Methods of using interleukin-3 (IL-3) mutant polypeptides for ex-vivo expansion of hematopoietic stem cells

Construction of pMON 5846 (FIG. 4) which Encodes [Met-(1-133) hIL-3 (Arg129)]

A plasmid containing the gene for the cDNA of hIL-3 cloned into pUC18 on an EcoRI to HindIII fragment was obtained from British Biotechnology Limited (Cambridge, England). This plasmid was designated pPO518. The purified plasmid DNA was cleaved by the restriction endonucleases NheI and BamHI. Approximately 0.5 micrograms of cleaved plasmid DNA was ligated to 1.0 picomoles of a pair of annealed oligonucleotides with the following sequence:

5′-CTAGCGATCTTTTAATAAGCTTG-3′ [SEQ ID NO: 1]

3′-GCTAGAAAATTATTCGAACCTAG-5′ [SEQ ID NO: 2]

The ligation mixture was used to transform competent JM101 cells to ampicillin resistance. Colonies were picked, and plasmid DNA was purified and subjected to restriction enzyme analysis. An isolate was identified in which the above oligonucleotide sequence had replaced the portion of the gene that encodes the extreme C-terminus. Within the new sequence was a new stop codon, TAA, and a recognition site for the enzyme HindIII. The new plasmid was designated pMON5846.

(a) Construction of Expression Vector Plasmid pMON2341

The plasmid pMON2341 was used to supply the particular replicon and expression elements used for construction of many of the plasmids used to produce hIL-3 and hIL-3 muteins in E. coli. These expression elements are described in the materials and methods section. pMON2341 is derived from pMON5515 (Olins et al., 1988) and from pMON2429. pMON2429 consists of the phage mp18 (Yanisch-Perron et al., 1985) with a BclI fragment carrying the chloramphenicol acetyl transferase (cat) gene from pBR328 (Covarrubias et al., 1981) inserted into the BamHI site. The cat gene in pMON2429 has been altered from that in pBR328 by site directed mutagenesis (Kunkel, 1985). The recognition sites for NcoI and EcoRI which occur in the native gene were altered so that these two restriction enzymes no longer recognize these sites. The changes did not alter the protein specified by the gene. Also, an NcoI site was introduced at the N-terminus of the coding sequence so that it overlaps the codon for initiator methionine.

The steps involved in construction of pMON2341 are listed below:

(1) The DNAs of pMON5515 and pMON2429 were treated with NcoI and HindIII. The fragments were ligated and used to transform competent E. coli to ampicillin resistance. From these colonies, some were identified that were chloramphenicol resistant. From one of these colonies, plasmid DNA was isolated in which the rat atriopeptigen gene of pMON5515 had been replaced by the NcoI to HindIII fragment containing the cat gene from pMON2429. This fragment contains the recognition sites for several restriction enzymes in the portion derived, from the multilinker region of mp18. The new plasmid was designated pMON2412.

(2) pMON2412 was treated with the enzyme ClaI which cleaves at one location in the pBR327 derived portion of the DNA. The protruding ends were rendered blunt by treatment with Klenow in the presence of nucleotide precursors. This DNA was mixed with an isolated 514 bp RsaI fragment derived from pEMBL8 (Dente et al., 1983). This RsaI fragment contains the origin of replication of phage f1. This ligation mixture was used to transform competent E. coli cells to ampicillin resistance. Among the plasmid DNAs isolated from these cells was pMON5578. This plasmid has the structure of pMON2412 with the f1 origin region inserted into the ClaI site. This in illustrated in the Figures and in Olins and Rangwala (1990).

(3) The DNA of pMON5578 was treated with restriction enzymes HindIII and MstII. The DNA was then treated with Klenow enzyme in the presence of nucleotide precursors to render the ends blunt. This treated DNA was ligated and used to transform competent E. coli to ampicillin resistance. From the ampicillin resistant colonies, one plasmid was recovered from which the portion between HindIII and MstII was absent. This deletion resulted in the removal of sequences from the plasmid which are recognized by a number of restriction endonuclease sites. The new plasmid was designated pMON5582.

(4) The DNA of pMON5582 was treated with SstII and BclI and ligated in the presence of annealed oligonucleotides with the sequences shown below.

5′-GGCAACAATTTCTACAAAACACTTGATACTGTATGAGCAT-3′-CGCCGTTGTTAAAGATGTTTTGTGAACTATGACATACTCGTAACAGTATAATTGCTTCAACAGAACAGATC-3′ [SEQ ID NO:3]

TGTCATATTAACGAAGTTGTCTTGT-5′ [SEQ ID NO:4]

This sequence encodes the essential elements of the recA promoter of E. coli including the transcription start site and the lexA repressor binding site (the operator) (Sancar et al., 1980). The plasmid recovered from the ligation mixes contained this recA promoter in place of the one In pMON5582 (and in pMON5515). The functionality of the recA promoter was illustrated by Olins and Rangwala (1990). The new plasmid was designated pMON5594.

(5) To eliminate the single EcoRI site in pMON5594, the DNA was treated with EcoRI, then with Klenow in the presence of nucleotide precursors to render the ends blunt and then the DNA was ligated. From this ligation mix a plasmid was recovered whose DNA was not cleaved with EcoRI. This plasmid was designated pMON5630.

(6) To alter the single recognition site for PstI, plasmid pMON5630 was subjected to site directed mutagenesis (Kunkel, 1985). The oligonucleotide used in this procedure has the sequence shown below.

5′-CCATTGCTGCCGGCATCGTGGTC-3′ [SEQ ID NO:5]

The result of the procedure was to construct pMON2341 which differs from pMON5630 in that the PstI site in the beta-lactamase gene was altered so that PstI no longer recognizes the site. The single nucleotide change does not alter the amino acid sequence of the beta-lactamase protein.

(b) Construction of pMON5847 (FIG. 5) which Encodes [Met-(1-133) hIL-3(Arg 129 )]

Plasmid pMON2341 was used to supply the replicon, promotor, ribosome binding site, transcription terminator and antibiotic resistance marker for the plasmids used to produce hIL-3 in E. coli from cDNA derived hIL-3 genes.

Plasmid pMON2341 was treated with restriction enzymes NcoI and HindIII. The restriction fragment containing the replication origin was purified. The DNA of plasmid pMON5846 was treated with NcoI and HindIII. The restriction fragment containing the hIL-3 gene was gel purified. These purified restriction fragments were mixed and ligated. The ligation mixture was used to transform competent JM101 cells to ampicillin resistance. Colonies were picked, and plasmid DNA was purified and analyzed using restriction enzymes. pMON5847 was identified as a plasmid with the replicon of pMON2341 and the hIL-3 gene in place of the chloramphenicol acetyl transferase gene. JM101 cells harboring this plasmid were cultured in M9 medium and treated with nalidixic acid as described above. Samples of the culture were examined for protein content. It was found that this hIL-3 mutein was produced at about 6% of total cell protein as measured on Coomassie stained polyacrylamide gels.

Construction of pMON5854 (FIG. 7) which Encodes [Met-(1-133) hIL-3(Arg 129 )]

To increase the accumulation of hIL-3 in E. coli, the coding sequence of the amino terminal portion of the protein was altered to more closely reflect the codon bias found in E. coli genes that produce high levels of proteins (Gouy and Gautier, 1982). To change the coding sequence for the amino terminal portion of the gene, a pair of synthetic oligonucleotides were inserted between the NcoI and HpaI sites within the coding sequence. About 0.5 micrograms of DNA of the plasmid pMON5847 (Example 2) was treated with NcoI and HpaI. This DNA was mixed with an annealed pair of oligonucleotides with the following sequence:

5′-CATGGCTCCAATGACTCAGACTACTTCTCTTAAGACT-3′-CGAGGTTACTGAGTCTGATGAAGAGAATTCTGATCTTGGGTT-3′ [SEQ ID NO:6]

AGAACCCAA-5′ [SEQ ID NO:7]

The fragments were ligated. The ligation mixture was used to transform competent JM101 to ampicillin resistance. Colonies were picked into broth. From the cultures plasmid DNA was made and examined for the presence of a DdeI site (CTNAG) which occurs in the synthetic sequence but not between the NcoI and HpaI sites in the sequence of pMON5847. The new recombinant plasmid was designated pMON5854. The nucleotide sequence of the DNA in the coding sequence of the amino terminal portion of the hIL-3 gene in pMON5854 was determined by DNA sequencing and found to be the same as that of the synthetic oligonucleotide used in ligation. Cultures of JM101 cells harboring this plasmid were grown and treated with nalidixic acid to induce production of the hIL-3 mutant protein. Analysis of the proteins on Coomassie gels showed that the accumulation of hIL-3 mutein was about 25% of total cell protein in cultures harboring pMON5854, significantly higher than it was in cultures harboring pMON5847.

Construction of pMON5887 (FIG. 12) which Encodes [Met-(1-125) hIL-3]

The plasmid DNA of pMON5854 (Example 3) was treated with EcoRI and HindIII and the larger fragment was gel purified. About 0.5 mitrogram of this DNA was ligated to 1 picomole of an annealed pair of oligonucleotides which encode amino acids 107 through 125 of hIL-3. The sequences of these oligonucleotides are shown below. EcoRI to HindIII

5′-AATTCCGTCOTAAMCTGACCTTCTATCTGAAAA-3′-GGCAGCATTTGACTGGAAGATAGACTTTTCCTTGGAGAACGCGCAGGCTCAACAGTAATA-3′ [SEQ ID NO;8]

GGAACCTCTTGCGCGTCCGAGTTGTCATTATTCGA-5′ [SEQ ID NO:9]

After ligation, the DNA was used to transform competent JM101 cells to ampicillin resistance. Colonies were picked into broth and plasmid DNA was isolated from each culture. Restriction analysis of the plasmid DNA showed the presence of an EcoRI to HindIII fragment smaller than that of pMON5854. The nucleotide sequence of the portion of the coding sequence between the EcoRI and HindIII sites was determined to confirm the accuracy of the replaced sequence. The new plasmid was designated pMON5887 encoding Met-(1-125) hIL-3 which has the following amino acid sequence:

[SEQ ID NO:10]

Met Ala Pro Met Thr Gln Thr Thr Ser Leu Lys Thr

Ser Trp Val Asn Cys Ser Asn Met Ile Asp Glu Ile

Ile Thr His Leu Lys Gln Pro Pro Leu Pro Leu Leu

Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp Ile

Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu

Ala Phe Asn Arg Ala Val Lys Ser Leu Gln Asn Ala

Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro

Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His

Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe

Arg Arg Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu

Asn Ala Gln Ala Gln Gln

Construction of pMON5967 which Encodes [Met-Ala-(15-125) hIL-3]

Plasmid DNA of pMON5887 isolated from E. coli GM48 (dam-) was cleaved with NcoI and ClaI and ligated to 1 picomole of an annealed pair of oligonucleotides, Nco I and ClaI, encoding amino acids [Met Ala (15-20) hIL-3]. The sequence of these oligonucleotides is shown below.

5′-CATGGCTAACTGCTCTAACATGAT-3′[SEQ ID NO:11]

3′-CGATTGACGAGATTGTACTAGC-5′[SEQ ID NO:12]

The resulting ligation mix was used to transform competent E. coli JM101 cells to ampicillin resistant colonies. Plasmid DNA was isolated from these cells and the size of the inserted fragment was determined to be smaller than that of pMON5887 by restriction analysis using NcoI and NsiI. The nucleotide sequence of the region between NcoI and ClaI was determined and found to be that of the synthetic oligonucleotides. The new plasmid was designated pMON5967 and cells containing it were induced for protein production. Sonicated cell pellets and supernatants were used for protein purification and bio-assay.

Construction of pMON5978 which Encodes [Met-Ala-(15-125) hIL-3]

Plasmid DNA of pMON5967 isolated from E. coli GM48(dam-) was cleaved with ClaI and NsiI and ligated to 1 picomole of an annealed assembly of six oligonucleotides encoding hIL-3 amino acids 20-70 (FIG. 2 ). This synthetic fragment encodes three unique restriction sites, EcoRV, XhoI and PstI. The sequence of these oligonucleotides is shown in FIG. 2 .

The resulting ligation mix was used to transform competent E. coli JM101 cells to ampicillin resistant colonies. Plasmid DNA was isolated and screened with XbaI and EcoRV for the presence of the new restriction site EcoRV. The DNA sequence of the region between ClaI and NsiI was determined and found to be the same as that of the synthetic oligonucleotides. The new plasmid was designated pMON5978, and cells containing it were induced for protein production. Sonicated cell pellets and supernatants were used for protein purification and bioassay.

Plasmid pMON5978 encodes [Met-Ala-(15-125) hIL-3] which has the following amino acid sequence:

[SEQ ID NO:13]

Met Ala Asn Cys Ser Asn Met Ile Asp Glu Ile Ile

Thr His Leu Lys Gln Pro Pro Leu Pro Leu Leu Asp

Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp Ile Leu

Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala

Phe Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser

Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro Cys

Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro

Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg

Arg Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn

Ala Gln Ala Gln Gln

Construction of pMON5898

Plasmid pMON5851 DNA was digested with restriction enzymes HindIII and NcoI resulting in a 3695 base pair NcoI,HindIII fragment. The genetic elements derived from pMON5851 are the beta-lactamase gene (AMP), pBR327 origin of replication, phage f1 origin of replication as the transcription terminator, AraBAD promoter, g10L ribosome binding site and the lamB secretion leader. The AraBAD promoter is identical to that described in plasmid pMON6235 and the lamB signal peptide sequence used is that shown in FIG. 8 fused to hIL-3 at the NcoI recognition site. Plasmid pMON5873 DNA was digested with restriction enzymes HindIII and NcoI resulting in a 408 base pair NcoI,HindIII fragment. The genetic element derived from pMON5873 is the hIL-3 gene (1-133). Clones containing the hIL-3 (1-133) gene contained a 408 base pair NcoI, HindIII restriction fragment. This construct was designated pMON5898.

Construction of pMON5987

Plasmid pMON6458 DNA was digested with restriction enzymes NcoI and HindIII, resulting in a 3940 base pair NcoI, HindIII fragment. The genetic elements derived from pMON6458 are the beta-lactamase gene (AMP), pBR327 origin of replication, phage f1 origin of replication as the transcription terminator, AraBAD promoter, g10L ribosome binding site and lamB secretion leader. Plasmid pMON5978 DNA was digested with NcoI and NsiI. The resulting 170 base pair NcoI, NsiI fragment encodes amino acids 15-71 of (15-125) hIL-3. Plasmid pMON5976 DNA was digested with NsiI and HindIII. The resulting 175 base pair NsiI, HindIII fragment encodes amino acids 72-125 of (15-125) hIL-3. The restriction fragments were ligated, and the ligation reaction mixture was used to transform E. coli K-12 strain JM101. Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and screened for the restriction sites EcoRV and NheI and DNA sequenced to confirm the correct insert.

Construction of pMON5988

The plasmid DNA of pMON5987 was digested with NheI and EcoRI, resulting in a 3903 base pair NheI, EcoRI fragment. The 3903 base pair NheI, EcoRI fragment was ligated to 1.0 picomoles of the following annealed oligonucleotides (oligo #3 and Oligo #4):

5′-CTAGCCACGGCCGCACCCACGCGACATCCAATCCATATCAA-3′-GGTGCCGGCGTGGGTGCGCTGTAGGTTAGGTATAGTTGGACGGTGACTGGAATG-3′ [SEQ ID NO:131]

CCTGCCACTGACCTTACAATT-5′ [SEQ ID NO:132]

The ligation reaction mixture was used to transform E. coli K-12 strain JM101 and transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm positive clones.

This plasmid was constructed to change alanine 101 to aspartic acid in the hIL-3 gene (15-125). The Ala 101 to Asp 101 change was confirmed by DNA sequencing. This plasmid was designated pMON5988 and encodes Peptide #1 [SEQ ID NO:65].

