GAAGACATGG ATATCCTGAT GGAACGAAAC 120
CTTCGAACTC CAAACCTGCT CGCATTCGTA AGGGCTGTCA AGCACTTAGA AAATGCATCA 180
GGTATTGAGG CAATTCTTCG TAATCTCCAA CCATGTCTGC CCTCTGCCAC GGCCGCACCC 240
TCTCGACATC CAATCATCAT CAAGGCAGGT GACTGGCAAG AATTCCGGGA AAAACTGACG 300
TTCTATCTGG TTACCCTTGA GCAAGCGCAG GAACAACAGT ACGTAATCGA GGGAAAGATT 360
TCCCCGGGTG GTGGTTCTGG CGGCGGCTCC AACATGGCTA ACTGCTCTAT AATGATCGAT 420
GAAATTATAC ATCACTTAAA GAGACCACCT AACCCTTTGC TGGACCCGAA CAACCTCAAT 480
TCTGAAGACA TGGATATCCT GATGGAACGA AACCTTCGAA CTCCAAACCT GCTCGCATTC 540
GTAAGGGCTG TCAAGCACTT AGAAAATGCA TCAGGTATTG AGGCAATTCT TCGTAATCTC 600
CAACCATGTC TGCCCTCTGC CACGGCCGCA CCCTCTCGAC ATCCAATCAT CATCAAGGCA 660
GGTGACTGGC AAGAATTCCG GGAAAAACTG ACGTTCTATC TGGTTACCCT TGAGCAAGCG 720
CAGGAACAAC AG 732
921 base pairs
nucleic acid
double
linear
DNA (genomic)
59
ATGGCTAACT GCTCTATAAT GATCGATGAA ATTATACATC ACTTAAAGAG ACCACCTAAC 60
CCTTTGCTGG ACCCGAACAA CCTCAATTCT GAAGACATGG ATATCCTGAT GGAACGAAAC 120
CTTCGAACTC CAAACCTGCT CGCATTCGTA AGGGCTGTCA AGCACTTAGA AAATGCATCA 180
GGTATTGAGG CAATTCTTCG TAATCTCCAA CCATGTCTGC CCTCTGCCAC GGCCGCACCC 240
TCTCGACATC CAATCATCAT CAAGGCAGGT GACTGGCAAG AATTCCGGGA AAAACTGACG 300
TTCTATCTGG TTACCCTTGA GCAAGCGCAG GAACAACAGT ACGTAATCGA GGGAAAGATT 360
TCCCCGGGTG GTGGTTCTGG CGGCGGCTCC AACATGGCTA CACCATTGGG CCCTGCCAGC 420
TCCCTGCCCC AGAGCTTCCT GCTCAAGTCT TTAGAGCAAG TGAGGAAGAT CCAGGGCGAT 480
GGCGCAGCGC TCCAGGAGAA GCTGTGTGCC ACCTACAAGC TGTGCCACCC CGAGGAGCTG 540
GTGCTGCTCG GACACTCTCT GGGCATCCCC TGGGCTCCCC TGAGCTCCTG CCCCAGCCAG 600
GCCCTGCAGC TGGCAGGCTG CTTGAGCCAA CTCCATAGCG GCCTTTTCCT CTACCAGGGG 660
CTCCTGCAGG CCCTGGAAGG GATATCCCCC GAGTTGGGTC CCACCTTGGA CACACTGCAG 720
›EXAMPLE 5 · 7 of 25
CTGGACGTCG CCGACTTTGC CACCACCATC TGGCAGCAGA TGGAAGAACT GGGAATGGCC 780
CCTGCCCTGC AGCCCACCCA GGGTGCCATG CCGGCCTTCG CCTCTGCTTT CCAGCGCCGG 840
GCAGGAGGGG TCCTGGTTGC TAGCCATCTG CAGAGCTTCC TGGAGGTGTC GTACCGCGTT 900
CTACGCCACC TTGCGCAGCC C 921
921 base pairs
nucleic acid
double
linear
DNA (genomic)
60
ATGGCTAACT GCTCTATAAT GATCGATGAA ATTATACATC ACTTAAAGAG ACCACCTAAC 60
CCTTTGCTGG ACCCGAACAA CCTCAATTCT GAAGACATGG ATATCCTGAT GGAACGAAAC 120
CTTCGAACTC CAAACCTGCT CGCATTCGTA AGGGCTGTCA AGCACTTAGA AAATGCATCA 180
GGTATTGAGG CAATTCTTCG TAATCTCCAA CCATGTCTGC CCTCTGCCAC GGCCGCACCC 240
TCTCGACATC CAATCATCAT CAAGGCAGGT GACTGGCAAG AATTCCGGGA AAAACTGACG 300
TTCTATCTGG TTACCCTTGA GCAAGCGCAG GAACAACAGT ACGTAGAGGG CGGTGGAGGC 360
TCCCCGGGTG GTGGTTCTGG CGGCGGCTCC AACATGGCTA CACCATTGGG CCCTGCCAGC 420
TCCCTGCCCC AGAGCTTCCT GCTCAAGTCT TTAGAGCAAG TGAGGAAGAT CCAGGGCGAT 480
GGCGCAGCGC TCCAGGAGAA GCTGTGTGCC ACCTACAAGC TGTGCCACCC CGAGGAGCTG 540
GTGCTGCTCG GACACTCTCT GGGCATCCCC TGGGCTCCCC TGAGCTCCTG CCCCAGCCAG 600
GCCCTGCAGC TGGCAGGCTG CTTGAGCCAA CTCCATAGCG GCCTTTTCCT CTACCAGGGG 660
CTCCTGCAGG CCCTGGAAGG GATATCCCCC GAGTTGGGTC CCACCTTGGA CACACTGCAG 720
CTGGACGTCG CCGACTTTGC CACCACCATC TGGCAGCAGA TGGAAGAACT GGGAATGGCC 780
CCTGCCCTGC AGCCCACCCA GGGTGCCATG CCGGCCTTCG CCTCTGCTTT CCAGCGCCGG 840
GCAGGAGGGG TCCTGGTTGC TAGCCATCTG CAGAGCTTCC TGGAGGTGTC GTACCGCGTT 900
CTACGCCACC TTGCGCAGCC C 921
732 base pairs
nucleic acid
double
linear
DNA (genomic)
61
ATGGCTAACT GCTCTATAAT GATCGATGAA ATTATACATC ACTTAAAGAG ACCACCTAAC 60
CCTTTGCTGG ACCCGAACAA CCTCAATTCT GAAGACATGG ATATCCTGAT GGAACGAAAC 120
CTTCGAACTC CAAACCTGCT CGCATTCGTA AGGGCTGTCA AGCACTTAGA AAATGCATCA 180
GGTATTGAGG CAATTCTTCG TAATCTCCAA CCATGTCTGC CCTCTGCCAC GGCCGCACCC 240
TCTCGACATC CAATCATCAT CAAGGCAGGT GACTGGCAAG AATTCCGGGA AAAACTGACG 300
TTCTATCTGG TTACCCTTGA GCAAGCGCAG GAACAACAGT ACGTAGAGGG CGGTGGAGGC 360
TCCCCGGGTG GTGGTTCTGG CGGCGGCTCC AACATGGCTA ACTGCTCTAT AATGATCGAT 420
GAAATTATAC ATCACTTAAA GAGACCACCT AACCCTTTGC TGGACCCGAA CAACCTCAAT 480
TCTGAAGACA TGGATATCCT GATGGAACGA AACCTTCGAA CTCCAAACCT GCTCGCATTC 540
GTAAGGGCTG TCAAGCACTT AGAAAATGCA TCAGGTATTG AGGCAATTCT TCGTAATCTC 600
CAACCATGTC TGCCCTCTGC CACGGCCGCA CCCTCTCGAC ATCCAATCAT CATCAAGGCA 660
GGTGACTGGC AAGAATTCCG GGAAAAACTG ACGTTCTATC TGGTTACCCT TGAGCAAGCG 720
CAGGAACAAC AG 732
777 base pairs
nucleic acid
double
linear
DNA (genomic)
62
ATGGCTAACT GCTCTATAAT GATCGATGAA ATTATACATC ACTTAAAGAG ACCACCTAAC 60
CCTTTGCTGG ACCCGAACAA CCTCAATTCT GAAGACATGG ATATCCTGAT GGAACGAAAC 120
CTTCGAACTC CAAACCTGCT CGCATTCGTA AGGGCTGTCA AGCACTTAGA AAATGCATCA 180
GGTATTGAGG CAATTCTTCG TAATCTCCAA CCATGTCTGC CCTCTGCCAC GGCCGCACCC 240
TCTCGACATC CAATCATCAT CAAGGCAGGT GACTGGCAAG AATTCCGGGA AAAACTGACG 300
TTCTATCTGG TTACCCTTGA GCAAGCGCAG GAACAACAGT ACGTAATCGA GGGAAGGATT 360
TCCCCGGGTG AACCGTCTGG TCCAATCTCT ACTATCAACC CGTCTCCTCC GTCTAAAGAA 420
TCTCATAAAT CTCCAAACAT GGCTAACTGC TCTATAATGA TCGATGAAAT TATACATCAC 480
TTAAAGAGAC CACCTAACCC TTTGCTGGAC CCGAACAACC TCAATTCTGA AGACATGGAT 540
ATCCTGATGG AACGAAACCT TCGAACTCCA AACCTGCTCG CATTCGTAAG GGCTGTCAAG 600
CACTTAGAAA ATGCATCAGG TATTGAGGCA ATTCTTCGTA ATCTCCAACC ATGTCTGCCC 660
TCTGCCACGG CCGCACCCTC TCGACATCCA ATCATCATCA AGGCAGGTGA CTGGCAAGAA 720
TTCCGGGAAA AACTGACGTT CTATCTGGTT ACCCTTGAGC AAGCGCAGGA ACAACAG 777
777 base pairs
nucleic acid
double
linear
DNA (genomic)
63
ATGGCTAACT GCTCTATAAT GATCGATGAA ATTATACATC ACTTAAAGAG ACCACCTAAC 60
CCTTTGCTGG ACCCGAACAA CCTCAATTCT GAAGACATGG ATATCCTGAT GGAACGAAAC 120
CTTCGAACTC CAAACCTGCT CGCATTCGTA AGGGCTGTCA AGCACTTAGA AAATGCATCA 180
GGTATTGAGG CAATTCTTCG TAATCTCCAA CCATGTCTGC CCTCTGCCAC GGCCGCACCC 240
TCTCGACATC CAATCATCAT CAAGGCAGGT GACTGGCAAG AATTCCGGGA AAAACTGACG 300
TTCTATCTGG TTACCCTTGA GCAAGCGCAG GAACAACAGT ACGTAATCGA GGGAAAGATT 360
TCCCCGGGTG AACCGTCTGG TCCAATCTCT ACTATCAACC CGTCTCCTCC GTCTAAAGAA 420
TCTCATAAAT CTCCAAACAT GGCTAACTGC TCTATAATGA TCGATGAAAT TATACATCAC 480
TTAAAGAGAC CACCTAACCC TTTGCTGGAC CCGAACAACC TCAATTCTGA AGACATGGAT 540
ATCCTGATGG AACGAAACCT TCGAACTCCA AACCTGCTCG CATTCGTAAG GGCTGTCAAG 600
CACTTAGAAA ATGCATCAGG TATTGAGGCA ATTCTTCGTA ATCTCCAACC ATGTCTGCCC 660
TCTGCCACGG CCGCACCCTC TCGACATCCA ATCATCATCA AGGCAGGTGA CTGGCAAGAA 720
TTCCGGGAAA AACTGACGTT CTATCTGGTT ACCCTTGAGC AAGCGCAGGA ACAACAG 777
777 base pairs
nucleic acid
double
linear
DNA (genomic)
64
ATGGCTAACT GCTCTATAAT GATCGATGAA ATTATACATC ACTTAAAGAG ACCACCTAAC 60
CCTTTGCTGG ACCCGAACAA CCTCAATTCT GAAGACATGG ATATCCTGAT GGAACGAAAC 120
CTTCGAACTC CAAACCTGCT CGCATTCGTA AGGGCTGTCA AGCACTTAGA AAATGCATCA 180
GGTATTGAGG CAATTCTTCG TAATCTCCAA CCATGTCTGC CCTCTGCCAC GGCCGCACCC 240
TCTCGACATC CAATCATCAT CAAGGCAGGT GACTGGCAAG AATTCCGGGA AAAACTGACG 300
TTCTATCTGG TTACCCTTGA GCAAGCGCAG GAACAACAGT ACGTAGAGGG CGGTGGAGGC 360
TCCCCGGGTG AACCGTCTGG TCCAATCTCT ACTATCAACC CGTCTCCTCC GTCTAAAGAA 420
TCTCATAAAT CTCCAAACAT GGCTAACTGC TCTATAATGA TCGATGAAAT TATACATCAC 480
TTAAAGAGAC CACCTAACCC TTTGCTGGAC CCGAACAACC TCAATTCTGA AGACATGGAT 540
ATCCTGATGG AACGAAACCT TCGAACTCCA AACCTGCTCG CATTCGTAAG GGCTGTCAAG 600
CACTTAGAAA ATGCATCAGG TATTGAGGCA ATTCTTCGTA ATCTCCAACC ATGTCTGCCC 660
TCTGCCACGG CCGCACCCTC TCGACATCCA ATCATCATCA AGGCAGGTGA CTGGCAAGAA 720
TTCCGGGAAA AACTGACGTT CTATCTGGTT ACCCTTGAGC AAGCGCAGGA ACAACAG 777
1047 base pairs
nucleic acid
double
linear
DNA (genomic)
65
ATGGCTAACT GCTCTATAAT GATCGATGAA ATTATACATC ACTTAAAGAG ACCACCTAAC 60
CCTTTGCTGG ACCCGAACAA CCTCAATTCT GAAGACATGG ATATCCTGAT GGAACGAAAC 120
CTTCGAACTC CAAACCTGCT CGCATTCGTA AGGGCTGTCA AGCACTTAGA AAATGCATCA 180
GGTATTGAGG CAATTCTTCG TAATCTCCAA CCATGTCTGC CCTCTGCCAC GGCCGCACCC 240
TCTCGACATC CAATCATCAT CAAGGCAGGT GACTGGCAAG AATTCCGGGA AAAACTGACG 300
TTCTATCTGG TTACCCTTGA GCAAGCGCAG GAACAACAGT ACGTAATCGA GGGAAGGATT 360
TCCCCCGGGC CTCCTGTCAA TGCTGGCGGC GGCTCTGGTG GTGGTTCTGG TGGCGGCTCT 420
GAGGGTGGCG GCTCTGAGGG TGGCGGTTCT GAGGGTGGCG GCTCTGAGGG TGGCGGTTCC 480
GGTGGCGGCT CCGGTTCCGG TGATTTTGAT TATGAAAACA TGGCTACACC ATTGGGCCCT 540
GCCAGCTCCC TGCCCCAGAG CTTCCTGCTC AAGTCTTTAG AGCAAGTGAG GAAGATCCAG 600
GGCGATGGCG CAGCGCTCCA GGAGAAGCTG TGTGCCACCT ACAAGCTGTG CCACCCCGAG 660
GAGCTGGTGC TGCTCGGACA CTCTCTGGGC ATCCCCTGGG CTCCCCTGAG CTCCTGCCCC 720
AGCCAGGCCC TGCAGCTGGC AGGCTGCTTG AGCCAACTCC ATAGCGGCCT TTTCCTCTAC 780
›EXAMPLE 5 · 8 of 25
CAGGGGCTCC TGCAGGCCCT GGAAGGGATA TCCCCCGAGT TGGGTCCCAC CTTGGACACA 840
CTGCAGCTGG ACGTCGCCGA CTTTGCCACC ACCATCTGGC AGCAGATGGA AGAACTGGGA 900
ATGGCCCCTG CCCTGCAGCC CACCCAGGGT GCCATGCCGG CCTTCGCCTC TGCTTTCCAG 960
CGCCGGGCAG GAGGGGTCCT GGTTGCTAGC CATCTGCAGA GCTTCCTGGA GGTGTCGTAC 1020
CGCGTTCTAC GCCACCTTGC GCAGCCC 1047
903 base pairs
nucleic acid
double
linear
DNA (genomic)
66
ATGGCTAACT GCTCTATAAT GATCGATGAA ATTATACATC ACTTAAAGAG ACCACCTAAC 60
CCTTTGCTGG ACCCGAACAA CCTCAATTCT GAAGACATGG ATATCCTGAT GGAACGAAAC 120
CTTCGAACTC CAAACCTGCT CGCATTCGTA AGGGCTGTCA AGCACTTAGA AAATGCATCA 180
GGTATTGAGG CAATTCTTCG TAATCTCCAA CCATGTCTGC CCTCTGCCAC GGCCGCACCC 240
TCTCGACATC CAATCATCAT CAAGGCAGGT GACTGGCAAG AATTCCGGGA AAAACTGACG 300
TTCTATCTGG TTACCCTTGA GCAAGCGCAG GAACAACAGT ACGTAATCGA GGGAAGGATT 360
TCCCCCGGGC CTCCTGTCAA TGCTGGCGGC GGCTCTGGTG GTGGTTCTGG TGGCGGCTCT 420
GAGGGTGGCG GCTCTGAGGG TGGCGGTTCT GAGGGTGGCG GCTCTGAGGG TGGCGGTTCC 480
GGTGGCGGCT CCGGTTCCGG TGATTTTGAT TATGAAAACA TGGCACCGGC TCGTTCCCCG 540
TCCCCGTCTA CCCAGCCGTG GGAACACGTG AATGCCATCC AGGAGGCCCG GCGTCTCCTG 600
AACCTGAGTA GAGACACTGC TGCTGAGATG AATGAAACAG TAGAAGTGAT ATCAGAAATG 660
TTTGACCTCC AGGAGCCGAC TTGCCTACAG ACCCGCCTGG AGCTGTACAA GCAGGGCCTG 720
CGGGGCAGCC TCACCAAGCT CAAGGGCCCC TTGACCATGA TGGCCAGCCA CTACAAGCAG 780
CACTGCCCTC CAACCCCGGA AACTTCCTGT GCAACCCAGA TTATCACCTT TGAAAGTTTC 840
AAAGAGAACC TGAAGGACTT CCTGCTTGTC ATCCCCTTTG ACTGCTGGGA GCCAGTCCAG 900
GAG 903
1017 base pairs
nucleic acid
double
linear
DNA (genomic)
67
ATGGCTAACT GCTCTATAAT GATCGATGAA ATTATACATC ACTTAAAGAG ACCACCTAAC 60
CCTTTGCTGG ACCCGAACAA CCTCAATTCT GAAGACATGG ATATCCTGAT GGAACGAAAC 120
CTTCGAACTC CAAACCTGCT CGCATTCGTA AGGGCTGTCA AGCACTTAGA AAATGCATCA 180
GGTATTGAGG CAATTCTTCG TAATCTCCAA CCATGTCTGC CCTCTGCCAC GGCCGCACCC 240
TCTCGACATC CAATCATCAT CAAGGCAGGT GACTGGCAAG AATTCCGGGA AAAACTGACG 300
TTCTATCTGG TTACCCTTGA GCAAGCGCAG GAACAACAGT ACGTAATCGA GGGAAGGATT 360
TCCCCCGGTG GCGGCGGCTC TGGTGGTGGT TCTGGTGGCG GCTCTGAGGG TGGCGGCTCT 420
GAGGGTGGCG GTTCTGAGGG TGGCGGCTCT GAGGGTGGCG GTTCCGGTGG CGGCTCCGGT 480
TCCGGTAACA TGGCTACACC ATTAGGCCCT GCCAGCTCCC TGCCCCAGAG CTTCCTGCTC 540
AAGTGCTTAG AGCAAGTGAG GAAGATCCAG GGCGATGGCG CAGCGCTCCA GGAGAAGCTG 600
TGTGCCACCT ACAAGCTGTG CCACCCCGAG GAGCTGGTGC TGCTCGGACA CTCTCTGGGC 660
ATCCCCTGGG CTCCCCTGAG CTCCTGCCCC AGCCAGGCCC TGCAGCTGGC AGGCTGCTTG 720
AGCCAACTCC ATAGCGGCCT TTTCCTCTAC CAGGGGCTCC TGCAGGCCCT GGAAGGGATA 780
TCCCCCGAGT TGGGTCCCAC CTTGGACACA CTGCAGCTGG ACGTCGCCGA CTTTGCCACC 840
ACCATCTGGC AGCAGATGGA AGAACTGGGA ATGGCCCCTG CCCTGCAGCC CACCCAGGGT 900
GCCATGCCGG CCTTCGCCTC TGCTTTCCAG CGCCGGGCAG GAGGGGTCCT GGTTGCTAGC 960
CATCTGCAGA GCTTCCTGGA GGTGTCGTAC CGCGTTCTAC GCCACCTTGC GCAGCCC 1017
966 base pairs
nucleic acid
double
linear
DNA (genomic)
68
ATGGCTAACT GCTCTATAAT GATCGATGAA ATTATACATC ACTTAAAGAG ACCACCTAAC 60
CCTTTGCTGG ACCCGAACAA CCTCAATTCT GAAGACATGG ATATCCTGAT GGAACGAAAC 120
CTTCGAACTC CAAACCTGCT CGCATTCGTA AGGGCTGTCA AGCACTTAGA AAATGCATCA 180
GGTATTGAGG CAATTCTTCG TAATCTCCAA CCATGTCTGC CCTCTGCCAC GGCCGCACCC 240
TCTCGACATC CAATCATCAT CAAGGCAGGT GACTGGCAAG AATTCCGGGA AAAACTGACG 300
TTCTATCTGG TTACCCTTGA GCAAGCGCAG GAACAACAGT ACGTAATCGA GGGAAGGATT 360
TCCCCGGGTG AACCGTCTGG TCCAATCTCT ACTATCAACC CGTCTCCTCC GTCTAAAGAA 420
TCTCATAAAT CTCCAAACAT GGCTACACCA TTAGGCCCTG CCAGCTCCCT GCCCCAGAGC 480
TTCCTGCTCA AGTGCTTAGA GCAAGTGAGG AAGATCCAGG GCGATGGCGC AGCGCTCCAG 540
GAGAAGCTGT GTGCCACCTA CAAGCTGTGC CACCCCGAGG AGCTGGTGCT GCTCGGACAC 600
TCTCTGGGCA TCCCCTGGGC TCCCCTGAGC TCCTGCCCCA GCCAGGCCCT GCAGCTGGCA 660
GGCTGCTTGA GCCAACTCCA TAGCGGCCTT TTCCTCTACC AGGGGCTCCT GCAGGCCCTG 720
GAAGGGATAT CCCCCGAGTT GGGTCCCACC TTGGACACAC TGCAGCTGGA CGTCGCCGAC 780
TTTGCCACCA CCATCTGGCA GCAGATGGAA GAACTGGGAA TGGCCCCTGC CCTGCAGCCC 840
ACCCAGGGTG CCATGCCGGC CTTCGCCTCT GCTTTCCAGC GCCGGGCAGG AGGGGTCCTG 900
GTTGCTAGCC ATCTGCAGAG CTTCCTGGAG GTGTCGTACC GCGTTCTACG CCACCTTGCG 960
CAGCCC 966
822 base pairs
nucleic acid
double
linear
DNA (genomic)
69
ATGGCTAACT GCTCTATAAT GATCGATGAA ATTATACATC ACTTAAAGAG ACCACCTAAC 60
CCTTTGCTGG ACCCGAACAA CCTCAATTCT GAAGACATGG ATATCCTGAT GGAACGAAAC 120
CTTCGAACTC CAAACCTGCT CGCATTCGTA AGGGCTGTCA AGCACTTAGA AAATGCATCA 180
GGTATTGAGG CAATTCTTCG TAATCTCCAA CCATGTCTGC CCTCTGCCAC GGCCGCACCC 240
TCTCGACATC CAATCATCAT CAAGGCAGGT GACTGGCAAG AATTCCGGGA AAAACTGACG 300
TTCTATCTGG TTACCCTTGA GCAAGCGCAG GAACAACAGT ACGTAATCGA GGGAAGGATT 360
TCCCCGGGTG AACCGTCTGG TCCAATCTCT ACTATCAACC CGTCTCCTCC GTCTAAAGAA 420
TCTCATAAAT CTCCAAACAT GGCACCGGCT CGTTCCCCGT CCCCGTCTAC CCAGCCGTGG 480
GAACACGTGA ATGCCATCCA GGAGGCCCGG CGTCTCCTGA ACCTGAGTAG AGACACTGCT 540
GCTGAGATGA ATGAAACAGT AGAAGTGATA TCAGAAATGT TTGACCTCCA GGAGCCGACT 600
TGCCTACAGA CCCGCCTGGA GCTGTACAAG CAGGGCCTGC GGGGCAGCCT CACCAAGCTC 660
AAGGGCCCCT TGACCATGAT GGCCAGCCAC TACAAGCAGC ACTGCCCTCC AACCCCGGAA 720
ACTTCCTGTG CAACCCAGAT TATCACCTTT GAAAGTTTCA AAGAGAACCT GAAGGACTTC 780
CTGCTTGTCA TCCCCTTTGA CTGCTGGGAG CCAGTCCAGG AG 822
966 base pairs
nucleic acid
double
linear
DNA (genomic)
70
ATGGCTAACT GCTCTATAAT GATCGATGAA ATTATACATC ACTTAAAGAG ACCACCTAAC 60
CCTTTGCTGG ACCCGAACAA CCTCAATTCT GAAGACATGG ATATCCTGAT GGAACGAAAC 120
CTTCGAACTC CAAACCTGCT CGCATTCGTA AGGGCTGTCA AGCACTTAGA AAATGCATCA 180
GGTATTGAGG CAATTCTTCG TAATCTCCAA CCATGTCTGC CCTCTGCCAC GGCCGCACCC 240
TCTCGACATC CAATCATCAT CAAGGCAGGT GACTGGCAAG AATTCCGGGA AAAACTGACG 300
TTCTATCTGG TTACCCTTGA GCAAGCGCAG GAACAACAGT ACGTAATCGA GGGAAAGATT 360
TCCCCGGGTG AACCGTCTGG TCCAATCTCT ACTATCAACC CGTCTCCTCC GTCTAAAGAA 420
TCTCATAAAT CTCCAAACAT GGCTACACCA TTAGGCCCTG CCAGCTCCCT GCCCCAGAGC 480
TTCCTGCTCA AGTGCTTAGA GCAAGTGAGG AAGATCCAGG GCGATGGCGC AGCGCTCCAG 540
GAGAAGCTGT GTGCCACCTA CAAGCTGTGC CACCCCGAGG AGCTGGTGCT GCTCGGACAC 600
TCTCTGGGCA TCCCCTGGGC TCCCCTGAGC TCCTGCCCCA GCCAGGCCCT GCAGCTGGCA 660
GGCTGCTTGA GCCAACTCCA TAGCGGCCTT TTCCTCTACC AGGGGCTCCT GCAGGCCCTG 720
GAAGGGATAT CCCCCGAGTT GGGTCCCACC TTGGACACAC TGCAGCTGGA CGTCGCCGAC 780
TTTGCCACCA CCATCTGGCA GCAGATGGAA GAACTGGGAA TGGCCCCTGC CCTGCAGCCC 840
ACCCAGGGTG CCATGCCGGC CTTCGCCTCT GCTTTCCAGC GCCGGGCAGG AGGGGTCCTG 900
GTTGCTAGCC ATCTGCAGAG CTTCCTGGAG GTGTCGTACC GCGTTCTACG CCACCTTGCG 960
CAGCCC 966
966 base pairs
nucleic acid
double
linear
DNA (genomic)
›EXAMPLE 5 · 9 of 25
71
ATGGCTAACT GCTCTATAAT GATCGATGAA ATTATACATC ACTTAAAGAG ACCACCTAAC 60
CCTTTGCTGG ACCCGAACAA CCTCAATTCT GAAGACATGG ATATCCTGAT GGAACGAAAC 120
CTTCGAACTC CAAACCTGCT CGCATTCGTA AGGGCTGTCA AGCACTTAGA AAATGCATCA 180
GGTATTGAGG CAATTCTTCG TAATCTCCAA CCATGTCTGC CCTCTGCCAC GGCCGCACCC 240
TCTCGACATC CAATCATCAT CAAGGCAGGT GACTGGCAAG AATTCCGGGA AAAACTGACG 300
TTCTATCTGG TTACCCTTGA GCAAGCGCAG GAACAACAGT ACGTAGAGGG CGGTGGAGGC 360
TCCCCGGGTG AACCGTCTGG TCCAATCTCT ACTATCAACC CGTCTCCTCC GTCTAAAGAA 420
TCTCATAAAT CTCCAAACAT GGCTACACCA TTAGGCCCTG CCAGCTCCCT GCCCCAGAGC 480
TTCCTGCTCA AGTGCTTAGA GCAAGTGAGG AAGATCCAGG GCGATGGCGC AGCGCTCCAG 540
GAGAAGCTGT GTGCCACCTA CAAGCTGTGC CACCCCGAGG AGCTGGTGCT GCTCGGACAC 600
TCTCTGGGCA TCCCCTGGGC TCCCCTGAGC TCCTGCCCCA GCCAGGCCCT GCAGCTGGCA 660
GGCTGCTTGA GCCAACTCCA TAGCGGCCTT TTCCTCTACC AGGGGCTCCT GCAGGCCCTG 720
GAAGGGATAT CCCCCGAGTT GGGTCCCACC TTGGACACAC TGCAGCTGGA CGTCGCCGAC 780
TTTGCCACCA CCATCTGGCA GCAGATGGAA GAACTGGGAA TGGCCCCTGC CCTGCAGCCC 840
ACCCAGGGTG CCATGCCGGC CTTCGCCTCT GCTTTCCAGC GCCGGGCAGG AGGGGTCCTG 900
GTTGCTAGCC ATCTGCAGAG CTTCCTGGAG GTGTCGTACC GCGTTCTACG CCACCTTGCG 960
CAGCCC 966
921 base pairs
nucleic acid
double
linear
DNA (genomic)
72
ATGGCTACAC CATTAGGCCC TGCCAGCTCC CTGCCCCAGA GCTTCCTGCT CAAGTGCTTA 60
GAGCAAGTGA GGAAGATCCA GGGCGATGGC GCAGCGCTCC AGGAGAAGCT GTGTGCCACC 120
TACAAGCTGT GCCACCCCGA GGAGCTGGTG CTGCTCGGAC ACTCTCTGGG CATCCCCTGG 180
GCTCCCCTGA GCTCCTGCCC CAGCCAGGCC CTGCAGCTGG CAGGCTGCTT GAGCCAACTC 240
CATAGCGGCC TTTTCCTCTA CCAGGGGCTC CTGCAGGCCC TGGAAGGGAT ATCCCCCGAG 300
TTGGGTCCCA CCTTGGACAC ACTGCAGCTG GACGTCGCCG ACTTTGCCAC CACCATCTGG 360
CAGCAGATGG AAGAACTGGG AATGGCCCCT GCCCTGCAGC CCACCCAGGG TGCCATGCCG 420
GCCTTCGCCT CTGCTTTCCA GCGCCGGGCA GGAGGGGTCC TGGTTGCTAG CCATCTGCAG 480
AGCTTCCTGG AGGTGTCGTA CCGCGTTCTA CGCCACCTTG CGCAGCCCTA CGTAATCGAG 540
GGAAGGATTT CCCCGGGTGG TGGTTCTGGC GGCGGCTCCA ACATGGCTAA CTGCTCTATA 600
ATGATCGATG AAATTATACA TCACTTAAAG AGACCACCTA ACCCTTTGCT GGACCCGAAC 660
AACCTCAATT CTGAAGACAT GGATATCCTG ATGGAACGAA ACCTTCGAAC TCCAAACCTG 720
CTCGCATTCG TAAGGGCTGT CAAGCACTTA GAAAATGCAT CAGGTATTGA GGCAATTCTT 780
CGTAATCTCC AACCATGTCT GCCCTCTGCC ACGGCCGCAC CCTCTCGACA TCCAATCATC 840
ATCAAGGCAG GTGACTGGCA AGAATTCCGG GAAAAACTGA CGTTCTATCT GGTTACCCTT 900
GAGCAAGCGC AGGAACAACA G 921
966 base pairs
nucleic acid
double
linear
DNA (genomic)
73
ATGGCTACAC CATTAGGCCC TGCCAGCTCC CTGCCCCAGA GCTTCCTGCT CAAGTGCTTA 60
GAGCAAGTGA GGAAGATCCA GGGCGATGGC GCAGCGCTCC AGGAGAAGCT GTGTGCCACC 120
TACAAGCTGT GCCACCCCGA GGAGCTGGTG CTGCTCGGAC ACTCTCTGGG CATCCCCTGG 180
GCTCCCCTGA GCTCCTGCCC CAGCCAGGCC CTGCAGCTGG CAGGCTGCTT GAGCCAACTC 240
CATAGCGGCC TTTTCCTCTA CCAGGGGCTC CTGCAGGCCC TGGAAGGGAT ATCCCCCGAG 300
TTGGGTCCCA CCTTGGACAC ACTGCAGCTG GACGTCGCCG ACTTTGCCAC CACCATCTGG 360
CAGCAGATGG AAGAACTGGG AATGGCCCCT GCCCTGCAGC CCACCCAGGG TGCCATGCCG 420
GCCTTCGCCT CTGCTTTCCA GCGCCGGGCA GGAGGGGTCC TGGTTGCTAG CCATCTGCAG 480
AGCTTCCTGG AGGTGTCGTA CCGCGTTCTA CGCCACCTTG CGCAGCCCTA CGTAATCGAG 540
GGAAGGATTT CCCCGGGTGA ACCGTCTGGT CCAATCTCTA CTATCAACCC GTCTCCTCCG 600
TCTAAAGAAT CTCATAAATC TCCAAACATG GCTAACTGCT CTATAATGAT CGATGAAATT 660
ATACATCACT TAAAGAGACC ACCTAACCCT TTGCTGGACC CGAACAACCT CAATTCTGAA 720
GACATGGATA TCCTGATGGA ACGAAACCTT CGAACTCCAA ACCTGCTCGC ATTCGTAAGG 780
GCTGTCAAGC ACTTAGAAAA TGCATCAGGT ATTGAGGCAA TTCTTCGTAA TCTCCAACCA 840
TGTCTGCCCT CTGCCACGGC CGCACCCTCT CGACATCCAA TCATCATCAA GGCAGGTGAC 900
TGGCAAGAAT TCCGGGAAAA ACTGACGTTC TATCTGGTTA CCCTTGAGCA AGCGCAGGAA 960
CAACAG 966
1047 base pairs
nucleic acid
double
linear
DNA (genomic)
74
ATGGCTACAC CATTAGGCCC TGCCAGCTCC CTGCCCCAGA GCTTCCTGCT CAAGTGCTTA 60
GAGCAAGTGA GGAAGATCCA GGGCGATGGC GCAGCGCTCC AGGAGAAGCT GTGTGCCACC 120
TACAAGCTGT GCCACCCCGA GGAGCTGGTG CTGCTCGGAC ACTCTCTGGG CATCCCCTGG 180
GCTCCCCTGA GCTCCTGCCC CAGCCAGGCC CTGCAGCTGG CAGGCTGCTT GAGCCAACTC 240
CATAGCGGCC TTTTCCTCTA CCAGGGGCTC CTGCAGGCCC TGGAAGGGAT ATCCCCCGAG 300
TTGGGTCCCA CCTTGGACAC ACTGCAGCTG GACGTCGCCG ACTTTGCCAC CACCATCTGG 360
CAGCAGATGG AAGAACTGGG AATGGCCCCT GCCCTGCAGC CCACCCAGGG TGCCATGCCG 420
GCCTTCGCCT CTGCTTTCCA GCGCCGGGCA GGAGGGGTCC TGGTTGCTAG CCATCTGCAG 480
AGCTTCCTGG AGGTGTCGTA CCGCGTTCTA CGCCACCTTG CGCAGCCCTA CGTAATCGAG 540
GGAAGGATTT CCCCCGGGCC TCCTGTCAAT GCTGGCGGCG GCTCTGGTGG TGGTTCTGGT 600
GGCGGCTCTG AGGGTGGCGG CTCTGAGGGT GGCGGTTCTG AGGGTGGCGG CTCTGAGGGT 660
GGCGGTTCCG GTGGCGGCTC CGGTTCCGGT GATTTTGATT ATGAAAACAT GGCTAACTGC 720
TCTATAATGA TCGATGAAAT TATACATCAC TTAAAGAGAC CACCTAACCC TTTGCTGGAC 780
CCGAACAACC TCAATTCTGA AGACATGGAT ATCCTGATGG AACGAAACCT TCGAACTCCA 840
AACCTGCTCG CATTCGTAAG GGCTGTCAAG CACTTAGAAA ATGCATCAGG TATTGAGGCA 900
ATTCTTCGTA ATCTCCAACC ATGTCTGCCC TCTGCCACGG CCGCACCCTC TCGACATCCA 960
ATCATCATCA AGGCAGGTGA CTGGCAAGAA TTCCGGGAAA AACTGACGTT CTATCTGGTT 1020
ACCCTTGAGC AAGCGCAGGA ACAACAG 1047
921 base pairs
nucleic acid
double
linear
DNA (genomic)
75
ATGGCTACAC CATTAGGCCC TGCCAGCTCC CTGCCCCAGA GCTTCCTGCT CAAGTGCTTA 60
GAGCAAGTGA GGAAGATCCA GGGCGATGGC GCAGCGCTCC AGGAGAAGCT GTGTGCCACC 120
TACAAGCTGT GCCACCCCGA GGAGCTGGTG CTGCTCGGAC ACTCTCTGGG CATCCCCTGG 180
GCTCCCCTGA GCTCCTGCCC CAGCCAGGCC CTGCAGCTGG CAGGCTGCTT GAGCCAACTC 240
CATAGCGGCC TTTTCCTCTA CCAGGGGCTC CTGCAGGCCC TGGAAGGGAT ATCCCCCGAG 300
TTGGGTCCCA CCTTGGACAC ACTGCAGCTG GACGTCGCCG ACTTTGCCAC CACCATCTGG 360
CAGCAGATGG AAGAACTGGG AATGGCCCCT GCCCTGCAGC CCACCCAGGG TGCCATGCCG 420
GCCTTCGCCT CTGCTTTCCA GCGCCGGGCA GGAGGGGTCC TGGTTGCTAG CCATCTGCAG 480
AGCTTCCTGG AGGTGTCGTA CCGCGTTCTA CGCCACCTTG CGCAGCCCTA CGTAATCGAG 540
GGAAGGATTT CCCCGGGTGG TGGTTCTGGC GGCGGCTCCA ACATGGCTAA CTGCTCTATA 600
ATGATCGATG AAATTATACA TCACTTAAAG AGACCACCTG CACCTTTGCT GGACCCGAAC 660
AACCTCAATG ACGAAGACGT CTCTATCCTG ATGGAACGAA ACCTTCGACT TCCAAACCTG 720
GAGAGCTTCG TAAGGGCTGT CAAGAACTTA GAAAATGCAT CAGGTATTGA GGCAATTCTT 780
CGTAATCTCC AACCATGTCT GCCCTCTGCC ACGGCCGCAC CCTCTCGACA TCCAATCATC 840
ATCAAGGCAG GTGACTGGCA AGAATTCCGG GAAAAACTGA CGTTCTATCT GGTTACCCTT 900
GAGCAAGCGC AGGAACAACA G 921
1047 base pairs
nucleic acid
double
linear
DNA (genomic)
76
ATGGCTACAC CATTAGGCCC TGCCAGCTCC CTGCCCCAGA GCTTCCTGCT CAAGTGCTTA 60
GAGCAAGTGA GGAAGATCCA GGGCGATGGC GCAGCGCTCC AGGAGAAGCT GTGTGCCACC 120
TACAAGCTGT GCCACCCCGA GGAGCTGGTG CTGCTCGGAC ACTCTCTGGG CATCCCCTGG 180
›EXAMPLE 5 · 10 of 25
GCTCCCCTGA GCTCCTGCCC CAGCCAGGCC CTGCAGCTGG CAGGCTGCTT GAGCCAACTC 240
CATAGCGGCC TTTTCCTCTA CCAGGGGCTC CTGCAGGCCC TGGAAGGGAT ATCCCCCGAG 300
TTGGGTCCCA CCTTGGACAC ACTGCAGCTG GACGTCGCCG ACTTTGCCAC CACCATCTGG 360
CAGCAGATGG AAGAACTGGG AATGGCCCCT GCCCTGCAGC CCACCCAGGG TGCCATGCCG 420
GCCTTCGCCT CTGCTTTCCA GCGCCGGGCA GGAGGGGTCC TGGTTGCTAG CCATCTGCAG 480
AGCTTCCTGG AGGTGTCGTA CCGCGTTCTA CGCCACCTTG CGCAGCCCTA CGTAATCGAG 540
GGAAGGATTT CCCCCGGGCC TCCTGTCAAT GCTGGCGGCG GCTCTGGTGG TGGTTCTGGT 600
GGCGGCTCTG AGGGTGGCGG CTCTGAGGGT GGCGGTTCTG AGGGTGGCGG CTCTGAGGGT 660
GGCGGTTCCG GTGGCGGCTC CGGTTCCGGT GATTTTGATT ATGAAAACAT GGCTAACTGC 720
TCTATAATGA TCGATGAAAT TATACATCAC TTAAAGAGAC CACCTGCACC TTTGCTGGAC 780
CCGAACAACC TCAATGACGA AGACGTCTCT ATCCTGATGG AACGAAACCT TCGACTTCCA 840