Construction of pMON5873 which encodes [Met-(1-133) hIL-3]

The gene obtained from British Biotechnology, Ltd. specified arginine at codon position 129. The amino acid specified in the native hIL-3 cDNA is serine. To produce a protein with the native sequence at this position, the portion of the coding sequence between the EcoRI site at codons 106 and 107 and the NheI site at codons 129 and 130 was replaced. Plasmid DNA of pMON5854 (Example 3) and pMON5853 (Example 64) were treated with EcoRI and NheI. The larger fragments of each were gel purified. These were ligated to a pair of an annealed oligonucleotides with the following sequences:

5′-AATTCCGTCGTAAACTGACCTTCTATCTGAAAACC-3′-GGCAGCATTTGACTGGAAGATAGACTTTTGGTTGGAGAACGCGCAGGCTCAACAGACCACTCTGTCG-3′ [SEQ ID NO: 136]

AACCTCTTGCGCGTCCGAGTTGTCTGGTGAGACAGCGATC-5′ [SEQ ID NO:137]

The ligation reaction mixtures were used to transform competent JM101 cells to ampicillin resistance. Colonies were picked into broth and grown. Plasmid DNA was isolated and screened for the presence of a new StyI recognition site present in the synthetic DNA and not in pMON5854 and pMON5853. The nucleotide sequence of the gene in the region between EcoRI and NheI was determined and found to be that of the synthetic oligonucleotides. The new plasmids were designated pMON5873 encoding [Met-(1-133) hIL-3] and pMON5872 encoding [Met-(15-133) hIL-3].

The plasmid, pMON5873, encodes Met-(1-133) hIL-3 which has the following amino acid sequence:

[SEQ ID NO:128]

Met Ala Pro Met Thr Gln Thr Thr Ser Leu Lys Thr

Ser Trp Val Asn Cys Ser Asn Met Ile Asp Glu Ile

Ile Thr His Leu Lys Gln Pro Pro Leu Pro Leu Leu

Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp Ile

Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu

Ala Phe Asn Arg Ala Val Lys Ser Leu Gln Asn Ala

Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro

Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His

Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe

Arg Arg Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu

Asn Ala Gln Ala Gln Gln Thr Thr Leu Ser Leu Ala

Ile Phe

Construction of pMON6458

Plasmid pMON6525 DNA was digested with restriction enzymes HindIII and SalI and the resulting 3172 base pair fragment was isolated from a 1% agarose gel by interception onto DEAE membrane. The genetic elements derived from pMON6525 are the beta-lactamase gene (AMP), pBR327 origin of replication, and phage f1 origin of replication as the transcription terminator. (The genetic elements derived from plasmid pMON6525 are identical to those in plasmid pMON2341 which could also be used to construct pMON6458.) Plasmid pMON6457 was, digested with restriction enzymes HindIII and SalI and the resulting 1117 base pair fragment was isolated by PAGE and crush and soak elution. The genetic elements derived from pMON6457 are the pAraBAD promoter, g10L ribosome binding site, lamB secretion leader and the (15-125) hIL-3 gene. The restriction fragments were ligated and the ligation reaction mixture was used to transform E. coli K-12 strain JM101. Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and the size of the inserted fragment was determined by restriction analysis employing restriction enzymes NcoI and HindIII in double digest. Clones containing the hIL-3 gene (encoding amino acids 15-125) contained a 345 base pair NcoI, HindIII restriction fragment. This construct was designated pMON6458. This plasmid was constructed to eliminate an EcoRI restriction site outside the hIL-3 gene coding region in plasmid pMON6457.

Construction of pMON6455

Plasmid pMON5905 DNA was digested with restriction enzymes HindIII and NcoI resulting in a 3936 base pair fragment. The genetic elements derived from pMON5905 are the beta-lactamase gene (AMP), pBR327 origin of replication, pAraBAD promoter, g10L ribosome binding site, lamB secretion leader and phage f1 origin of replication as the transcription terminator. The following genetic elements; beta-lactamase gene (AMP), pBR327 origin of replication, g10L ribosome binding site and phage f1 origin of replication as the transcription terminator, derived from plasmid pMON5905 are identical to those in plasmid pMON5594 which could also be used to construct pMON6455. The AraBAD promoter is identical to that described in pMON6235. The lamB signal peptide sequence used in pMON6455 is that shown in FIG. 8 fused to hIL-3 (15-125) at the NcoI site. Plasmid pMON5887 DNA was digested with restriction enzymes HindIII and NcoI, resulting in a 384 base pair NcoI, HindIII fragment. The restriction fragments were ligated, and the ligation reaction mixture was used to transform into E. coli K-12 strain JM101. Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and the size of the inserted fragment was determined by restriction analysis employing restriction enzymes NcoI and HindIII in double digest. Positive clones containing the hIL-3 gene (encoding amino acids 1-125) contained a 384 base pair NcoI, HindIII restriction fragment. This construct was designated pMON6455.

Construction of pMON6456

Plasmid pMON5905 DNA was digested with restriction enzymes HindIII and NcoI resulting in a 3936 base pair fragment. The genetic elements derived from pMONS905 are the beta-lactamase gene (AMP), pBR327 origin of replication, phage f1 origin of replication as the transcription terminator, pAraBAD promoter, g10L ribosome binding site and the lamB secretion leader. Plasmid pMON5871 was digested with restriction enzymes HindIII and NcoI, resulting in a 330base pair NcoI, HindIII fragment. The genetic element derived from pMON5871 encompassed the bases encoding the (1-107) hIL-3 gene. The restriction fragments were ligated, and the ligation reaction mixture was used to transform E. coli K-12 strain JM101. Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and the size of the inserted fragment was determined by restriction analysis employing restriction enzymes NcoI and HindIII in double digest. Clones containing the hIL-3 gene (encoding amino acids 1-107) contained a 330 base pair NcoI, HindIII restriction fragment. This construct was designated pMON6456.

Construction of pMON6457

Plasmid pMON6455 DNA grown in E. coli strain GM48 (dam-)was digested with restriction enzymes NcoI and ClaI, resulting in a 4293 base pair NcoI, ClaI fragment. The restriction fragment was ligated to 1.0 picomoles of annealed oligonucleotides (Oligo #5 and Oligo #6) with the following sequence coding for Met Ala 14-20 hIL-3:

5′-CATGGCTAACTGCTCTAACATGAT-3′[SEQ ID NO:151]

3′-CGATTGACCAGATTGTACTAGC-5′[SEQ ID NO:152]

The resulting DNA was transformed into E. coli K-12 strain JM101 and transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and the size of the inserted fragment was determined by restriction analysis employing restriction enzymes XbaI and EcoRI in double digest. Positive clones containing the hIL-3 gene (encoding aa 15-125 of hIL-3) contained a 433 base pair XbaI, EcoRI restriction fragment and were DNA sequenced. This construct was designated pMON6457. This plasmid was constructed to delete the first 14 amino acids of hIL-3. The coding sequence of the resulting gene begins as follows:

The first two amino acids (Methionine, Alanine) create an NcoI restriction site and a signal peptidase cleavage site between the lamB signal peptide and (15-125) hIL-3. Plasmid pMON6457 encodes (15-125) hIL-3 which has the amino acid sequence designated SEQ ID NO 65.

Construction of pMON6235

One of the DNA fragments used to create this plasmid was generated by site-directed mutagenesis employing PCR techniques described previously using the following oligonucleotides, Oligo #51(A) [SEQ ID NO:155] and Oligo #52(A) [SEQ ID NO:156], were used as primers in this procedure. The template for the PCR reaction was E. coli strain W3110 chromosomal DNA, prepared as described in Maniatis (1982). The oligonucleotide primers were designed to amplify the AraBAD promoter (Greenfield et al., 1978). The resulting DNA product was digested with the restriction enzymes SacII and BglII. The reaction mixture was purified as described previously. Plasmid, pMON5594, DNA was digested with SacII and BglII, resulting in a 4416 base pair SacII,BglII restriction fragment which contains the following genetic elements; beta-lactamase gene (AMP), pBR327 origin of replication, G10L ribosome binding site, phage f1 origin of replication as the transcription terminator and the chloramphenicol acetyl transferase (cat) gene. The 4416 base pair SacII,BglII restriction fragment from pMON5594 was ligated to the PCR-generated SacII, BglII DNA fragment. The ligation mixture was used to transform E. coli K-12 strain JM101. Positive clones contained a 323 base pair SacII,BglII fragment and were DNA sequenced to confirm that the SacII,BglII fragment was the AraBAD promoter. This construct was designated pMON6235.

Construction of pMON6460

One of the DNA fragments to construct this plasmid was generated by site-directed mutagenesis employing PCR techniques described previously using the oligonucleotides, Oligo #7 [SEQ ID NO: 26] and Oligo #8 [SEQ ID NO: 27] as primers. The template for the PCR reaction was plasmid pMON6458 DNA. The resulting DNA product was digested with the restriction enzymes NcoI and EcoRI. Upon completion, the digest was heated at 70° C. for 15 minutes to inactivate the enzymes. The restriction fragment was purified by phenol/chloroform extraction and precipitation with equal volume isopropanol in the presence of 2M NH 4 OAc. The oligonucleotide, Oligo #8, introduces two stop codons (TAA) after amino acid 93 of hIL-3 and Creates a SalI restriction endonuclease recognition sequence. The NcoI, EcoRI restriction fragment from pMON6458 was ligated to the PCR-generated NcoI, EcoRI restriction fragment. Positive clones containing the above mentioned changes released a 1023 base pair SalI fragment. This construct was designated pMON6460. This plasmid was constructed to serve as the template for the creation of single amino acid substitution variants at positions 94, 95, 96 and 97 of hIL-3.

Construction of pMON6461

One of the DNA fragments to create this plasmid was generated by site-directed mutagenesis employing PCR techniques described previously using the following oligonucleotide, Oligo #7 [SEQ. ID NO: 26] and Oligo #9 [SEQ. ID NO: 28], as primers. The template for the PCR reaction was plasmid pMON6458 DNA. The resulting DNA product was digested with the restriction enzymes NcoI and EcoRI. The oligonucleotide, Oligo #9, introduces two stop codons (TAA) after amino acid 97 of hIL-3 and creates a SalI restriction endonuclease recognition sequence. The NcoI, EcoRI restriction fragment from pMON5458 was ligated to the PCR-generated NcoI, EcoRI DNA fragment. Positive clones containing the above mentioned changes released a 1035 base pair SalI fragment. This construct was designated pMON6461. This plasmid was constructed to serve as the template for the creation of single amino acid substitution variants at positions 98, 99, 100 and 101 of hIL-3.

Construction of pMON6462

One of the DNA fragments to create this plasmid was generated by site-directed mutagenesis employing PCR techniques described previously using the following oligonucleotide, Oligo #7 [SEQ. ID NO: 26] and Oligo #10 [SEQ. ID NO: 31], as primers. The template for the PCR reaction was plasmid pMON6458 DNA. The resulting DNA product was digested with the restriction enzymes NcoI and EcoRI. The oligonucleotide, Oligo #10 [SEQ. ID NO: 31] introduces two stop codons (TAA) after amino acid 101 of hIL-3 and creates a SalI restriction endonuclease recognition sequence. The NcoI, EcoRI restriction fragment from pMON5458 was ligated to the PCR-generated NcoI, EcoRI DNA fragment. Positive clones containing the above mentioned changes released a 1047 base pair SalI fragment. This construct was designated pMON6462. This plasmid was constructed to serve as the template for the creation of single amino acid substitution variants at positions 102, 103, 104 and 105 of hIL-3.

Construction of Single Amino Acid Substitution Libraries at Positions 94, 95, 96 and 97

One of the DNA fragments used to construct the plasmids containing single amino acid substitution at positions 94, 95, 96 and 97 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction plasmid pMON6460 DNA was the template and the oligonucleotide, Oligo #7 [SEQ. ID NO: 26], was used as the primer at the N-terminus, The degenerate oligonucleotides, Oligo #11 [SEQ. ID NO: 32], Oligo #12 [SEQ. ID NO: 33], Oligo #13 [SEQ. ID NO: 34] and Oligo #14 [SEQ. ID NO: 35], were the primers at the C-terminus. These oligonucleotides are 32-fold degenerate, with G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 94, 95, 96 and 97 of hIL-3 respectively. These degenerate oligonucleotide primers theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at a single position. The degenerate oligonucleotides (Oligo #11 [SEQ. ID NO: 32], Oligo #12 [SEQ. ID NO: 33], Oligo #13 [SEQ. ID NO: 34] and Oligo #14 [SEQ. ID NO: 35]) replace the twelve bases introduced into pMON6460, that encode the two stop codons (TAA) after amino acid 93 of hIL-3 and the SalI recognition sequence. At the other 9 bases the DNA sequence was restored to encode the native hIL-3 protein sequence. The resulting PCR-generated DNA products were digested with the restriction enzymes NcoI and EcoRI. The 4008 bp NcoI, EcoRI restriction fragment from pMON6460 was ligated to the PCR-generated NcoI, EcoRI DNA fragments. Plasmid DNA from individual colonies was isolated as described previously and screened by DNA dot blot differential hybridization using the oligonucleotide, Oligo #15 [SEQ. ID NO: 36], as the probe which had been labeled with P 32 . Clones shown to be positive by hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

Construction of Single Amino Acid Substitution Libraries at Positions 98. 99, 100 and 101

Single amino acid substitutions variants were constructed at position 98, 99, 100 and 101 as described previously, with the following changes. In the PCR reaction the template was plasmid pMON6461 DNA and the oligonucleotide, Oligo #7 [SEQ. ID NO: 26], was used as the primer at the N-terminus. The degenerate oligonucleotides, Oligo #16 [SEQ. ID NO: 37], Oligo #17 [SEQ. ID NO: 38], Oligo #18 [SEQ. ID NO: 39] and Oligo #19 [SEQ. ID NO: 40], were used as primers at the C-terminus. The resulting PCR-generated DNA products were purified and digested with restriction enzymes NcoI and EcoRI. The 4008 bp NcoI, EcoRI restriction fragment from pMON6461 was ligated to the PCR-generated DNA NcoI, EcoRI restriction fragment. Single colonies were screened by DNA dot blot differential hybridization using the oligonucleotide, Oligo #20 [SEQ. ID NO: 41], as the probe. Clones shown to be positive by hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

Construction of Single Amino Acid Substitution Libraries at Positions 102, 103, 104 and 105

Single amino acid substitutions variants were constructed at position 102, 103, 104 and 105 as described previously, with the following changes. The template was pMON6462 and the oligonucleotide, Oligo #7 [SEQ. ID NO: 26], was used as the primer at the N-terminus. The degenerate oligonucleotides, Oligo #21 [SEQ. ID NO: 42], Oligo #22 [SEQ. ID NO: 43], Oligo #23 [SEQ. ID NO: 44] and Oligo #24 [SEQ. ID NO: 45] were used as primers at the C-terminus. The resulting PCR-generated DNA products were purified and digested with restriction enzymes, NcoI and EcoRI. The 4008 bp NcoI, EcoRI restriction fragment from pMON6462 was ligated to the PCR-generated NcoI, EcoRI restriction fragment. Single colonies were screened by DNA dot blot differential hybridization using the oligonucleotide, Oligo #25 [SEQ. ID NO: 46], as the probe. Clones shown to be positive by hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

Construction of Plasmid pMON6464

Amino acids 17-22 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON6458 DNA was the template in the PCR reaction using the oligonucleotides, Oligo #26 and Oligo #27 as primers. The resulting PCR-generated DNA products were purified and digested with NcoI and EcoRI. The 4008 bp NcoI, EcoRI restriction fragment from pMON6458 was ligated to the PCR-generated NcoI, EcoRI restriction fragment. Positive clones contained a 263 base pair NcoI, EcoRI restriction fragment in which the bases encoding amino acids 17-22 of hIL-3 have been deleted. pMON6464 was made to serve as the template for the creation of single amino acid substitution variants at positions 17, 18, 19, 20, 21 and 22 of hIL-3.