AACCTGGAGA GCTTCGTAAG GGCTGTCAAG AACTTAGAAA ATGCATCAGG TATTGAGGCA 900
ATTCTTCGTA ATCTCCAACC ATGTCTGCCC TCTGCCACGG CCGCACCCTC TCGACATCCA 960
ATCATCATCA AGGCAGGTGA CTGGCAAGAA TTCCGGGAAA AACTGACGTT CTATCTGGTT 1020
ACCCTTGAGC AAGCGCAGGA ACAACAG 1047
966 base pairs
nucleic acid
double
linear
DNA (genomic)
77
ATGGCTACAC CATTAGGCCC TGCCAGCTCC CTGCCCCAGA GCTTCCTGCT CAAGTGCTTA 60
GAGCAAGTGA GGAAGATCCA GGGCGATGGC GCAGCGCTCC AGGAGAAGCT GTGTGCCACC 120
TACAAGCTGT GCCACCCCGA GGAGCTGGTG CTGCTCGGAC ACTCTCTGGG CATCCCCTGG 180
GCTCCCCTGA GCTCCTGCCC CAGCCAGGCC CTGCAGCTGG CAGGCTGCTT GAGCCAACTC 240
CATAGCGGCC TTTTCCTCTA CCAGGGGCTC CTGCAGGCCC TGGAAGGGAT ATCCCCCGAG 300
TTGGGTCCCA CCTTGGACAC ACTGCAGCTG GACGTCGCCG ACTTTGCCAC CACCATCTGG 360
CAGCAGATGG AAGAACTGGG AATGGCCCCT GCCCTGCAGC CCACCCAGGG TGCCATGCCG 420
GCCTTCGCCT CTGCTTTCCA GCGCCGGGCA GGAGGGGTCC TGGTTGCTAG CCATCTGCAG 480
AGCTTCCTGG AGGTGTCGTA CCGCGTTCTA CGCCACCTTG CGCAGCCCTA CGTAATCGAG 540
GGAAGGATTT CCCCGGGTGA ACCGTCTGGT CCAATCTCTA CTATCAACCC GTCTCCTCCG 600
TCTAAAGAAT CTCATAAATC TCCAAACATG GCTAACTGCT CTATAATGAT CGATGAAATT 660
ATACATCACT TAAAGAGACC ACCTGCACCT TTGCTGGACC CGAACAACCT CAATGACGAA 720
GACGTCTCTA TCCTGATGGA ACGAAACCTT CGACTTCCAA ACCTGGAGAG CTTCGTAAGG 780
GCTGTCAAGA ACTTAGAAAA TGCATCAGGT ATTGAGGCAA TTCTTCGTAA TCTCCAACCA 840
TGTCTGCCCT CTGCCACGGC CGCACCCTCT CGACATCCAA TCATCATCAA GGCAGGTGAC 900
TGGCAAGAAT TCCGGGAAAA ACTGACGTTC TATCTGGTTA CCCTTGAGCA AGCGCAGGAA 960
CAACAG 966
921 base pairs
nucleic acid
double
linear
DNA (genomic)
78
ATGGCTACAC CATTAGGCCC TGCCAGCTCC CTGCCCCAGA GCTTCCTGCT CAAGTGCTTA 60
GAGCAAGTGA GGAAGATCCA GGGCGATGGC GCAGCGCTCC AGGAGAAGCT GTGTGCCACC 120
TACAAGCTGT GCCACCCCGA GGAGCTGGTG CTGCTCGGAC ACTCTCTGGG CATCCCCTGG 180
GCTCCCCTGA GCTCCTGCCC CAGCCAGGCC CTGCAGCTGG CAGGCTGCTT GAGCCAACTC 240
CATAGCGGCC TTTTCCTCTA CCAGGGGCTC CTGCAGGCCC TGGAAGGGAT ATCCCCCGAG 300
TTGGGTCCCA CCTTGGACAC ACTGCAGCTG GACGTCGCCG ACTTTGCCAC CACCATCTGG 360
CAGCAGATGG AAGAACTGGG AATGGCCCCT GCCCTGCAGC CCACCCAGGG TGCCATGCCG 420
GCCTTCGCCT CTGCTTTCCA GCGCCGGGCA GGAGGGGTCC TGGTTGCTAG CCATCTGCAG 480
AGCTTCCTGG AGGTGTCGTA CCGCGTTCTA CGCCACCTTG CGCAGCCCTA CGTAGAGGGC 540
GGTGGAGGCT CCCCGGGTGG TGGTTCTGGC GGCGGCTCCA ACATGGCTAA CTGCTCTATA 600
ATGATCGATG AAATTATACA TCACTTAAAG AGACCACCTG CACCTTTGCT GGACCCGAAC 660
AACCTCAATG ACGAAGACGT CTCTATCCTG ATGGAACGAA ACCTTCGACT TCCAAACCTG 720
GAGAGCTTCG TAAGGGCTGT CAAGAACTTA GAAAATGCAT CAGGTATTGA GGCAATTCTT 780
CGTAATCTCC AACCATGTCT GCCCTCTGCC ACGGCCGCAC CCTCTCGACA TCCAATCATC 840
ATCAAGGCAG GTGACTGGCA AGAATTCCGG GAAAAACTGA CGTTCTATCT GGTTACCCTT 900
GAGCAAGCGC AGGAACAACA G 921
966 base pairs
nucleic acid
double
linear
DNA (genomic)
79
ATGGCTACAC CATTAGGCCC TGCCAGCTCC CTGCCCCAGA GCTTCCTGCT CAAGTGCTTA 60
GAGCAAGTGA GGAAGATCCA GGGCGATGGC GCAGCGCTCC AGGAGAAGCT GTGTGCCACC 120
TACAAGCTGT GCCACCCCGA GGAGCTGGTG CTGCTCGGAC ACTCTCTGGG CATCCCCTGG 180
GCTCCCCTGA GCTCCTGCCC CAGCCAGGCC CTGCAGCTGG CAGGCTGCTT GAGCCAACTC 240
CATAGCGGCC TTTTCCTCTA CCAGGGGCTC CTGCAGGCCC TGGAAGGGAT ATCCCCCGAG 300
TTGGGTCCCA CCTTGGACAC ACTGCAGCTG GACGTCGCCG ACTTTGCCAC CACCATCTGG 360
CAGCAGATGG AAGAACTGGG AATGGCCCCT GCCCTGCAGC CCACCCAGGG TGCCATGCCG 420
GCCTTCGCCT CTGCTTTCCA GCGCCGGGCA GGAGGGGTCC TGGTTGCTAG CCATCTGCAG 480
AGCTTCCTGG AGGTGTCGTA CCGCGTTCTA CGCCACCTTG CGCAGCCCTA CGTAGAGGGC 540
GGTGGAGGCT CCCCGGGTGA ACCGTCTGGT CCAATCTCTA CTATCAACCC GTCTCCTCCG 600
TCTAAAGAAT CTCATAAATC TCCAAACATG GCTAACTGCT CTATAATGAT CGATGAAATT 660
ATACATCACT TAAAGAGACC ACCTGCACCT TTGCTGGACC CGAACAACCT CAATGACGAA 720
GACGTCTCTA TCCTGATGGA ACGAAACCTT CGACTTCCAA ACCTGGAGAG CTTCGTAAGG 780
GCTGTCAAGA ACTTAGAAAA TGCATCAGGT ATTGAGGCAA TTCTTCGTAA TCTCCAACCA 840
TGTCTGCCCT CTGCCACGGC CGCACCCTCT CGACATCCAA TCATCATCAA GGCAGGTGAC 900
TGGCAAGAAT TCCGGGAAAA ACTGACGTTC TATCTGGTTA CCCTTGAGCA AGCGCAGGAA 960
CAACAG 966
921 base pairs
nucleic acid
double
linear
DNA (genomic)
80
ATGGCTACAC CATTGGGCCC TGCCAGCTCC CTGCCCCAGA GCTTCCTGCT CAAGTCTTTA 60
GAGCAAGTGA GGAAGATCCA GGGCGATGGC GCAGCGCTCC AGGAGAAGCT GTGTGCCACC 120
TACAAGCTGT GCCACCCCGA GGAGCTGGTG CTGCTCGGAC ACTCTCTGGG CATCCCCTGG 180
GCTCCCCTGA GCTCCTGCCC CAGCCAGGCC CTGCAGCTGG CAGGCTGCTT GAGCCAACTC 240
CATAGCGGCC TTTTCCTCTA CCAGGGGCTC CTGCAGGCCC TGGAAGGGAT ATCCCCCGAG 300
TTGGGTCCCA CCTTGGACAC ACTGCAGCTG GACGTCGCCG ACTTTGCCAC CACCATCTGG 360
CAGCAGATGG AAGAACTGGG AATGGCCCCT GCCCTGCAGC CCACCCAGGG TGCCATGCCG 420
GCCTTCGCCT CTGCTTTCCA GCGCCGGGCA GGAGGGGTCC TGGTTGCTAG CCATCTGCAG 480
AGCTTCCTGG AGGTGTCGTA CCGCGTTCTA CGCCACCTTG CGCAGCCCTA CGTAGAGGGC 540
GGTGGAGGCT CCCCGGGTGG TGGTTCTGGC GGCGGCTCCA ACATGGCTAA CTGCTCTATA 600
ATGATCGATG AAATTATACA TCACTTAAAG AGACCACCTG CACCTTTGCT GGACCCGAAC 660
AACCTCAATG ACGAAGACGT CTCTATCCTG ATGGAACGAA ACCTTCGACT TCCAAACCTG 720
GAGAGCTTCG TAAGGGCTGT CAAGAACTTA GAAAATGCAT CAGGTATTGA GGCAATTCTT 780
CGTAATCTCC AACCATGTCT GCCCTCTGCC ACGGCCGCAC CCTCTCGACA TCCAATCATC 840
ATCAAGGCAG GTGACTGGCA AGAATTCCGG GAAAAACTGA CGTTCTATCT GGTTACCCTT 900
GAGCAAGCGC AGGAACAACA G 921
966 base pairs
nucleic acid
double
linear
DNA (genomic)
81
ATGGCTACAC CATTGGGCCC TGCCAGCTCC CTGCCCCAGA GCTTCCTGCT CAAGTCTTTA 60
GAGCAAGTGA GGAAGATCCA GGGCGATGGC GCAGCGCTCC AGGAGAAGCT GTGTGCCACC 120
TACAAGCTGT GCCACCCCGA GGAGCTGGTG CTGCTCGGAC ACTCTCTGGG CATCCCCTGG 180
GCTCCCCTGA GCTCCTGCCC CAGCCAGGCC CTGCAGCTGG CAGGCTGCTT GAGCCAACTC 240
CATAGCGGCC TTTTCCTCTA CCAGGGGCTC CTGCAGGCCC TGGAAGGGAT ATCCCCCGAG 300
TTGGGTCCCA CCTTGGACAC ACTGCAGCTG GACGTCGCCG ACTTTGCCAC CACCATCTGG 360
›EXAMPLE 5 · 11 of 25
CAGCAGATGG AAGAACTGGG AATGGCCCCT GCCCTGCAGC CCACCCAGGG TGCCATGCCG 420
GCCTTCGCCT CTGCTTTCCA GCGCCGGGCA GGAGGGGTCC TGGTTGCTAG CCATCTGCAG 480
AGCTTCCTGG AGGTGTCGTA CCGCGTTCTA CGCCACCTTG CGCAGCCCTA CGTAGAGGGC 540
GGTGGAGGCT CCCCGGGTGA ACCGTCTGGT CCAATCTCTA CTATCAACCC GTCTCCTCCG 600
TCTAAAGAAT CTCATAAATC TCCAAACATG GCTAACTGCT CTATAATGAT CGATGAAATT 660
ATACATCACT TAAAGAGACC ACCTGCACCT TTGCTGGACC CGAACAACCT CAATGACGAA 720
GACGTCTCTA TCCTGATGGA ACGAAACCTT CGACTTCCAA ACCTGGAGAG CTTCGTAAGG 780
GCTGTCAAGA ACTTAGAAAA TGCATCAGGT ATTGAGGCAA TTCTTCGTAA TCTCCAACCA 840
TGTCTGCCCT CTGCCACGGC CGCACCCTCT CGACATCCAA TCATCATCAA GGCAGGTGAC 900
TGGCAAGAAT TCCGGGAAAA ACTGACGTTC TATCTGGTTA CCCTTGAGCA AGCGCAGGAA 960
CAACAG 966
777 base pairs
nucleic acid
double
linear
DNA (genomic)
82
ATGGCTAACT GCTCTATAAT GATCGATGAA ATTATACATC ACTTAAAGAG ACCACCTGCA 60
CCTTTGCTGG ACCCGAACAA CCTCAATGAC GAAGACGTCT CTATCCTGAT GGAACGAAAC 120
CTTCGACTTC CAAACCTGGA GAGCTTCGTA AGGGCTGTCA AGAACTTAGA AAATGCATCA 180
GGTATTGAGG CAATTCTTCG TAATCTCCAA CCATGTCTGC CCTCTGCCAC GGCCGCACCC 240
TCTCGACATC CAATCATCAT CAAGGCAGGT GACTGGCAAG AATTCCGGGA AAAACTGACG 300
TTCTATCTGG TTACCCTTGA GCAAGCGCAG GAACAACAGT ACGTAATCGA GGGAAGGATT 360
TCCCCGGGTG AACCGTCTGG TCCAATCTCT ACTATCAACC CGTCTCCTCC GTCTAAAGAA 420
TCTCATAAAT CTCCAAACAT GGCTAACTGC TCTATAATGA TCGATGAAAT TATACATCAC 480
TTAAAGAGAC CACCTGCACC TTTGCTGGAC CCGAACAACC TCAATGACGA AGACGTCTCT 540
ATCCTGATGG AACGAAACCT TCGACTTCCA AACCTGGAGA GCTTCGTAAG GGCTGTCAAG 600
AACTTAGAAA ATGCATCAGG TATTGAGGCA ATTCTTCGTA ATCTCCAACC ATGTCTGCCC 660
TCTGCCACGG CCGCACCCTC TCGACATCCA ATCATCATCA AGGCAGGTGA CTGGCAAGAA 720
TTCCGGGAAA AACTGACGTT CTATCTGGTT ACCCTTGAGC AAGCGCAGGA ACAACAG 777
984 base pairs
nucleic acid
double
linear
DNA (genomic)
83
ATGGCTAACT GCTCTATAAT GATCGATGAA ATTATACATC ACTTAAAGAG ACCACCTGCA 60
CCTTTGCTGG ACCCGAACAA CCTCAATGAC GAAGACGTCT CTATCCTGAT GGAACGAAAC 120
CTTCGACTTC CAAACCTGGA GAGCTTCGTA AGGGCTGTCA AGAACTTAGA AAATGCATCA 180
GGTATTGAGG CAATTCTTCG TAATCTCCAA CCATGTCTGC CCTCTGCCAC GGCCGCACCC 240
TCTCGACATC CAATCATCAT CAAGGCAGGT GACTGGCAAG AATTCCGGGA AAAACTGACG 300
TTCTATCTGG TTACCCTTGA GCAAGCGCAG GAACAACAGT ACGTAATCGA GGGAAGGATT 360
TCCCCGGGTG AACCGTCTGG TCCAATCTCT ACTATCAACC CGTCTCCTCC GTCTAAAGAA 420
TCTCATAAAT CTCCAAACAT GGCTACACCA TTGGGCCCTG CCAGCTCCCT GCCCCAGAGC 480
TTCCTGCTCA AGTCTTTAGA GCAAGTGAGG AAGATCCAGG GCGATGGCGC AGCGCTCCAG 540
GAGAAGCTGT GTGCCACCTA CAAGCTGTGC CACCCCGAGG AGCTGGTGCT GCTCGGACAC 600
TCTCTGGGCA TCCCCTGGGC TCCCCTGAGC TCCTGCCCCA GCCAGGCCCT GCAGCTGGCA 660
GGCTGCTTGA GCCAACTCCA TAGCGGCCTT TTCCTCTACC AGGGGCTCCT GCAGGCCCTG 720
GAAGGGATAT CCCCCGAGTT GGGTCCCACC TTGGACACAC TGCAGCTGGA CGTCGCCGAC 780
TTTGCCACCA CCATCTGGCA GCAGATGGAA GAACTGGGAA TGGCCCCTGC CCTGCAGCCC 840
ACCCAGGGTG CCATGCCGGC CTTCGCCTCT GCTTTCCAGC GCCGGGCAGG AGGGGTCCTG 900
GTTGCTAGCC ATCTGCAGAG CTTCCTGGAG GTGTCGTACC GCGTTCTACG CCACCTTGCG 960
CAGCCCTGAT AAGGATCCGA ATTC 984
921 base pairs
nucleic acid
double
linear
DNA (genomic)
84
ATGGCTAACT GCTCTATAAT GATCGATGAA ATTATACATC ACTTAAAGAG ACCACCTGCA 60
CCTTTGCTGG ACCCGAACAA CCTCAATGAC GAAGACGTCT CTATCCTGAT GGAACGAAAC 120
CTTCGACTTC CAAACCTGGA GAGCTTCGTA AGGGCTGTCA AGAACTTAGA AAATGCATCA 180
GGTATTGAGG CAATTCTTCG TAATCTCCAA CCATGTCTGC CCTCTGCCAC GGCCGCACCC 240
TCTCGACATC CAATCATCAT CAAGGCAGGT GACTGGCAAG AATTCCGGGA AAAACTGACG 300
TTCTATCTGG TTACCCTTGA GCAAGCGCAG GAACAACAGT ACGTAATCGA GGGAAGGATT 360
TCCCCGGGTG GTGGTTCTGG CGGCGGCTCC AACATGGCTA CACCATTAGG CCCTGCCAGC 420
TCCCTGCCCC AGAGCTTCCT GCTCAAGTGC TTAGAGCAAG TGAGGAAGAT CCAGGGCGAT 480
GGCGCAGCGC TCCAGGAGAA GCTGTGTGCC ACCTACAAGC TGTGCCACCC CGAGGAGCTG 540
GTGCTGCTCG GACACTCTCT GGGCATCCCC TGGGCTCCCC TGAGCTCCTG CCCCAGCCAG 600
GCCCTGCAGC TGGCAGGCTG CTTGAGCCAA CTCCATAGCG GCCTTTTCCT CTACCAGGGG 660
CTCCTGCAGG CCCTGGAAGG GATATCCCCC GAGTTGGGTC CCACCTTGGA CACACTGCAG 720
CTGGACGTCG CCGACTTTGC CACCACCATC TGGCAGCAGA TGGAAGAACT GGGAATGGCC 780
CCTGCCCTGC AGCCCACCCA GGGTGCCATG CCGGCCTTCG CCTCTGCTTT CCAGCGCCGG 840
GCAGGAGGGG TCCTGGTTGC TAGCCATCTG CAGAGCTTCC TGGAGGTGTC GTACCGCGTT 900
CTACGCCACC TTGCGCAGCC C 921
921 base pairs
nucleic acid
double
linear
DNA (genomic)
85
ATGGCTAACT GCTCTATAAT GATCGATGAA ATTATACATC ACTTAAAGAG ACCACCTGCA 60
CCTTTGCTGG ACCCGAACAA CCTCAATGAC GAAGACGTCT CTATCCTGAT GGAACGAAAC 120
CTTCGACTTC CAAACCTGGA GAGCTTCGTA AGGGCTGTCA AGAACTTAGA AAATGCATCA 180
GGTATTGAGG CAATTCTTCG TAATCTCCAA CCATGTCTGC CCTCTGCCAC GGCCGCACCC 240
TCTCGACATC CAATCATCAT CAAGGCAGGT GACTGGCAAG AATTCCGGGA AAAACTGACG 300
TTCTATCTGG TTACCCTTGA GCAAGCGCAG GAACAACAGT ACGTAATCGA GGGAAGGATT 360
TCCCCGGGTG GTGGTTCTGG CGGCGGCTCC AACATGGCTA CACCATTGGG CCCTGCCAGC 420
TCCCTGCCCC AGAGCTTCCT GCTCAAGTCT TTAGAGCAAG TGAGGAAGAT CCAGGGCGAT 480
GGCGCAGCGC TCCAGGAGAA GCTGTGTGCC ACCTACAAGC TGTGCCACCC CGAGGAGCTG 540
GTGCTGCTCG GACACTCTCT GGGCATCCCC TGGGCTCCCC TGAGCTCCTG CCCCAGCCAG 600
GCCCTGCAGC TGGCAGGCTG CTTGAGCCAA CTCCATAGCG GCCTTTTCCT CTACCAGGGG 660
CTCCTGCAGG CCCTGGAAGG GATATCCCCC GAGTTGGGTC CCACCTTGGA CACACTGCAG 720
CTGGACGTCG CCGACTTTGC CACCACCATC TGGCAGCAGA TGGAAGAACT GGGAATGGCC 780
CCTGCCCTGC AGCCCACCCA GGGTGCCATG CCGGCCTTCG CCTCTGCTTT CCAGCGCCGG 840
GCAGGAGGGG TCCTGGTTGC TAGCCATCTG CAGAGCTTCC TGGAGGTGTC GTACCGCGTT 900
CTACGCCACC TTGCGCAGCC C 921
732 base pairs
nucleic acid
double
linear
DNA (genomic)
86
ATGGCTAACT GCTCTATAAT GATCGATGAA ATTATACATC ACTTAAAGAG ACCACCTGCA 60
CCTTTGCTGG ACCCGAACAA CCTCAATGAC GAAGACGTCT CTATCCTGAT GGAACGAAAC 120
CTTCGACTTC CAAACCTGGA GAGCTTCGTA AGGGCTGTCA AGAACTTAGA AAATGCATCA 180
GGTATTGAGG CAATTCTTCG TAATCTCCAA CCATGTCTGC CCTCTGCCAC GGCCGCACCC 240
TCTCGACATC CAATCATCAT CAAGGCAGGT GACTGGCAAG AATTCCGGGA AAAACTGACG 300
TTCTATCTGG TTACCCTTGA GCAAGCGCAG GAACAACAGT ACGTAATCGA GGGAAGGATT 360
TCCCCGGGTG GTGGTTCTGG CGGCGGCTCC AACATGGCTA ACTGCTCTAT AATGATCGAT 420
GAAATTATAC ATCACTTAAA GAGACCACCT GCACCTTTGC TGGACCCGAA CAACCTCAAT 480
GACGAAGACG TCTCTATCCT GATGGAACGA AACCTTCGAC TTCCAAACCT GGAGAGCTTC 540
GTAAGGGCTG TCAAGAACTT AGAAAATGCA TCAGGTATTG AGGCAATTCT TCGTAATCTC 600
CAACCATGTC TGCCCTCTGC CACGGCCGCA CCCTCTCGAC ATCCAATCAT CATCAAGGCA 660
GGTGACTGGC AAGAATTCCG GGAAAAACTG ACGTTCTATC TGGTTACCCT TGAGCAAGCG 720
CAGGAACAAC AG 732
921 base pairs
nucleic acid
double
linear
DNA (genomic)
›EXAMPLE 5 · 12 of 25
87
ATGGCTAACT GCTCTATAAT GATCGATGAA ATTATACATC ACTTAAAGAG ACCACCTGCA 60
CCTTTGCTGG ACCCGAACAA CCTCAATGAC GAAGACGTCT CTATCCTGAT GGAACGAAAC 120
CTTCGACTTC CAAACCTGGA GAGCTTCGTA AGGGCTGTCA AGAACTTAGA AAATGCATCA 180
GGTATTGAGG CAATTCTTCG TAATCTCCAA CCATGTCTGC CCTCTGCCAC GGCCGCACCC 240
TCTCGACATC CAATCATCAT CAAGGCAGGT GACTGGCAAG AATTCCGGGA AAAACTGACG 300
TTCTATCTGG TTACCCTTGA GCAAGCGCAG GAACAACAGT ACGTAGAGGG CGGTGGAGGC 360
TCCCCGGGTG GTGGTTCTGG CGGCGGCTCC AACATGGCTA CACCATTGGG CCCTGCCAGC 420
TCCCTGCCCC AGAGCTTCCT GCTCAAGTCT TTAGAGCAAG TGAGGAAGAT CCAGGGCGAT 480
GGCGCAGCGC TCCAGGAGAA GCTGTGTGCC ACCTACAAGC TGTGCCACCC CGAGGAGCTG 540
GTGCTGCTCG GACACTCTCT GGGCATCCCC TGGGCTCCCC TGAGCTCCTG CCCCAGCCAG 600
GCCCTGCAGC TGGCAGGCTG CTTGAGCCAA CTCCATAGCG GCCTTTTCCT CTACCAGGGG 660
CTCCTGCAGG CCCTGGAAGG GATATCCCCC GAGTTGGGTC CCACCTTGGA CACACTGCAG 720
CTGGACGTCG CCGACTTTGC CACCACCATC TGGCAGCAGA TGGAAGAACT GGGAATGGCC 780
CCTGCCCTGC AGCCCACCCA GGGTGCCATG CCGGCCTTCG CCTCTGCTTT CCAGCGCCGG 840
GCAGGAGGGG TCCTGGTTGC TAGCCATCTG CAGAGCTTCC TGGAGGTGTC GTACCGCGTT 900
CTACGCCACC TTGCGCAGCC C 921
732 base pairs
nucleic acid
double
linear
DNA (genomic)
88
ATGGCTAACT GCTCTATAAT GATCGATGAA ATTATACATC ACTTAAAGAG ACCACCTGCA 60
CCTTTGCTGG ACCCGAACAA CCTCAATGAC GAAGACGTCT CTATCCTGAT GGAACGAAAC 120
CTTCGACTTC CAAACCTGGA GAGCTTCGTA AGGGCTGTCA AGAACTTAGA AAATGCATCA 180
GGTATTGAGG CAATTCTTCG TAATCTCCAA CCATGTCTGC CCTCTGCCAC GGCCGCACCC 240
TCTCGACATC CAATCATCAT CAAGGCAGGT GACTGGCAAG AATTCCGGGA AAAACTGACG 300
TTCTATCTGG TTACCCTTGA GCAAGCGCAG GAACAACAGT ACGTAGAGGG CGGTGGAGGC 360
TCCCCGGGTG GTGGTTCTGG CGGCGGCTCC AACATGGCTA ACTGCTCTAT AATGATCGAT 420
GAAATTATAC ATCACTTAAA GAGACCACCT GCACCTTTGC TGGACCCGAA CAACCTCAAT 480
GACGAAGACG TCTCTATCCT GATGGAACGA AACCTTCGAC TTCCAAACCT GGAGAGCTTC 540
GTAAGGGCTG TCAAGAACTT AGAAAATGCA TCAGGTATTG AGGCAATTCT TCGTAATCTC 600
CAACCATGTC TGCCCTCTGC CACGGCCGCA CCCTCTCGAC ATCCAATCAT CATCAAGGCA 660
GGTGACTGGC AAGAATTCCG GGAAAAACTG ACGTTCTATC TGGTTACCCT TGAGCAAGCG 720
CAGGAACAAC AG 732
966 base pairs
nucleic acid
double
linear
DNA (genomic)
89
ATGGCTAACT GCTCTATAAT GATCGATGAA ATTATACATC ACTTAAAGAG ACCACCTGCA 60
CCTTTGCTGG ACCCGAACAA CCTCAATGAC GAAGACGTCT CTATCCTGAT GGAACGAAAC 120
CTTCGACTTC CAAACCTGGA GAGCTTCGTA AGGGCTGTCA AGAACTTAGA AAATGCATCA 180
GGTATTGAGG CAATTCTTCG TAATCTCCAA CCATGTCTGC CCTCTGCCAC GGCCGCACCC 240
TCTCGACATC CAATCATCAT CAAGGCAGGT GACTGGCAAG AATTCCGGGA AAAACTGACG 300
TTCTATCTGG TTACCCTTGA GCAAGCGCAG GAACAACAGT ACGTAGAGGG CGGTGGAGGC 360
TCCCCGGGTG AACCGTCTGG TCCAATCTCT ACTATCAACC CGTCTCCTCC GTCTAAAGAA 420
TCTCATAAAT CTCCAAACAT GGCTACACCA TTGGGCCCTG CCAGCTCCCT GCCCCAGAGC 480
TTCCTGCTCA AGTCTTTAGA GCAAGTGAGG AAGATCCAGG GCGATGGCGC AGCGCTCCAG 540
GAGAAGCTGT GTGCCACCTA CAAGCTGTGC CACCCCGAGG AGCTGGTGCT GCTCGGACAC 600
TCTCTGGGCA TCCCCTGGGC TCCCCTGAGC TCCTGCCCCA GCCAGGCCCT GCAGCTGGCA 660
GGCTGCTTGA GCCAACTCCA TAGCGGCCTT TTCCTCTACC AGGGGCTCCT GCAGGCCCTG 720
GAAGGGATAT CCCCCGAGTT GGGTCCCACC TTGGACACAC TGCAGCTGGA CGTCGCCGAC 780
TTTGCCACCA CCATCTGGCA GCAGATGGAA GAACTGGGAA TGGCCCCTGC CCTGCAGCCC 840
ACCCAGGGTG CCATGCCGGC CTTCGCCTCT GCTTTCCAGC GCCGGGCAGG AGGGGTCCTG 900
GTTGCTAGCC ATCTGCAGAG CTTCCTGGAG GTGTCGTACC GCGTTCTACG CCACCTTGCG 960
CAGCCC 966
777 base pairs
nucleic acid
double
linear
DNA (genomic)
90
ATGGCTAACT GCTCTATAAT GATCGATGAA ATTATACATC ACTTAAAGAG ACCACCTGCA 60
CCTTTGCTGG ACCCGAACAA CCTCAATGAC GAAGACGTCT CTATCCTGAT GGAACGAAAC 120
CTTCGACTTC CAAACCTGGA GAGCTTCGTA AGGGCTGTCA AGAACTTAGA AAATGCATCA 180
GGTATTGAGG CAATTCTTCG TAATCTCCAA CCATGTCTGC CCTCTGCCAC GGCCGCACCC 240
TCTCGACATC CAATCATCAT CAAGGCAGGT GACTGGCAAG AATTCCGGGA AAAACTGACG 300
TTCTATCTGG TTACCCTTGA GCAAGCGCAG GAACAACAGT ACGTAGAGGG CGGTGGAGGC 360
TCCCCGGGTG AACCGTCTGG TCCAATCTCT ACTATCAACC CGTCTCCTCC GTCTAAAGAA 420
TCTCATAAAT CTCCAAACAT GGCTAACTGC TCTATAATGA TCGATGAAAT TATACATCAC 480
TTAAAGAGAC CACCTGCACC TTTGCTGGAC CCGAACAACC TCAATGACGA AGACGTCTCT 540
ATCCTGATGG AACGAAACCT TCGACTTCCA AACCTGGAGA GCTTCGTAAG GGCTGTCAAG 600
AACTTAGAAA ATGCATCAGG TATTGAGGCA ATTCTTCGTA ATCTCCAACC ATGTCTGCCC 660
TCTGCCACGG CCGCACCCTC TCGACATCCA ATCATCATCA AGGCAGGTGA CTGGCAAGAA 720
TTCCGGGAAA AACTGACGTT CTATCTGGTT ACCCTTGAGC AAGCGCAGGA ACAACAG 777
AATTCCGGGA AAAACTGACG TTCTATCTGG TTACCCTTGA G 41
CTGCGCTTGC TCAAGGGTAA CCAGATAGAA CGTCAGTTTT TCCCGG 46
CAAGCGCAGG AACAACAGTA CGTAATCGAG GGAAGGATT 39
ACCCGGGGAA ATCCTTCCCT CGATTACGTA CTGTTGTTC 39
TCCCCGGGTG GTGGTTCTGG CGGCGGCTCC AACATGTAAG GTACCGCATG CAAGCTTAGA 60
TCT 63
AGCTAGATCT AAGCTTGCAT GCGGTACCTT ACATGTTGGA GCCGCCGCCA GAACCACC 58
CCGGGTGAAC CGTCTGGTCC AATCTCTACT ATCAACCCGT CTCCTCCGTC TAAAGAATCT 60
CATAAATCTC CAAA 74
CATGTTTGGA GATTTATGAG ATTCTTTAGA CGGAGGAGAC GGGTTGATAG TAGAGATTGG 60
ACCAGACGGT TCAC 74
CTAGCCATCT GCAGAGCTTC CTGGAGGTGT CGTACCGCGT TCTACGCCAC CTTGCGCAGC 60
CCTACGTA 68
AGCTTACGTA GGGCTGCGCA AGGTGGCGTA GAACGCGGTA CGACACCTCC AGGAAGCTCT 60
GCAGATGG 68
GTAATCGAGG GAAAGATTTC C 21
CCGGGGAAAT CTTTCCCTCG ATTAC 25
GTAGAGGGCG GTGGAGGCTC C 21
CCGGGGAGCC TCCACCGCCC TCTAC 25
CATGGCACCA GCAAGATCAC CATCACCATC AACTCAACCT TGGGAACATG TGAATGCC 58
CATTCACATG TTCCCAAGGT TGAGTTGATG GTGATGGTGA TCTTGCTGGT GC 52
CTGCCAGCTC CCTGCCCCAG AGCTTCCTGC TCAAGTCTTT AGAGCAAGTG AGGAAGATCC 60
AGGGCG 66
CTGGATCTTC CTCACTTGCT CTAAAGACTT GAGCAGGAAG CTCTGGGGCA GGGAGCTGGC 60
AGGGCC 66
AGCTTACCTG CCATGGCTCC AGTACCACCA GGTGAAGATT CCAAAGAT 48
TTGGAATCTT CACCTGGTGG TACTGGAGCC ATGGCAGGTA 40
AGCTTCCATG GCTACCCCCC TGGGCC 26
CAGGGGGGTA GCCATGGA 18
CATGGCTACA CCATTGGGCC 20
CAATGGTGTA GC 12
CATGGCTACA CCATTAGGAC 20
TAATGGTGTA GC 12
CCTGTCAACC CGGGCGGCGG CTCTGGTGGT 30
TCATAATACA TGTTACCGGA ACGGAGCCGC C 31
ATCGTCTGAC CTCCCGGGAC CTCCTGTCAA TGCT 34
AGCGTTTGAC ATGTTTTCAT AATCAAAATC 30
307 amino acids
amino acid
linear
protein
121
Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys
1 5 10 15
Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn Ser Glu Asp
20 25 30
Met Asp Ile Leu Met Glu Arg Asn Leu Arg Thr Pro Asn Leu Leu Ala
35 40 45
Phe Val Arg Ala Val Lys His Leu Glu Asn Ala Ser Gly Ile Glu Ala
50 55 60
Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro
65 70 75 80
Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg
85 90 95
Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln
›EXAMPLE 5 · 13 of 25
100 105 110
Gln Tyr Val Ile Glu Gly Arg Ile Ser Pro Gly Gly Gly Ser Gly Gly
115 120 125
Gly Ser Asn Met Ala Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln
130 135 140
Ser Phe Leu Leu Lys Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp
145 150 155 160
Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His
165 170 175
Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala
180 185 190
Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu
195 200 205
Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala
210 215 220
Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln
225 230 235 240
Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu
245 250 255
Leu Gly Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala
260 265 270
Phe Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser
275 280 285
His Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu
290 295 300
Ala Gln Pro
305
307 amino acids
amino acid
linear
protein
122
Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys
1 5 10 15
Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn Ser Glu Asp
20 25 30
Met Asp Ile Leu Met Glu Arg Asn Leu Arg Thr Pro Asn Leu Leu Ala
35 40 45
Phe Val Arg Ala Val Lys His Leu Glu Asn Ala Ser Gly Ile Glu Ala
50 55 60
Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro
65 70 75 80
Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg
85 90 95
Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln
100 105 110
Gln Tyr Val Ile Glu Gly Arg Ile Ser Pro Gly Gly Gly Ser Gly Gly
115 120 125
Gly Ser Asn Met Ala Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln
130 135 140
Ser Phe Leu Leu Lys Ser Leu Glu Gln Val Arg Lys Ile Gln Gly Asp
145 150 155 160
Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His
165 170 175
Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala
180 185 190
Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu
195 200 205
Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala
210 215 220
Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln
225 230 235 240
Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu
245 250 255
Leu Gly Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala
260 265 270
Phe Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser
275 280 285
His Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu
290 295 300
Ala Gln Pro
305
307 amino acids
amino acid
linear
protein
123
Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys
1 5 10 15
Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn Ser Glu Asp
20 25 30
Met Asp Ile Leu Met Glu Arg Asn Leu Arg Thr Pro Asn Leu Leu Ala
35 40 45
Phe Val Arg Ala Val Lys His Leu Glu Asn Ala Ser Gly Ile Glu Ala
50 55 60
Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro
65 70 75 80
Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg
85 90 95
Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln
100 105 110
Gln Tyr Val Ile Glu Gly Lys Ile Ser Pro Gly Gly Gly Ser Gly Gly
115 120 125
Gly Ser Asn Met Ala Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln
130 135 140
Ser Phe Leu Leu Lys Ser Leu Glu Gln Val Arg Lys Ile Gln Gly Asp
145 150 155 160
Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His