Construction of Plasmid pMON6465

Amino acids 23-28 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON6458 DNA was the template in the reaction using the oligonucleotides, Oligo # 26 and Oligo #28, as primers. The resulting PCR-generated DNA product was purified and digested with NcoI and EcoRI. The 4008 bp NcoI, EcoRI restriction fragment from pMON6458 was ligated to the PCR-generated NcoI, EcoRI restriction fragment. Positive clones contained a 263 base pair NcoI, EcoRI restriction fragment in which the bases encoding amino acids 23-28 of hIL-3 have been deleted. pMON6465 was made to serve as the template for the creation of single amino acid substitution variants at positions 23, 24, 25, 26, 27 and 28 of hIL-3.

Construction of Plasmid pMON6466

Amino acids 29-34 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON6458 DNA was the template in the reaction using the oligonucleotides, Oligo #26 and Oligo #29 as the primers. The resulting PCR-generated DNA product was purified and digested with NcoI and EcoRI. The 4008 bp NcoI, EcoRI restriction fragment from pMON6458 was ligated to the PCR-generated NcoI, EcoRI restriction fragment. Positive clones contained a 263 base pair NcoI, EcoRI restriction fragment in which the bases encoding amino acids 29-34 of hIL-3 have been deleted. pMON6466 was made to serve as the template for the creation of single amino acid substitution variants at positions 29, 30, 31, 32, 33 and 34 of hIL-3.

Construction of Plasmid pMON6467

Amino acids 35-40 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON5988 DNA was the template in the reaction using the oligonucleotides, Oligo #7 and Oligo #30, as primers. The resulting PCR-generated DNA product was purified and digested with NcoI and EcoRV. The NcoI, EcoRV restriction fragment from pMON5988 was ligated to the PCR-generated NcoI, EcoRV restriction fragment. Positive clones contained a 81 base pair NcoI, EcoRV restriction fragment in which the bases encoding amino acids 35-40 of hIL-3 have been deleted. pMON6467 was made to serve as the template for the creation of single amino acid substitution variants at positions 35, 36, 37, 38, 39 and 40 of hIL-3.

Construction of Plasmid pMON6468

Amino acids 41-46 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON5988 DNA was the template in the reaction using the oligonucleotides, Oligo #7 and Oligo #31, as the primers. The resulting PCR-generated DNA product was purified and digested with NcoI and XhoI. The NcoI, XhoI restriction fragment from pMON5988 was ligated to the PCR-generated NcoI, XhoI restriction fragment. Positive clones contained a 119 base pair NcoI, XhoI restriction fragment in which the bases encoding amino acids 41-46 of hIL-3 have been deleted. pMON6468 was made to serve as the template for the creation of single amino acid substitution variants at positions 41, 42, 43, 44, 45 and 46 of hIL-3.

Construction of Plasmid pMON6469

Amino acids 47-52 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON5988 DNA was the template in the reaction using the oligonucleotides, Oligo #7 and Oligo #32, as the primers. The resulting PCR-generated DNA product was purified and digested with NcoI and XhoI. The NcoI, XhoI restriction fragment from pMON5988 was ligated to the PCR-generated NcoI, XhoI restriction fragment. Positive clones contained a 119 base pair NcoI, XhoI restriction fragment in which the bases encoding amino acids 47-52 of hIL-3 have been deleted. pMON6469 was made to serve as the template for the creation of single amino acid substitution variants at positions 47, 48, 49, 50, 51 and 52 of hIL-3.

Construction of Plasmid pMON6470

Amino acids 53-58 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid, pMON5988, DNA was the template in the reaction using the oligonucleotides, Oligo #7 and Oligo #33, as primers. The resulting PCR-generated DNA product was purified and digested with NcoI and NsiI. The NcoI, NsiI restriction fragment from pMON5988 was ligated to the PCR-generated NcoI, NsiI restriction fragment. Positive clones contained a 152 base pair NcoI, NsiI restriction fragment in which the bases encoding amino acids 53-58 of hIL-3 have been deleted. pMON6470 was made to serve as the template for the creation of single amino acid substitution variants at positions 53, 54, 55, 56, 57 and 58 of hIL-3.

Construction of Plasmid pMON6471

Amino acids 59-64 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON5988 DNA was the template in the reaction using the oligonucleotides, Oligo #7 and Oligo #34, as the primers. The resulting PCR-generated DNA product was purified and digested with NcoI and NsiI. The NcoI, NsiI restriction fragment from pMON5988 was ligated to the PCR-generated NcoI, NsiI restriction fragment. Positive clones contained a 152 base pair NcoI, NsiI restriction fragment in which the bases encoding amino acids 59-64 of hIL-3 have been deleted. pMON6471 was made to serve as the template for the creation of single amino acid substitution variants at positions 59, 60, 61, 62, 63 and 64 of hIL-3.

Construction of Plasmid pMON6472

Amino acids 65-70 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON5988 DNA was the template in the reaction using the oligonucleotides, Oligo #26 and Oligo #35, as primers. The resulting PCR-generated DNA product was purified and digested with EcoRI and XhoI. The EcoRI, XhoI restriction fragment from pMON5988 was ligated to the PCR-generated EcoRI, XhoI restriction fragment. Positive clones contained a 126 base pair EcoRI, XhoI restriction fragment in which the bases encoding amino acids 65-70 of hIL-2 have been deleted. pMON6472 was made to serve as the template for the creation of single amino acid substitution variants at positions 65, 66, 67, 68, 69 and 70 of hIL-3.

Construction of Plasmid pMON6473

Amino acids 71-76 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid, pMON5988, DNA was the template in the reaction using the oligonucleotides, Oligo #26 and Oligo #36, as primers. The resulting PCR-generated DNA product was and digested with PstI and EcoRI. The PstI, EcoRI restriction fragment from pMON5988 was ligated to the PCR-generated PstI, EcoRI restriction fragment. Restriction analysis was with NcoI, NsiI and EcoRI in a triple digest. Positive clones contained a 263 base pair NcoI, EcoRI restriction fragment, in which the bases encoding amino acids 71-76 of hIL-3 have been deleted, and lost the NsiI restriction site. pMON6473 was made to serve as the template for the creation of single amino acid substitution variants at positions 71, 72, 73, 74, 75 and 76 of hIL-3.

Construction of Plasmid pMON6474

Amino acids 77-82 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON5988 DNA was the template in the reaction using the oligonucleotides, Oligo #26 and Oligo #37, as primers. The resulting PCR-generated DNA product was purified and digested with PstI and EcoRI. The PstI, EcoRI restriction fragment from pMON5988 was ligated to the PCR-generated PstI, EcoRI restriction fragment. Restriction analysis was with NcoI, NsiI and EcoRI in a triple digest. Positive clones contained a 170 base pair NcoI, NsiI restriction fragment and a 93 base pair NsiI, EcoRI restriction fragment in which the bases encoding amino acids 77-82 of hIL-3 have been deleted. pMON6474 was made to serve as the template for the creation of single amino acid substitution variants at positions 77, 78, 79, 80, 81 and 82 of hIL-3.

Construction of Plasmid pMON6475

Amino acids 83-88 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON5988 DNA was the template in the reaction using the oligonucleotides, Oligo #26 and Oligo #38, as primers. The resulting PCR-generated DNA product was digested with PstI and EcoRI. The PstI, EcoRI restriction fragment from pMON5988 was ligated to the PCR-generated PstI, EcoRI restriction fragment. Restriction analysis was with NcoI, NsiI and EcoRI in a triple digest. Positive clones contained a 170 base pair NcoI, NsiI restriction fragment and a 93 base pair NsiI, EcoRI restriction fragment in which the bases encoding amino acids 83-88 of hIL-3 have been deleted. pMON6475 was made to serve as the template for the creation of single amino acid substitution variants at positions 83, 84, 85, 86, 87 and 88 of hIL-3.

Construction of Plasmid pMON6476

Amino acids 88-93 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON6458 DNA was the template in the reaction using the oligonucleotides, Oligo #7 and Oligo #39, as primers. The resulting PCR-generated DNA product was purified and digested with NcoI and EcoRI. The NcoI, EcoRI restriction fragment from pMON6458 was ligated to the PCR-generated NcoI, EcoRI restriction fragment. Positive clones contained a 263 base pair NcoI, EcoRI restriction fragment in which the-bases encoding amino acids 88-93 of hIL-3 have been deleted. pMON6476 was made to serve as the template for the creation of single amino acid substitution variants at positions 88, 89, 90, 91, 92 and 93 of hIL-3.

Construction of Plasmid pMON6477

Amino acids 106-111 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON6458 DNA was the template in the reaction using the oligonucleotides, Oligo #7 and Oligo #40, as primers. The resulting PCR-generated DNA fragment was purified and digested with NcoI and HindIII. The NcoI, HindIII restriction fragment from pMON6458 was ligated to the PCR-generated NcoI, HindIII restriction fragment. Positive clones contained a 327 base pair NcoI, HindIII restriction fragment in which the bases encoding amino acids 106-111 of hIL-3 have been deleted. pMON6477 was made to serve as the template for the creation of single amino acid substitution variants at positions 106, 107, 108, 109, 110 and 111 of hIL-3.

Construction of Plasmid pMON6478

Amino acids 112-117 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON6458 DNA was the template in the reaction using the oligonucleotides, Oligo #7 and Oligo #41, as primers. The resulting PCR-generated DNA product was purified and digested with NcoI and HindIII. The 4008 bp NcoI, HindIII restriction fragment from pMON6458 was ligated to the PCR-generated NcoI, HindIII restriction fragment. Positive clones contained a 327 base pair NcoI, HindIII restriction fragment in which the bases encoding amino acids 112-117 of hIL-3 have been deleted. pMON6478 was made to serve as the template for the creation of single amino acid substitution variants at positions 112, 113, 114, 115, 116 and 117 of hIL-3.

Construction of Plasmid pMON6479

Amino acids 118-123 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON6458 DNA was the template in the reaction using the oligonucleotides, oligo #7 and Oligo #42, as primers. The resulting PCR-generated DNA product was purified and digested with NcoI and HindIII. The NcoI, HindIII restriction fragment from pMON6458 was ligated to the PCR-generated NcoI, HindIII restriction fragment. Positive clones contained a 327 base pair NcoI, HindIII restriction fragment in which the bases encoding amino acids 118-123 of hIL-3 have been deleted. pMON6479 was made to serve as the template for the creation of single amino acid substitution variants at positions 118, 119, 120, 121, 122 and 123 of hIL-3.

Construction of Single Amino Acid Substitution Libraries at Positions 17, 18, 19, 20, 21 and 22

One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 17, 18, 19, 20, 21 and 22 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6464 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #43, Oligo #44, Oligo #45, Oligo #46, Oligo #47 and Oligo #48 were the primers at the C-terminus. The oligonucleotide, Oligo #26, was used as the primer at the N-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6464. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 17, 18, 19, 20, 21 and 22 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA product was digested with NcoI and EcoRV. Plasmid pMON6464 DNA was digested with restriction enzymes NcoI and EcoRV resulting in a 4190 base pair fragment which was ligated to the PCR-generated NcoI, EcoRV restriction fragments. Plasmid DNA was isolated and screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #139, which had been labeled with P 32 . Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

Construction of Single Amino Acid Substitution Libraries at Positions 23, 24, 25, 26, 27 and 28

One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 23, 24, 25, 26, 27 and 28 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6465 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #49, Oligo #50, Oligo #51, Oligo #52, Oligo #53 and Oligo #54 were the primers at the C-terminus. The oligonucleotide, Oligo #26, was used as the primer at the N-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6465. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 23, 24, 25, 26, 27 and 28 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA products were purified and digested with restriction enzymes NcoI and EcoRV. Plasmid pMON6465 DNA was digested with restriction enzymes NcoI and EcoRV and the resulting 4190 base pair fragment was ligated to the PCR-generated NcoI, EcoRV DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #140, which had been labeled with P 32 . Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

Construction of Single Amino Acid Substitution Libraries at Positions 29, 30, 31, 32, 33 and 34

One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 29, 30, 31, 32, 33 and 34 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6466 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #55, Oligo #56, Oligo #57, Oligo #58, Oligo #59 and Oligo #60 were the primers at the C-terminus. The oligonucleotide Oligo #26 was used as the primer at the N-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6466. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 29, 30, 31, 32, 33 and 34 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA products were purified and digested with the restriction enzymes NcoI and EcoRV. Plasmid pMON6466 DNA was digested with restriction enzymes NcoI and EcoRV and the resulting 4190 base pair fragment was ligated to the PCR-generated NcoI, EcoRV DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #141, which had been labeled with P 32 . Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

Construction of Single Amino Acid Substitution Libraries at Positions 35, 36, 37, 38, 39 and 40

One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 35, 36, 37, 38, 39 and 40 of hIL-3 were generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6467 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #61, Oligo #62, Oligo #63, Oligo #64, Oligo #65 and Oligo #66 were the primers at the C-terminus. The oligonucleotide, Oligo #7, was used as the primer at the N-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6467. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 35, 36, 37, 38, 39 and 40 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored and at the other position, 32 different codons substitutions were created at positions independently. The resulting PCR-generated DNA products were purified and digested with the restriction enzymes NcoI and EcoRV. Plasmid pMON6467 DNA was digested with restriction enzymes NcoI and EcoRV and the resulting 4190 base pair fragment was ligated to the PCR-generated NcoI, EcoRV DNA fragments.

Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #142, which had been labeled with p32. Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

Construction of Single Amino Acid Substitution Libraries at Positions 41, 42, 43, 44, 45 and 46

One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 41, 42, 43, 44, 45 and 46 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6468 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #67, Oligo #68, Oligo #69, Oligo #70, Oligo #71 and Oligo #72 were the primers at the C-terminus. The oligonucleotide, Oligo #7, was used as the primer at the N-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6468. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 41, 42, 43, 44, 45 and 46 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA products were purified and digested with the restriction enzymes NcoI and XhoI. Plasmid pMON6468 DNA was digested with restriction enzymes NcoI and XhoI and the resulting 4152 base pair fragment was ligated to the PCR-generated NcoI, XhoI DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #143, which had been labeled with P 32 . Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

Construction of Single Amino Acid Substitution Libraries at Positions 47, 48, 49, 50, 51 and 52

One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 47, 48, 49, 50, 51 and 52 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6469 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #73, Oligo #74, Oligo #75, Oligo #76, Oligo #77 and Oligo #78 , were the primers at the C-terminus. The oligonucleotide, Oligo #7, was used as the primer at the N-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6469. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 47, 48, 49, 50, 51 and 52 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA products were purified and digested with the restriction enzymes NcoI and XhoI. Plasmid pMON6469 DNA was digested with restriction enzymes NcoI and XhoI and the resulting 4152 base pair fragment was ligated to the PCR-generated NcoI, XhoI DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #143, which had been labeled with P 32 . Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

Construction of Single Amino Acid Substitution Libraries at Positions 53, 54, 55, 56, 57 and 58

One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 53, 54, 55, 56, 57 and 58 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6470 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #79, Oligo #80, Oligo #81, Oligo #82, Oligo #83 and Oligo #84 , were the primers at the C-terminus. The oligonucleotide, Oligo #7, was used as the primer at the N-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6470. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 53, 54, 55, 56, 57 and 58 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA products were purified and digested with the restriction enzymes NcoI and NsiI. Plasmid pMON6470 DNA was digested with restriction enzymes NcoI and NsiI and the resulting 4119 base pair fragment was ligated to the PCR-generated NcoI, NsiI DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #145, which had been labeled with P 32 . Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

Construction of Single Amino Acid Substitution Libraries at Positions 59, 60, 61, 62, 63 and 64

One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 59, 60, 61, 62, 63 and 64 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6471 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #85, Oligo #86, Oligo #87, Oligo #88, Oligo #89 and Oligo #90 , were the primers at the C-terminus. The oligonucleotide, Oligo #7, was used as the primer at the N-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6471. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 59, 60, 61, 62, 63 and 64 of hIL-3 respectively, These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA products were purified and digested with the restriction enzymes NcoI and NsiI. Plasmid pMON6471 DNA was digested with restriction enzymes NcoI and NsiI and the resulting 4119 base pair fragment was ligated to the PCR-generated NcoI, NsiI DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #146, which had been labeled with P 32 . Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