165 170 175
Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala
180 185 190
Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu
195 200 205
Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala
210 215 220
Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln
225 230 235 240
Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu
245 250 255
Leu Gly Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala
260 265 270
Phe Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser
275 280 285
His Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu
290 295 300
Ala Gln Pro
305
307 amino acids
amino acid
linear
protein
124
Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys
1 5 10 15
Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn Ser Glu Asp
20 25 30
Met Asp Ile Leu Met Glu Arg Asn Leu Arg Thr Pro Asn Leu Leu Ala
35 40 45
Phe Val Arg Ala Val Lys His Leu Glu Asn Ala Ser Gly Ile Glu Ala
50 55 60
Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro
65 70 75 80
Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg
85 90 95
Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln
100 105 110
Gln Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly Gly Gly Ser Gly Gly
115 120 125
Gly Ser Asn Met Ala Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln
130 135 140
Ser Phe Leu Leu Lys Ser Leu Glu Gln Val Arg Lys Ile Gln Gly Asp
145 150 155 160
Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His
165 170 175
Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala
180 185 190
Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu
195 200 205
Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala
210 215 220
Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln
225 230 235 240
Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu
245 250 255
Leu Gly Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala
260 265 270
Phe Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser
275 280 285
His Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu
290 295 300
Ala Gln Pro
305
244 amino acids
amino acid
linear
protein
125
Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys
1 5 10 15
Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn Ser Glu Asp
20 25 30
Met Asp Ile Leu Met Glu Arg Asn Leu Arg Thr Pro Asn Leu Leu Ala
35 40 45
Phe Val Arg Ala Val Lys His Leu Glu Asn Ala Ser Gly Ile Glu Ala
50 55 60
Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro
65 70 75 80
Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg
85 90 95
Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln
100 105 110
Gln Tyr Val Ile Glu Gly Arg Ile Ser Pro Gly Gly Gly Ser Gly Gly
115 120 125
Gly Ser Asn Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His
130 135 140
His Leu Lys Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn
›EXAMPLE 5 · 14 of 25
145 150 155 160
Ser Glu Asp Met Asp Ile Leu Met Glu Arg Asn Leu Arg Thr Pro Asn
165 170 175
Leu Leu Ala Phe Val Arg Ala Val Lys His Leu Glu Asn Ala Ser Gly
180 185 190
Ile Glu Ala Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr
195 200 205
Ala Ala Pro Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln
210 215 220
Glu Phe Arg Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala
225 230 235 240
Gln Glu Gln Gln
244 amino acids
amino acid
linear
protein
126
Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys
1 5 10 15
Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn Ser Glu Asp
20 25 30
Met Asp Ile Leu Met Glu Arg Asn Leu Arg Thr Pro Asn Leu Leu Ala
35 40 45
Phe Val Arg Ala Val Lys His Leu Glu Asn Ala Ser Gly Ile Glu Ala
50 55 60
Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro
65 70 75 80
Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg
85 90 95
Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln
100 105 110
Gln Tyr Val Ile Glu Gly Lys Ile Ser Pro Gly Gly Gly Ser Gly Gly
115 120 125
Gly Ser Asn Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His
130 135 140
His Leu Lys Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn
145 150 155 160
Ser Glu Asp Met Asp Ile Leu Met Glu Arg Asn Leu Arg Thr Pro Asn
165 170 175
Leu Leu Ala Phe Val Arg Ala Val Lys His Leu Glu Asn Ala Ser Gly
180 185 190
Ile Glu Ala Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr
195 200 205
Ala Ala Pro Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln
210 215 220
Glu Phe Arg Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala
225 230 235 240
Gln Glu Gln Gln
244 amino acids
amino acid
linear
protein
127
Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys
1 5 10 15
Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn Ser Glu Asp
20 25 30
Met Asp Ile Leu Met Glu Arg Asn Leu Arg Thr Pro Asn Leu Leu Ala
35 40 45
Phe Val Arg Ala Val Lys His Leu Glu Asn Ala Ser Gly Ile Glu Ala
50 55 60
Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro
65 70 75 80
Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg
85 90 95
Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln
100 105 110
Gln Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly Gly Gly Ser Gly Gly
115 120 125
Gly Ser Asn Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His
130 135 140
His Leu Lys Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn
145 150 155 160
Ser Glu Asp Met Asp Ile Leu Met Glu Arg Asn Leu Arg Thr Pro Asn
165 170 175
Leu Leu Ala Phe Val Arg Ala Val Lys His Leu Glu Asn Ala Ser Gly
180 185 190
Ile Glu Ala Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr
195 200 205
Ala Ala Pro Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln
210 215 220
Glu Phe Arg Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala
225 230 235 240
Gln Glu Gln Gln
322 amino acids
amino acid
linear
protein
128
Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys
1 5 10 15
Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn Ser Glu Asp
20 25 30
Met Asp Ile Leu Met Glu Arg Asn Leu Arg Thr Pro Asn Leu Leu Ala
35 40 45
Phe Val Arg Ala Val Lys His Leu Glu Asn Ala Ser Gly Ile Glu Ala
50 55 60
Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro
65 70 75 80
Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg
85 90 95
Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln
100 105 110
Gln Tyr Val Ile Glu Gly Arg Ile Ser Pro Gly Glu Pro Ser Gly Pro
115 120 125
Ile Ser Thr Ile Asn Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser
130 135 140
Pro Asn Met Ala Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser
145 150 155 160
Phe Leu Leu Lys Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly
165 170 175
Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro
180 185 190
Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro
195 200 205
Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser
210 215 220
Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu
225 230 235 240
Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu
245 250 255
Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu
260 265 270
Gly Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe
275 280 285
Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His
290 295 300
Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala
305 310 315 320
Gln Pro
322 amino acids
amino acid
linear
protein
129
Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys
1 5 10 15
Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn Ser Glu Asp
20 25 30
Met Asp Ile Leu Met Glu Arg Asn Leu Arg Thr Pro Asn Leu Leu Ala
35 40 45
Phe Val Arg Ala Val Lys His Leu Glu Asn Ala Ser Gly Ile Glu Ala
50 55 60
Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro
65 70 75 80
Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg
85 90 95
Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln
100 105 110
Gln Tyr Val Ile Glu Gly Lys Ile Ser Pro Gly Glu Pro Ser Gly Pro
115 120 125
Ile Ser Thr Ile Asn Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser
130 135 140
Pro Asn Met Ala Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser
145 150 155 160
Phe Leu Leu Lys Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly
165 170 175
Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro
180 185 190
Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro
195 200 205
Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser
210 215 220
Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu
225 230 235 240
Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu
245 250 255
Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu
260 265 270
Gly Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe
275 280 285
Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His
290 295 300
Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala
305 310 315 320
Gln Pro
322 amino acids
amino acid
linear
protein
130
Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys
1 5 10 15
Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn Ser Glu Asp
20 25 30
Met Asp Ile Leu Met Glu Arg Asn Leu Arg Thr Pro Asn Leu Leu Ala
›EXAMPLE 5 · 15 of 25
35 40 45
Phe Val Arg Ala Val Lys His Leu Glu Asn Ala Ser Gly Ile Glu Ala
50 55 60
Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro
65 70 75 80
Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg
85 90 95
Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln
100 105 110
Gln Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly Glu Pro Ser Gly Pro
115 120 125
Ile Ser Thr Ile Asn Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser
130 135 140
Pro Asn Met Ala Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser
145 150 155 160
Phe Leu Leu Lys Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly
165 170 175
Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro
180 185 190
Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro
195 200 205
Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser
210 215 220
Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu
225 230 235 240
Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu
245 250 255
Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu
260 265 270
Gly Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe
275 280 285
Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His
290 295 300
Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala
305 310 315 320
Gln Pro
259 amino acids
amino acid
linear
protein
131
Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys
1 5 10 15
Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn Ser Glu Asp
20 25 30
Met Asp Ile Leu Met Glu Arg Asn Leu Arg Thr Pro Asn Leu Leu Ala
35 40 45
Phe Val Arg Ala Val Lys His Leu Glu Asn Ala Ser Gly Ile Glu Ala
50 55 60
Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro
65 70 75 80
Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg
85 90 95
Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln
100 105 110
Gln Tyr Val Ile Glu Gly Arg Ile Ser Pro Gly Glu Pro Ser Gly Pro
115 120 125
Ile Ser Thr Ile Asn Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser
130 135 140
Pro Asn Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His
145 150 155 160
Leu Lys Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn Ser
165 170 175
Glu Asp Met Asp Ile Leu Met Glu Arg Asn Leu Arg Thr Pro Asn Leu
180 185 190
Leu Ala Phe Val Arg Ala Val Lys His Leu Glu Asn Ala Ser Gly Ile
195 200 205
Glu Ala Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala
210 215 220
Ala Pro Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu
225 230 235 240
Phe Arg Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln
245 250 255
Glu Gln Gln
259 amino acids
amino acid
linear
protein
132
Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys
1 5 10 15
Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn Ser Glu Asp
20 25 30
Met Asp Ile Leu Met Glu Arg Asn Leu Arg Thr Pro Asn Leu Leu Ala
35 40 45
Phe Val Arg Ala Val Lys His Leu Glu Asn Ala Ser Gly Ile Glu Ala
50 55 60
Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro
65 70 75 80
Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg
85 90 95
Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln
100 105 110
Gln Tyr Val Ile Glu Gly Lys Ile Ser Pro Gly Glu Pro Ser Gly Pro
115 120 125
Ile Ser Thr Ile Asn Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser
130 135 140
Pro Asn Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His
145 150 155 160
Leu Lys Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn Ser
165 170 175
Glu Asp Met Asp Ile Leu Met Glu Arg Asn Leu Arg Thr Pro Asn Leu
180 185 190
Leu Ala Phe Val Arg Ala Val Lys His Leu Glu Asn Ala Ser Gly Ile
195 200 205
Glu Ala Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala
210 215 220
Ala Pro Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu
225 230 235 240
Phe Arg Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln
245 250 255
Glu Gln Gln
259 amino acids
amino acid
linear
protein
133
Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys
1 5 10 15
Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn Ser Glu Asp
20 25 30
Met Asp Ile Leu Met Glu Arg Asn Leu Arg Thr Pro Asn Leu Leu Ala
35 40 45
Phe Val Arg Ala Val Lys His Leu Glu Asn Ala Ser Gly Ile Glu Ala
50 55 60
Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro
65 70 75 80
Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg
85 90 95
Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln
100 105 110
Gln Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly Glu Pro Ser Gly Pro
115 120 125
Ile Ser Thr Ile Asn Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser
130 135 140
Pro Asn Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His
145 150 155 160
Leu Lys Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn Ser
165 170 175
Glu Asp Met Asp Ile Leu Met Glu Arg Asn Leu Arg Thr Pro Asn Leu
180 185 190
Leu Ala Phe Val Arg Ala Val Lys His Leu Glu Asn Ala Ser Gly Ile
195 200 205
Glu Ala Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala
210 215 220
Ala Pro Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu
225 230 235 240
Phe Arg Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln
245 250 255
Glu Gln Gln
307 amino acids
amino acid
linear
protein
134
Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys
1 5 10 15
Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn Leu Asn Asp Glu Asp
20 25 30
Val Ser Ile Leu Met Asp Arg Asn Leu Arg Leu Pro Asn Leu Glu Ser
35 40 45
Phe Val Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Gly Ile Glu Ala
50 55 60
Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro
65 70 75 80
Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg
85 90 95
Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln
100 105 110
Gln Tyr Val Ile Glu Gly Arg Ile Ser Pro Gly Gly Gly Ser Gly Gly
115 120 125
Gly Ser Asn Met Ala Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln
130 135 140
Ser Phe Leu Leu Lys Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp
145 150 155 160
Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His
165 170 175
Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala
180 185 190
Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu
195 200 205
Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala
›EXAMPLE 5 · 16 of 25
210 215 220
Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln
225 230 235 240
Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu
245 250 255
Leu Gly Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala
260 265 270
Phe Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser
275 280 285
His Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu
290 295 300
Ala Gln Pro
305
307 amino acids
amino acid
linear
protein
135
Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys
1 5 10 15
Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn Leu Asn Asp Glu Asp
20 25 30
Val Ser Ile Leu Met Asp Arg Asn Leu Arg Leu Pro Asn Leu Glu Ser
35 40 45
Phe Val Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Gly Ile Glu Ala
50 55 60
Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro
65 70 75 80
Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg
85 90 95
Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln
100 105 110
Gln Tyr Val Ile Glu Gly Arg Ile Ser Pro Gly Gly Gly Ser Gly Gly
115 120 125
Gly Ser Asn Met Ala Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln
130 135 140
Ser Phe Leu Leu Lys Ser Leu Glu Gln Val Arg Lys Ile Gln Gly Asp
145 150 155 160
Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His
165 170 175
Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala
180 185 190
Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu
195 200 205
Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala
210 215 220
Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln
225 230 235 240
Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu
245 250 255
Leu Gly Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala
260 265 270
Phe Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser
275 280 285
His Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu
290 295 300
Ala Gln Pro
305
244 amino acids
amino acid
linear
protein
136
Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys
1 5 10 15
Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn Leu Asn Asp Glu Asp
20 25 30
Val Ser Ile Leu Met Asp Arg Asn Leu Arg Leu Pro Asn Leu Glu Ser
35 40 45
Phe Val Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Gly Ile Glu Ala
50 55 60
Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro
65 70 75 80
Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg
85 90 95
Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln
100 105 110
Gln Tyr Val Ile Glu Gly Arg Ile Ser Pro Gly Gly Gly Ser Gly Gly
115 120 125
Gly Ser Asn Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His
130 135 140
His Leu Lys Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn Leu Asn
145 150 155 160
Asp Glu Asp Val Ser Ile Leu Met Asp Arg Asn Leu Arg Leu Pro Asn
165 170 175
Leu Glu Ser Phe Val Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Gly
180 185 190
Ile Glu Ala Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr
195 200 205
Ala Ala Pro Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln
210 215 220
Glu Phe Arg Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala
225 230 235 240
Gln Glu Gln Gln
259 amino acids
amino acid
linear
protein
137
Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys
1 5 10 15
Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn Leu Asn Asp Glu Asp
20 25 30
Val Ser Ile Leu Met Asp Arg Asn Leu Arg Leu Pro Asn Leu Glu Ser
35 40 45
Phe Val Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Gly Ile Glu Ala
50 55 60
Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro
65 70 75 80
Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg
85 90 95
Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln
100 105 110
Gln Tyr Val Ile Glu Gly Arg Ile Ser Pro Gly Glu Pro Ser Gly Pro
115 120 125
Ile Ser Thr Ile Asn Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser
130 135 140
Pro Asn Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His
145 150 155 160
Leu Lys Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn Leu Asn Asp
165 170 175
Glu Asp Val Ser Ile Leu Met Asp Arg Asn Leu Arg Leu Pro Asn Leu
180 185 190
Glu Ser Phe Val Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Gly Ile
195 200 205
Glu Ala Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala
210 215 220
Ala Pro Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu
225 230 235 240
Phe Arg Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln
245 250 255
Glu Gln Gln
322 amino acids
amino acid
linear
protein
138
Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys
1 5 10 15
Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn Leu Asn Asp Glu Asp
20 25 30
Val Ser Ile Leu Met Asp Arg Asn Leu Arg Leu Pro Asn Leu Glu Ser
35 40 45
Phe Val Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Gly Ile Glu Ala
50 55 60
Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro
65 70 75 80
Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg
85 90 95
Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln
100 105 110
Gln Tyr Val Ile Glu Gly Arg Ile Ser Pro Gly Glu Pro Ser Gly Pro
115 120 125
Ile Ser Thr Ile Asn Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser
130 135 140
Pro Asn Met Ala Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser
145 150 155 160
Phe Leu Leu Lys Ser Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly
165 170 175
Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro
180 185 190
Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro
195 200 205
Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser
210 215 220
Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu
225 230 235 240
Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu
245 250 255
Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu
260 265 270
Gly Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe
275 280 285
Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His
290 295 300
Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala
305 310 315 320
Gln Pro
349 amino acids
amino acid
linear
protein
139
Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys
1 5 10 15
Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn Ser Glu Asp
20 25 30
Met Asp Ile Leu Met Glu Arg Asn Leu Arg Thr Pro Asn Leu Leu Ala
›EXAMPLE 5 · 17 of 25
35 40 45
Phe Val Arg Ala Val Lys His Leu Glu Asn Ala Ser Gly Ile Glu Ala
50 55 60
Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro
65 70 75 80
Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg
85 90 95
Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln
100 105 110
Gln Tyr Val Ile Glu Gly Arg Ile Ser Pro Gln Pro Pro Val Asn Ala
115 120 125
Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Glu Gly Gly Gly
130 135 140
Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser
145 150 155 160
Gly Gly Gly Ser Gly Ser Gly Asp Phe Asp Tyr Glu Asn Met Ala Thr
165 170 175
Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys Ser
180 185 190
Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu
195 200 205
Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu
210 215 220
Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro
225 230 235 240
Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser Gly
245 250 255
Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser Pro
260 265 270
Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp Phe
275 280 285
Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro Ala
290 295 300
Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gln
305 310 315 320
Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe Leu
325 330 335
Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro
340 345
GGATCCACCA TGAGCCGCCT GCCCGTCCTG CTCCTGCTCC AACTCCTGGT CCGCCCCGCC 60
ATGG 64
259 amino acids
amino acid
linear
protein
141
Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys
1 5 10 15
Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn Ser Glu Asp
20 25 30
Met Asp Ile Leu Met Glu Arg Asn Leu Arg Thr Pro Asn Leu Leu Ala
35 40 45
Phe Val Arg Ala Val Lys His Leu Glu Asn Ala Ser Gly Ile Glu Ala
50 55 60
Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro
65 70 75 80
Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg
85 90 95
Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln
100 105 110
Gln Tyr Val Ile Glu Gly Arg Ile Ser Pro Gly Gly Gly Ser Gly Gly
115 120 125
Gly Ser Asn Met Ala Pro Ala Arg Ser Pro Ser Pro Ser Thr Gln Pro
130 135 140
Trp Glu His Val Asn Ala Ile Gln Glu Ala Arg Arg Leu Leu Asn Leu
145 150 155 160
Ser Arg Asp Thr Ala Ala Glu Met Asn Glu Thr Val Glu Val Ile Ser
165 170 175
Glu Met Phe Asp Leu Gln Glu Pro Thr Cys Leu Gln Thr Arg Leu Glu
180 185 190
Leu Tyr Lys Gln Gly Leu Arg Gly Ser Leu Thr Lys Leu Lys Gly Pro
195 200 205
Leu Thr Met Met Ala Ser His Tyr Lys Gln His Cys Pro Pro Thr Pro
210 215 220
Glu Thr Ser Cys Ala Thr Gln Ile Ile Thr Phe Glu Ser Phe Lys Glu
225 230 235 240
Asn Leu Lys Asp Phe Leu Leu Val Ile Pro Phe Asp Cys Trp Glu Pro
245 250 255
Val Gln Glu
301 amino acids
amino acid
linear
protein
142
Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys
1 5 10 15
Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn Ser Glu Asp
20 25 30
Met Asp Ile Leu Met Glu Arg Asn Leu Arg Thr Pro Asn Leu Leu Ala
35 40 45
Phe Val Arg Ala Val Lys His Leu Glu Asn Ala Ser Gly Ile Glu Ala
50 55 60
Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro
65 70 75 80
Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg
85 90 95
Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln
100 105 110
Gln Tyr Val Ile Glu Gly Arg Ile Ser Pro Gln Pro Pro Val Asn Ala
115 120 125
Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Glu Gly Gly Gly
130 135 140
Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser
145 150 155 160
Gly Gly Gly Ser Gly Ser Gly Asp Phe Asp Tyr Glu Asn Met Ala Pro
165 170 175
Ala Arg Ser Pro Ser Pro Ser Thr Gln Pro Trp Glu His Val Asn Ala
180 185 190
Ile Gln Glu Ala Arg Arg Leu Leu Asn Leu Ser Arg Asp Thr Ala Ala
195 200 205
Glu Met Asn Glu Thr Val Glu Val Ile Ser Glu Met Phe Asp Leu Gln
210 215 220
Glu Pro Thr Cys Leu Gln Thr Arg Leu Glu Leu Tyr Lys Gln Gly Leu
225 230 235 240
Arg Gly Ser Leu Thr Lys Leu Lys Gly Pro Leu Thr Met Met Ala Ser
245 250 255
His Tyr Lys Gln His Cys Pro Pro Thr Pro Glu Thr Ser Cys Ala Thr
260 265 270
Gln Ile Ile Thr Phe Glu Ser Phe Lys Glu Asn Leu Lys Asp Phe Leu
275 280 285
Leu Val Ile Pro Phe Asp Cys Trp Glu Pro Val Gln Glu
290 295 300
335 amino acids
amino acid
linear
protein
143
Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys
1 5 10 15
Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn Ser Glu Asp
20 25 30
Met Asp Ile Leu Met Glu Arg Asn Leu Arg Thr Pro Asn Leu Leu Ala
35 40 45
Phe Val Arg Ala Val Lys His Leu Glu Asn Ala Ser Gly Ile Glu Ala
50 55 60
Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro
65 70 75 80
Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg
85 90 95
Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln
100 105 110
Gln Tyr Val Pro Val Asn Ala Gly Gly Gly Ser Gly Gly Gly Ser Gly
115 120 125
Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly
130 135 140
Gly Ser Glu Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Gly Asn Met
145 150 155 160
Ala Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu
165 170 175
Lys Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu
180 185 190
Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu
195 200 205
Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser
210 215 220
Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His
225 230 235 240
Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile
245 250 255
Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala
260 265 270
Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala
275 280 285
Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala
290 295 300
Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser
305 310 315 320
Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro
325 330 335
274 amino acids
amino acid
linear
protein
144
Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys
1 5 10 15
Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn Ser Glu Asp
20 25 30
Met Asp Ile Leu Met Glu Arg Asn Leu Arg Thr Pro Asn Leu Leu Ala
35 40 45
Phe Val Arg Ala Val Lys His Leu Glu Asn Ala Ser Gly Ile Glu Ala
›EXAMPLE 5 · 18 of 25
50 55 60
Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro
65 70 75 80
Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg
85 90 95
Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln
100 105 110
Gln Tyr Val Ile Glu Gly Arg Ile Ser Pro Gly Glu Pro Ser Gly Pro
115 120 125
Ile Ser Thr Ile Asn Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser
130 135 140
Pro Asn Met Ala Pro Ala Arg Ser Pro Ser Pro Ser Thr Gln Pro Trp
145 150 155 160
Glu His Val Asn Ala Ile Gln Glu Ala Arg Arg Leu Leu Asn Leu Ser
165 170 175
Arg Asp Thr Ala Ala Glu Met Asn Glu Thr Val Glu Val Ile Ser Glu
180 185 190
Met Phe Asp Leu Gln Glu Pro Thr Cys Leu Gln Thr Arg Leu Glu Leu
195 200 205
Tyr Lys Gln Gly Leu Arg Gly Ser Leu Thr Lys Leu Lys Gly Pro Leu
210 215 220
Thr Met Met Ala Ser His Tyr Lys Gln His Cys Pro Pro Thr Pro Glu
225 230 235 240
Thr Ser Cys Ala Thr Gln Ile Ile Thr Phe Glu Ser Phe Lys Glu Asn
245 250 255
Leu Lys Asp Phe Leu Leu Val Ile Pro Phe Asp Cys Trp Glu Pro Val
260 265 270
Gln Glu
317 amino acids
amino acid
linear
protein
145
Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys
1 5 10 15
Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn Ser Glu Asp
20 25 30
Met Asp Ile Leu Met Glu Arg Asn Leu Arg Thr Pro Asn Leu Leu Ala
35 40 45
Phe Val Arg Ala Val Lys His Leu Glu Asn Ala Ser Gly Ile Glu Ala
50 55 60
Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro
65 70 75 80
Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg
85 90 95
Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln
100 105 110
Gln Tyr Val Ile Glu Gly Arg Ile Ser Pro Gly Gly Gly Ser Gly Gly
115 120 125
Gly Ser Asn Met Ala Pro Val Pro Pro Gly Glu Asp Ser Lys Asp Val
130 135 140
Ala Ala Pro His Arg Gln Pro Leu Thr Ser Ser Glu Arg Ile Asp Lys
145 150 155 160
Gln Ile Arg Tyr Ile Leu Asp Gly Ile Ser Ala Leu Arg Lys Glu Thr
165 170 175
Cys Asn Lys Ser Asn Met Cys Glu Ser Ser Lys Glu Ala Leu Ala Glu
180 185 190
Asn Asn Leu Asn Leu Pro Lys Met Ala Glu Lys Asp Gly Cys Phe Gln
195 200 205
Ser Gly Phe Asn Glu Glu Thr Cys Leu Val Lys Ile Ile Thr Gly Leu
210 215 220
Leu Glu Phe Glu Val Tyr Leu Glu Tyr Leu Gln Asn Arg Phe Glu Ser
225 230 235 240
Ser Glu Glu Gln Ala Arg Ala Val Gln Met Ser Thr Lys Val Leu Ile
245 250 255
Gln Phe Leu Gln Lys Lys Ala Lys Asn Leu Asp Ala Ile Thr Thr Pro
260 265 270
Asp Pro Thr Thr Asn Ala Ser Leu Leu Thr Lys Leu Gln Ala Gln Asn
275 280 285
Gln Trp Leu Gln Asp Met Thr Thr His Leu Ile Leu Arg Ser Phe Lys
290 295 300
Glu Phe Leu Gln Ser Ser Leu Arg Ala Leu Arg Gln Met
305 310 315
307 amino acids
amino acid
linear
protein
146
Met Ala Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu
1 5 10 15
Leu Lys Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala
20 25 30
Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu
35 40 45
Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser
50 55 60
Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu
65 70 75 80
His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly
85 90 95
Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val
100 105 110
Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met
115 120 125
Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser
130 135 140
Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln
145 150 155 160
Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro
165 170 175
Tyr Val Ile Glu Gly Arg Ile Ser Pro Gly Gly Gly Ser Gly Gly Gly
180 185 190
Ser Asn Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His
195 200 205
Leu Lys Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn Ser
210 215 220
Glu Asp Met Asp Ile Leu Met Glu Arg Asn Leu Arg Thr Pro Asn Leu
225 230 235 240
Leu Ala Phe Val Arg Ala Val Lys His Leu Glu Asn Ala Ser Gly Ile
245 250 255
Glu Ala Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala
260 265 270
Ala Pro Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu
275 280 285
Phe Arg Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln
290 295 300
Glu Gln Gln
305
307 amino acids
amino acid
linear
protein
147
Met Ala Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu
1 5 10 15
Leu Lys Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala
20 25 30
Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu
35 40 45
Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser
50 55 60
Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu
65 70 75 80
His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly
85 90 95
Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val
100 105 110
Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met
115 120 125
Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser
130 135 140
Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln
145 150 155 160
Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro
165 170 175
Tyr Val Ile Glu Gly Arg Ile Ser Pro Gly Gly Gly Ser Gly Gly Gly
180 185 190
Ser Asn Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His
195 200 205
Leu Lys Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn Leu Asn Asp
210 215 220
Glu Asp Val Ser Ile Leu Met Asp Arg Asn Leu Arg Leu Pro Asn Leu
225 230 235 240
Glu Ser Phe Val Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Gly Ile
245 250 255
Glu Ala Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala
260 265 270
Ala Pro Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu
275 280 285
Phe Arg Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln
290 295 300
Glu Gln Gln
305
337 amino acids
amino acid
linear
protein
148
Met Ala Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu
1 5 10 15
Leu Lys Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala
20 25 30
Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu
35 40 45
Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser
50 55 60
Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu
65 70 75 80
His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly
85 90 95
Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val
100 105 110
Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met
›EXAMPLE 5 · 19 of 25
115 120 125
Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser
130 135 140
Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln
145 150 155 160
Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro
165 170 175
Tyr Val Pro Gln Pro Pro Val Asn Ala Gly Gly Gly Ser Gly Gly Gly
180 185 190
Ser Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu
195 200 205
Gly Gly Gly Ser Glu Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Gly
210 215 220
Asp Phe Asp Tyr Glu Asn Met Ala Asn Cys Ser Ile Met Ile Asp Glu
225 230 235 240
Ile Ile His His Leu Lys Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn
245 250 255
Asn Leu Asn Asp Glu Asp Val Ser Ile Leu Met Asp Arg Asn Leu Arg
260 265 270
Leu Val Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Gly Ile Glu Ala
275 280 285
Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro
290 295 300
Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg
305 310 315 320
Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln
325 330 335
Gln
322 amino acids
amino acid
linear
protein
149
Met Ala Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu
1 5 10 15
Leu Lys Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala
20 25 30
Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu
35 40 45
Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser
50 55 60
Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu
65 70 75 80
His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly
85 90 95
Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val
100 105 110
Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met
115 120 125
Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser
130 135 140
Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln
145 150 155 160
Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro
165 170 175
Tyr Val Ile Glu Gly Arg Ile Ser Pro Gly Glu Pro Ser Gly Pro Ile
180 185 190
Ser Thr Ile Asn Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro
195 200 205
Asn Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu
210 215 220
Lys Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn Leu Asn Asp Glu
225 230 235 240
Asp Val Ser Ile Leu Met Asp Arg Asn Leu Arg Leu Pro Asn Leu Glu
245 250 255
Ser Phe Val Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Gly Ile Glu
260 265 270
Ala Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala
275 280 285
Pro Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe
290 295 300
Arg Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu
305 310 315 320
Gln Gln
322 amino acids
amino acid
linear
protein
150
Met Ala Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu
1 5 10 15
Leu Lys Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala
20 25 30
Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu
35 40 45
Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser
50 55 60
Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu
65 70 75 80
His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly
85 90 95
Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val
100 105 110
Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met
115 120 125
Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser
130 135 140
Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln
145 150 155 160
Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro
165 170 175
Tyr Val Ile Glu Gly Arg Ile Ser Pro Gly Glu Pro Ser Gly Pro Ile
180 185 190
Ser Thr Ile Asn Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro
195 200 205
Asn Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu
210 215 220
Lys Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn Ser Glu
225 230 235 240
Asp Met Asp Ile Leu Met Glu Arg Asn Leu Arg Thr Pro Asn Leu Leu
245 250 255
Ala Phe Val Arg Ala Val Lys His Leu Glu Asn Ala Ser Gly Ile Glu
260 265 270
Ala Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala
275 280 285
Pro Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe
290 295 300
Arg Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu
305 310 315 320
Gln Gln
349 amino acids
amino acid
linear
protein
151
Met Ala Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu
1 5 10 15
Leu Lys Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala
20 25 30
Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu
35 40 45
Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser
50 55 60
Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu
65 70 75 80
His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly
85 90 95
Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val
100 105 110
Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met
115 120 125
Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser
130 135 140
Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln
145 150 155 160
Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro
165 170 175
Tyr Val Ile Glu Gly Arg Ile Ser Pro Gln Pro Pro Val Asn Ala Gly
180 185 190
Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Glu Gly Gly Gly Ser
195 200 205
Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Gly
210 215 220
Gly Gly Ser Gly Ser Gly Asp Phe Asp Tyr Glu Asn Met Ala Asn Cys
225 230 235 240
Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys Arg Pro Pro Asn
245 250 255
Pro Leu Leu Asp Pro Asn Asn Leu Asn Ser Glu Asp Met Asp Ile Leu
260 265 270
Met Glu Arg Asn Leu Arg Thr Pro Asn Leu Leu Ala Phe Val Arg Ala
275 280 285
Val Lys His Leu Glu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn
290 295 300
Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His Pro
305 310 315 320
Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu Lys Leu Thr
325 330 335
Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln Gln
340 345
307 amino acids
amino acid
linear
protein
152
Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys
1 5 10 15
Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn Leu Asn Asp Glu Asp
20 25 30
Val Ser Ile Leu Met Asp Arg Asn Leu Arg Leu Pro Asn Leu Glu Ser
35 40 45
Phe Val Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Gly Ile Glu Ala
›EXAMPLE 5 · 20 of 25
50 55 60
Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro
65 70 75 80
Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg
85 90 95
Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln
100 105 110
Gln Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly Gly Gly Ser Gly Gly
115 120 125
Gly Ser Asn Met Ala Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln
130 135 140
Ser Phe Leu Leu Lys Ser Leu Glu Gln Val Arg Lys Ile Gln Gly Asp
145 150 155 160
Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His
165 170 175
Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala
180 185 190
Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu
195 200 205
Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala
210 215 220
Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln
225 230 235 240
Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu
245 250 255
Leu Gly Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala
260 265 270
Phe Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser
275 280 285
His Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu
290 295 300
Ala Gln Pro
305
244 amino acids
amino acid
linear
protein
153
Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys
1 5 10 15
Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn Leu Asn Asp Glu Asp
20 25 30
Val Ser Ile Leu Met Asp Arg Asn Leu Arg Leu Pro Asn Leu Glu Ser
35 40 45
Phe Val Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Gly Ile Glu Ala
50 55 60
Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro
65 70 75 80
Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg
85 90 95
Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln
100 105 110
Gln Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly Gly Gly Ser Gly Gly
115 120 125
Gly Ser Asn Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His
130 135 140
His Leu Lys Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn Leu Asn
145 150 155 160
Asp Glu Asp Val Ser Ile Leu Met Asp Arg Asn Leu Arg Leu Pro Asn
165 170 175
Leu Glu Ser Phe Val Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Gly
180 185 190
Ile Glu Ala Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr
195 200 205
Ala Ala Pro Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln
210 215 220
Glu Phe Arg Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala
225 230 235 240
Gln Glu Gln Gln
322 amino acids
amino acid
linear
protein
154
Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys
1 5 10 15
Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn Leu Asn Asp Glu Asp
20 25 30
Val Ser Ile Leu Met Asp Arg Asn Leu Arg Leu Pro Asn Leu Glu Ser
35 40 45
Phe Val Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Gly Ile Glu Ala