Construction of Single Amino Acid Substitution Libraries at Positions 65, 66, 67, 68, 69 and 70

One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 65, 66, 67, 68, 69 and 70 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6472 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #91, Oligo #92, Oligo #93, Oligo #94, Oligo #95 and Oligo #96 , were the primers at the N-terminus. The oligonucleotide, Oligo 26, was used as the primer at the C-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6472. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 65, 66, 67, 68, 69 and 70 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA products were purified and digested with the restriction enzymes EcoRI and XhoI. Plasmid pMON6472 DNA was digested with restriction enzymes EcoRI and XhoI and the resulting 4145 base pair fragment was ligated to the PCR-generated EcoRI, XhoI DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #147, which had been labeled with P 32 . Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

Construction of Single Amino Acid Substitution Libraries at Positions 71, 72, 73, 74, 75 and 76

One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 71, 72, 73, 74, 75 and 76 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6473 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #97, Oligo #98, Oligo #99, Oligo #100, Oligo #101 and Oligo #102 , were the primers at the N-terminus. The oligonucleotide, Oligo #26, was used as the primer at the C-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6473. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 71, 72, 73, 74, 75 and 76 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA fragments were purified and digested with the restriction enzymes EcoRI and PstI. Plasmid pMON6473 DNA was digested with restriction enzymes EcoRI and PstI and the resulting 4171 base pair fragment was ligated to the PCR-generated EcoRI, PstI DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #148, which had been labeled with P 32 . Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

Construction of Single Amino Acid Substitution Libraries at Positions 77, 78, 79, 80, 81 and 82

One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 77, 78, 79, 80, 81 and 82 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the reaction the plasmid pMON6474 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #103, Oligo #104, Oligo #105, Oligo #106, Oligo #107 and Oligo #108, were the primers at the N-terminus. The oligonucleotide, Oligo #26, was used as the primer at the C-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6474. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 77, 78, 79, 80, 81 and 82 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA products were purified and digested with the restriction enzymes EcoRI and PstI as described previosly. Plasmid pMON6474 DNA was digested with restriction enzymes EcoRI and PstI and the resulting 4171 base pair fragment was ligated to the PCR-generated EcoRI, PstI DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #149, which had been labeled with P 32 . Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

Construction of Single Amino Acid Substitution Libraries at Positions 83, 84, 85, 86, 87 and 88

One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 83, 84, 85, 86, 87 and 88 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6475 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #109, Oligo #110, Oligo #111, Oligo #112, Oligo #113 and Oligo #114, were the primers at the N-terminus. The oligonucleotide, Oligo #26, was used an the primer at the C-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6475. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 83, 84, 85, 86, 87 and 88 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA producs were purified and digested with the restriction enzymes EcoRI and PstI. Plasmid pMON6475 DNA was digested with restriction enzymes EcoRI and PstI and the resulting 4171 base pair fragment was ligated to the PCR-generated EcoRI, PstI DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #150, which had been labeled with P 32 . Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

Construction of Single Amino Acid Substitution Libraries at Positions 88, 89, 90, 91, 92 and 93

One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 88, 89, 90, 91, 92 and 93 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6476 DNA was the template and the following degenerate oligonucleotides, Oligo #114, Oligo #115, Oligo #116, Oligo #117, Oligo #118 and Oligo #119, were the primers at the C-terminus. The oligonucleotide, Oligo #7, was used as the primer at the N-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6476. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 88, 89, 90, 91, 92 and 93 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA products were purified and digested with the restriction enzymes EcoRI and NcoI. Plasmid pMON6476 DNA was digested with restriction enzymes EcoRI and NcoI and the resulting 4008 base pair fragment was ligated to the PCR-generated EcoRI, NcoI DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #151, which had been labeled with P 32 . Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

Construction of Single Amino Acid Substitution Libraries at Positions 106, 107, 108, 109, 110 and 111

One of the DNA fragments used to construct the plasmids containing the single amino acid substitutions at positions 106, 107, 108, 109, 110 and 111 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously in two sequential PCR reactions. In the first PCR reaction, plasmid pMON6477 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #120, Oligo #121, Oligo #122, Oligo #123, Oligo #124 and Oligo #125 were the primers at the C-terminus. The oligonucleotide, Oligo #7 was the primer at the N-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON9477. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 106, 107, 108, 109, 110 and 111 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The DNA generated in this PCR reaction was purified by phenol/chloroform extraction and precipitation with equal volume isopropanol in the presence of 2M NH 4 OAc to remove any primer that was not extended. This DNA was then used as a primer in the second PCR reaction.

In the second PCR reaction plasmid pMON6477 DNA was the template, the DNA product generated in the first PCR reaction (described above) was the primer at the N-terminus and the oligonucleotide, Oligo #126 (DNA sequence shown in Table 1), was the primer at the C-terminus. The resulting PCR-generated DNA products were purified and digested with the restriction enzymes HindIII and NcoI. Plasmid pMON6477 was digested with restriction enzymes HindIII and NcoI and the resulting 3944 base pair fragment was ligated to the PCR-generated HindIII, NcoI DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #152, which had been labeled with P 32 . Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

Construction of Single Amino Acid Substitution Libraries at Positions 112, 113, 114, 115, 116 and 117

One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 112, 113, 114, 115, 116 and 117 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6478 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #127, Oligo #128, Oligo #129, Oligo #130, Oligo #131 and Oligo #132, were the primers at the C-terminus. The oligonucleotide, Oligo #7, was used as the primer at the N-terminus, The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6478. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 112, 113, 114, 115, 116 and 117 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA products were purified and digested with the restriction enzymes HindIII and NcoI. Plasmid pMON6478 was digested with restriction enzymes HindIII and NcoI and the resulting 3944 base pair fragment was ligated to the PCR-generated HindIII, NcoI DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #153, which had been labeled with P 32 . Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

Construction of Single Amino Acid Substitution Libraries at Positions 118, 119, 120, 121, 122 and 123

One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 118, 119, 120, 121, 122 and 123 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6479 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #133, Oligo #134, Oligo #135, oligo #136, Oligo #137 and Oligo #138, were the primers at the C-terminus. The oligonucleotide, Oligo #7, was used as the primer at the N-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6479. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 118, 119, 120, 121, 122 and 123 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA products were purified and digested with the restriction enzymes HindIII and NcoI. Plasmid pMON6479 DNA was digested with restriction enzymes HindIII and NcoI and the resulting 3944 base pair fragment was ligated to the PCR-generated HindIII, NcoI DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #154, which had been labeled with P 32 . Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

Construction of pMON13358

Plasmid pMON5978 DNA (Example 6) was digested with restriction enzymes NsiI and EcoRI and the resulting 3853 base pair NsiI,EcoRI fragment contains the following genetic elements; beta-lactamase gene (AMP), pBR327 origin of replication, phage f1 origin of replication as the transcription terminator, recA promoter, g10L ribosome binding site and the bases encoding amino acids 15-71 and 106-125 of (15-125) hIL-3. The 3853 base pair NsiI,EcoRI restriction fragment from pMON5978 was ligated to the following annealed complementary oligonucleotides.

Oligo #15(A) [SEQ ID NO: 29]

Oligo #16(A) [SEQ ID NO: 30]

In the resulting plasmid the 111 bases between the NsiI and EcoRI restriction sites in the (15-125) hIL-3 gene are replaced with 24 bases from the above mentioned oligonucleotides. This linker also creates a NdeI recognition sequence.

Construction of pMON13304

Plasmid pMON13358 DNA is digested with restriction enzymes PstI and EcoRI and the resulting 3846 base pair PstI,EcoRI fragment contains the following genetic elements; beta-lactamase gene (AMP), pBR327 origin of replication, phage f1 origin of replication as the transcription terminator, recA promoter, g10L ribosome binding site and the bases encoding amino acids 15-69 and 106-125 of (15-125) hIL-3. The 3846 base pair NsiI,EcoRI restriction fragment from pMON13358 is ligated to the following annealed complementary oligonucleotides.

When assembled, the oligonucleotides create PstI and EcoRI restriction ends and the DNA sequence that encodes amino acids 70-105 of (15-125) hIL-3 with the following amino acid substitutions; 98I and 100R. The codons encoding amino acids 70-105 of (15-125) hIL-3 are those found in the hIL-3 cDNA sequence except at those positions where amino acid substitutions were made. The plasmid, pMON13304, encodes the (15-125) hIL-3 variant with the following amino acid sequence:

Peptide #A1 [SEQ ID NO:66]

Construction of pMON13305

Plasmid pMON13358 DNA is digested with restriction enzymes PstI and EcoRI and the resulting 3846 base pair PstI, EcoRI fragment contains the following genetic elements; beta-lactamase gene (AMP), pBR327 origin of replication, phage f1 origin of replication as the transcription terminator, recA promoter, g10L ribosome binding site and the bases encoding amino acids 15-69 and 106-125 of (15-125) hIL-3. The 3846 base pair NsiI,EcoRI restriction fragment from pMON13358 is ligated to the following annealed complementary oligonucleotides.

When assembled, the oligonucleotides create PstI and EcoRI restriction ends and the DNA sequence that encodes amino acids 70-105 of (15-125) hIL-3 with the following amino acid substitutions; 95R, 98I and 100R. The codons encoding amino acids 70-105 of (15-125) hIL-3 are those found in the hIL-3 cDNA sequence except at those positions where amino acid substitutions were made. The plasmid, pMON13305, encodes the (15-125) hIL-3 variant with the following amino acid sequence:

Peptide #A2 [SEQ ID NO:67]

Construction of pMON13286

Plasmid pMON5978 DNA was digested with restriction enzymes NcoI and EcoRV and the resulting 3865 base pair NcoI,EcoRV fragment contains the following genetic elements; beta-lactamase gene (AMP), pBR327 origin of replication, phage f1 origin of replication as the transcription terminator, precA promoter, g10L ribosome binding site and the bases encoding amino acids 47-125 of (1-125) hIL-3. The 3865 base pair NcoI,EcoRV restriction fragment from pMON5978 was ligated to the following annealed complementary oligonucleotides.

When assembled, the oligonucleotides create NcoI and EcoRV restriction ends and the DNA sequence that encodes amino acids 15-46 of (15-125) hIL-3 with the following amino acid substitutions; 42D, 45M and 46S. The codons encoding amino acids 15-46 of (15-125) hIL-3 are those found in the hIL-3 cDNA sequence except at those positions where amino acid substitutions were made. The plasmid, pMON13286, encodes the (15-125) hIL-3 variant with the following amino acid sequence:

Construction of pMON5853 (FIG. 6) which Encodes [Met-(15-133) hIL-3 (Arg129)]

Plasmid DNA of pMON5847 (Example 2) was treated with NcoI. The restriction enzyme was inactivated by heat treatment (65° C. for 10 minutes). The DNA was then treated with large fragment of DNA polymerase I (Klenow) in the presence of all four nucleotide precursors. This produces DNA termini with non-overlapping ends. After 5 minutes at 37° C., the polymerase was inactivated by heat treatment at 65° C. for 10 minutes. The DNA was then treated with HpaI, an enzyme which produces non-overlapping termini. The DNA was ethanol precipitated and ligated. The ligation reaction mixture was used to transform competent JM101 cells to ampicillin resistance. Colonies were picked and plasmid DNA was analyzed by restriction analysis. A plasmid designated pMON5853 was identified as one containing a deletion of the amino terminal 14 codons of the hIL-3 gene. The DNA sequence for the junction of the ribosome binding site to the (15-133) hIL-3 gene was determined to be the following:

The lower line contains the one-letter code for the amino acids specified by the coding sequence of the amino terminus of the 15-133 hIL-3 gene. These are methionine, asparagine, cysteine, serine and asparagine.

When cultures of JM101 cells harboring this plasmid were induced with nalidixic acid, it was found that hIL-3 (15-133) accumulated at levels higher than hIL-3 (pMON5847).

The plasmid, pMON5853, encodes Met-(15-133) hIL-3 (Arg 129 ) which has the following amino acid sequence:

Met Asn Cys Ser Asn Met Ile Asp Glu Ile Ile

Ile Thr His Leu Lys Gln Pro Pro Leu Pro Leu Leu

Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp Ile

Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu

Ala Phe Asn Arg Ala Val Lys Ser Leu Gln Asn Ala

Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro

Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His

Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe

Arg Arg Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu

Asn Ala Gln Ala Gln Gln Thr Thr Leu Arg Leu Ala

Ile Phe [SEQ ID NO:135]

Formula XI shown below is a representation of a [(15-125) hIL-3 mutein] with numbers in bold type added above the amino acids to represent the position at which the amino acid below the bolded number appears in native (1-133) hIL-3 [e.g. the amino acid at position 1 of Formula XI corresponds to the Asn which appears at position 15 in native (1-133) hIL-3]. The number shown in bold indicates the amino acids that correspond to the native IL-3(1-133). The non-bold members below the amino acids sequences are for Seq Id reference numbers. When the muteins are expressed the initial amino acid may be preceded by Met- or Met-Ala-.

Table 6 shows (15-125) hIL-3 muteins of the present invention which have one (and in some cases two) amino acid substitutions in the (15-125) hIL-3 polypeptide and which were constructed as described in the Examples. The mutants in Table 6 were secreted into the periplasmic space in E. coli. The periplasmic content was released by osmotic shock and the material in the crude osmotic shock fraction was screened for growth promoting activity. Biological activity is the growth promoting activity of AML cells relative to (15-125) hIL-3 (pMON6458 or pMMON5988). The numbers in parentheses indicate the number of repeat assays. When a variant was assayed more than once the standard deviation is indicated. An “-” indicates that the hIL3 variant protein level was less than 1.0 μg/ml and was not screened for growth promoting activity.

The mutants in Table 6 were made as described in the Examples, particularly Examples 19, 20, 21 and 38 to 53.

It will be apparent to those skilled in the art that other codons besides those shown in Table 6 can also code for the substituted amino acids in the hIL-3 muteins. The present invention includes the DNAs encoding the mutant hIL-3 polypeptides of the invention including the various codons which can code for the parental and substituted amino acids of the hIL-3 muteins of the invention due to the degeneracy of the genetic code.

hIL-3 (15-125) variant genes encoding the variants listed in Table 6 can also be expressed from intracellular expression vectors to produce large quantities of the variant protein which can be purified and assayed for biological activity. The hIL-3 variant genes, from Table 6, can be excised from the secretion expression vector, as a 345 base pair NcoI/HindIII fragment and ligated into an appropriate intracellular expression vector, such as pMON2341 digested with NcoI and HindIII. Examples of variants transferred to pMON2341 in this manner are shown in Table 7. Two examples of such a transfer are described. in the construction of pMON13215 (EXAMPLE 64) and pMON13252 (EXAMPLE 65).