50 55 60
Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro
65 70 75 80
Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg
85 90 95
Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln
100 105 110
Gln Tyr Val Ile Glu Gly Gly Gly Ser Pro Gly Glu Pro Ser Gly Pro
115 120 125
Ile Ser Thr Ile Asn Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser
130 135 140
Pro Asn Met Ala Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser
145 150 155 160
Phe Leu Leu Lys Ser Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly
165 170 175
Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro
180 185 190
Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro
195 200 205
Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser
210 215 220
Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu
225 230 235 240
Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu
245 250 255
Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu
260 265 270
Gly Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe
275 280 285
Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His
290 295 300
Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala
305 310 315 320
Gln Pro
259 amino acids
amino acid
linear
protein
155
Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys
1 5 10 15
Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn Leu Asn Asp Glu Asp
20 25 30
Val Ser Ile Leu Met Asp Arg Asn Leu Arg Leu Pro Asn Leu Glu Ser
35 40 45
Phe Val Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Gly Ile Glu Ala
50 55 60
Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro
65 70 75 80
Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg
85 90 95
Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln
100 105 110
Gln Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly Glu Pro Ser Gly Pro
115 120 125
Ile Ser Thr Ile Asn Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser
130 135 140
Pro Asn Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His
145 150 155 160
Leu Lys Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn Leu Asn Asp
165 170 175
Glu Asp Val Ser Ile Leu Met Asp Arg Asn Leu Arg Leu Pro Asn Leu
180 185 190
Glu Ser Phe Val Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Gly Ile
195 200 205
Glu Ala Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala
210 215 220
Ala Pro Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu
225 230 235 240
Phe Arg Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln
245 250 255
Glu Gln Gln
322 amino acids
amino acid
linear
protein
156
Met Ala Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu
1 5 10 15
Leu Lys Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala
20 25 30
Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu
35 40 45
Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser
50 55 60
Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gly Leu
65 70 75 80
His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly
85 90 95
Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val
100 105 110
Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met
115 120 125
Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser
130 135 140
Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln
145 150 155 160
Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro
165 170 175
Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly Glu Pro Ser Gly Pro Ile
180 185 190
Ser Thr Ile Asn Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro
›EXAMPLE 5 · 21 of 25
195 200 205
Asn Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu
210 215 220
Lys Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn Leu Asn Asp Glu
225 230 235 240
Asp Val Ser Ile Leu Met Asp Arg Asn Leu Arg Leu Pro Asn Leu Glu
245 250 255
Ser Phe Val Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Gly Ile Glu
260 265 270
Ala Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala
275 280 285
Pro Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe
290 295 300
Arg Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu
305 310 315 320
Gln Gln
322 amino acids
amino acid
linear
protein
157
Met Ala Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu
1 5 10 15
Leu Lys Ser Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala
20 25 30
Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu
35 40 45
Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser
50 55 60
Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gly Leu
65 70 75 80
His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly
85 90 95
Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val
100 105 110
Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met
115 120 125
Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser
130 135 140
Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln
145 150 155 160
Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro
165 170 175
Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly Glu Pro Ser Gly Pro Ile
180 185 190
Ser Thr Ile Asn Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro
195 200 205
Asn Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu
210 215 220
Lys Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn Leu Asn Asp Glu
225 230 235 240
Asp Val Ser Ile Leu Met Asp Arg Asn Leu Arg Leu Pro Asn Leu Glu
245 250 255
Ser Phe Val Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Gly Ile Glu
260 265 270
Ala Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala
275 280 285
Pro Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe
290 295 300
Arg Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu
305 310 315 320
Gln Gln
307 amino acids
amino acid
linear
protein
158
Met Ala Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu
1 5 10 15
Leu Lys Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala
20 25 30
Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu
35 40 45
Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser
50 55 60
Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gly Leu
65 70 75 80
His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly
85 90 95
Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val
100 105 110
Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met
115 120 125
Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser
130 135 140
Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln
145 150 155 160
Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro
165 170 175
Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly Gly Gly Ser Gly Gly Gly
180 185 190
Ser Asn Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His
195 200 205
Leu Lys Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn Leu Asn Asp
210 215 220
Glu Asp Val Ser Ile Leu Met Asp Arg Asn Leu Arg Leu Pro Asn Leu
225 230 235 240
Glu Ser Phe Val Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Gly Ile
245 250 255
Glu Ala Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala
260 265 270
Ala Pro Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu
275 280 285
Phe Arg Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln
290 295 300
Glu Gln Gln
305
307 amino acids
amino acid
linear
protein
159
Met Ala Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu
1 5 10 15
Leu Lys Ser Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala
20 25 30
Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu
35 40 45
Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser
50 55 60
Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gly Leu
65 70 75 80
His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly
85 90 95
Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val
100 105 110
Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met
115 120 125
Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser
130 135 140
Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln
145 150 155 160
Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro
165 170 175
Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly Gly Gly Ser Gly Gly Gly
180 185 190
Ser Asn Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His
195 200 205
Leu Lys Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn Leu Asn Asp
210 215 220
Glu Asp Val Ser Ile Leu Met Asp Arg Asn Leu Arg Leu Pro Asn Leu
225 230 235 240
Glu Ser Phe Val Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Gly Ile
245 250 255
Glu Ala Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala
260 265 270
Ala Pro Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu
275 280 285
Phe Arg Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln
290 295 300
Glu Gln Gln
305
128 amino acids
amino acid
linear
protein
160
Met Ala Pro Ala Arg Ser Pro Ser Pro Ser Thr Gln Pro Trp Glu His
1 5 10 15
Val Asn Ala Ile Gln Glu Ala Arg Arg Leu Leu Asn Leu Ser Arg Asp
20 25 30
Thr Ala Ala Glu Met Asn Glu Thr Val Glu Val Ile Ser Glu Met Phe
35 40 45
Asp Leu Gln Glu Pro Thr Cys Leu Gln Thr Arg Leu Glu Leu Tyr Lys
50 55 60
Gln Gly Leu Arg Gly Ser Leu Thr Lys Leu Lys Gly Pro Leu Thr Met
65 70 75 80
Met Ala Ser His Tyr Lys Gln His Cys Pro Pro Thr Pro Glu Thr Ser
85 90 95
Cys Ala Thr Gln Ile Ile Thr Phe Glu Ser Phe Lys Glu Asn Leu Lys
100 105 110
Asp Phe Leu Leu Val Ile Pro Phe Asp Cys Trp Glu Pro Val Gln Glu
115 120 125
176 amino acids
amino acid
linear
protein
161
Met Ala Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu
1 5 10 15
Leu Lys Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala
20 25 30
Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu
35 40 45
Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser
50 55 60
Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu
65 70 75 80
His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly
›EXAMPLE 5 · 22 of 25
85 90 95
Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val
100 105 110
Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met
115 120 125
Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser
130 135 140
Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln
145 150 155 160
Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro
165 170 175
176 amino acids
amino acid
linear
protein
162
Met Ala Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu
1 5 10 15
Leu Lys Ser Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala
20 25 30
Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu
35 40 45
Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser
50 55 60
Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu
65 70 75 80
His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly
85 90 95
Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val
100 105 110
Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met
115 120 125
Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser
130 135 140
Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln
145 150 155 160
Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro
165 170 175
186 amino acids
amino acid
linear
protein
163
Met Ala Pro Val Pro Pro Gly Glu Asp Ser Lys Asp Val Ala Ala Pro
1 5 10 15
His Arg Gln Pro Leu Thr Ser Ser Glu Arg Ile Asp Lys Gln Ile Arg
20 25 30
Tyr Ile Leu Asp Gly Ile Ser Ala Leu Arg Lys Glu Thr Cys Asn Lys
35 40 45
Ser Asn Met Cys Glu Ser Ser Lys Glu Ala Leu Ala Glu Asn Asn Leu
50 55 60
Asn Leu Pro Lys Met Ala Glu Lys Asp Gly Cys Phe Gln Ser Gly Phe
65 70 75 80
Asn Glu Glu Thr Cys Leu Val Lys Ile Ile Thr Gly Leu Leu Glu Phe
85 90 95
Glu Val Tyr Leu Glu Tyr Leu Gln Asn Arg Phe Glu Ser Ser Glu Glu
100 105 110
Gln Ala Arg Ala Val Gln Met Ser Thr Lys Val Leu Ile Gln Phe Leu
115 120 125
Gln Lys Lys Ala Lys Asn Leu Asp Ala Ile Thr Thr Pro Asp Pro Thr
130 135 140
Thr Asn Ala Ser Leu Leu Thr Lys Leu Gln Ala Gln Asn Gln Trp Leu
145 150 155 160
Gln Asp Met Thr Thr His Leu Ile Leu Arg Ser Phe Lys Glu Phe Leu
165 170 175
Gln Ser Ser Leu Arg Ala Leu Arg Gln Met
180 185
155 amino acids
amino acid
linear
protein
164
Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys
1 5 10 15
Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gln Cys Pro
20 25 30
Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe
35 40 45
Ser Leu Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala Gln Asp
50 55 60
Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg
65 70 75 80
Gln Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser
85 90 95
Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr
100 105 110
Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala
115 120 125
Ile Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg Phe Leu
130 135 140
Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg
145 150 155
286 amino acids
amino acid
linear
protein
165
Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys
1 5 10 15
Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn Ser Glu Asp
20 25 30
Met Asp Ile Leu Met Glu Arg Asn Leu Arg Thr Pro Asn Leu Leu Ala
35 40 45
Phe Val Arg Ala Val Lys His Leu Glu Asn Ala Ser Gly Ile Glu Ala
50 55 60
Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro
65 70 75 80
Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg
85 90 95
Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln
100 105 110
Gln Tyr Val Ile Glu Gly Arg Ile Ser Pro Gly Gly Gly Ser Gly Gly
115 120 125
Gly Ser Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val
130 135 140
Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser
145 150 155 160
Gln Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala
165 170 175
Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys
180 185 190
Ala Gln Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met
195 200 205
Ala Ala Arg Gln Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly
210 215 220
Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu
225 230 235 240
Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp
245 250 255
Pro Asn Ala Ile Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val
260 265 270
Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg
275 280 285
286 amino acids
amino acid
linear
protein
166
Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys
1 5 10 15
Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn Ser Glu Asp
20 25 30
Met Asp Ile Leu Met Glu Arg Asn Leu Arg Thr Pro Asn Leu Leu Ala
35 40 45
Phe Val Arg Ala Val Lys His Leu Glu Asn Ala Ser Gly Ile Glu Ala
50 55 60
Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro
65 70 75 80
Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg
85 90 95
Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln
100 105 110
Gln Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly Gly Gly Ser Gly Gly
115 120 125
Gly Ser Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val
130 135 140
Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser
145 150 155 160
Gln Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala
165 170 175
Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys
180 185 190
Ala Gln Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met
195 200 205
Ala Ala Arg Gln Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly
210 215 220
Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu
225 230 235 240
Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp
245 250 255
Pro Asn Ala Ile Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val
260 265 270
Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg
275 280 285
286 amino acids
amino acid
linear
protein
167
Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys
1 5 10 15
Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gln Cys Pro
20 25 30
Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe
35 40 45
Ser Leu Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala Gln Asp
50 55 60
Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg
65 70 75 80
Gln Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser
›EXAMPLE 5 · 23 of 25
85 90 95
Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr
100 105 110
Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala
115 120 125
Ile Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg Phe Leu
130 135 140
Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Tyr Val Ile Glu Gly
145 150 155 160
Arg Ile Ser Pro Gly Gly Gly Ser Gly Gly Gly Ser Asn Met Ala Asn
165 170 175
Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys Arg Pro Pro
180 185 190
Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn Ser Glu Asp Met Asp Ile
195 200 205
Leu Met Glu Arg Asn Leu Arg Thr Pro Asn Leu Leu Ala Phe Val Arg
210 215 220
Ala Val Lys His Leu Glu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg
225 230 235 240
Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His
245 250 255
Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu Lys Leu
260 265 270
Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln Gln
275 280 285
290 amino acids
amino acid
linear
protein
168
Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys
1 5 10 15
Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gln Cys Pro
20 25 30
Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe
35 40 45
Ser Leu Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala Gln Asp
50 55 60
Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg
65 70 75 80
Gln Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser
85 90 95
Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr
100 105 110
Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala
115 120 125
Ile Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg Phe Leu
130 135 140
Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe His Ala Tyr
145 150 155 160
Val Glu Gly Gly Gly Gly Ser Pro Gly Gly Gly Ser Gly Gly Gly Ser
165 170 175
Asn Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu
180 185 190
Lys Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn Ser Glu
195 200 205
Asp Met Asp Ile Leu Met Glu Arg Asn Leu Arg Thr Pro Asn Leu Leu
210 215 220
Ala Phe Val Arg Ala Val Lys His Leu Glu Asn Ala Ser Gly Ile Glu
225 230 235 240
Ala Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala
245 250 255
Pro Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe
260 265 270
Arg Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu
275 280 285
Gln Gln
290
ACGTCCATGG CNTCNCCNGC NCCNCCTGCT TGTGACCTCC GAGTC 45
AATAGCTGAA TTCTTACCCT TCCTGAGACA GATT 34
TGACAAGCTT ACCTGACGCA GAGGGTGGAC CCT 33
ATGCACGAAT TCCCTGACGC AGAGGGTGGA 30
AATTCCATGC ATAC 14
GGTACGTATG 10
561 base pairs
nucleic acid
double
linear
DNA (genomic)
175
ATGGCTCCAG TACCACCAGG TGAAGATTCC AAAGATGTGG CCGCCCCACA CAGACAGCCA 60
CTCACCTCTT CAGAACGAAT TGACAAACAA ATTCGGTACA TCCTCGACGG GATATCAGCC 120
CTGAGAAAGG AGACATGTAA CAAGAGTAAC ATGTGTGAAA GCAGCAAAGA GGCGCTAGCA 180
GAAAACAACC TGAACCTTCC AAAGATGGCT GAAAAAGATG GATGCTTCCA ATCCGGATTC 240
AATGAGGAGA CTTGCCTGGT GAAAATCATC ACTGGTCTTT TGGAGTTTGA GGTATACCTC 300
GAGTACCTCC AGAACAGATT TGAGAGTAGT GAGGAACAAG CCAGAGCTGT GCAGATGTCG 360
ACAAAAGTCC TGATCCAGTT CCTGCAGAAA AAGGCAAAGA ATCTAGATGC AATAACCACC 420
CCTGACCCAA CCACAAATGC ATCCCTGCTG ACGAAGCTGC AGGCACAGAA CCAGTGGCTG 480
CAGGACATGA CAACTCATCT CATTCTGCGC AGCTTTAAGG AGTTCCTGCA GTCCAGCCTG 540
AGGGCTCTTC GGCAAATGTA G 561
402 base pairs
nucleic acid
double
linear
DNA (genomic)
176
ATGGCACCGG CTCGTTCCCC GTCCCCGTCT ACCCAGCCGT GGGAACACGT GAATGCCATC 60
CAGGAGGCCC GGCGTCTCCT GAACCTGAGT AGAGACACTG CTGCTGAGAT GAATGAAACA 120
GTAGAAGTGA TATCAGAAAT GTTTGACCTC CAGGAGCCGA CTTGCCTACA GACCCGCCTG 180
GAGCTGTACA AGCAGGGCCT GCGGGGCAGC CTCACCAAGC TCAAGGGCCC CTTGACCATG 240
ATGGCCAGCC ACTACAAGCA GCACTGCCCT CCAACCCCGG AAACTTCCTG TGCAACCCAG 300
ATTATCACCT TTGAAAGTTT CAAAGAGAAC CTGAAGGACT TCCTGCTTGT CATCCCCTTT 360
GACTGCTGGG AGCCAGTCCA GGAGTGATAA GGATCCGAAT TC 402
546 base pairs
nucleic acid
double
linear
DNA (genomic)
177
ATGGCTACAC CATTAGGCCC TGCCAGCTCC CTGCCCCAGA GCTTCCTGCT CAAGTGCTTA 60
GAGCAAGTGA GGAAGATCCA GGGCGATGGC GCAGCGCTCC AGGAGAAGCT GTGTGCCACC 120
TACAAGCTGT GCCACCCCGA GGAGCTGGTG CTGCTCGGAC ACTCTCTGGG CATCCCCTGG 180
GCTCCCCTGA GCTCCTGCCC CAGCCAGGCC CTGCAGCTGG CAGGCTGCTT GAGCCAACTC 240
CATAGCGGCC TTTTCCTCTA CCAGGGGCTC CTGCAGGCCC TGGAAGGGAT ATCCCCCGAG 300
TTGGGTCCCA CCTTGGACAC ACTGCAGCTG GACGTCGCCG ACTTTGCCAC CACCATCTGG 360
CAGCAGATGG AAGAACTGGG AATGGCCCCT GCCCTGCAGC CCACCCAGGG TGCCATGCCG 420
GCCTTCGCCT CTGCTTTCCA GCGCCGGGCA GGAGGGGTCC TGGTTGCTAG CCATCTGCAG 480
AGCTTCCTGG AGGTGTCGTA CCGCGTTCTA CGCCACCTTG CGCAGCCCTG ATAAGGATCC 540
GAATTC 546
546 base pairs
nucleic acid
double
linear
DNA (genomic)
178
ATGGCTACAC CATTAGGACC TGCCAGCTCC CTGCCCCAGA GCTTCCTGCT CAAGTGCTTA 60
GAGCAAGTGA GGAAGATCCA GGGCGATGGC GCAGCGCTCC AGGAGAAGCT GTGTGCCACC 120
TACAAGCTGT GCCACCCCGA GGAGCTGGTG CTGCTCGGAC ACTCTCTGGG CATCCCCTGG 180
GCTCCCCTGA GCTCCTGCCC CAGCCAGGCC CTGCAGCTGG CAGGCTGCTT GAGCCAACTC 240
CATAGCGGCC TTTTCCTCTA CCAGGGGCTC CTGCAGGCCC TGGAAGGGAT ATCCCCCGAG 300
TTGGGTCCCA CCTTGGACAC ACTGCAGCTG GACGTCGCCG ACTTTGCCAC CACCATCTGG 360
CAGCAGATGG AAGAACTGGG AATGGCCCCT GCCCTGCAGC CCACCCAGGG TGCCATGCCG 420
GCCTTCGCCT CTGCTTTCCA GCGCCGGGCA GGAGGGGTCC TGGTTGCTAG CCATCTGCAG 480
AGCTTCCTGG AGGTGTCGTA CCGCGTTCTA CGCCACCTTG CGCAGCCCTG ATAAGGATCC 540
GAATTC 546
546 base pairs
nucleic acid
double
linear
DNA (genomic)
179