Construction of pMON13215

Plasmid, pMON2341, DNA was digested with restriction enzymes NcoI and HindIII resulting in a 3619 base pair NcoI/HindIII fragment. The genetic elements derived from pMON2341 are the beta-lactamase gene (AMP), pBR327 origin of replication, F1 phage origin of replication as the transcription terminator, precA, g10L ribosome binding site. The plasmid encoding the hIL-3 (15-125) Trp (116) variant, from Table 6 wag digested with NcoT and HindIII resulting in a 345 base pair NcoI/HindIII fragment. The 345 Base pair NcoI/HindIII fragment was ligated with the 3619 base pair fragment from pMON2341 and the ligation reaction mixture was used to transform E.coli K-12 strain JM101. Plasmid DNA was isolated and screened by restriction anaylsis using NcoI and HindIII. Positive clones contained a 345 base pair NcoI/HindIII. This construct was designated PMON132I5. The plasmid, pMON13215, encodes the (15-125) hIL-3 variant with the following amino acid sequence:

PEPTIDE A9; (15-125)HIL-3 TRP (116) PMON13215

Construction of pMON13252

Plasmid, pMON2341, DNA was digested with restriction enzymes NcoI and HindIII resulting in a 3619 base pair NcoI/HindIII fragment. The genetic elements derived from pMON2341 are the beta-lactamase gene (AMP), pBR327 origin of replication F1 phage origin of replication as the transcription terminator, precA, g10L ribosome binding site. The plasmid encoding the hIL-3 (15-125) Asp ( 50) variant, from Table 6, was digested with NcoI and HindIII resulting in a 345 base pair NcoI/HindIII fragment. This 345 Base pair NcoI/HindIII fragment was ligated with the 3619 base pair fragment from pMON2341 and the ligation reaction mixture was used to transform E. coli K-12 strain JM101. Plasmid DNA was isolated and screened by restriction analysis using NcoI and HindIII. Positive clones contained a 345 base pair NcoI/HindIII. This construct was designated pMON13252. The plasmid, pMON13252, encodes the (15-125) hIL-3 variant with the following amino acid sequence:

PEPTIDE A10; (15-125)HIL-3 ASP (50) pMON13252

Table 7 shows the biological activity of (15-125) hIL-3 mutant polypeptides of the present invention expressed from Intracellular expression vectors. Upon expression these muteins may have Met- or Met-Ala-preceding the initial (15-125) hIL-3 amino acid. The relative biological activity of IL-3 mutants is calculated by dividing the EC 50 (1-133) hIL-3 by the EC 50 of the mutant.

The variants in Table 8 were constructed by cassette mutagenesis using methods described in the Materials and Methods and the Examples contained herein, particularly Examples 54-57. Parental plasmid DNA (Table 8), digested with the appropriate restriction enzymes (Table 8), was ligated with the indicated annealed pairs of complementary oligonucleotides (Table 8). The assembled oligonucleotides create appropriate restriction ends and a portion of the (15-125) hIL-3 gene sequence Individual isolates were screened by restriction analysis and DNA sequenced to confirm that the desired changes in the (15-125) hIL-3 variant gene were made. The oligonucleotides create change(s) in the (15-125) hIL-3 gene which enclode the corresponding amino acid substitution in the variant polypeptide (Table 8). The amino acids substitutions in polypeptide #1 (SEQ ID NO:65) are indicated in Table 8.

It will be apparent to those skilled in the art that other codons besides those shown in Table 8 can also code for the substituted amino acids in the hIL-3 muteins. The present invention includes the DNAs encoding the mutant hIL-3 polypeptides of the invention including the various codons which can code for the parental and substituted amino acids of the hIL-3 muteins of the invention due to the degeneracy of the genetic code.

hIL-3 (15-125) variant genes encoding the variants listed in Table 8 can also be expressed from intracellular expression vectors to produce large quantities of the variant protein which can be purified and assayed for biological activity. The hIL-3 variant genes, from Table 8, can be excised from the secretion expression vector, as a 345 base pair NcoI/HindIII fragment and ligated into an appropriate intrecellular expression vector, such as pMON2341 digested with NcoI and HindIII.

Table 9 shows the biological activity of (15-125) hIL-3 muteins of the present invention which have one amino acid substitutions in the (15-125) hIL-3 polypeptide and which were constructed as described in Example 66. The mutants in Table 9 were secreted into the periplasmic space in E. coli. The periplasmic content was released by osmotic shock and the material in the crude osmotic shock fraction was screened for growth promoting activity. Biological activity is the growth promoting activity of AML cells relative to (1-125) hIL-3 (pMON6458 or pMMON5988). The relative biological activity of IL-3 mutants is calculated by dividing the EC 50 (1-133) hIL-3 by the EC 50 of the mutant. The numbers in parentheses indicate the number of repeat assays. When a variant was assayed more than once the standard deviation is indicated. An “*” 0 indicates that the hIL3 variant protein level was less than 1.0 μg/ml and was not screened for growth promoting activity.

CTAGCGATCT TTTAATAAGC TTG 23

23 base pairs

nucleic acid

single

linear

DNA (synthetic)

2

GATCCAAGCT TATTAAAAGA TCG 23

69 base pairs

nucleic acid

single

linear

DNA (synthetic)

3

GGCAACAATT TCTACAAAAC ACTTGATACT GTATGAGCAT ACAGTATAAT TGCTTCAACA 60

GAACAGATC 69

67 base pairs

nucleic acid

single

linear

DNA (synthetic)

4

TGTTCTGTTG AAGCAATTAT ACTGTATGCT CATACAGTAT CAAGTGTTTT GTAGAAATTG 60

TTGCCGC 67

23 base pairs

nucleic acid

single

linear

DNA (synthetic)

5

CCATTGCTGC CGGCATCGTG GTC 23

46 base pairs

nucleic acid

single

linear

DNA (synthetic)

6

CATGGCTCCA ATGACTCAGA CTACTTCTCT TAAGACTTCT TGGGTT 46

42 base pairs

nucleic acid

single

linear

DNA (synthetic)

7

AACCCAAGAA GTCTTAAGAG AAGTAGTCTG AGTCATTGGA GC 42

64 base pairs

nucleic acid

single

linear

DNA (synthetic)

8

AATTCCGTCG TAAACTGACC TTCTATCTGA AAACCTTGGA GAACGCGCAG GCTCAACAGT 60

AATA 64

64 base pairs

nucleic acid

single

linear

DNA (synthetic)

9

AGCTTATTAC TGTTGAGCCT GCGCGTTCTC CAAGGTTTTC AGATAGAAGG TCAGTTTACG 60

ACGG 64

126 amino acids

amino acid

linear

peptide

10

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

115 120 125

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

11

CATGGCTAAC TGCTCTAACA TGAT 24

22 base pairs

nucleic acid

single

linear

DNA (synthetic)

12

CGATCATGTT AGAGCAGTTA GC 22

113 amino acids

amino acid

linear

peptide

13

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 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

27 amino acids

amino acid

linear

peptide

14

Met Met Ile Thr Leu Arg Lys Leu Pro Leu Ala Val Ala Val Ala Ala

1 5 10 15

Gly Val Met Ser Ala Gln Ala Met Ala Asn Cys

20 25

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 Xaa Xaa Glu Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa

20 25 30

Xaa Xaa Xaa Xaa Xaa Xaa Asn Leu Xaa Xaa Glu Xaa Xaa Xaa Xaa Leu

35 40 45

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Leu Xaa Xaa Xaa Xaa Xaa Xaa

50 55 60

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa

65 70 75 80

Xaa Xaa Xaa Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg Xaa Xaa

85 90 95

Xaa Xaa Xaa Xaa Xaa Xaa Asp Xaa Xaa Xaa Phe Xaa Xaa Lys Leu Xaa

100 105 110

Phe Xaa Xaa Xaa Xaa Leu 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 Xaa Xaa Glu Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa

1 5 10 15

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Leu Xaa Xaa Glu Xaa Xaa Xaa

20 25 30

Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Leu Xaa Xaa Xaa Xaa

35 40 45

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu

50 55 60

Xaa Xaa Xaa Xaa Xaa Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg

65 70 75 80

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp Xaa Xaa Xaa Phe Xaa Xaa Lys

85 90 95

Leu Xaa Phe Xaa Xaa Xaa Xaa Leu 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

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 Arg Lys

85 90 95

Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln

100 105 110

58 base pairs

nucleic acid

single

linear

DNA (synthetic)

24

CTAGCCACGG CCGCACCCAC GCGACATCCA ATCCATATCA AGGACGGTGA CTGGAATG 58

58 base pairs

nucleic acid

single

linear

DNA (synthetic)

25

TTAACATTCC AGTCACCGTC CTTGATATGG ATTGGATGTC GCGTGGGTGC GGCCGTGG 58

20 base pairs

nucleic acid

single

linear

DNA (synthetic)

26

TGTCTGCTCA GGCCATGGCT 20

67 base pairs

nucleic acid

single

linear

DNA (synthetic)

27

GCGCGAATTC ATTCCAGTCA CCGTCCTTGA TATGGTCGAC TTATTACGTG GGTGCGGCCG 60

TGGCTAG 67

55 base pairs

nucleic acid

single

linear

DNA (synthetic)

28

GCGCGAATTC ATTCCAGTCA CCGTCGACTT ATTAGATTGG ATGTCGCGTG GGTGC 55

16 base pairs

nucleic acid

single

linear

DNA (synthetic)

29

TGAACCATAT GTCAGG 16

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

30

AATTCCTGAC ATATGGTTCA TGCA 24

43 base pairs

nucleic acid

single

linear

DNA (synthetic)

31

GCGCGAATTC GTCGACTTAT TAGTCCTTGA TATGGATTGG ATG 43

67 base pairs

nucleic acid

single

linear

DNA (synthetic)

32

GCGCGAATTC ATTCCAGTCA CCGTCCTTGA TATGGATTGG ATGSNNCGTG GGTGCGGCCG 60

TGGCTAG 67

64 base pairs

nucleic acid

single

linear

DNA (synthetic)

33

GCGCGAATTC ATTCCAGTCA CCGTCCTTGA TATGGATTGG SNNTCGCGTG GGTGCGGCCG 60

TGGC 64

61 base pairs

nucleic acid

single

linear

DNA (synthetic)

34

GCGCGAATTC ATTCCAGTCA CCGTCCTTGA TATGGATSNN ATGTCGCGTG GGTGCGGCCG 60

T 61

58 base pairs

nucleic acid

single

linear

DNA (synthetic)

35

GCGCGAATTC ATTCCAGTCA CCGTCCTTGA TATGSNNTGG ATGTCGCGTG GGTGCGGC 58

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

36

GATATGGATT GGATGTCGCG TGGG 24

55 base pairs

nucleic acid

single

linear

DNA (synthetic)

37

GCGCGAATTC ATTCCAGTCA CCGTCCTTGA TSNNGATTGG ATGTCGCGTG GGTGC 55

52 base pairs

nucleic acid

single

linear

DNA (synthetic)

38

GCGCGAATTC ATTCCAGTCA CCGTCCTTSN NATGGATTGG ATGTCGCGTG GG 52

49 base pairs

nucleic acid

single

linear

DNA (synthetic)

39

GCGCGAATTC ATTCCAGTCA CCGTCSNNGA TATGGATTGG ATGTCGCGT 49

46 base pairs

nucleic acid

single

linear

DNA (synthetic)

40

GCGCGAATTC ATTCCAGTCA CCSNNCTTGA TATGGATTGG ATGTCG 46

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

41

GTCACCGTCC TTGATATGGA TTGG 24

43 base pairs

nucleic acid

single

linear

DNA (synthetic)

42

GCGCGAATTC ATTCCAGTCS NNGTCCTTGA TATGGATTGG ATG 43

40 base pairs

nucleic acid

single

linear

DNA (synthetic)

43

GCGCGAATTC ATTCCASNNA CCGTCCTTGA TATGGATTGG 40

37 base pairs

nucleic acid

single

linear

DNA (synthetic)

44

GCGCGAATTC ATTSNNGTCA CCGTCCTTGA TATGGAT 37

34 base pairs

nucleic acid

single

linear

DNA (synthetic)

45

GCGCGAATTC SNNCCAGTCA CCGTCCTTGA TATG 34

25 base pairs

nucleic acid

single

linear

DNA (synthetic)

46

GAATTCATTC CAGTCACCGT TCCTT 25

26 base pairs

nucleic acid

single

linear

DNA (synthetic)

47

CGCGCGGAAT TCATTCCAGT CACCGT 26

42 base pairs

nucleic acid

single

linear

DNA (synthetic)

48

CGCGCGCCAT GGCTAACTGC ATTATAACAC ACACTTAAAG CA 42

58 base pairs

nucleic acid

single

linear

DNA (synthetic)

49

CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAACA GCCACCTTTG CCTTTGCT 58

76 base pairs

nucleic acid

single

linear

DNA (synthetic)

50

CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC TTAAAGCTGG 60

ACTTCAACAA CCTCAA 76

50 base pairs

nucleic acid

single

linear

DNA (synthetic)

51

GCGCGCGATA TCTTGGTCTT CTTCACCATT CAGCGGCAGC GGTGGCTGCT 50

65 base pairs

nucleic acid

single

linear

DNA (synthetic)

52

GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATC AGGATGAGGT TGTTGAAGTC 60

CAGCA 65

47 base pairs

nucleic acid

single

linear

DNA (synthetic)

53

GCGCGCCTCG AGGTTTGGAC GACGAAGATC TTGGTCTTCA CCATTGA 47

68 base pairs

nucleic acid

single

linear

DNA (synthetic)

54

GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AATGCCTCGT TATTTTCCAT 60

CAGGATAT 68

50 base pairs

nucleic acid

single

linear

DNA (synthetic)

55

GCGCGCTGAT GCATTCTGCA GAGACTTGAC GAGGTTTGGA CGACGAAGGT 50

47 base pairs

nucleic acid

single

linear

DNA (synthetic)

56

GCGCGCCTCG AGGCATTCAA CCGTGCTGCA TCAGCAATTG AGAGCAT 47

35 base pairs

nucleic acid

single

linear

DNA (synthetic)

57

GCGCGCCTGC AGAATATTCT TAAAAATCTC CTGCC 35

53 base pairs

nucleic acid

single

linear

DNA (synthetic)

58

GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCCCATGTC TGCCGCTAGC CAC 53

71 base pairs

nucleic acid

single

linear

DNA (synthetic)

59

GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATCTCCT GACGGCCGCA 60

CCCACGCGAC A 71

67 base pairs

nucleic acid

single

linear

DNA (synthetic)

60

CGCGCGGAAT TCATTCCAGT CACCGTCCTT GATATGGATT GGATGTCGCA GGGCAGACAT 60

GGCAGGA 67

78 base pairs

nucleic acid

single

linear

DNA (synthetic)

61

CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCCAA GGTTTTCAGA TAGAAGGTAT 60

TCCAGTCACC GTCCTTGA 78

60 base pairs

nucleic acid

single

linear

DNA (synthetic)

62

CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCCAA CAGTTTACGA CGGAATTCAT 60

42 base pairs

nucleic acid

single

linear

DNA (synthetic)

63

CGCGCGAAGC TTATTACTGT TGGGTTTTCA GATAGAAGGT CA 42

58 base pairs

nucleic acid

single

linear

DNA (synthetic)

64

CGCGCGCCAT GGCTAACTGC NNSAACATGA TCGATGAAAT TATAACACAC TTAAAGCA 58

111 amino acids

amino acid

linear

peptide

65

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 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

66

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 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 Ile Ile Arg 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

67

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 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 Arg Pro Ile Ile Ile Arg 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

339 base pairs

nucleic acid

double

linear

DNA (genomic)

68

ATGGCTAACT GCTCTAACAT GATCGATGAA ATCATCACCC ACCTGAAGCA GCCACCGCTG 60

CCGCTGCTGG ACTTCAACAA CCTCAATGAC GAAGACATGT CTATCCTGAT GGAAAATAAC 120

CTTCGTCGTC CAAACCTCGA GGCATTCAAC CGTGCTGTCA AGTCTCTGCA GAATGCATCA 180

GCAATTGAGA GCATTCTTAA AAATCTCCTG CCATGTCTGC CCCTGGCCAC GGCCGCACCC 240

ACGCGACATC CAATCCATAT CAAGGACGGT GACTGGAATG AATTCCGTCG TAAACTGACC 300

TTCTATCTGA AAACCTTGGA GAACGCGCAG GCTCAACAG 339

113 amino acids

amino acid

linear

peptide

69

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 Asp Glu Asp

20 25 30

Met Ser 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 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

45 base pairs

nucleic acid

single

linear

DNA (synthetic)

70

CTTTAAGTGT GTTATAATTT CGTTGATCAT GTTAGAGCAG TTAGC 45

40 base pairs

nucleic acid

single

linear

DNA (synthetic)

71

CATGGCTAAC TGCTCTAACA TGATCCAAGA AATTATAACA 40

45 base pairs

nucleic acid

single

linear

DNA (synthetic)

72

CTTTAAGTGT GTTATAATTT CTTGGATCAT GTTAGAGCAG TTAGC 45

40 base pairs

nucleic acid

single

linear

DNA (synthetic)

73

CATGGCTAAC TGCTCTAACA TGATCGAAGA AATTATAACA 40

76 base pairs

nucleic acid

single

linear

DNA (synthetic)

74

CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACANNS TTAAAGCAGC 60

CACCTTTGCC TTTGCT 76

76 base pairs

nucleic acid

single

linear

DNA (synthetic)

75

CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC NNSAAGCAGC 60

CACCTTTGCC TTTGCT 76

76 base pairs

nucleic acid

single

linear

DNA (synthetic)

76

CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC TTANNSCAGC 60

CACCTTTGCC TTTGCT 76

94 base pairs

nucleic acid

single

linear

DNA (synthetic)

77

CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC TTAAAGNNSC 60

CACCTTTGCC TTTGCTGGAC TTCAACAACC TCAA 94

94 base pairs

nucleic acid

single

linear

DNA (synthetic)

78

CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC TTAAAGCAGN 60

NSCCTTTGCC TTTGCTGGAC TTCAACAACC TCAA 94

94 base pairs

nucleic acid

single

linear

DNA (synthetic)

79

CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC TTAAAGCAGC 60

CANNSTTGCC TTTGCTGGAC TTCAACAACC TCAA 94

94 base pairs

nucleic acid

single

linear

DNA (synthetic)

80

CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC TTAAAGCAGC 60

CACCTNNSCC TTTGCTGGAC TTCAACAACC TCAA 94

94 base pairs

nucleic acid

single

linear

DNA (synthetic)

81

CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC TTAAAGCAGC 60

CACCTTTGNN STTGCTGGAC TTCAACAACC TCAA 94

94 base pairs

nucleic acid

single

linear

DNA (synthetic)

82

CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC TTAAAGCAGC 60

CACCTTTGCC TNNSCTGGAC TTCAACAACC TCAA 94

65 base pairs

nucleic acid

single

linear

DNA (synthetic)

83

GCGCGCGATA TCTTGGTCTT CACCATTGAG GTTGTTGAAG TCSNNCAGCG GCAGCGGTGG 60

CTGCT 65

65 base pairs

nucleic acid

single

linear

DNA (synthetic)

84

GCGCGCGATA TCTTGGTCTT CACCATTGAG GTTGTTGAAS NNCAGCAGCG GCAGCGGTGG 60

CTGCT 65

65 base pairs

nucleic acid

single

linear

DNA (synthetic)

85

GCGCGCGATA TCTTGGTCTT CACCATTGAG GTTGTTSNNG TCCAGCAGCG GCAGCGGTGG 60

CTGCT 65

65 base pairs

nucleic acid

single

linear

DNA (synthetic)

86

GCGCGCGATA TCTTGGTCTT CACCATTGAG GTTSNNGAAG TCCAGCAGCG GCAGCGGTGG 60

CTGCT 65

65 base pairs

nucleic acid

single

linear

DNA (synthetic)

87

GCGCGCGATA TCTTGGTCTT CACCATTGAG SNNGTTGAAG TCCAGCAGCG GCAGCGGTGG 60

CTGCT 65

65 base pairs

nucleic acid

single

linear

DNA (synthetic)

88

GCGCGCGATA TCTTGGTCTT CACCATTSNN GTTGTTGAAG TCCAGCAGCG GCAGCGGTGG 60

CTGCT 65

83 base pairs

nucleic acid

single

linear

DNA (synthetic)

89

GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATC AGGATATCTT GGTCTTCACC 60

SNNGAGGTTG TTGAAGTCCA GCA 83

83 base pairs

nucleic acid

single

linear

DNA (synthetic)

90

GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATC AGGATATCTT GGTCTTCSNN 60

ATTGAGGTTG TTGAAGTCCA GCA 83

83 base pairs

nucleic acid

single

linear

DNA (synthetic)

91

GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATC AGGATATCTT GGTCSNNACC 60

ATTGAGGTTG TTGAAGTCCA GCA 83

83 base pairs

nucleic acid

single

linear

DNA (synthetic)

92

GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATC AGGATATCTT GSNNTTCACC 60

ATTGAGGTTG TTGAAGTCCA GCA 83

83 base pairs

nucleic acid

single

linear

DNA (synthetic)

93

GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATC AGGATATCSN NGTCTTCACC 60

ATTGAGGTTG TTGAAGTCCA GCA 83

83 base pairs

nucleic acid

single

linear

DNA (synthetic)

94

GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATC AGGATSNNTT GGTCTTCACC 60

ATTGAGGTTG TTGAAGTCCA GCA 83

65 base pairs

nucleic acid

single

linear

DNA (synthetic)

95

GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATC AGSNNATCTT GGTCTTCACC 60

ATTGA 65

65 base pairs

nucleic acid

single

linear

DNA (synthetic)

96

GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATS NNGATATCTT GGTCTTCACC 60

ATTGA 65

65 base pairs

nucleic acid

single

linear

DNA (synthetic)

97

GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCSNNC AGGATATCTT GGTCTTCACC 60

ATTGA 65

65 base pairs

nucleic acid

single

linear

DNA (synthetic)

98

GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTSNNCATC AGGATATCTT GGTCTTCACC 60

ATTGA 65

65 base pairs

nucleic acid

single

linear

DNA (synthetic)

99

GCGCGCCTCG AGGTTTGGAC GACGAAGGTT SNNTTCCATC AGGATATCTT GGTCTTCACC 60

ATTGA 65

65 base pairs

nucleic acid

single

linear

DNA (synthetic)

100

GCGCGCCTCG AGGTTTGGAC GACGAAGSNN ATTTTCCATC AGGATATCTT GGTCTTCACC 60

ATTGA 65

86 base pairs

nucleic acid

single

linear

DNA (synthetic)

101

GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AATGCCTCGA GGTTTGGACG 60

ACGSNNGTTA TTTTCCATCA GGATAT 86

86 base pairs

nucleic acid

single

linear

DNA (synthetic)

102

GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AATGCCTCGA GGTTTGGACG 60

SNNAAGGTTA TTTTCCATCA GGATAT 86

86 base pairs

nucleic acid

single

linear

DNA (synthetic)

103

GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AATGCCTCGA GGTTTGGSNN 60

ACGAAGGTTA TTTTCCATCA GGATAT 86

86 base pairs

nucleic acid

single

linear

DNA (synthetic)

104

GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AATGCCTCGA GGTTSNNACG 60

ACGAAGGTTA TTTTCCATCA GGATAT 86

86 base pairs

nucleic acid

single

linear

DNA (synthetic)

105

GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AATGCCTCGA GSNNTGGACG 60

ACGAAGGTTA TTTTCCATCA GGATAT 86

86 base pairs

nucleic acid

single

linear

DNA (synthetic)

106

GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AATGCCTCSN NGTTTGGACG 60

ACGAAGGTTA TTTTCCATCA GGATAT 86

68 base pairs

nucleic acid

single

linear

DNA (synthetic)

107

GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AATGCSNNGA GGTTTGGACG 60

ACGAAGGT 68

68 base pairs

nucleic acid

single

linear

DNA (synthetic)

108

GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AASNNCTCGA GGTTTGGACG 60

ACGAAGGT 68

68 base pairs

nucleic acid

single

linear

DNA (synthetic)

109

GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTS NNTGCCTCGA GGTTTGGACG 60

ACGAAGGT 68

68 base pairs

nucleic acid

single

linear

DNA (synthetic)

110

GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGSNNG AATGCCTCGA GGTTTGGACG 60

ACGAAGGT 68

68 base pairs

nucleic acid

single

linear

DNA (synthetic)

111

GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCSNNGTTG AATGCCTCGA GGTTTGGACG 60

ACGAAGGT 68

68 base pairs

nucleic acid

single

linear

DNA (synthetic)

112

GCGCGCTGAT GCATTCTGCA GAGACTTGAC SNNACGGTTG AATGCCTCGA GGTTTGGACG 60

ACGAAGGT 68

65 base pairs

nucleic acid

single

linear

DNA (synthetic)

113

GCGCGCCTCG AGGCATTCAA CCGTGCTNNS AAGTCTCTGC AGAATGCATC AGCAATTGAG 60

AGCAT 65

65 base pairs

nucleic acid

single

linear

DNA (synthetic)

114

GCGCGCCTCG AGGCATTCAA CCGTGCTGTC NNSTCTCTGC AGAATGCATC AGCAATTGAG 60

AGCAT 65

65 base pairs

nucleic acid

single

linear

DNA (synthetic)

115

GCGCGCCTCG AGGCATTCAA CCGTGCTGTC AAGNNSCTGC AGAATGCATC AGCAATTGAG 60

AGCAT 65

65 base pairs

nucleic acid

single

linear

DNA (synthetic)

116

GCGCGCCTCG AGGCATTCAA CCGTGCTGTC AAGTCTNNSC AGAATGCATC AGCAATTGAG 60

AGCAT 65

65 base pairs

nucleic acid

single

linear

DNA (synthetic)

117

GCGCGCCTCG AGGCATTCAA CCGTGCTGTC AAGTCTCTGN NSAATGCATC AGCAATTGAG 60

AGCAT 65

65 base pairs

nucleic acid

single

linear

DNA (synthetic)

118

GCGCGCCTCG AGGCATTCAA CCGTGCTGTC AAGTCTCTGC AGNNSGCATC AGCAATTGAG 60

AGCAT 65

53 base pairs

nucleic acid

single

linear

DNA (synthetic)

119

GCGCGCCTGC AGAATNNSTC AGCAATTGAG AGCATTCTTA AAAATCTCCT GCC 53

53 base pairs

nucleic acid

single

linear

DNA (synthetic)

120

GCGCGCCTGC AGAATGCANN SGCAATTGAG AGCATTCTTA AAAATCTCCT GCC 53

53 base pairs

nucleic acid

single

linear

DNA (synthetic)

121

GCGCGCCTGC AGAATGCATC ANNSATTGAG AGCATTCTTA AAAATCTCCT GCC 53

53 base pairs

nucleic acid

single

linear

DNA (synthetic)

122

GCGCGCCTGC AGAATGCATC AGCANNSGAG AGCATTCTTA AAAATCTCCT GCC 53

53 base pairs

nucleic acid

single

linear

DNA (synthetic)

123

GCGCGCCTGC AGAATGCATC AGCAATTNNS AGCATTCTTA AAAATCTCCT GCC 53

53 base pairs

nucleic acid

single

linear

DNA (synthetic)

124

GCGCGCCTGC AGAATGCATC AGCAATTGAG NNSATTCTTA AAAATCTCCT GCC 53

71 base pairs

nucleic acid

single

linear

DNA (synthetic)

125

GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCNNSCTTA AAAATCTCCT GCCATGTCTG 60

CCGCTAGCCA C 71

71 base pairs

nucleic acid

single

linear

DNA (synthetic)

126

GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTNNSA AAAATCTCCT GCCATGTCTG 60

CCGCTAGCCA C 71

71 base pairs

nucleic acid

single

linear

DNA (synthetic)

127

GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTN NSAATCTCCT GCCATGTCTG 60

CCGCTAGCCA C 71

134 amino acids

amino acid

linear

peptide

128

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

Ala Pro Met Thr Gln Thr Thr Ser Leu Lys Thr Ser Trp Val Asn Cys

1 5 10 15

Ser Xaa Met Ile Asp Glu Ile Ile Xaa His Leu Lys Xaa Pro Pro Xaa

20 25 30

Pro Leu Leu Asp Xaa Asn Asn Leu Asn Xaa Glu Asp Xaa Asp Ile Leu

35 40 45

Met Glu Xaa Asn Leu Arg Xaa Pro Asn Leu Xaa Xaa Phe Xaa Arg Ala

50 55 60

Val Lys Xaa Leu Xaa Asn Ala Ser Xaa Ile Glu Xaa Ile Leu Xaa Asn

65 70 75 80

Leu Xaa Pro Cys Leu Pro Xaa Ala Thr Ala Ala Pro Xaa Arg His Pro

85 90 95

Ile Xaa Ile Lys Xaa Gly Asp Trp Xaa Glu Phe Arg Xaa Lys Leu Thr

100 105 110

Phe Tyr Leu Xaa Thr Leu Glu Xaa Ala Gln Xaa Gln Gln Thr Thr Leu

115 120 125

Ser Leu Ala Ile Phe

130

Asn Cys Ser Xaa Met Ile Asp Glu Ile Ile Xaa His Leu Lys Xaa Pro

1 5 10 15

Pro Xaa Pro Leu Leu Asp Xaa Asn Asn Leu Asn Xaa Glu Asp Xaa Asp

20 25 30

Ile Leu Met Glu Xaa Asn Leu Arg Xaa Pro Asn Leu Xaa Xaa Phe Xaa

35 40 45

Arg Ala Val Lys Xaa Leu Xaa Asn Ala Ser Xaa Ile Glu Xaa Ile Leu

50 55 60

Xaa Asn Leu Xaa Pro Cys Leu Pro Xaa Ala Thr Ala Ala Pro Xaa Arg

65 70 75 80

His Pro Ile Xaa Ile Lys Xaa Gly Asp Trp Xaa Glu Phe Arg Xaa Lys

85 90 95

Leu Thr Phe Tyr Leu Xaa Thr Leu Glu Xaa Ala Gln Xaa Gln Gln

100 105 110

58 base pairs

nucleic acid

single

linear

DNA (synthetic)

131

CTAGCCACGG CCGCACCCAC GCGACATCCA ATCCATATCA AGGACGGTGA CTGGAATG 58

58 base pairs

nucleic acid

single

linear

DNA (synthetic)

132

TTAACATTCC AGTCACCGTC CTTGATATGG ATTGGATGTC GCGTGGGTGC GGCCGTGG 58

29 base pairs

nucleic acid

double

linear

DNA (genomic)

133

AAGGAGATAT ATCCATGAAC TGCTCTAAC 29

5 amino acids

amino acid

linear

peptide

134

Met Asn Cys Ser Asn

1 5

120 amino acids

amino acid

linear

peptide

135

Met Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln

1 5 10 15

Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln

20 25 30

Asp Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe

35 40 45

Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser Ile

50 55 60

Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr

65 70 75 80

Arg His Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg

85 90 95

Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln

100 105 110

Thr Thr Leu Arg Leu Ala Ile Phe

115 120

71 base pairs

nucleic acid

single

linear

DNA (synthetic)

136

AATTCCGTCG TAAACTGACC TTCTATCTGA AAACCTTGGA GAACGCGCAG GCTCAACAGA 60

CCACTCTGTC G 71

71 base pairs

nucleic acid

single

linear

DNA (synthetic)

137

CTAGCGACAG AGTGGTCTGT TGAGCCTGCG CGTTCTCCAA GGTTTTCAGA TAGAAGGTCA 60

GTTTACGACG G 71

71 base pairs

nucleic acid

single

linear

DNA (synthetic)

138

GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AANNSCTCCT GCCATGTCTG 60

CCGCTAGCCA C 71

71 base pairs

nucleic acid

single

linear

DNA (synthetic)

139

GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATNNSCT GCCATGTCTG 60

CCGCTAGCCA C 71

71 base pairs

nucleic acid

single

linear

DNA (synthetic)

140

GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATCTCNN SCCATGTCTG 60

CCGCTAGCCA C 71

89 base pairs

nucleic acid

single

linear

DNA (synthetic)

141

GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATCTCCT GNNSTGTCTG 60

CCGCTAGCCA CGGCCGCACC CACGCGACA 89

89 base pairs

nucleic acid

single

linear

DNA (synthetic)

142

GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATCTCCT GCCANNSCTG 60

CCGCTAGCCA CGGCCGCACC CACGCGACA 89

89 base pairs

nucleic acid

single

linear

DNA (synthetic)

143

GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATCTCCT GCCATGTNNS 60

CCGCTAGCCA CGGCCGCACC CACGCGACA 89

408 base pairs

nucleic acid

double

linear

DNA (genomic)