ATGGCTACAC CATTGGGCCC TGCCAGCTCC CTGCCCCAGA GCTTCCTGCT CAAGTCTTTA 60
GAGCAAGTGA GGAAGATCCA GGGCGATGGC GCAGCGCTCC AGGAGAAGCT GTGTGCCACC 120
TACAAGCTGT GCCACCCCGA GGAGCTGGTG CTGCTCGGAC ACTCTCTGGG CATCCCCTGG 180
GCTCCCCTGA GCTCCTGCCC CAGCCAGGCC CTGCAGCTGG CAGGCTGCTT GAGCCAACTC 240
CATAGCGGCC TTTTCCTCTA CCAGGGGCTC CTGCAGGCCC TGGAAGGGAT ATCCCCCGAG 300
TTGGGTCCCA CCTTGGACAC ACTGCAGCTG GACGTCGCCG ACTTTGCCAC CACCATCTGG 360
CAGCAGATGG AAGAACTGGG AATGGCCCCT GCCCTGCAGC CCACCCAGGG TGCCATGCCG 420
GCCTTCGCCT CTGCTTTCCA GCGCCGGGCA GGAGGGGTCC TGGTTGCTAG CCATCTGCAG 480
AGCTTCCTGG AGGTGTCGTA CCGCGTTCTA CGCCACCTTG CGCAGCCCTG ATAAGGATCC 540
GAATTC 546
465 base pairs
nucleic acid
double
linear
DNA (genomic)
180
ATGGCGTCTC CGGCGCCGCC TGCTTGTGAC CTCCGAGTCC TCAGTAAACT GCTTCGTGAC 60
TCCCATGTCC TTCACAGCAG ACTGAGCCAG TGCCCAGAGG TTCACCCTTT GCCTACACCT 120
GTCCTGCTGC CTGCTGTGGA CTTTAGCTTG GGAGAATGGA AAACCCAGAT GGAGGAGACC 180
›EXAMPLE 5 · 24 of 25
AAGGCACAGG ACATTCTGGG AGCAGTGACC CTTCTGCTGG AGGGAGTGAT GGCAGCACGG 240
GGACAACTGG GACCCACTTG CCTCTCATCC CTCCTGGGGC AGCTTTCTGG ACAGGTCCGT 300
CTCCTCCTTG GGGCCCTGCA GAGCCTCCTT GGAACCCAGC TTCCTCCACA GGGCAGGACC 360
ACAGCTCACA AGGATCCCAA TGCCATCTTC CTGAGCTTCC AACACCTGCT CCGAGGAAAG 420
GTGCGTTTCC TGATGCTTGT AGGAGGGTCC ACCCTCTGCG TCAGG 465
143 base pairs
nucleic acid
double
linear
DNA (genomic)
181
CCTGTCAACC CGGGCGGCGG CTCTGGTGGT GGTTCTGGTG GCGGCTCTGA GGGTGGCGGC 60
TCTGAGGGTG GCGGTTCTGA GGGTGGCGGC TCTGAGGGTG GCGGTTCCGG TGGCGGCTCC 120
GGTTCCGGTA ACATGTATTA TGA 143
180 base pairs
nucleic acid
double
linear
DNA (genomic)
182
ATCGTCTGAC CTCCCGGGCC TCCTGTCAAT GCTGGCGGCG GCTCTGGTGG TGGTTCTGGT 60
GGCGGCTCTG AGGGTGGCGG CTCTGAGGGT GGCGGTTCTG AGGGTGGCGG CTCTGAGGGT 120
GGCGGTTCCG GTGGCGGCTC CGGTTCCGGT GATTTTGATT ATGAAAACAT GTCAAACGCT 180
858 base pairs
nucleic acid
double
linear
DNA (genomic)
183
ATGGCTAACT GCTCTATAAT GATCGATGAA ATTATACATC ACTTAAAGAG ACCACCTAAC 60
CCTTTGCTGG ACCCGAACAA CCTCAATTCT GAAGACATGG ATATCCTGAT GGAACGAAAC 120
CTTCGAACTC CAAACCTGCT CGCATTCGTA AGGGCTGTCA AGCACTTAGA AAATGCATCA 180
GGTATTGAGG CAATTCTTCG TAATCTCCAA CCATGTCTGC CCTCTGCCAC GGCCGCACCC 240
TCTCGACATC CAATCATCAT CAAGGCAGGT GACTGGCAAG AATTCCGGGA AAAACTGACG 300
TTCTATCTGG TTACCCTTGA GCAAGCGCAG GAACAACAGT ACGTAATCGA GGGAAGGATT 360
TCCCCGGGTG GTGGTTCTGG CGGCGGCTCC AACATGGCGT CTCCGGCGCC GCCTGCTTGT 420
GACCTCCGAG TCCTCAGTAA ACTGCTTCGT GACTCCCATG TCCTTCACAG CAGACTGAGC 480
CAGTGCCCAG AGGTTCACCC TTTGCCTACA CCTGTCCTGC TGCCTGCTGT GGACTTTAGC 540
TTGGGAGAAT GGAAAACCCA GATGGAGGAG ACCAAGGCAC AGGACATTCT GGGAGCAGTG 600
ACCCTTCTGC TGGAGGGAGT GATGGCAGCA CGGGGACAAC TGGGACCCAC TTGCCTCTCA 660
TCCCTCCTGG GGCAGCTTTC TGGACAGGTC CGTCTCCTCC TTGGGGCCCT GCAGAGCCTC 720
CTTGGAACCC AGCTTCCTCC ACAGGGCAGG ACCACAGCTC ACAAGGATCC CAATGCCATC 780
TTCCTGAGCT TCCAACACCT GCTCCGAGGA AAGGTGCGTT TCCTGATGCT TGTAGGAGGG 840
TCCACCCTCT GCGTCAGG 858
858 base pairs
nucleic acid
double
linear
DNA (genomic)
184
ATGGCTAACT GCTCTATAAT GATCGATGAA ATTATACATC ACTTAAAGAG ACCACCTAAC 60
CCTTTGCTGG ACCCGAACAA CCTCAATTCT GAAGACATGG ATATCCTGAT GGAACGAAAC 120
CTTCGAACTC CAAACCTGCT CGCATTCGTA AGGGCTGTCA AGCACTTAGA AAATGCATCA 180
GGTATTGAGG CAATTCTTCG TAATCTCCAA CCATGTCTGC CCTCTGCCAC GGCCGCACCC 240
TCTCGACATC CAATCATCAT CAAGGCAGGT GACTGGCAAG AATTCCGGGA AAAACTGACG 300
TTCTATCTGG TTACCCTTGA GCAAGCGCAG GAACAACAGT ACGTAGAGGG CGGTGGAGGC 360
TCCCCGGGTG GTGGTTCTGG CGGCGGCTCC AACATGGCGT CTCCGGCGCC GCCTGCTTGT 420
GACCTCCGAG TCCTCAGTAA ACTGCTTCGT GACTCCCATG TCCTTCACAG CAGACTGAGC 480
CAGTGCCCAG AGGTTCACCC TTTGCCTACA CCTGTCCTGC TGCCTGCTGT GGACTTTAGC 540
TTGGGAGAAT GGAAAACCCA GATGGAGGAG ACCAAGGCAC AGGACATTCT GGGAGCAGTG 600
ACCCTTCTGC TGGAGGGAGT GATGGCAGCA CGGGGACAAC TGGGACCCAC TTGCCTCTCA 660
TCCCTCCTGG GGCAGCTTTC TGGACAGGTC CGTCTCCTCC TTGGGGCCCT GCAGAGCCTC 720
CTTGGAACCC AGCTTCCTCC ACAGGGCAGG ACCACAGCTC ACAAGGATCC CAATGCCATC 780
TTCCTGAGCT TCCAACACCT GCTCCGAGGA AAGGTGCGTT TCCTGATGCT TGTAGGAGGG 840
TCCACCCTCT GCGTCAGG 858
852 base pairs
nucleic acid
double
linear
DNA (genomic)
185
ATGGCGTCTC CGGCGCCGCC TGCTTGTGAC CTCCGAGTCC TCAGTAAACT GCTTCGTGAC 60
TCCCATGTCC TTCACAGCAG ACTGAGCCAG TGCCCAGAGG TTCACCCTTT GCCTACACCT 120
GTCCTGCTGC CTGCTGTGGA CTTTAGCTTG GGAGAATGGA AAACCCAGAT GGAGGAGACC 180
AAGGCACAGG ACATTCTGGG AGCAGTGACC CTTCTGCTGG AGGGAGTGAT GGCAGCACGG 240
GGACAACTGG GACCCACTTG CCTCTCATCC CTCCTGGGGC AGCTTTCTGG ACAGGTCCGT 300
CTCCTCCTTG GGGCCCTGCA GAGCCTCCTT GGAACCCAGC TTCCTCCACA GGGCAGGACC 360
ACAGCTCACA AGGATCCCAA TGCCATCTTC CTGAGCTTCC AACACCTGCT CCGAGGAAAG 420
GTGCGTTTCC TGATGCTTGT AGGAGGGTCC ACCCTCTGCG TCAGGATCGA GGGAAGGATT 480
TCCCCGGGTG GTGGTTCTGG CGGCGGCTCC AACATGGCTA ACTGCTCTAT AATGATCGAT 540
GAAATTATAC ATCACTTAAA GAGACCACCT AACCCTTTGC TGGACCCGAA CAACCTCAAT 600
TCTGAAGACA TGGATATCCT GATGGAACGA AACCTTCGAA CTCCAAACCT GCTCGCATTC 660
GTAAGGGCTG TCAAGCACTT AGAAAATGCA TCAGGTATTG AGGCAATTCT TCGTAATCTC 720
CAACCATGTC TGCCCTCTGC CACGGCCGCA CCCTCTCGAC ATCCAATCAT CATCAAGGCA 780
GGTGACTGGC AAGAATTCCG GGAAAAACTG ACGTTCTATC TGGTTACCCT TGAGCAAGCG 840
CAGGAACAAC AG 852
870 base pairs
nucleic acid
double
linear
DNA (genomic)
186
ATGGCGTCTC CGGCGCCGCC TGCTTGTGAC CTCCGAGTCC TCAGTAAACT GCTTCGTGAC 60
TCCCATGTCC TTCACAGCAG ACTGAGCCAG TGCCCAGAGG TTCACCCTTT GCCTACACCT 120
GTCCTGCTGC CTGCTGTGGA CTTTAGCTTG GGAGAATGGA AAACCCAGAT GGAGGAGACC 180
AAGGCACAGG ACATTCTGGG AGCAGTGACC CTTCTGCTGG AGGGAGTGAT GGCAGCACGG 240
GGACAACTGG GACCCACTTG CCTCTCATCC CTCCTGGGGC AGCTTTCTGG ACAGGTCCGT 300
CTCCTCCTTG GGGCCCTGCA GAGCCTCCTT GGAACCCAGC TTCCTCCACA GGGCAGGACC 360
ACAGCTCACA AGGATCCCAA TGCCATCTTC CTGAGCTTCC AACACCTGCT CCGAGGAAAG 420
GTGCGTTTCC TGATGCTTGT AGGAGGGTCC ACCCTCTGCG TCAGGGAATT CCATGCATAC 480
GTAGAGGGCG GTGGAGGCTC CCCGGGTGGT GGTTCTGGCG GCGGCTCCAA CATGGCTAAC 540
TGCTCTATAA TGATCGATGA AATTATACAT CACTTAAAGA GACCACCTAA CCCTTTGCTG 600
GACCCGAACA ACCTCAATTC TGAAGACATG GATATCCTGA TGGAACGAAA CCTTCGAACT 660
CCAAACCTGC TCGCATTCGT AAGGGCTGTC AAGCACTTAG AAAATGCATC AGGTATTGAG 720
GCAATTCTTC GTAATCTCCA ACCATGTCTG CCCTCTGCCA CGGCCGCACC CTCTCGACAT 780
CCAATCATCA TCAAGGCAGG TGACTGGCAA GAATTCCGGG AAAAACTGAC GTTCTATCTG 840
GTTACCCTTG AGCAAGCGCA GGAACAACAG 870
18 amino acids
amino acid
linear
peptide
187
Met Ser Arg Leu Pro Val Leu Leu Leu Leu Gln Leu Leu Val Arg Pro
1 5 10 15
Ala Met
18 amino acids
amino acid
linear
peptide
188
Tyr Val Ile Glu Gly Arg Ile Ser Pro Gly Gly Gly Ser Gly Gly Gly
1 5 10 15
Ser Asn
18 amino acids
amino acid
linear
peptide
189
Tyr Val Ile Glu Gly Lys Ile Ser Pro Gly Gly Gly Ser Gly Gly Gly
1 5 10 15
Ser Asn
18 amino acids
amino acid
linear
peptide
190
Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly Gly Gly Ser Gly Gly Gly
1 5 10 15
Ser Asn
33 amino acids
amino acid
linear
peptide
191
Tyr Val Ile Glu Gly Arg Ile Ser Pro Gly Glu Pro Ser Gly Pro Ile
1 5 10 15
Ser Thr Ile Asn Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro
20 25 30
Asn
33 amino acids
amino acid
linear
peptide
192
Tyr Val Ile Glu Gly Lys Ile Ser Pro Gly Glu Pro Ser Gly Pro Ile
1 5 10 15
Ser Thr Ile Asn Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro
20 25 30
Asn
33 amino acids
amino acid
linear
peptide
193
Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly Glu Pro Ser Gly Pro Ile
1 5 10 15
Ser Thr Ile Asn Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro
›EXAMPLE 5 · 25 of 25
20 25 30
Asn
49 amino acids
amino acid
linear
peptide
194
Tyr Val Ile Glu Gly Arg Ile Ser Pro Gly Gly Gly Ser Gly Gly Gly
1 5 10 15
Ser Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu
20 25 30
Gly Gly Gly Ser Glu Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Gly
35 40 45
Asn
60 amino acids
amino acid
linear
peptide
195
Tyr Val Ile Glu Gly Arg Ile Ser Pro Gln Pro Pro Val Asn Ala Gly
1 5 10 15
Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Glu Gly Gly Gly Ser
20 25 30
Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Gly
35 40 45
Gly Gly Ser Gly Ser Gly Asp Phe Asp Tyr Glu Asn
50 55 60
22 amino acids
amino acid
linear
peptide
196
Glu Phe His Ala Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly Gly Gly
1 5 10 15
Ser Gly Gly Gly Ser Asn
20
›Tables in the description — 12
| pMON | R 1 | R 2 | (relative potency) |
|---|---|---|---|
| pMON3987 | 13288 | G-CSF | 0.35 ± 0.11 |
| pMON3988 | 13288 | 13288 | 0.64 ± 0.13 |
| pMON3989 | 13288 | GM-CSF | 0.6 ± 0.09 |
| pMON3995 | G-CSF | 13288 | 0.41 ± 0.44 |
| pMON3997 | 13288 | G-CSF | 0.26 (n = 1) |
| pMON3998 | 13288 | GM-CSF | 0.21 (n = 1) |
| pMON26406 | 13288 | G-CSF | 0.37 ± 0.30 |
| pMON26433 | G-CSF | 13288 | 0.79 ± 0.35 |
| pMON26415 | 13288 | G-CSF Ser17 | 0.46 ± 0.08 |
| pMON26416 | G-CSF | 13416 | 0.43 ± 0.02 |
| pMON26425 | 13288 | 13288 | 1.32 ± 0.41 |
| pMON26427 | 13288 | 13288 | 1.41 ± 0.91 |
| pMON26420 | 13416 | 13416 | 2.09 ± 0.52 |
| pMON26430 | 13288 | G-CSF | 1.04 ± 0.69 |
| pMON26429 | 13288 | 13288 | 1.88 ± 0.09 |
| pMON26431 | 13288 | 13288 | 0.66 ± 0.26 |
| Growth | Donor | Donor | Donor | Donor | Donor | |
|---|---|---|---|---|---|---|
| Assay | Factor | # 1 | # 2 | # 3 | # 4 | # 5 |
| Proliferation Index | pMON13056 | 39.4 | 73.4 | ND | 5.7 | 5.7 |
| of CD34 + Cell | pMON13056 + | 135 | 206 | 37.4 | 17.4 | 6.4 |
| Cultures at Day 12 | SCF | |||||
| pMON13148 | 21.4 | 23.8 | ND | ND | ND | |
| pMON13148 + | 88.1 | 117.7 | ND | ND | ND | |
| SCF | ||||||
| native | 9 | 4.1 | 10.7 | 1 | 1.4 | |
| hIL-3 | ||||||
| native | 70.5 | 61.3 | 62.3 | 22.6 | 12.2 | |
| hIL-3 + | ||||||
| SCF | ||||||
| Percentage CD15 + | pMON13056 | 57 | 39.6 | 61.1 | 56 | 67.5 |
| Cells at Day 12 of | pMON13056 + | 70.8 | 45.4 | 72.7 | 46.3 | 87.4 |
| CD34 + Cultures | SCF | |||||
| pMON13148 | 47.3 | 58.6 | ND | ND | ND | |
| pMON13418 + | 38.7 | 31.7 | ND | ND | ND | |
| SCF | ||||||
| native | 25.6 | 10.5 | 43.3 | 26 | 18.2 | |
| hIL-3 | ||||||
| native | 17.7 | 11.5 | 55 | 12.4 | 24.2 | |
| hIL-3 + | ||||||
| SCF | ||||||
| Percentage CD41 + | pMON13056 | 12.6 | 16.5 | 18.2 | 3.4 | 4.6 |
| Cells at Day 12 of | pMON13056 + | 7.4 | 8.3 | 5.5 | 4.8 | 1.8 |
| CD34 + Cultures | SCF | |||||
| pMON13148 | 6 | 9.1 | ND | ND | ND | |
| pMON13148 + | 14.1 | 8.3 | ND | ND | ND | |
| SCF | ||||||
| native | 18.9 | 14.1 | 13.7 | 4.2 | 5.5 | |
| hIL-3 | ||||||
| native | 15.3 | 10.7 | 12.9 | 7.4 | 15 | |
| hIL-3 + | ||||||
| SCF | ||||||
| Total Number of | pMON13056 | 20 | 49 | ND | 0.8 | 1 |
| Megakaryoctes (E + 05) | pMON13056 + | 40 | 68 | 8.2 | 3.4 | 0.5 |
| In Day 10-12 | SCF | |||||
| Cultures | pMON13148 | 5.2 | 8.7 | ND | ND | ND |
| pMON13148 + | 50 | 52 | ND | ND | ND | |
| SCF | ||||||
| native | 6.8 | 2.3 | 5.9 | 0.2 | 0.3 | |
| hIL-3 | ||||||
| native | 43 | 26 | 32 | 16 | 7.4 | |
| hIL-3 + | ||||||
| SCF | ||||||
| Colony Forming Unit | pMON13056 | 0.9 | 3.2 | ND | 0.2 | 0.1 |
| Granulocyte | pMON13056 + | 1 | 3 | 0.7 | 1.1 | 0.04 |
| Macrophage (CFU-GM) | SCF | |||||
| Index | pMON13148 | 0.5 | 0.8 | ND | ND | ND |
| pMON13148 + | 1.2 | 3.2 | ND | ND | ND | |
| SCF | ||||||
| native | 0.2 | 0.06 | 0.03 | 0.03 | 0.03 | |
| hIL-3 | ||||||
| native | 1.9 | 1.1 | 0.3 | 0.6 | 0.3 | |
| hIL-3 + | ||||||
| SCF | ||||||
| Colony Forning | pMON13056 | 1.4 | 5.1 | ND | 0.2 | 0.2 |
| Unit-Index | pMON13056 + | 1.3 | 4.3 | 0.3 | 1.1 | 0.2 |
| SCF | ||||||
| pMON13148 | 0.7 | 1.1 | ND | ND | ND | |
| PMON13148 + | 1.6 | 5.2 | ND | ND | ND | |
| SCF | ||||||
| native | 0.2 | 0.1 | 0.03 | 0.03 | 0.04 | |
| hIL-3 | ||||||
| native | 2.7 | 1.5 | 0.3 | 0.5 | 0.4 | |
| hIL-3 + | ||||||
| SCF |
| Treatment | Donor 1 | Donor 2 |
| native IL-3 | 42 × 10E4 | 169 × 10E4 |
| pMON13288 | 114 × 10E4 | 259 × 10E4 |
| G-CSF | 14 × 10E4 | 32 × 10E4 |
| pMON13288 | 194 × 10E4 | 609 × 10E4 |
| and G-CSF | ||
| pMON13056 | 219 × 10E4 | 621 × 10E4 |
| Abbreviated Designation | Amino Acid | ||
| A | Ala | Alanine | |
| C | Cys | Cysteine | |
| D | Asp | Aspartic acid | |
| E | Glu | Glutamic acid | |
| F | Phe | Phenylalanine | |
| G | Gly | Glycine | |
| H | His | Histidine | |
| I | Ile | Isoleucine | |
| K | Lys | Lysine | |
| L | Leu | Leucine | |
| M | Met | Methionine | |
| N | Asn | Asparagine | |
| P | Pro | Proline | |
| Q | Gln | Glutamine | |
| R | Arg | Arginine | |
| S | Ser | Serine | |
| T | Thr | Threonine | |
| V | Val | Valine | |
| W | Trp | Tryptophan | |
| Y | Tyr | Tyrosine |
| 133 amino acids | amino acid | linear | ||
| peptide | ||||
| Modified-site | /note= “Met- may or may not precede the | amino acid in position 1” | ||
| Modified-site | 17 | /note= “Xaa at position 17 is Ser, | Lys, Gly, Asp, Met, Gln, or Arg” | |
| Modified-site | 18 | /note= “Xaa at position 18 is Asn, | His, Leu, Ile, Phe, Arg, or Gln” | |
| Modified-site | 19 | /note= “Xaa at positiion 19 is Met, | Phe, Ile, Arg, Gly, Ala, or Cys” | |
| Modified-site | 20 | /note= “Xaa at position 20 is Ile, | Cys, Gln, Glu, Arg, Pro, or Ala” | |
| Modified-site | 21 | /note= “Xaa at position 21 is Asp, | Phe, Lys, Arg, Ala, Gly, Glu, Gln, Asn, Thr, Ser, | or Val” |
| Modified-site | 22 | /note= “Xaa at position 22 is Glu, | Trp, Pro, Ser, Ala, His, Asp, Asn, Gln, Leu, Val, | or Gly” |
| Modified-site | 23 | /note= “Xaa at position 23 is Ile, | Val, Ala, Leu, Gly, Trp, Lys, Phe, Ser, or | Arg” |
| Modified-site | 24 | /note= “Xaa at position 24 is Ile, | Gly, Val, Arg, Ser, Phe, or Leu” | |
| Modified-site | 25 | /note= “Xaa at position 25 is Thr, | His, Gly, Gln, Arg, Pro, or Ala” | |
| Modified-site | 26 | /note= “Xaa at position 26 is His, | Thr, Phe, Gly, Arg, Ala, or Trp” | |
| Modified-site | 27 | /note= “Xaa at position 27 is Leu, | Gly, Arg, Thr, Ser, or Ala” | |
| Modified-site | 28 | /note= “Xaa at position 28 is Lys, | Arg, Leu, Gln, Gly, Pro, Val, or Trp” | |
| Modified-site | 29 | /note= “Xaa at position 29 is Gln, | Asn, Leu, Pro, Arg, or Val” | |
| Modified-site | 30 | /note= “Xaa at position 30 is Pro, | His, Thr, Gly, Asp, Gln, Ser, Leu, or Lys” | |
| Modified-site | 31 | /note= “Xaa at position 31 is Pro, | Asp, Gly, Ala, Arg, Leu, or Gln” | |
| Modified-site | 32 | /note= “Xaa at position 32 is Leu, | Val, Arg, Gln, Asn, Gly, Ala, or Glu” | |
| Modified-site | 33 | /note= “Xaa at position 33 is Pro, | Leu, Gln, Ala, Thr, or Glu” | |
| Modified-site | 34 | /note= “Xaa at position 34 is Leu, | Val, Gly, Ser, Lys, Glu, Gln, Thr, Arg, Ala, Phe, | Ile, or Met” |
| Modified-site | 35 | /note= “Xaa at position 35 is Leu, | Ala, Gly, Asn, Pro, Gln, or Val” | |
| Modified-site | 36 | /note= “Xaa at position 36 is Asp, | Leu, or Val” | |
| Modified-site | 37 | /note= “Xaa at position 37 is Phe, | Ser, Pro, Trp, or Ile” | |
| Modified-site | 38 | /note= “Xaa at position 38 is Asn, | or Ala” | |
| Modified-site | 40 | /note= “Xaa at position 40 is Leu, | Trp, or Arg” | |
| Modified-site | 41 | /note= “Xaa at position 41 is Asn, | Cys, Arg, Leu, His, Met, or Pro” | |
| Modified-site | 42 | /note= “Xaa at position 42 is Gly, | Asp, Ser, Cys, Asn, Lys, Thr, Leu, Val, Glu, Phe, Tyr, | Ile, Met, or Ala” |
| Modified-site | 43 | /note= “Xaa at position 43 is Glu, | Asn, Tyr, Leu, Phe, Asp, Ala, Cys, Gln, Arg, Thr, Gly, | or Ser” |
| Modified-site | 44 | /note= “Xaa at position 44 is Asp, | Ser, Leu, Arg, Lys, Thr, Met, Trp, Glu, Asn, Gln, Ala, | or Pro” |
| Modified-site | 45 | /note= “Xaa at position 45 is Gln, | Pro, Phe, Val, Met, Leu, Thr, Lys, Trp, Asp, Asn, Arg, | Ser, Ala, Ile, Glu, or His” |
| Modified-site | 46 | /note= “Xaa at position 46 is Asp, | Phe, Ser, Thr, Cys, Glu, Asn, Gln, Lys, His, Ala, Tyr, | Ile, Val, or Gly” |
| Modified-site | 47 | /note= “Xaa at position 47 is Ile, | Gly, Val, Ser, Arg, Pro, or His” | |
| Modified-site | 48 | /note= “Xaa at position 48 is Leu, | Ser, Cys, Arg, Ile, His, Phe, Glu, Lys, Thr, Ala, Met, | Val, or Asn” |
| Modified-site | 49 | /note= “Xaa at position 49 is Met, | Arg, Ala, Gly, Pro, Asn, His, or Asp” | |
| Modified-site | 50 | /note= “Xaa at position 50 is Glu, | Leu, Thr, Asp, Tyr, Lys, Asn, Ser, Ala, Ile, Val, His, | Phe, Met, or Gln” |
| Modified-site | 51 | /note= “Xaa at position 51 is Asn, | Arg, Met, Pro, Ser, Thr, or His” | |
| Modified-site | 52 | /note= “Xaa at position 52 is Asn, | His, Arg, Leu, Gly, Ser, or Thr” | |
| Modified-site | 53 | /note= “Xaa at position 53 is | Leu, Thr, Ala, Gly, Glu, Pro, Lys, Ser, or Met” | |
| Modified-site | 54 | /note= “Xaa at position 54 is Arg, | Asp, Ile, Ser, Val, Thr, Gln, Asn, Lys, His, Ala, | or Leu” |
| Modified-site | 55 | /note= “Xaa at position 55 is Arg, | Thr, Val, Ser, Leu, or Gly” | |
| Modified-site | 56 | /note= “Xaa at position 56 is Pro, | Gly, Cys, Ser, Gln, Glu, Arg, His, Thr, Ala, Tyr, | Phe, Leu, Val, or Lys” |
| Modified-site | 57 | /note= “Xaa at position 57 is Asn | or Gly” | |
| Modified-site | 58 | /note= “Xaa at position 58 is Leu, | Ser, Asp, Arg, Gln, Val, or Cys” | |
| Modified-site | 59 | /note= “Xaa at position 59 is Glu, | Tyr, His, Leu, Pro, or Arg” | |
| Modified-site | 60 | /note= “Xaa at position 60 is Ala, | Ser, Pro, Tyr, Asn, or Thr” | |
| Modified-site | 61 | /note= “Xaa at position 61 is Phe, | Asn, Glu, Pro, Lys, Arg, or Ser” | |
| Modified-site | 62 | /note= “Xaa at position 62 is Asn, | His, Val, Arg, Pro, Thr, Asp, or Ile” | |
| Modified-site | 63 | /note= “Xaa at position 63 is Arg, | Tyr, Trp, Lys, Ser, His, Pro, or Val” | |
| Modified-site | 64 | /note= “Xaa at position 64 is Ala, | Asn, Pro, Ser, or Lys” | |
| Modified-site | 65 | /note= “Xaa at position 65 is Val, | Thr, Pro, His, Leu, Phe, or Ser” | |
| Modified-site | 66 | /note= “Xaa at position 66 is Lys, | Ile, Arg, Val, Asn, Glu, or Ser” | |
| Modified-site | 67 | /note= “Xaa at position 67 is Ser, | Ala, Phe, Val, Gly, Asn, Ile, Pro, or His” | |
| Modified-site | 68 | /note= “Xaa at position 68 is Leu, | Val, Trp, Ser, Ile, Phe, Thr, or His” | |
| Modified-site | 69 | /note= “Xaa at position 69 is Gln, | Ala, Pro, Thr, Glu, Arg, Trp, Gly, or Leu” | |
| Modified-site | 70 | /note= “Xaa at position 70 is Asn, | Leu, Val, Trp, Pro, or Ala” | |
| Modified-site | 71 | /note= “Xaa at position 71 is | Ala, Met, Leu, Pro, Arg, Glu, Thr, Gln, Trp, | or Asn” |
| Modified-site | 72 | /note= “Xaa at position 72 is Ser, | Glu, Met, Ala, His, Asn, Arg, or Asp” | |
| Modified-site | 73 | /note= “Xaa at position 73 is Ala, | Glu, Asp, Leu, Ser, Gly, Thr, or Arg” | |
| Modified-site | 74 | /note= “Xaa at position 74 is Ile, | Met, Thr, Pro, Arg, Gly, or Ala” | |
| Modified-site | 75 | /note= “Xaa at position 75 is | Glu, Lys, Gly, Asp, Pro, Trp, Arg, Ser, Gln, | or Leu” |
| Modified-site | 76 | /note= “Xaa at position 76 is Ser, | Val, Ala, Asn, Trp, Glu, Pro, Gly, or Asp” | |
| Modified-site | 77 | /note= “Xaa at position 77 is Ile, | Ser, Arg, Thr, or Leu” | |
| Modified-site | 78 | /note= “Xaa at position 78 is Leu, | Ala, Ser, Glu, Phe, Gly, or Arg” | |
| Modified-site | 79 | /note= “Xaa at position 79 is Lys, Thr, | Asn, Met, Arg, Ile, Gly, or Asp” | |
| Modified-site | 80 | /note= “Xaa at position 80 is Asn, | Trp, Val, Gly, Thr, Leu, Glu, or Arg” | |
| Modified-site | 81 | /note= “Xaa at position 81 is Leu, | Gln, Gly, Ala, Trp, Arg, Val, or Lys” | |
| Modified-site | 82 | /note= “Xaa at position 82 is Leu, | Gln, Lys, Trp, Arg, Asp, Glu, Asn, His, Thr, Ser, Ala, | Tyr, Phe, Ile, Met, or Val” |
| Modified-site | 83 | /note= “Xaa at position 83 is Pro, | Ala, Thr, Trp, Arg, or Met” | |
| Modified-site | 84 | /note= “Xaa at position 84 is Cys, | Glu, Gly, Arg, Met, or Val” | |
| Modified-site | 85 | /note= “Xaa at position 85 is Leu, | Asn, Val, or Gln” | |
| Modified-site | 86 | /note= “Xaa at position 86 is Pro, | Cys, Arg, Ala, or Lys” | |
| Modified-site | 87 | /note= “Xaa at position 87 is Leu, | Ser, Trp, or Gly” | |
| Modified-site | 88 | /note= “Xaa at position 88 is Ala, | Lys, Arg, Val, or Trp” | |
| Modified-site | 89 | /note= “Xaa at position 89 is Thr, | Asp, Cys, Leu, Val, Glu, His, Asn, or Ser” | |
| Modified-site | 90 | /note= “Xaa at position 90 is Ala, | Pro, Ser, Thr, Gly, Asp, Ile, or Met” | |
| Modified-site | 91 | /note= “Xaa at position 91 is Ala, | Pro, Ser, Thr, Phe, Leu, Asp, or His” | |
| Modified-site | 92 | /note= “Xaa at position 92 is Pro, | Phe, Arg, Ser, Lys, His, Ala, Gly, Ile, or Leu” | |
| Modified-site | 93 | /note= “Xaa at position 93 is Thr, | Asp, Ser, Asn, Pro, Ala, Leu, or Arg” | |
| Modified-site | 94 | /note= “Xaa at position 94 is Arg, | Ile, Ser, Glu, Leu, Val, Gln, Lys, His, Ala, or Pro” | |
| Modified-site | 95 | /note= “Xaa at position 95 is His, | Gln, Pro, Arg, Val, Leu, Gly, Thr, Asn, Lys, Ser, Ala, | Trp, Phe, Ile, or Tyr” |
| Modified-site | 96 | /note= “Xaa at position 96 is Pro, | Lys, Tyr, Gly, Ile, or Thr” | |
| Modified-site | 97 | /note= “Xaa at position 97 is Ile, | Val, Lys, Ala, or Asn” | |
| Modified-site | 98 | /note= “Xaa at position 98 is His, | Ile, Asn, Leu, Asp, Ala, Thr, Glu, Gln, Ser, Phe, Met, | Val, Lys, Arg, Tyr, or Pro” |
| Modified-site | 99 | /note= “Xaa at position 99 is Ile, | Leu, Arg, Asp, Val, Pro, Gln, Gly, Ser, Phe, | or His” |
| Modified-site | 100 | /note= “Xaa at position 100 is | Lys, Tyr, Leu, His, Arg, Ile, Ser, Gln, or Pro” | |