144

ATGGCTCCAA TGACTCAGAC TACTTCTCTT AAGACTTCTT GGGTTAACTG CTCTAACATG 60

ATCGATGAAA TTATAACACA CTTAAAGCAG CCACCTTTGC CTTTGCTGGA CTTCAACAAC 120

CTCAATGGGG AAGACCAAGA CATTCTGATG GAAAATAACC TTCGAAGGCC AAACCTGGAG 180

GCATTCAACA GGGCTGTCAA GAGTTTACAG AATGCATCAG CAATTGAGAG CATTCTTAAA 240

AATCTCCTGC CATGTCTGCC CCTGGCCACG GCCGCACCCA CGCGACATCC AATCCATATC 300

AAGGACGGTG ACTGGAATGA ATTCCGTCGT AAACTGACCT TCTATCTGAA AACCTTGGAG 360

AACGCGCAGG CTCAACAGAC CACTCTGTCG CTAGCGATCT TTTAATAA 408

ATC GAT GAA ATC ATC ACC CAC CTG AAG CAG CCA CCG CTG CCG CTG CTG 48

Ile Asp Glu Ile Ile Thr His Leu Lys Gln Pro Pro Leu Pro Leu Leu

1 5 10 15

GAC TTC AAC AAC CTC AAT GGT GAA GAC CAA GAT ATC CTG ATG GAA AAT 96

Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp Ile Leu Met Glu Asn

20 25 30

AAC CTT CGT CGT CCA AAC CTC GAG GCA TTC AAC CGT GCT GTC AAG TCT 144

Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser

35 40 45

CTG CAG AAT GCA T 157

Leu Gln Asn Ala

50

52 amino acids

amino acid

linear

protein

146

Ile Asp Glu Ile Ile Thr His Leu Lys Gln Pro Pro Leu Pro Leu Leu

1 5 10 15

Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp Ile Leu Met Glu Asn

20 25 30

Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser

35 40 45

Leu Gln Asn Ala

50

414 base pairs

nucleic acid

double

linear

DNA (genomic)

147

CCATGGCTCC AATGACTCAG ACTACTTCTC TTAAGACTTC TTGGGTTAAC TGCTCTAACA 60

TGATCGATGA AATTATAACA CACTTAAAGC AGCCACCTTT GCCTTTGCTG GACTTCAACA 120

ACCTCAATGG GGAAGACCAA GACATTCTGA TGGAAAATAA CCTTCGAAGG CCAAACCTGG 180

AGGCATTCAA CAGGGCTGTC AAGAGTTTAC AGAATGCATC AGCAATTGAG AGCATTCTTA 240

AAAATCTCCT GCCATGTCTG CCCCTGGCCA CGGCCGCACC CACGCGACAT CCAATCCATA 300

TCAAGGACGG TGACTGGAAT GAATTCCGTC GTAAACTGAC CTTCTATCTG AAAACCTTGG 360

AGAACGCGCA GGCTCAACAG ACCACTCTGT CGCTAGCGAT CTTTTAATAA GCTT 414

414 base pairs

nucleic acid

double

linear

DNA (genomic)

148

AAGCTTATTA AAAGATCGCT AGCGACAGAG TGGTCTGTTG AGCCTGCGCG TTCTCCAAGG 60

TTTTCAGATA GAAGGTCAGT TTACGACGGA ATTCATTCCA GTCACCGTCC TTGATATGGA 120

TTGGATGTCG CGTGGGTGCG GCCGTGGCCA GGGGCAGACA TGGCAGGAGA TTTTTAAGAA 180

TGCTCTCAAT TGCTGATGCA TTCTGTAAAC TCTTGACAGC CCTGTTGAAT GCCTCCAGGT 240

TTGGCCTTCG AAGGTTATTT TCCATCAGAA TGTCTTGGTC TTCCCCATTG AGGTTGTTGA 300

AGTCCAGCAA AGGCAAAGGT GGCTGCTTTA AGTGTGTTAT AATTTCATCG ATCATGTTAG 360

AGCAGTTAAC CCAAGAAGTC TTAAGAGAAG TAGTCTGAGT CATTGGAGCC ATGG 414

81 base pairs

nucleic acid

double

linear

DNA (genomic)

149

ATGATGATTA CTCTGCGCAA ACTTCCTCTG GCGGTTGCCG TCGCAGCGGG CGTAATGTCT 60

GCTCAGGCCA TGGCTAACTG C 81

81 base pairs

nucleic acid

double

linear

DNA (genomic)

150

GCAGTTAGCC ATGGCCTGAG CAGACATTAC GCCCGCTGCG ACGGCAACCG CCAGAGGAAG 60

TTTGCGCAGA GTAATCATCA T 81

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

151

CATGGCTAAC TGCTCTAACA TGAT 24

22 base pairs

nucleic acid

single

linear

DNA (synthetic)

152

CGATCATGTT AGAGCAGTTA GC 22

12 base pairs

nucleic acid

double

linear

DNA (genomic)

153

ATGGCTAACT GC 12

4 amino acids

amino acid

linear

peptide

154

Met Ala Asn Cys

1

34 base pairs

nucleic acid

single

linear

DNA (synthetic)

155

GCCGATACCG CGGCATACTC CCACCATTCA GAGA 34

33 base pairs

nucleic acid

single

linear

DNA (synthetic)

156

GCCGATAAGA TCTAAAACGG GTATGGAGAA ACA 33

89 base pairs

nucleic acid

single

linear

DNA (synthetic)

157

GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATCTCCT GCCATGTCTG 60

NNSCTAGCCA CGGCCGCACC CACGCGACA 89

89 base pairs

nucleic acid

single

linear

DNA (synthetic)

158

GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATCTCCT GCCATGTCTG 60

CCGNNSGCCA CGGCCGCACC CACGCGACA 89

89 base pairs

nucleic acid

single

linear

DNA (synthetic)

159

GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATCTCCT GCCATGTCTG 60

CCGCTANNSA CGGCCGCACC CACGCGACA 89

86 base pairs

nucleic acid

single

linear

DNA (synthetic)

160

CGCGCGGAAT TCATTCCAGT CACCGTCCTT GATATGGATT GGATGTCGCG TGGGTGCGGC 60

SNNGGCCAGG GGCAGACATG GCAGGA 86

86 base pairs

nucleic acid

single

linear

DNA (synthetic)

161

CGCGCGGAAT TCATTCCAGT CACCGTCCTT GATATGGATT GGATGTCGCG TGGGTGCSNN 60

CGTGGCCAGG GGCAGACATG GCAGGA 86

86 base pairs

nucleic acid

single

linear

DNA (synthetic)

162

CGCGCGGAAT TCATTCCAGT CACCGTCCTT GATATGGATT GGATGTCGCG TGGGSNNGGC 60

CGTGGCCAGG GGCAGACATG GCAGGA 86

86 base pairs

nucleic acid

single

linear

DNA (synthetic)

163

CGCGCGGAAT TCATTCCAGT CACCGTCCTT GATATGGATT GGATGTCGCG TSNNTGCGGC 60

CGTGGCCAGG GGCAGACATG GCAGGA 86

86 base pairs

nucleic acid

single

linear

DNA (synthetic)

164

CGCGCGGAAT TCATTCCAGT CACCGTCCTT GATATGGATT GGATGTCGSN NGGGTGCGGC 60

CGTGGCCAGG GGCAGACATG GCAGGA 86

48 base pairs

nucleic acid

single

linear

DNA (synthetic)

165

TTTCAGATAG AAGGTCAGTT TACGACGGAA SNNATTCCAG TCACCGTC 48

48 base pairs

nucleic acid

single

linear

DNA (synthetic)

166

TTTCAGATAG AAGGTCAGTT TACGACGSNN TTCATTCCAG TCACCGTC 48

48 base pairs

nucleic acid

single

linear

DNA (synthetic)

167

TTTCAGATAG AAGGTCAGTT TACGSNNGAA TTCATTCCAG TCACCGTC 48

48 base pairs

nucleic acid

single

linear

DNA (synthetic)

168

TTTCAGATAG AAGGTCAGTT TSNNACGGAA TTCATTCCAG TCACCGTC 48

48 base pairs

nucleic acid

single

linear

DNA (synthetic)

169

TTTCAGATAG AAGGTCAGSN NACGACGGAA TTCATTCCAG TCACCGTC 48

48 base pairs

nucleic acid

single

linear

DNA (synthetic)

170

TTTCAGATAG AAGGTSNNTT TACGACGGAA TTCATTCCAG TCACCGTC 48

23 base pairs

nucleic acid

single

linear

DNA (synthetic)

171

CGCGCGAAGC TTATTACTGT TGA 23

78 base pairs

nucleic acid

single

linear

DNA (synthetic)

172

CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCCAA GGTTTTCAGA TAGAASNNCA 60

GTTTACGACG GAATTCAT 78

78 base pairs

nucleic acid

single

linear

DNA (synthetic)

173

CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCCAA GGTTTTCAGA TASNNGGTCA 60

GTTTACGACG GAATTCAT 78

78 base pairs

nucleic acid

single

linear

DNA (synthetic)

174

CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCCAA GGTTTTCAGS NNGAAGGTCA 60

GTTTACGACG GAATTCAT 78

78 base pairs

nucleic acid

single

linear

DNA (synthetic)

175

CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCCAA GGTTTTSNNA TAGAAGGTCA 60

GTTTACGACG GAATTCAT 78

78 base pairs

nucleic acid

single

linear

DNA (synthetic)

176

CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCCAA GGTSNNCAGA TAGAAGGTCA 60

GTTTACGACG GAATTCAT 78

78 base pairs

nucleic acid

single

linear

DNA (synthetic)

177

CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCCAA SNNTTTCAGA TAGAAGGTCA 60

GTTTACGACG GAATTCAT 78

60 base pairs

nucleic acid

single

linear

DNA (synthetic)

178

CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCSNN GGTTTTCAGA TAGAAGGTCA 60

60 base pairs

nucleic acid

single

linear

DNA (synthetic)

179

CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTSNNCAA GGTTTTCAGA TAGAAGGTCA 60

60 base pairs

nucleic acid

single

linear

DNA (synthetic)

180

CGCGCGAAGC TTATTACTGT TGAGCCTGCG CSNNCTCCAA GGTTTTCAGA TAGAAGGTCA 60

60 base pairs

nucleic acid

single

linear

DNA (synthetic)

181

CGCGCGAAGC TTATTACTGT TGAGCCTGSN NGTTCTCCAA GGTTTTCAGA TAGAAGGTCA 60

60 base pairs

nucleic acid

single

linear

DNA (synthetic)

182

CGCGCGAAGC TTATTACTGT TGAGCSNNCG CGTTCTCCAA GGTTTTCAGA TAGAAGGTCA 60

60 base pairs

nucleic acid

single

linear

DNA (synthetic)

183

CGCGCGAAGC TTATTACTGT TGSNNCTGCG CGTTCTCCAA GGTTTTCAGA TAGAAGGTCA 60

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

184

TGCTCTAACA TGATCGATGA AATT 24

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

185

GAAATTATAA CACACTTAAA GCAG 24

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

186

AAGCAGCCAC CTTTGCCTTT GCTG 24

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

187

AAGCAGCCAC CGCTGCCGCT GCTG 24

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

188

CTCAATGGTG AAGACCAAGA TATC 24

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

189

GATATCCTGA TGGAAAATAA CCTT 24

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

190

AACCTTCGTC GTCCAAACCT CGAG 24

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

191

CTCGAGGCAT TCAACCGTGC TGTC 24

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

192

GCTGTCAAGT CTCTGCAGAA TGCA 24

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

193

AATGCATCAG CAATTGAGAG CATT 24

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

194

AGCATTCTTA AAAATCTCCT GCCA 24

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

195

CTGCCATGTC TGCCCCTGGC CACG 24

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

196

CTGGCCACGG CCGCACCCAC GCGA 24

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

197

AATGAATTCC GTCGTAAACT GACC 24

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

198

CTGACCTTCT ATCTGAAAAC CTTG 24

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

199

ACCTTGGAGA ACGCGCAGGC TCAA 24

22 base pairs

nucleic acid

single

linear

DNA (synthetic)

200

GAATGCATCA GCAATTGAGA GC 22

20 base pairs

nucleic acid

single

linear

DNA (synthetic)

201

AATTGCTGAT GCATTCTGCA 20

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

202

ATTCTTAAAA ATCTCCTGCC ATGT 24

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

203

CAGGAGATTT TTAAGAATGC TCTC 24

30 base pairs

nucleic acid

single

linear

DNA (synthetic)

204

CTGCCCCTGG CCACGGCCGC ACCCACGCGA 30

30 base pairs

nucleic acid

single

linear

DNA (synthetic)

205

GGGTGCGGCC GTGGCCAGGG GCAGACATGG 30

34 base pairs

nucleic acid

single

linear

DNA (synthetic)

206

CATCCAATCA TCATCCGTGA CGGTGACTGG AATG 34

44 base pairs

nucleic acid

single

linear

DNA (synthetic)

207

AATTCATTCC AGTCACCGTC ACGGATGATG ATTGGATGTC GCGT 44

34 base pairs

nucleic acid

single

linear

DNA (synthetic)

208

CGCCCAATCA TCATCCGTGA CGGTGACTGG AATG 34

44 base pairs

nucleic acid

single

linear

DNA (synthetic)

209

AATTCATTCC AGTCACCGTC ACGGATGATG ATTGGGCGTC GCGT 44

40 base pairs

nucleic acid

single

linear

DNA (synthetic)

210

CATGGCTAAC TGCTCTAACA TGATCGATGA AATTATAACA 40

45 base pairs

nucleic acid

single

linear

DNA (synthetic)

211

CTTTAAGTGT GTTATAATTT CATCGATCAT GTTAGAGCAG TTAGC 45

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

212

CACTTAAAGC AGCCACCTTT GCCTTTGCTG GACTTC 36

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

213

GAGGTTGTTG AAGTCCAGCA AAGGCAAAGG TGGCTG 36

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

214

AACAACCTCA ATGACGAAGA CATGTCT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

215

AGACATGTCT TCGTCATT 18

339 base pairs

nucleic acid

double

linear

DNA (genomic)

216

ATGGCTAACT GCTCTAACAT GATCGATGAA ATCATCACCC ACCTGAAGCA GCCACCGCTG 60

CCGCTGCTGG ACTTCAACAA CCTCAATGGT GAAGACCAAG ATATCCTGAT GGAAAATAAC 120

CTTCGTCGTC CAAACCTCGA GGCATTCAAC CGTGCTGTCA ACTCTCTGCA GAATGCATCA 180

GCAATTGAGA GCATTCTTAA AAATCTCCTG CCATGTCTGC CCCTGGCCAC GGCCGCACCC 240

ACGCGACATC CAATCCATAT CAAGGACGGT GACTGGAATG AATTCCGTCG TAAACTGACC 300

TTCTATCTGA AAACCTTGGA GAACGCGCAG GCTCAACAG 339

111 amino acids

amino acid

linear

peptide

217

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 Arg Lys

85 90 95

Leu Thr Phe Tyr Leu Trp Thr Leu Glu Asn Ala Gln Ala Gln Gln

100 105 110

111 amino acids

amino acid

linear

peptide

218

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 Asp 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

45 base pairs

nucleic acid

single

linear

DNA (synthetic)

219

CTTTAAGTGT GTTATAATTT CTTCGATCAT GTTAGAGCAG TTAGC 45

339 base pairs

nucleic acid

double

linear

DNA (genomic)

220

ATGGCTAACT GCTCTAACAT GATCGATGAA ATCATCACCC ACCTGAAGCA GCCACCGCTG 60

CCGCTGCTGG ACTTCAACAA CCTCAATGGT GAAGACCAAG ATATCCTGAT GGAAAATAAC 120

CTTCGTCGTC CAAACCTCGA GGCATTCAAC CGTGCTGTCA ACTCTCTGCA GAATGCATCA 180

GCAATTGAGA GCATTCTTAA AAATCTCCTG CCATGTCTGC CCCTGGCCAC GGCCGCACCC 240

ACGCGACATC CAATCCATAT CAAGGACGGT GACTGGAATG AATTCCGTCG TAAACTGACC 300

TTCTATCTGT GGACCTTGGA GAACGCGCAG GCTCAACAG 339

40 base pairs

nucleic acid

single

linear

DNA (synthetic)