| Modified-site | 101 | /note= “Xaa at position 101 is | Asp, Pro, Met, Lys, His, Thr, Val, Tyr, Glu, Asn, Ser, | Ala, Gly, Ile, Leu, or Gln” |
| Modified-site | 102 | /note= “Xaa at position 102 is Gly, | Leu, Glu, Lys, Ser, Tyr, or Pro” | |
| Modified-site | 103 | /note= “Xaa at position 103 is Asp, | or Ser” | |
| Modified-site | 104 | /note= “Xaa at position 104 is | Trp, Val, Cys, Tyr, Thr, Met, Pro, Leu, Gln, Lys, Ala, | Phe, or Gly” |
| Modified-site | 105 | /note= “Xaa at position 105 is | Asn, Pro, Ala, Phe, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, | Asp, or His” |
| Modified-site | 106 | /note= “Xaa at position 106 is Glu, | Ser, Ala, Lys, Thr, Ile, Gly, or Pro” | |
| Modified-site | 108 | /note= “Xaa at position 108 is Arg, | Lys, Asp, Leu, Thr, Ile, Gln, His, Ser, Ala, or Pro” | |
| Modified-site | 109 | /note= “Xaa at position 109 is Arg, | Thr, Pro, Glu, Tyr, Leu, Ser, or Gly” | |
| Modified-site | 110 | /note= “Xaa at position 110 is Lys, | Ala, Asn, Thr, Leu, Arg, Gln, His, Glu, Ser, or Trp” | |
| Modified-site | 111 | /note= “Xaa at position 111 is Leu, | Ile, Arg, Asp, or Met” | |
| Modified-site | 112 | /note= “Xaa at position 112 is Thr, | Val, Gln, Tyr, Glu, His, Ser, or Phe” | |
| Modified-site | 113 | /note= “Xaa at position 113 is Phe, | Ser, Cys, His, Gly, Trp, Tyr, Asp, Lys, Leu, Ile, Val, | or Asn” |
| Modified-site | 114 | /note= “Xaa at position 114 is Tyr, | Cys, His, Ser, Trp, Arg, or Leu” | |
| Modified-site | 115 | /note= “Xaa at position 115 is | Leu, Asn, Val, Pro, Arg, Ala, His, Thr, Trp, or | Met” |
| Modified-site | 116 | /note= “Xaa at position 116 is Lys, | Leu, Pro, Thr, Met, Asp, Val, Glu, Arg, Trp, Ser, | Asn, His, Ala, Tyr, Phe, Gln, or Ile” |
| Modified-site | 117 | /note= “Xaa at position 117 is Thr, | Ser, Asn, Ile, Trp, Lys, or Pro” | |
| Modified-site | 118 | /note= “Xaa at position 118 is Leu, | Ser, Pro, Ala, Glu, Cys, Asp, or Tyr” | |
| Modified-site | 119 | /note= “Xaa at position 119 is Glu, | Ser, Lys, Pro, Leu, Thr, Tyr, or Arg” | |
| Modified-site | 120 | /note= “Xaa at position 120 is Asn, | Ala, Pro, Leu, His, Val, or Gln” | |
| Modified-site | 121 | /note= “Xaa at position 121 is Ala, | Ser, Ile, Asn, Pro, Lys, Asp, or Gly” | |
| Modified-site | 122 | /note= “Xaa at position 122 is | Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, | or Cys” |
| Modified-site | 123 | /note= “Xaa at position 123 is Ala, | Met, Glu, His, Ser, Pro, Tyr, or Leu” | |
| 1 |
| 133 amino acids | amino acid | linear | ||
| peptide | ||||
| Modified-site | /note= “Met- may or may not precede | the amino acid in position 1” | ||
| Modified-site | 17 | /note= “Xaa at position 17 is Ser, | Gly, Asp, Met, or Gln” | |
| Modified-site | 18 | /note= “Xaa at position 18 is Asn, | His, or Ile” | |
| Modified-site | 19 | /note= “Xaa at position 19 is Met | or Ile” | |
| Modified-site | 21 | /note=“Xaa at position 21 is Asp | or Glu” | |
| Modified-site | 23 | /note= “Xaa at position 23 is Ile, | Ala, Leu, or Gly” | |
| Modified-site | 24 | /note= “Xaa at position 24 is Ile, | Val, or Leu” | |
| Modified-site | 25 | /note= “Xaa at position 25 is Thr, | His, Gln, or Ala” | |
| Modified-site | 26 | /note= “Xaa at position 26 is His | or Ala” | |
| Modified-site | 29 | /note= “Xaa at position 29 is Gln, | Asn, or Val” | |
| Modified-site | 30 | /note= “Xaa at position 30 is Pro, | Gly, or Gln” | |
| Modified-site | 31 | /note= “Xaa at position 31 is Pro, | Asp, Gly, or Gln” | |
| Modified-site | 32 | /note= “Xaa at position 32 is Leu, | Arg, Gln, Asn, Gly, Ala, or Glu” | |
| Modified-site | 33 | /note= “Xaa at position 33 is Pro | or Glu” | |
| Modified-site | 34 | /note= “Xaa at position 34 is Leu, | Val, Gly, Ser, Lys, Ala, Arg, Gln, Glu, Ile, Phe, | Thr, or Met” |
| Modified-site | 35 | /note= “Xaa at position 35 is Leu, | Ala, Asn, Pro, Gln, or Val” | |
| Modified-site | 37 | /note= “Xaa at position 37 is Phe, | Ser, Pro, or Trp” | |
| Modified-site | 38 | /note=“Xaa at position 38 is Asn | or Ala” | |
| Modified-site | 42 | /note= “Xaa at position 42 is Gly, | Asp, Ser, Cys, Ala, Asn, Ile, Leu, Met, Tyr, | or Arg” |
| Modified-site | 44 | /note=“Xaa at position 44 is Asp | or Glu” | |
| Modified-site | 45 | /note= “Xaa at position 45 is Gln, | Val, Met, Leu, Thr, Ala, Asn, Glu, Ser, or Lys” | |
| Modified-site | 46 | /note= “Xaa at position 46 is Asp, | Phe, Ser, Thr, Ala, Asn, Gln, Glu, His, Ile, | Lys, Tyr, Val, or Cys” |
| Modified-site | 50 | /note= “Xaa at position 50 is Glu, | Ala, Asn, Ser, or Asp” | |
| Modified-site | 51 | /note= “Xaa at position 51 is Asn, | Arg, Met, Pro, Ser, Thr, or His” | |
| Modified-site | 54 | /note=“Xaa at position 54 is Arg | or Ala” | |
| Modified-site | 55 | /note= “Xaa at position 55 is Arg, | Thr, Val, Leu, or Gly” | |
| Modified-site | 56 | /note= “Xaa at position 56 is Pro, | Gly, Ser, Gln, Ala, Arg, Asn, Glu, Leu, Thr, Val, | or Lys” |
| Modified-site | 60 | /note= “Xaa at position 60 is Ala | or Ser” | |
| Modified-site | 62 | /note= “Xaa at position 62 is Asn, | Pro, Thr, or Ile” | |
| Modified-site | 63 | /note= “Xaa at position 63 is Arg | or Lys” | |
| Modified-site | 64 | /note= “Xaa at position 64 is Ala | or Asn” | |
| Modified-site | 65 | /note= “Xaa at position 65 is Val | or Thr” | |
| Modified-site | 66 | /note= “Xaa at position 66 is Lys | or Arg” | |
| Modified-site | 67 | /note= “Xaa at position 67 is Ser | Phe or His” | |
| Modified-site | 68 | /note= “Xaa at position 68 is Leu, | Ile, Phe, or His” | |
| Modified-site | 69 | /note= “Xaa at position 69 is Gln, | Ala, Pro, Thr, Glu, Arg, or Gly” | |
| Modified-site | 71 | /note= “Xaa at position 71 is Ala, | Pro, or Arg” | |
| Modified-site | 72 | /note= “Xaa at position 72 is Ser, | Glu, Arg, or Asp” | |
| Modified-site | 73 | /note= “Xaa at position 73 is Ala | or Leu” | |
| Modified-site | 76 | /note= “Xaa at position 76 is Ser, | Val, Ala, Asn, Glu, Pro, or Gly” | |
| Modified-site | 77 | /note= “Xaa at position 77 is Ile | or Leu” | |
| Modified-site | 79 | /note= “Xaa at position 79 is | Lys, Thr, Gly, Asn, Met, Arg, Ile, or Asp” | |
| Modified-site | 80 | /note= “Xaa at position 80 is Asn, | Gly, Glu, or Arg” | |
| Modified-site | 82 | /note= “Xaa at position 82 is Leu, | Gln, Trp, Arg, Asp, Ala, Asn, Glu, His, Ile, | Met, Phe, Ser, Thr, Tyr, or Val” |
| Modified-site | 83 | /note= “Xaa at position 83 is Pro | or Thr” | |
| Modified-site | 85 | /note= “Xaa at position 85 is Leu | or Val” | |
| Modified-site | 87 | /note= “Xaa at position 87 is Leu | or Ser” | |
| Modified-site | 88 | /note= “Xaa at position 88 is Ala | or Trp” | |
| Modified-site | 91 | /note= “Xaa at position 91 is Ala | or Pro” | |
| Modified-site | 93 | /note= “Xaa at position 93 is Thr, | Asp, Ser, Pro, Ala, Leu, or Arg” | |
| Modified-site | 95 | /note= “Xaa at position 95 is His, | Pro, Arg, Val, Leu, Gly, Asn, Phe, Ser, or Thr” | |
| Modified-site | 96 | /note= “Xaa at position 96 is Pro | or Tyr” | |
| Modified-site | 97 | /note= “Xaa at position 97 is Ile | or Val” | |
| Modified-site | 98 | /note= “Xaa at position 98 is His, | Ile, Asn, Leu, Ala, Thr, Arg, Gln, Lys, | Met, Ser, Tyr, Val, or Pro” |
| Modified-site | 99 | /note= “Xaa at position 99 is Ile, | Leu, or Val” | |
| Modified-site | 100 | /note= “Xaa at position 100 is Lys, | Arg, Ile, Gln, Pro, or Ser” | |
| Modified-site | 101 | /note= “Xaa at position 101 is Asp, | Pro, Met, Lys, Thr, His, Asn, Ile, Leu, or Tyr” | |
| Modified-site | 104 | /note= “Xaa at position 104 is Trp | or Leu” | |
| Modified-site | 105 | /note= “Xaa at position 105 is | Asn, Pro, Ala, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, | Asp, or His” |
| Modified-site | 106 | /note= “Xaa at position 106 is Glu | or Gly” | |
| Modified-site | 108 | /note=“Xaa at position 108 is Arg, | Ala, or Ser” | |
| Modified-site | 109 | /note= “Xaa at position 109 is Arg, | Thr, Glu, Leu, or Ser” | |
| Modified-site | 112 | /note= “Xaa at position 112 is Thr, | Val, or Gln” | |
| Modified-site | 114 | /note= “Xaa at position 114 is Tyr | or Trp” | |
| Modified-site | 115 | /note= “Xaa at position 115 is Leu | or Ala” | |
| Modified-site | 116 | /note= “Xaa at position 116 is Lys, | Thr, Val, Trp, Ser, Ala, His, Met, Phe, Tyr, or Ile” | |
| Modified-site | 117 | /note= “Xaa at position 117 is Thr | or Ser” | |
| Modified-site | 120 | /note= “Xaa at position 120 is Asn, | Pro, Leu, His, Val, or Gln” | |
| Modified-site | 121 | /note= “Xaa at position 121 is Ala, | Ser, Ile, Asn, Pro, Asp, or Gly” | |
| Modified-site | 122 | /note= “Xaa at position 122 is | Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, | or Cys” |
| Modified-site | 123 | /note= “Xaa at position 123 is Ala, | Met, Glu, His, Ser, Pro, Tyr, or Leu” | |
| 2 |
| 133 amino acids | amino acid | linear | ||
| peptide | ||||
| Modified-site | /note= “Met- may or may not precede | the amino acid in position 1” | ||
| Modified-site | 17 | /note= “Xaa at position 17 is Ser, | Gly, Asp, or Gln” | |
| Modified-site | 18 | /note= “Xaa at position 18 is Asn, | His, or Ile” | |
| Modified-site | 23 | /note= “Xaa at position 23 is Ile, | Ala, Leu, or Gly” | |
| Modified-site | 25 | /note= “Xaa at position 25 is Thr, | His, or Gln” | |
| Modified-site | 26 | /note= “Xaa at position 26 is His | or Ala” | |
| Modified-site | 29 | /note=“Xaa at position 29 is Gln | or Asn” | |
| Modified-site | 30 | /note= “Xaa at position 30 is Pro | or Gly” | |
| Modified-site | 32 | /note= “Xaa at position 32 is Leu, | Arg, Asn, or Ala” | |
| Modified-site | 34 | /note= “Xaa at position 34 is Leu, | Val, Ser, Ala, Arg, Gln, Glu, Ile, Phe, Thr, or Met” | |
| Modified-site | 35 | /note= “Xaa at position 35 is Leu, | Ala, Asn, or Pro” | |
| Modified-site | 38 | /note= “Xaa at position 38 is Asn | or Ala” | |
| Modified-site | 42 | /note= “Xaa at position 42 is Gly, | Asp, Ser, Ala, Asn, Ile, Leu, Met, Tyr, or Arg” | |
| Modified-site | 45 | /note= “Xaa at position 45 is Gln, | Val, Met, Leu, Ala, Asn, Glu, or Lys” | |
| Modified-site | 46 | /note= “Xaa at position 46 is Asp, | Phe, Ser, Gln, Glu, His, Val, or Thr” | |
| Modified-site | 50 | /note= “Xaa at position 50 is Glu, | Asn, Ser, or Asp” | |
| Modified-site | 51 | /note= “Xaa at position 51 is Asn, | Arg, Pro, Thr, or His” | |
| Modified-site | 55 | /note= “Xaa at position 55 is Arg, | Leu, or Gly” | |
| Modified-site | 56 | /note= “Xaa at position 56 is Pro, | Gly, Ser, Ala, Asn, Val, Leu, or Gln” | |
| Modified-site | 62 | /note= “Xaa at position 62 is Asn, | Pro, or Thr” | |
| Modified-site | 64 | /note= “Xaa at position 64 is Ala | or Asn” | |
| Modified-site | 65 | /note= “Xaa at position 65 is Val | or Thr” | |
| Modified-site | 67 | /note= “Xaa at position 67 is Ser | or Phe” | |
| Modified-site | 68 | /note= “Xaa at position 68 is Leu | or Phe” | |
| Modified-site | 69 | /note= “Xaa at position 69 is Gln, | Ala, Glu, or Arg” | |
| Modified-site | 76 | /note= “Xaa at position 76 is Ser, | Val, Asn, Pro, or Gly” | |
| Modified-site | 77 | /note= “Xaa at position 77 is Ile | or Leu” | |
| Modified-site | 79 | /note= “Xaa at position 79 is Lys, | Asn, Met, Arg, Ile, or Gly” | |
| Modified-site | 80 | /note= “Xaa at position 80 is Asn, | Gly, Glu, or Arg” | |
| Modified-site | 82 | /note= “Xaa at position 82 is Leu, | Gln, Trp, Arg, Asp, Asn, Glu, His, Met, Phe, Ser, | Thr, Tyr, or Val” |
| Modified-site | 87 | /note= “Xaa at position 87 is Leu | or Ser” | |
| Modified-site | 88 | /note= “Xaa at position 88 is Ala | or Trp” | |
| Modified-site | 91 | /note= “Xaa at position 91 is Ala | or Pro” | |
| Modified-site | 93 | /note= “Xaa at position 93 is Thr, | Asp, or Ala” | |
| Modified-site | 95 | /note= “Xaa at position 95 is His, | Pro, Arg, Val, Gly, Asn, Ser, or Thr” | |
| Modified-site | 98 | /note= “Xaa at position 98 is His, | Ile, Asn, Ala, Thr, Gln, Glu, Lys, Met, Ser, Tyr, | Val, or Leu” |
| Modified-site | 99 | /note= “Xaa at position 99 is Ile | or Leu” | |
| Modified-site | 100 | /note= “Xaa at position 100 is Lys | or Arg” | |
| Modified-site | 101 | /note= “Xaa at position 101 is Asp, | Pro, Met, Lys, Thr, His, Asn, Ile, Leu, or Tyr” | |
| Modified-site | 105 | /note= “Xaa at position 105 is Asn, | Pro, Ser, Ile, or Asp” | |
| Modified-site | 108 | /note= “Xaa at position 108 is Arg, Ala, | or Ser” | |
| Modified-site | 109 | /note= “Xaa at position 109 is Arg, | Thr, Glu, Leu, or Ser” | |
| Modified-site | 112 | /note= “Xaa at position 112 is Thr | or Gln” | |
| Modified-site | 116 | /note= “Xaa at position 116 is Lys, | Val, Trp, Ala, His, Phe, Tyr, or Ile” | |
| Modified-site | 117 | /note= “Xaa at position 117 is Thr | or Ser” | |
| Modified-site | 120 | /note= “Xaa at position 120 is Asn, | Pro, Leu, His, Val, or Gln” | |
| Modified-site | 121 | /note= “Xaa at position 121 is Ala, | Ser, Ile, Pro, or Asp” | |
| Modified-site | 122 | /note= “Xaa at position 122 is Gln, | Met, Trp, Phe, Pro, His, Ile, or Tyr” | |
| Modified-site | 123 | /note= “Xaa at position 123 is Ala, | Met, Glu, Ser, or Leu” | |
| 3 |
| 111 amino acids | amino acid | linear | ||
| peptide | ||||
| Modified-site | /note= “Met- or Met-Ala- may or may | not precede the amino acid in position 1” | ||
| Modified-site | /note= “Xaa at position 3 is Ser, | Lys, Gly, Asp, Met, Gln, or Arg” | ||
| Modified-site | /note= “Xaa at position 4 is Asn, | His, Leu, Ile, Phe, Arg, or Gln” | ||
| Modified-site | /note= “Xaa at position 5 is Met, | Phe, Ile, Arg, Gly, Ala, or Cys” | ||
| Modified-site | /note= “Xaa at position 6 is Ile, | Cys, Gln, Glu, Arg, Pro, or Ala” | ||
| Modified-site | /note= “Xaa at position 7 is Asp, | Phe, Lys, Arg, Ala, Gly, Glu, Gln, Asn, Thr, Ser, | or Val” | |
| Modified-site | /note= “Xaa at position 8 is Glu, | Trp, Pro, Ser, Ala, His, Asp, Asn, Gln, Leu, Val, | or Gly” | |
| Modified-site | /note= “Xaa at position 9 is | Ile, Val, Ala, Leu, Gly, Trp, Lys, Phe, Ser or Arg” | ||
| Modified-site | 10 | /note= “Xaa at position 10 is Ile, | Gly, Val, Arg, Ser, Phe, or Leu” | |
| Modified-site | 11 | /note= “Xaa at position 11 is Thr, | His, Gly, Gln, Arg, Pro, or Ala” | |
| Modified-site | 12 | /note= “Xaa at position 12 is His, | Thr, Phe, Gly, Arg, Ala, or Trp” | |
| Modified-site | 13 | /note= “Xaa at position 13 is Leu, | Gly, Arg, Thr, Ser, or Ala” | |
| Modified-site | 14 | /note= “Xaa at position 14 is Lys, | Arg, Leu, Gln, Gly, Pro, Val, or Trp” | |
| Modified-site | 15 | /note= “Xaa at position 15 is Gln, | Asn, Leu, Pro, Arg, or Val” | |
| Modified-site | 16 | /note= “Xaa at position 16 is Pro, | His, Thr, Gly, Asp, Gln, Ser, Leu, or Lys” | |
| Modified-site | 17 | /note= “Xaa at position 17 is Pro, | Asp, Gly, Ala, Arg, Leu, or Gln” | |
| Modified-site | 18 | /note= “Xaa at position 18 is Leu, | Val, Arg, Gln, Asn, Gly, Ala, or Glu” | |
| Modified-site | 19 | /note= “Xaa at position 19 is Pro, | Leu, Gln, Ala, Thr, or Glu” | |
| Modified-site | 20 | /note= “Xaa at position 20 is Leu, | Val, Gly, Ser, Lys, Glu, Gln, Thr, Arg, Ala, Phe, | Ile, or Met” |
| Modified-site | 21 | /note= “Xaa at position 21 is Leu, | Ala, Gly, Asn, Pro, Gln, or Val” | |
| Modified-site | 22 | /note= “Xaa at position 22 is Asp, | Leu, or Val” | |
| Modified-site | 23 | /note= “Xaa at position 23 is Phe, | Ser, Pro, Trp, or Ile” | |
| Modified-site | 24 | /note= “Xaa at position 24 is Asn | or Ala” | |
| Modified-site | 26 | /note= “Xaa at position 26 is Leu, | Trp, or Arg” | |
| Modified-site | 27 | /note= “Xaa at position 27 is Asn, | Cys, Arg, Leu, His, Met, or Pro” | |
| Modified-site | 28 | /note= “Xaa at position 28 is Gly, | Asp, Ser, Cys, Ala, Lys, Asn, Thr, Leu, Val, Glu, | Phe, Tyr, Ile, or Met” |
| Modified-site | 29 | /note= “Xaa at position 29 is Glu, | Asn, Tyr, Leu, Phe, Asp, Ala, Cys, Gln, Arg, Thr, | Gly, or Ser” |
| Modified-site | 30 | /note= “Xaa at position 30 is Asp, | Ser, Leu, Arg, Lys, Thr, Met, Trp, Glu, Asn, Gln, | Ala, or Pro” |
| Modified-site | 31 | /note= “Xaa at position 31 is Gln, | Pro, Phe, Val, Met, Leu, Thr, Lys, Asp, Asn, Arg, | Ser, Ala, Ile, Glu, His, or Trp” |
| Modified-site | 32 | /note= “Xaa at position 32 is Asp, | Phe, Ser, Thr, Cys, Glu, Asn, Gln, Lys, His, Ala, | Tyr, Ile, Val, or Gly” |
| Modified-site | 33 | /note= “Xaa at position 33 is Ile, | Gly, Val, Ser, Arg, Pro, or His” | |
| Modified-site | 34 | /note= “Xaa at position 34 is Leu, | Ser, Cys, Arg, Ile, His, Phe, Glu, Lys, Thr, Ala, | Met, Val, or Asn” |
| Modified-site | 35 | /note= “Xaa at position 35 is Met, | Arg, Ala, Gly, Pro, Asn, His, or Asp” | |
| Modified-site | 36 | /note= “Xaa at position 36 is Glu, | Leu, Thr, Asp, Tyr, Lys, Asn, Ser, Ala, Ile, Val, | His, Phe, Met, or Gln” |
| Modified-site | 37 | /note= “Xaa at position 37 is Asn, | Arg, Met, Pro, Ser, Thr, or His” | |
| Modified-site | 38 | /note= “Xaa at position 38 is Asn, | His, Arg, Leu, Gly, Ser, or Thr” | |
| Modified-site | 39 | /note= “Xaa at position 39 is | Leu, Thr, Ala, Gly, Glu, Pro, Lys, Ser, or Met” | |
| Modified-site | 40 | /note= “Xaa at position 40 is Arg, | Asp, Ile, Ser, Val, Thr, Gln, Asn, Lys, His, | Ala, or Leu” |
| Modified-site | 41 | /note= “Xaa at position 41 is Arg, | Thr, Val, Ser, Leu, or Gly” | |
| Modified-site | 42 | /note= “Xaa at position 42 is Pro, | Gly, Cys, Ser, Gln, Glu, Arg, His, Thr, Ala, Tyr, | Phe, Leu, Val, or Lys” |
| Modified-site | 43 | /note= “Xaa at position 43 is Asn | or Gly” | |
| Modified-site | 44 | /note= “Xaa at position 44 is Leu, | Ser, Asp, Arg, Gln, Val, or Cys” | |
| Modified-site | 45 | /note= “Xaa at position 45 is Glu, | Tyr, His, Leu, Pro, or Arg” | |
| Modified-site | 46 | /note= “Xaa at position 46 is Ala, | Ser, Pro, Tyr, Asn, or Thr” | |
| Modified-site | 47 | /note= “Xaa at position 47 is Phe, | Asn, Glu, Pro, Lys, Arg, or Ser” | |
| Modified-site | 48 | /note= “Xaa at position 48 is Asn, | His, Val, Arg, Pro, Thr, Asp, or Ile” | |
| Modified-site | 49 | /note= “Xaa at position 49 is Arg, | Tyr, Trp, Lys, Ser, His, Pro, or Val” | |
| Modified-site | 50 | /note= “Xaa at position 50 is Ala, | Asn, Pro, Ser, or Lys” | |
| Modified-site | 51 | /note= “Xaa at position 51 is Val, | Thr, Pro, His, Leu, Phe, or Ser” | |
| Modified-site | 52 | /note= “Xaa at position 52 is Lys, | Ile, Arg, Val, Asn, Glu, or Ser” | |
| Modified-site | 53 | /note= “Xaa at position 53 is Ser, | Ala, Phe, Val, Gly, Asn, Ile, Pro, or His” | |
| Modified-site | 54 | /note= “Xaa at position 54 is Leu, | Val, Trp, Ser, Ile, Phe, Thr, or His” | |
| Modified-site | 55 | /note= “Xaa at position 55 is Gln, | Ala, Pro, Thr, Glu, Arg, Trp, Gly, or Leu” | |
| Modified-site | 56 | /note= “Xaa at position 56 is Asn, | Leu, Val, Trp, Pro, or Ala” | |
| Modified-site | 57 | /note= “Xaa at position 57 is Ala, | Met, Leu, Pro, Arg, Glu, Thr, Gln, Trp, or Asn” | |
| Modified-site | 58 | /note= “Xaa at position 58 is Ser, | Glu, Met, Ala, His, Asn, Arg, or Asp” | |
| Modified-site | 59 | /note= “Xaa at position 59 is Ala, | Glu, Asp, Leu, Ser, Gly, Thr, or Arg” | |
| Modified-site | 60 | /note= “Xaa at position 60 is Ile, | Met, Thr, Pro, Arg, Gly, Ala” | |
| Modified-site | 61 | /note= “Xaa at position 61 is | Glu, Lys, Gly, Asp, Pro, Trp, Arg, Ser, Gln, | or Leu” |
| Modified-site | 62 | /note= “Xaa at position 62 is Ser, | Val, Ala, Asn, Trp, Glu, Pro, Gly, or Asp” | |
| Modified-site | 63 | /note= “Xaa at position 63 is Ile, | Ser, Arg, Thr, or Leu” | |
| Modified-site | 64 | /note= “Xaa at position 64 is Leu, | Ala, Ser, Glu, Phe, Gly, or Arg” | |
| Modified-site | 65 | /note= “Xaa at position 65 is Lys, | Thr, Gly, Asn, Met, Arg, Ile, or Asp” | |
| Modified-site | 66 | /note= “Xaa at position 66 is Asn, | Trp, Val, Gly, Thr, Leu, Glu, or Arg” | |
| Modified-site | 67 | /note= “Xaa at position 67 is Leu, | Gln, Gly, Ala, Trp, Arg, Val, or Lys” | |
| Modified-site | 68 | /note= “Xaa at position 68 is Leu, | Gln, Lys, Trp, Arg, Asp, Glu, Asn, His, Thr, Ser, Ala, | Tyr, Phe, Ile, Met, or Val” |
| Modified-site | 69 | /note= “Xaa at position 69 is Pro, | Ala, Thr, Trp, Arg, or Met” | |
| Modified-site | 70 | /note= “Xaa at position 70 is Cys, | Glu, Gly, Arg, Met, or Val” | |
| Modified-site | 71 | /note= “Xaa at position 71 is Leu, | Asn, Val, or Gln” | |
| Modified-site | 72 | /note= “Xaa at position 72 is Pro, | Cys, Arg, Ala, or Lys” | |
| Modified-site | 73 | /note= “Xaa at position 73 is Leu, | Ser, Trp, or Gly” | |
| Modified-site | 74 | /note= “Xaa at position 74 is Ala, | Lys, Arg, Val, or Trp” | |
| Modified-site | 75 | /note= “Xaa at position 75 is Thr, | Asp, Cys, Leu, Val, Glu, His, Asn, or Ser” | |
| Modified-site | 76 | /note= “Xaa at position 76 is Ala, | Pro, Ser, Thr, Gly, Asp, Ile, or Met” | |
| Modified-site | 77 | /note= “Xaa at position 77 is Ala, | Pro, Ser, Thr, Phe, Leu, Asp, or His” | |
| Modified-site | 78 | /note= “Xaa at position 78 is Pro, | Phe, Arg, Ser, Lys, His, Ala, Gly, Ile, or Leu” | |
| Modified-site | 79 | /note= “Xaa at position 79 is Thr, | Asp, Ser, Asn, Pro, Ala, Leu, or Arg” | |
| Modified-site | 80 | /note= “Xaa at position 80 is Arg, | Ile, Ser, Glu, Leu, Val, Gln, Lys, His, Ala, or Pro” | |
| Modified-site | 81 | /note= “Xaa at position 81 is His, | Gln, Pro, Arg, Val, Leu, Gly, Thr, Asn, Lys, Ser, | Ala, Trp, Phe, Ile, or Tyr” |
| Modified-site | 82 | /note= “Xaa at position 82 is Pro, | Lys, Tyr, Gly, Ile, or Thr” | |
| Modified-site | 83 | /note= “Xaa at position 83 is Ile, | Val, Lys, Ala, or Asn” | |
| Modified-site | 84 | /note= “Xaa at position 84 is His, | Ile, Asn, Leu, Asp, Ala, Thr, Glu, Gln, Ser, | Phe, Met, Val, Lys, Arg, Tyr, or Pro” |
| Modified-site | 85 | /note= “Xaa at position 85 is | Ile, Leu, Arg, Asp, Val, Pro, Gln, Gly, Ser, | Phe, or His” |
| Modified-site | 86 | /note= “Xaa at position 86 is | Lys, Tyr, Leu, His, Arg, Ile, Ser, Gln, or Pro” | |
| Modified-site | 87 | /note= “Xaa at position 87 is | Asp, Pro, Met, Lys, His, Thr, Val, Tyr, Glu, Asn, | Ser, Ala, Gly, Ile, Leu, or Gln” |
| Modified-site | 88 | /note= “Xaa at position 88 Gly, | Leu, Glu, Lys, Ser, Tyr, or Pro” | |
| Modified-site | 89 | /note= “Xaa at position 89 is Asp | or Ser” | |
| Modified-site | 90 | /note= “Xaa at position 90 is | Trp, Val, Cys, Tyr, Thr, Met, Pro, Leu, Gln, Lys, | Ala, Phe, or Gly” |
| Modified-site | 91 | /note= “Xaa at position 91 is | Asn, Pro, Ala, Phe, Ser, Trp, Gln, Tyr, Leu, Lys, | Ile, Asp, or His” |
| Modified-site | 92 | /note= “Xaa at position 92 is Glu, | Ser, Ala, Lys, Thr, Ile, Gly, or Pro” | |
| Modified-site | 94 | /note= “Xaa at position 94 is Arg, | Lys, Asp, Leu, Thr, Ile, Gln, His, Ser, Ala, or Pro” | |
| Modified-site | 95 | /note= “Xaa at position 95 is Arg, | Thr, Pro, Glu, Tyr, Leu, Ser, or Gly” | |
| Modified-site | 96 | /note= “Xaa at position 96 is Lys, | Asn, Thr, Leu, Gln, Arg, His, Glu, Ser, Ala, | or Trp” |
| Modified-site | 97 | /note= “Xaa at position 97 is Leu, | Ile, Arg, Asp, or Met” | |
| Modified-site | 98 | /note= “Xaa at position 98 is Thr, | Val, Gln, Tyr, Glu, His, Ser, or Phe” | |
| Modified-site | 99 | /note= “Xaa at position 99 is Phe, | Ser, Cys, His, Gly, Trp, Tyr, Asp, Lys, Leu, Ile, | Val, or Asn” |
| Modified-site | 100 | /note= “Xaa at position 100 is Tyr, | Cys, His, Ser, Trp, Arg, or Leu” | |
| Modified-site | 101 | /note= “Xaa at position 101 is Leu, | Asn, Val, Pro, Arg, Ala, His, Thr, Trp, or Met” | |
| Modified-site | 102 | /note= “Xaa at position 102 is | Lys, Leu, Pro, Thr, Met, Asp, Val, Glu, Arg, Trp, | Ser, Asn, His, Ala, Tyr, Phe, Gln, or Ile” |
| Modified-site | 103 | /note= “Xaa at position 103 is Thr, | Ser, Asn, Ile, Trp, Lys, or Pro” | |
| Modified-site | 104 | /note= “Xaa at position 104 is Leu, | Ser, Pro, Ala, Glu, Cys, Asp, or Tyr” | |
| Modified-site | 105 | /note= “Xaa at position 105 is Glu, | Ser, Lys, Pro, Leu, Thr, Tyr, or Arg” | |
| Modified-site | 106 | /note= “Xaa at position 106 is Asn, | Ala, Pro, Leu, His, Val or Gln” | |
| Modified-site | 107 | /note= “Xaa at position 107 is Ala, | Ser, Ile, Asn, Pro, Lys, Asp, or Gly” | |
| Modified-site | 108 | /note= “Xaa at position 108 is | Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, | or Cys” |
| Modified-site | 109 | /note= “Xaa at position 109 is Ala, | Met, Glu, His, Ser, Pro, Tyr, or Leu” | |
| 4 |
| 111 amino acids | amino acid | linear | ||
| peptide | ||||
| Modified-site | /note= “Met- or Met-Ala- may or may | not precede the amino acid in position 1” | ||
| Modified-site | /note= “Xaa at position 3 is Ser, | Gly, Asp, Met, or Gln” | ||
| Modified-site | /note= “Xaa at position 4 is Asn, | His, or Ile” | ||
| Modified-site | /note= “Xaa at position 5 is Met | or Ile” | ||
| Modified-site | /note= “Xaa at position 7 is Asp or | Glu” | ||
| Modified-site | /note= “Xaa at position 9 is Ile, | Ala, Leu, or Gly” | ||
| Modified-site | 10 | /note= “Xaa at position 10 is Ile, | Val, or Leu” | |
| Modified-site | 11 | /note= “Xaa at position 11 is Thr, | His, Gln, or Ala” | |
| Modified-site | 12 | /note= “Xaa at position 12 is His | or Ala” | |
| Modified-site | 15 | /note= “Xaa at position 15 is Gln, | Asn, or Val” | |
| Modified-site | 16 | /note= “Xaa at position 16 is Pro, | Gly, or Gln” | |
| Modified-site | 17 | /note= “Xaa at position 17 is Pro, | Asp, Gly, or Gln” | |
| Modified-site | 18 | /note= “Xaa at position 18 is Leu, | Arg, Gln, Asn, Gly, Ala, or Glu” | |
| Modified-site | 19 | /note= “Xaa at position 19 is Pro | or Glu” | |
| Modified-site | 20 | /note= “Xaa at position 20 is Leu, | Val, Gly, Ser, Lys, Ala, Arg, Gln, Glu, Ile, Phe, | Thr, or Met” |
| Modified-site | 21 | /note= “Xaa at position 21 is Leu, | Ala, Asn, Pro, Gln, or Val” | |
| Modified-site | 23 | /note= “Xaa at position 23 is Phe, | Ser, Pro, or Trp” | |
| Modified-site | 24 | /note= “Xaa at position 24 is Asn | or Ala” | |
| Modified-site | 28 | /note= “Xaa at position 28 is Gly, | Asp, Ser, Cys, Ala, Asn, Ile, Leu, Met, Tyr, or Arg” | |
| Modified-site | 30 | /note= “Xaa at position 30 is Asp | or Glu” | |
| Modified-site | 31 | /note= “Xaa at position 31 is Gln, | Val, Met, Leu, Thr, Ala, Asn, Glu, Ser, or Lys” | |
| Modified-site | 32 | /note= “Xaa at position 32 is Asp, | Phe, Ser, Thr, Ala, Asn, Gln, Glu, His, Ile, Lys, | Tyr, Val, or Cys” |
| Modified-site | 36 | /note= “Xaa at position 36 is Glu, | Ala, Asn, Ser, or Asp” | |
| Modified-site | 37 | /note= “Xaa at position 37 is Asn, | Arg, Met, Pro, Ser, Thr, or His” | |
| Modified-site | 40 | /note= “Xaa at position 40 is Arg | or Ala” | |
| Modified-site | 41 | /note= “Xaa at position 41 is Arg, | Thr, Val, Leu, or Gly” | |
| Modified-site | 42 | /note= “Xaa at position 42 is Pro, | Gly, Ser, Gln, Ala, Arg, Asn, Glu, Leu, Thr, Val, | or Lys” |
| Modified-site | 46 | /note= “Xaa at position 46 is Ala | or Ser” | |
| Modified-site | 48 | /note= “Xaa at position 48 is Asn, | Pro, Thr, or Ile” | |
| Modified-site | 49 | /note= “Xaa at position 49 is Arg | or Lys” | |
| Modified-site | 50 | /note= “Xaa at position 50 is Ala | or Asn” | |
| Modified-site | 51 | /note= “Xaa at position 51 is Val | or Thr” | |
| Modified-site | 52 | /note= “Xaa at position 52 is Lys | or Arg” | |
| Modified-site | 53 | /note= “Xaa at position 53 is Ser, | Phe, or His” | |
| Modified-site | 54 | /note= “Xaa at position 54 is Leu, | Ile, Phe, or His” | |
| Modified-site | 55 | /note= “Xaa at position 55 is Gln, | Ala, Pro, Thr, Glu, Arg, or Gly” | |
| Modified-site | 57 | /note= “Xaa at position 57 is Ala, | Pro, or Arg” | |
| Modified-site | 58 | /note= “Xaa at position 58 is Ser, | Glu, Arg, or Asp” | |
| Modified-site | 59 | /note= “Xaa at position 59 is Ala | or Leu” | |
| Modified-site | 62 | /note= “Xaa at position 62 is Ser, | Val, Ala, Asn, Glu, Pro, or Gly” | |
| Modified-site | 63 | /note= “Xaa at position 63 is Ile | or Leu” | |
| Modified-site | 65 | /note= “Xaa at position 65 is Lys, | Thr, Gly, Asn, Met, Arg, Ile, or Asp” | |
| Modified-site | 66 | /note= “Xaa at position 66 is Asn, | Gly, Glu, or Arg” | |
| Modified-site | 68 | /note= “Xaa at position 68 is Leu, | Gln, Trp, Arg, Asp, Ala, Asn, Glu, His, Ile, Met, | Phe, Ser, Thr, Tyr, or Val” |
| Modified-site | 69 | /note= “Xaa at position 69 is Pro | or Thr” | |
| Modified-site | 71 | /note= “Xaa at position 71 is Leu | or Val” | |
| Modified-site | 73 | /note= “Xaa at position 73 is Leu | or Ser” | |
| Modified-site | 74 | /note= “Xaa at position 74 is Ala | or Trp” | |
| Modified-site | 77 | /note= “Xaa at position 77 is Ala | or Pro” | |
| Modified-site | 79 | /note= “Xaa at position 79 is Thr, | Asp, Ser, Pro, Ala, Leu, or Arg” | |
| Modified-site | 81 | /note= “Xaa at position 81 is His, | Pro, Arg, Val, Leu, Gly, Asn, Phe, Ser, or Thr” | |
| Modified-site | 82 | /note= “Xaa at position 82 is Pro | or Tyr” | |
| Modified-site | 83 | /note= “Xaa at position 83 is Ile | or Val” | |
| Modified-site | 84 | /note= “Xaa at position 84 is His, | Ile, Asn, Leu, Ala, Thr, Arg, Gln, Lys, | Met, Ser, Tyr, Val, or Pro” |
| Modified-site | 85 | /note= “Xaa at position 85 is Ile, | Leu, or Val” | |
| Modified-site | 86 | /note= “Xaa at position 86 is Lys, | Arg, Ile, Gln, Pro, or Ser” | |
| Modified-site | 87 | /note= “Xaa at position 87 is Asp, | Pro, Met, Lys, His, Thr, Asn, Ile, Leu, or Tyr” | |
| Modified-site | 90 | /note= “Xaa at position 90 is Trp | or Leu” | |
| Modified-site | 91 | /note=“Xaa at position 91 is Asn, | Pro, Ala, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp, | or His” |
| Modified-site | 92 | /note= “Xaa at position 92 is Glu | or Gly” | |
| Modified-site | 94 | /note= “Xaa at position 94 is Arg, | Ala, or Ser” | |
| Modified-site | 95 | /note= “Xaa at position 95 is Arg, | Thr, Glu, Leu, or Ser” | |
| Modified-site | 98 | /note= “Xaa at position 98 is Thr, | Val, or Gln” | |
| Modified-site | 100 | /note= “Xaa at position 100 is Tyr | or Trp” | |
| Modified-site | 101 | /note= “Xaa at position 101 is Leu | or Ala” | |
| Modified-site | 102 | /note= “Xaa at position 102 is Lys, | Thr, Val, Trp, Ser, Ala, His, Met, Phe, Tyr, or Ile” | |
| Modified-site | 103 | /note= “Xaa at position 103 is Thr | or Ser” | |
| Modified-site | 106 | /note= “Xaa at position 106 is Asn, | Pro, Leu, His, Val, or Gln” | |
| Modified-site | 107 | /note= “Xaa at position 107 is Ala, | Ser, Ile, Asn, Pro, Asp, or Gly” | |
| Modified-site | 108 | /note= “Xaa at position 108 is Gln, | Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys” | |
| Modified-site | 109 | /note= “Xaa at position 109 is Ala, | Met, Glu, His, Ser, Pro, Tyr, or Leu” | |
| 5 |
| 111 amino acids | amino acid | linear | ||
| peptide | ||||
| Modified-site | /note= “Met- or Met-Ala- may or may | not precede the amino acid in position 1” | ||
| Modified-site | /note= “Xaa at position 3 is Ser, | Gly, Asp, or Gln” | ||
| Modified-site | /note= “Xaa at position 4 is Asn, | His, or Ile” | ||
| Modified-site | /note= “Xaa at position 9 is Ile, | Ala, Leu, or Gly” | ||
| Modified-site | 11 | /note= “Xaa at position 11 is Thr, | His, or Gln” | |
| Modified-site | 12 | /note= “Xaa at position 12 is His | or Ala” | |
| Modified-site | 15 | /note= “Xaa at position 15 is Gln | or Asn” | |
| Modified-site | 16 | /note= “Xaa at position 16 is Pro | or Gly” | |
| Modified-site | 18 | /note= “Xaa at position 18 is Leu, | Arg, Asn, or Ala” | |
| Modified-site | 20 | /note= “Xaa at position 20 is Leu, | Val, Ser, Ala, Arg, Gln, Glu, Ile, Phe, Thr, or Met” | |
| Modified-site | 21 | /note= “Xaa at position 21 is Leu, | Ala, Asn, or Pro” | |
| Modified-site | 24 | /note= “Xaa at position 24 is Asn | or Ala” | |
| Modified-site | 28 | /note= “Xaa at position 28 is Gly, | Asp, Ser, Ala, Asn, Ile, Leu, Met, Tyr, or Arg” | |
| Modified-site | 31 | /note= “Xaa at position 31 is Gln, | Val, Met, Leu, Ala, Asn, Glu, or Lys” | |
| Modified-site | 32 | /note= “Xaa at position 32 is Asp, | Phe, Ser, Ala, Gln, Glu, His, Val, or Thr” | |
| Modified-site | 36 | /note= “Xaa at position 36 is Glu, | Asn, Ser, or Asp” | |
| Modified-site | 37 | /note= “Xaa at position 37 is Asn, | Arg, Pro, Thr, or His” | |
| Modified-site | 41 | /note= “Xaa at position 41 is Arg, | Leu, or Gly” | |
| Modified-site | 42 | /note= “Xaa at position 42 is Pro, | Gly, Ser, Ala, Asn, Val, Leu, or Gln” | |
| Modified-site | 48 | /note= “Xaa at position 48 is Asn, | Pro, or Thr” | |
| Modified-site | 50 | /note= “Xaa at position 50 is Ala | or Asn” | |
| Modified-site | 51 | /note= “Xaa at position 51 is Val | or Thr” | |
| Modified-site | 53 | /note= “Xaa at position 53 is Ser | or Phe” | |
| Modified-site | 54 | /note= “Xaa at position 54 is Leu | or Phe” | |
| Modified-site | 55 | /note= “Xaa at position 55 is Gln, | Ala, Glu, or Arg” | |
| Modified-site | 62 | /note= “Xaa at position 62 is Ser, | Val, Asn, Pro, or Gly” | |
| Modified-site | 63 | /note= “Xaa at position 63 is Ile | or Leu” | |
| Modified-site | 65 | /note= “Xaa at position 65 is Lys, | Asn, Met, Arg, Ile, or Gly” | |
| Modified-site | 66 | /note= “Xaa at position 66 is Asn, | Gly, Glu, or Arg” | |
| Modified-site | 68 | /note= “Xaa at position 68 is Leu, | Gln, Trp, Arg, Asp, Asn, Glu, His, Met, Phe, Ser, | Thr, Tyr, or Val” |
| Modified-site | 73 | /note= “Xaa at position 73 is Leu | or Ser” | |
| Modified-site | 74 | /note= “Xaa at position 74 is Ala | or Trp” | |
| Modified-site | 77 | /note= “Xaa at position 77 is Ala | or Pro” | |
| Modified-site | 79 | /note= “Xaa at position 79 is Thr, | Asp, or Ala” | |
| Modified-site | 81 | /note= “Xaa at position 81 is His, | Pro, Arg, Val, Gly, Asn, Ser, or Thr” | |
| Modified-site | 84 | /note= “Xaa at position 84 is His, | Ile, Asn, Leu, Ala, Thr, Arg, Gln, Glu, Lys, Met, | Ser, Tyr, or Val” |
| Modified-site | 85 | /note= “Xaa at position 85 is Ile | or Leu” | |
| Modified-site | 86 | /note= “Xaa at position 86 is Lys | or Arg” | |
| Modified-site | 87 | /note= “Xaa at position 87 is Asp, | Pro, Met, Lys, His, Pro, Asn, Ile, Leu, or Tyr” | |
| Modified-site | 91 | /note= “Xaa at position 91 is Asn, | Pro, Ser, Ile, or Asp” | |
| Modified-site | 94 | /note=“Xaa at position 94 is Arg, | Ala, or Ser” | |
| Modified-site | 95 | /note= “Xaa at position 95 is Arg, | Thr, Glu, Leu, or Ser” | |
| Modified-site | 98 | /note= “Xaa at position 98 is Thr | or Gln” | |
| Modified-site | 102 | /note= “Xaa at position 102 is Lys, | Val, Trp, or Ile” | |
| Modified-site | 103 | /note= “Xaa at position 103 is Thr, | Ala, His, Phe, Tyr, or Ser” | |
| Modified-site | 106 | /note= “Xaa at position 106 is Asn, | Pro, Leu, His, Val, or Gln” | |
| Modified-site | 107 | /note= “Xaa at position 107 is Ala, | Ser, Ile, Pro, or Asp” | |
| Modified-site | 108 | /note= “Xaa at position 108 is Gln, | Met, Trp, Phe, Pro, His, Ile, or Tyr” | |
| Modified-site | 109 | /note= “Xaa at position 109 is Ala, | Met, Glu, Ser, or Leu” | |
| 6 |
| 133 amino acids | amino acid | linear | |
| peptide | |||
| Modified-site | /note= “Met- may or may not precede | the amino acid in position 1” | |
| Modified-site | 18 | /note= “Xaa at position 18 is Asn | or Ile” |
| Modified-site | 19 | /note= “Xaa at position 19 is Met, | Ala, or Ile” |
| Modified-site | 20 | /note= “Xaa at position 20 is Ile, | Pro, or Leu” |
| Modified-site | 23 | /note= “Xaa at position 23 is Ile, | Ala, or Leu” |
| Modified-site | 25 | /note= “Xaa at position 25 is Thr | or His” |
| Modified-site | 29 | /note= “Xaa at position 29 is Gln, | Arg, Val, or Ile” |
| Modified-site | 32 | /note= “Xaa at position 32 is Leu, | Ala, Asn, or Arg” |
| Modified-site | 34 | /note= “Xaa at position 34 is Leu | or Ser” |
| Modified-site | 37 | /note= “Xaa at position 37 is Phe, | Pro, or Ser” |
| Modified-site | ; 38 | /note= “Xaa at position 38 is Asn | or Ala” |
| Modified-site | 42 | /note= “Xaa at position 42 is Gly, | Ala, Ser, Asp, or Asn” |
| Modified-site | 45 | /note= “Xaa at position 45 is Gln, | Val, or Met” |
| Modified-site | 46 | /note= “Xaa at position 46 is Asp | or Ser” |
| Modified-site | 49 | /note= “Xaa at position 49 is Met, | Ile, Leu, or Asp” |
| Modified-site | 50 | /note= “Xaa at position 50 is Glu | or Asp” |
| Modified-site | 51 | /note= “Xaa at position 51 is Asn, | Arg, or Ser” |
| Modified-site | 55 | /note= “Xaa at position 55 is Arg, | Leu, or Thr” |
| Modified-site | 56 | /note= “Xaa at position 56 is Pro | or Ser” |
| Modified-site | 59 | /note= “Xaa at position 59 is Glu | or Leu” |
| Modified-site | 60 | /note= “Xaa at position 60 is Ala | or Ser” |
| Modified-site | 62 | /note= “Xaa at position 62 is Asn | Val, or Pro” |
| Modified-site | 63 | /note= “Xaa at position 63 is Arg | or His” |
| Modified-site | 65 | /note= “Xaa at position 65 is Val | or Ser” |
| Modified-site | 67 | /note= “Xaa at position 67 is Ser, | Asn, His, or Gln” |
| Modified-site | 69 | /note= “Xaa at position 69 is Gln | or Glu” |
| Modified-site | 73 | /note= “Xaa at position 73 is Ala | or Gly” |
| Modified-site | 76 | /note= “Xaa at position 76 is Ser, | Ala, or Pro” |
| Modified-site | 79 | /note= “Xaa at position 79 is Lys, | Arg, or Ser” |
| Modified-site | 82 | /note= “Xaa at position 82 is Leu, | Glu, Val, or Trp” |
| Modified-site | 85 | /note= “Xaa at position 85 is Leu | or Val” |
| Modified-site | 87 | /note= “Xaa at position 87 is Leu, | Ser, or Tyr” |
| Modified-site | 88 | /note= “Xaa at position 88 is Ala | or Trp” |
| Modified-site | 91 | /note= “Xaa at position 91 is Ala | or Pro” |
| Modified-site | 93 | /note= “Xaa at position 93 is Pro | or Ser” |
| Modified-site | 95 | /note= “Xaa at position 95 is His | or Thr” |
| Modified-site | 98 | /note= “Xaa at position 98 is His, | Ile, or Thr” |
| Modified-site | 100 | /note= “Xaa at position 100 is Lys | or Arg” |
| Modified-site | 101 | /note= “Xaa at position 101 is Asp, | Ala, or Met” |
| Modified-site | 105 | /note= “Xaa at position 105 is Asn | or Glu” |
| Modified-site | 109 | /note= “Xaa at position 109 is Arg, | Glu, or Leu” |
| Modified-site | 112 | /note= “Xaa at position 112 is Thr | or Gln” |
| Modified-site | 116 | /note= “Xaa at position 116 is Lys, | Val, Trp, or Ser” |
| Modified-site | 117 | /note= “Xaa at position 117 is Thr | or Ser” |
| Modified-site | 120 | /note= “Xaa at position 120 is Asn, | Gln, or His” |
| Modified-site | 123 | /note= “Xaa at position 123 is Ala | or Glu” |
| 7 |
| 111 amino acids | amino acid | linear | |
| peptide | |||
| Modified-site | /note= “Met- or Met-Ala may or may | not precede the amino acid in position 1” | |
| Modified-site | /note= “Xaa at position 4 is Asn or | Ile” | |
| Modified-site | /note= “Xaa at position 5 is Met, | Ala, or Ile” | |
| Modified-site | /note= “Xaa at position 6 is Ile, | Pro, or Leu” | |
| Modified-site | /note= “Xaa at position 9 is Ile, | Ala, or Leu” | |
| Modified-site | 11 | /note= “Xaa at position 11 is Thr | or His” |
| Modified-site | 15 | /note= “Xaa at position 15 is Gln, | Arg, Val, or Ile” |
| Modified-site | 18 | /note= “Xaa at position 18 is Leu, | Ala, Asn, or Arg” |
| Modified-site | 20 | /note= “Xaa at position 20 is Leu | or Ser” |
| Modified-site | 23 | /note= “Xaa at position 23 is Phe, | Pro, or Ser” |
| Modified-site | 24 | /note= “Xaa at position 24 is Asn | or Ala” |
| Modified-site | 28 | /note= “Xaa at position 28 is Gly, | Ala, Ser, Asp, or Asn” |
| Modified-site | 31 | /note= “Xaa at position 31 is Gln, | Val, or Met” |
| Modified-site | 32 | /note= “Xaa at position 32 is Asp | or Ser” |
| Modified-site | 35 | /note= “Xaa at position 35 is Met, | Ile, or Asp” |
| Modified-site | 36 | /note= “Xaa at position 36 is Glu | or Asp” |
| Modified-site | 37 | /note= “Xaa at position 37 is Asn, | Arg, or Ser” |
| Modified-site | 41 | /note= “Xaa at position 41 is Arg, | Leu, or Thr” |
| Modified-site | 42 | /note= “Xaa at position 42 is Pro | or Ser” |
| Modified-site | 45 | /note= “Xaa at position 45 is Glu | or Leu” |
| Modified-site | 46 | /note= “Xaa at position 46 is Ala | or Ser” |
| Modified-site | 48 | /note= “Xaa at position 48 is Asn, | Val, or Pro” |
| Modified-site | 49 | /note= “Xaa at position 49 is Arg | or His” |
| Modified-site | 51 | /note= “Xaa at position 51 is Val | or Ser” |
| Modified-site | 53 | /note= “Xaa at position 53 is Ser, | Asn, His, or Gln” |
| Modified-site | 55 | /note= “Xaa at position 55 is Gln | or Glu” |
| Modified-site | 59 | /note= “Xaa at position 59 is Ala | or Gly” |
| Modified-site | 62 | /note= “Xaa at position 62 is Ser, | Ala, or Pro” |
| Modified-site | 65 | /note= “Xaa at position 65 is Lys, | Arg, or Ser” |
| Modified-site | 67 | /note= “Xaa at position 67 is Leu, | Glu, or Val” |
| Modified-site | 68 | /note= “Xaa at position 68 is Leu, | Glu, Val, or Trp” |
| Modified-site | 71 | /note= “Xaa at position 71 is Leu | or Val” |
| Modified-site | 73 | /note= “Xaa at position 73 is Leu, | Ser, or Tyr” |
| Modified-site | 74 | /note= “Xaa at position 74 is Ala | or Trp” |
| Modified-site | 77 | /note= “Xaa at position 77 is Ala | or Pro” |
| Modified-site | 79 | /note= “Xaa at position 79 is Pro | or Ser” |
| Modified-site | 81 | /note= “Xaa at position 81 is His | or Thr” |
| Modified-site | 84 | /note= “Xaa at position 84 is His, | Ile, or Thr” |
| Modified-site | 86 | /note= “Xaa at position 86 is Lys | or Arg” |
| Modified-site | 87 | /note= “Xaa at position 87 is Asp, | Ala, or Met” |
| Modified-site | 91 | /note= “Xaa at position 91 is Asn | or Glu” |
| Modified-site | 95 | /note= “Xaa at position 95 is Arg, | Glu, or Leu” |
| Modified-site | 98 | /note= “Xaa at position 98 is Thr | or Gln” |
| Modified-site | 102 | /note= “Xaa at position 102 is Lys, | Val, Trp, or Ser” |
| Modified-site | 103 | /note= “Xaa at position 103 is Thr | or Ser” |
| Modified-site | 106 | /note= “Xaa at position 106 is Asn, | Gln, or His” |
| Modified-site | 109 | /note= “Xaa at position 109 is Ala | or Glu” |
| 8 |
Claims as granted
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10 codes- A61K38/20
- A61K38/00
- C12N5/08
- C12P21/04
- C07K14/54
- C12N15/24
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