221

CATGGCTAAC TGCTCTAACA TGATCAACGA AATTATAACA 40

58 base pairs

nucleic acid

single

linear

DNA (synthetic)

222

CGCGCGCCAT GGCTAACTGC TCTNNSATGA TCGATGAAAT TATAACACAC TTAAAGCA 58

58 base pairs

nucleic acid

single

linear

DNA (synthetic)

223

CGCGCGCCAT GGCTAACTGC TCTAACNNSA TCGATGAAAT TATAACACAC TTAAAGCA 58

58 base pairs

nucleic acid

single

linear

DNA (synthetic)

224

CGCGCGCCAT GGCTAACTGC TCTAACATGN NSGATGAAAT TATAACACAC TTAAAGCA 58

58 base pairs

nucleic acid

single

linear

DNA (synthetic)

225

CGCGCGCCAT GGCTAACTGC TCTAACATGA TCNNSGAAAT TATAACACAC TTAAAGCA 58

58 base pairs

nucleic acid

single

linear

DNA (synthetic)

226

CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATNNSAT TATAACACAC TTAAAGCA 58

76 base pairs

nucleic acid

single

linear

DNA (synthetic)

227

CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAANN SATAACACAC TTAAAGCAGC 60

CACCTTTGCC TTTGCT 76

76 base pairs

nucleic acid

single

linear

DNA (synthetic)

228

CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TNNSACACAC TTAAAGCAGC 60

CACCTTTGCC TTTGCT 76

76 base pairs

nucleic acid

single

linear

DNA (synthetic)

229

CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATANNSCAC TTAAAGCAGC 60

CACCTTTGCC TTTGCT 76

40 base pairs

nucleic acid

single

linear

DNA (synthetic)

230

CATGGCTAAC TGCTCTAACA TGATCAGCGA AATTATAACA 40

45 base pairs

nucleic acid

single

linear

DNA (synthetic)

231

CTTTAAGTGT GTTATAATTT CGCTGATCAT GTTAGAGCAG TTAGC 45

40 base pairs

nucleic acid

single

linear

DNA (synthetic)

232

CATGGCTAAC TGCTCTAACA TGATCACCGA AATTATAACA 40

45 base pairs

nucleic acid

single

linear

DNA (synthetic)

233

CTTTAAGTGT GTTATAATTT CCGTGATCAT GTTAGAGCAG TTAGC 45

40 base pairs

nucleic acid

single

linear

DNA (synthetic)

234

CATGGCTAAC TGCTCTAACA TGATCGATAA CATTATAACA 40

45 base pairs

nucleic acid

single

linear

DNA (synthetic)

235

CTTTAAGTGT GTTATAATGT TATCGATCAT GTTAGAGCAG TTAGC 45

40 base pairs

nucleic acid

single

linear

DNA (synthetic)

236

CATGGCTAAC TGCTCTAACA TGATCGATGA CATTATAACA 40

45 base pairs

nucleic acid

single

linear

DNA (synthetic)

237

CTTTAAGTGT GTTATAATGT CATCGATCAT GTTAGAGCAG TTAGC 45

40 base pairs

nucleic acid

single

linear

DNA (synthetic)

238

CATGGCTAAC TGCTCTAACA TGATCGATCA GATTATAACA 40

45 base pairs

nucleic acid

single

linear

DNA (synthetic)

239

CTTTAAGTGT GTTATAATCT GATCGATCAT GTTAGAGCAG TTAGC 45

40 base pairs

nucleic acid

single

linear

DNA (synthetic)

240

CATGGCTAAC TGCTCTAACA TGATCGATCT GATTATAACA 40

45 base pairs

nucleic acid

single

linear

DNA (synthetic)

241

CTTTAAGTGT GTTATAATCA GATCGATCAT GTTAGAGCAG TTAGC 45

40 base pairs

nucleic acid

single

linear

DNA (synthetic)

242

CATGGCTAAC TGCTCTAACA TGATCGATGT TATTATAACA 40

45 base pairs

nucleic acid

single

linear

DNA (synthetic)

243

CTTTAAGTGT GTTATAATAA CATCGATCAT GTTAGAGCAG TTAGC 45

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

244

CACTTAAAGC AGCCACCTTT GCCTGCTCTG GACTTC 36

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

245

GAGGTTGTTG AAGTCCAGAG CAGGCAAAGG TGGCTG 36

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

246

CACTTAAAGC AGCCACCTTT GCCTCGTCTG GACTTC 36

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

247

GAGGTTGTTG AAGTCCAGAC GAGGCAAAGG TGGCTG 36

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

248

CACTTAAAGC AGCCACCTTT GCCTCAGCTG GACTTC 36

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

249

GAGGTTGTTG AAGTCCAGCT GAGGCAAAGG TGGCTG 36

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

250

CACTTAAAGC AGCCACCTTT GCCTGAACTG GACTTC 36

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

251

GAGGTTGTTG AAGTCCAGCT CAGGCAAAGG TGGCTG 36

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

252

CACTTAAAGC AGCCACCTTT GCCTATCCTG GACTTC 36

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

253

GAGGTTGTTG AAGTCCAGGA TAGGCAAAGG TGGCTG 36

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

254

CACTTAAAGC AGCCACCTTT CCCTTTCCTG GACTTC 36

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

255

GAGGTTGTTG AAGTCCAGGA AAGGCAAAGG TGGCTG 36

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

256

CACTTAAAGC AGCCACCTTT GCCTACCCTG GACTTC 36

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

257

GAGGTTGTTG AAGTCCAGGG TAGGCAAAGG TGGCTG 36

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

258

AACAACCTCA ATCGTGAAGA CCAAGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

259

ATCTTGGTCT TCACGATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

260

AACAACCTCA ATAACGAAGA CCAAGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

261

ATCTTGGTCT TCGTTATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

262

AACAACCTCA ATGAAGAAGA CCAAGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

263

ATCTTGGTCT TCTTCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

264

AACAACCTCA ATATCGAAGA CCAAGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

265

ATCTTGGTCT TCGATATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

266

AACAACCTCA ATCTGGAAGA CCAAGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

267

ATCTTGGTCT TCCAGATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

268

AACAACCTCA ATAAAGAAGA CCAAGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

269

ATCTTGGTCT TCTTTATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

270

AACAACCTCA ATATGGAAGA CCAAGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

271

ATCTTGGTCT TCCATATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

272

AACAACCTCA ATTTCGAAGA CCAAGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

273

ATCTTGGTCT TCGAAATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

274

AACAACCTCA ATACCGAAGA CCAAGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

275

ATCTTGGTCT TCGGTATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

276

AACAACCTCA ATTACGAAGA CCAAGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

277

ATCTTGGTCT TCGTAATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

278

AACAACCTCA ATGTTGAAGA CCAAGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

279

ATCTTGGTCT TCAACATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

280

AACAACCTCA ATGGGCGTGA CCAAGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

281

ATCTTGGTCT CGCCCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

282

AACAACCTCA ATGGGCAGGA CCAAGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

283

ATCTTGGTCC TGCCCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

284

AACAACCTCA ATGGGGGTGA CCAAGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

285

ATCTTGGTCA CCCCCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

286

AACAACCTCA ATGGGACCGA CCAAGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

287

ATCTTGGTCG GTCCCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

288

AACAACCTCA ATGGGGAAGC TCAAGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

289

ATCTTGAGCT TCCCCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

290

AACAACCTCA ATGGGGAAAA CCAAGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

291

ATCTTGGTTT TCCCCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

292

AACAACCTCA ATGGGGAACA GCAAGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

293

ATCTTGCTGT TCCCCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

294

AACAACCTCA ATGGGGAAGA ACAAGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

295

ATCTTGTTCT TCCCCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

296

AACAACCTCA ATGGGGAAGA CGCTGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

297

ATCAGCGTCT TCCCCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

298

AACAACCTCA ATGGGGAAGA CCGTGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

299

ATCACGGTCT TCCCCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

300

AACAACCTCA ATGGGGAAGA CAACGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

301

ATCGTTGTCT TCCCCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

302

AACAACCTCA ATGGGGAAGA CGACGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

303

ATCGTCGTCT TCCCCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

304

AACAACCTCA ATGGTGAAGA CGAAGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

305

ATCTTCGTCT TCCCCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

306

AACAACCTCA ATGGTGAAGA CCACGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

307

ATCGTGGTCT TCCCCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

308

AACAACCTCA ATGGGGAAGA CATCGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

309

ATCGATGTCT TCCCCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

310

AACAACCTCA ATGGGGAAGA CTCCGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

311

ATCGGAGTCT TCCCCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

312

AACAACCTCA ATGGGGAAGA CCAAGCT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

313

AGCTTGGTCT TCCCCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

314

AACAACCTCA ATGGGGAAGA CCAAAAC 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

315

GTTTTGGTCT TCCCCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

316

AACAACCTCA ATGGGGAAGA CCAACAG 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

317

CTGTTGGTCT TCCCCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

318

AACAACCTCA ATGGGGAAGA CCAAGAA 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

319

TTCTTGGTCT TCCCCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

320

AACAACCTCA ATGGGGAAGA CCAACAC 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

321

GTGTTGGTCT TCCCCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

322

AACAACCTCA ATGGGGAAGA CCAAATC 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

323

GATTTGGTCT TCCCCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

324

AACAACCTCA ATGGGGAAGA CCAACTG 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

325

CAGTTGGTCT TCCCCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

326

AACAACCTCA ATGGGGAAGA CCAAAAA 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

327

TTTTTGGTCT TCCCCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

328

AACAACCTCA ATGGGGAAGA CCAATAC 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

329

GTATTGGTCT TCCCCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

330

AACAACCTCA ATGGGGAAGA CCAAGTT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

331

AACTTGGTCT TCCCCATT 18

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

332

ATCGCTATGG AAAATAACCT TCGAAGGCCA AACCTG 36

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

333

CCTTCGAAGG TTATTTTCCA TAGCGAT 27

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

334

ATCGAAATGG AAAATAACCT TCGAAGGCCA AACCTG 36

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

335

CCTTCGAAGG TTATTTTCCA TTTCGAT 27

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

336

ATCAAAATGG AAAATAACCT TCGAAGGCCA AACCTG 36

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

337

CCTTCGAAGG TTATTTTCCA TTTTGAT 27

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

338

ATCATGATGG AAAATAACCT TCGAAGGCCA AACCTG 36

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

339

CCTTCGAAGG TTATTTTCCA TCATGAT 27

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

340

ATCACCATGG AAAATAACCT TCGAAGGCCA AACCTG 36

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

341

CCTTCGAAGG TTATTTTCCA TGGTGAT 27

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

342

ATCGTTATGG AAAATAACCT TCGAAGGCCA AACCTG 36

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

343

CCTTCGAAGG TTATTTTCCA TAACGAT 27

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

344

ATCCTGATGC ACAATAACCT TCGAAGGCCA AACCTG 36

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

345

CCTTCGAAGG TTATTGTGCA TCAGGAT 27

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

346

ATCCTGATGA TGAATAACCT TCGAAGGCCA AACCTG 36

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

347

CCTTCGAAGG TTATTCATCA TCAGGAT 27

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

348

ATCCTGATGT TCAATAACCT TCGAAGGCCA AACCTG 36

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

349

CCTTCGAAGG TTATTGAACA TCAGGAT 27

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

350

ATCCTGATGG CTAATAACCT TCGAAGGCCA AACCTG 36

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

351

CCTTCGAAGG TTATTAGCCA TCAGGAT 27

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

352

ATCCTGATGA ACAATAACCT TCGAAGGCCA AACCTG 36

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

353

CCTTCGAAGG TTATTGTTCA TCAGGAT 27

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

354

ATCCTGATGA TCAATAACCT TCGAAGGCCA AACCTG 36

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

355

CCTTCGAAGG TTATTGATCA TCAGGAT 27

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

356

ATCCTGATGA AAAATAACCT TCGAAGGCCA AACCTG 36

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

357

CCTTCGAAGG TTATTTTTCA TCAGGAT 27

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

358

ATCCTGATGT CCAATAACCT TCGAAGGCCA AACCTG 36

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

359

CCTTCGAAGG TTATTGGACA TCAGGAT 27

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

360

ATCCTGATGG TTAATAACCT TCGAAGGCCA AACCTG 36

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

361

CCTTCGAAGG TTATTAACCA TCAGGAT 27

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

362

ATCCTGATGG AAAATAACCT TGCTAGGCCA AACCTG 36

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

363

CCTAGCAAGG TTATTTTCCA TCAGGAT 27

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

364

ATCCTGATGG AAAATAACCT TAACAGGCCA AACCTG 36

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

365

CCTGTTAAGG TTATTTTCCA TCAGGAT 27

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

366

ATCCTGATGG AAAATAACCT TCACAGGCCA AACCTG 36

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

367

CCTGTGAAGG TTATTTTCCA TCAGGAT 27

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

368

ATCCTGATGG AAAATAACCT TAAAAGGCCA AACCTG 36

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

369

CCTTTTAAGG TTATTTTCCA TCAGGAT 27

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

370

ATCCTGATGG AAAATAACCT TCGAAGGGCT AACCTG 36

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

371

CCTGTTGAAT GCCTCCAGGT TAGC 24

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

372

ATCCTGATGG AAAATAACCT TCGAAGGCGT AACCTG 36

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

373

CCTGTTGAAT GCCTCCAGGT TACG 24

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

374

ATCCTGATGG AAAATAACCT TCGAAGGAAC AACCTG 36

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

375

CCTGTTGAAT GCCTCCAGGT TGTT 24

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

376

ATCCTGATGG AAAATAACCT TCGAAGGGAA AACCTG 36

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

377

CCTGTTGAAT GCCTCCAGGT TTTC 24

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

378

ATCCTGATGG AAAATAACCT TCGAAGGCAC AACCTG 36

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

379

CCTGTTGAAT GCCTCCAGGT TGTG 24

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

380

ATCCTGATGG AAAATAACCT TCGAAGGCTG AACCTG 36

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

381

CCTGTTGAAT GCCTCCAGGT TCAG 24

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

382

ATCCTGATGG AAAATAACCT TCGAAGGTTC AACCTG 36

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

383

CCTGTTGAAT GCCTCCAGGT TGAA 24

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

384

ATCCTGATGG AAAATAACCT TCGAAGGACC AACCTG 36

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

385

CCTGTTGAAT GCCTCCAGGT TGGT 24

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

386

ATCCTGATGG AAAATAACCT TCGAAGGTAC AACCTG 36

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

387

CCTGTTGAAT GCCTCCAGGT TGTA 24

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

388

ATCCTGATGG AAAATAACCT TCGAAGGGTT AACCTG 36

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

389

CCTGTTGAAT GCCTCCAGGT TAAC 24

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

390

AAAAATCTCG CTCCATGT 18

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

391

AGCGAGATTT TTAAGAAT 18

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

392

AAAAATCTCA ACCCATGT 18

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

393

GTTGAGATTT TTAAGAAT 18

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

394

AAAAATCTCG AACCATGT 18

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

395

TTCGAGATTT TTAAGAAT 18

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

396

AAAAATCTCC ACCCATGT 18

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

397

GTGGAGATTT TTAAGAAT 18

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

398

AAAAATCTCA TCCCATGT 18

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

399

GATGAGATTT TTAAGAAT 18

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

400

AAAAATCTCA TGCCATGT 18

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

401

CATGAGATTT TTAAGAAT 18

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

402

AAAAATCTCT TCCCATGT 18

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

403

GAAGAGATTT TTAAGAAT 18

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

404

AAAAATCTCT CCCCATGT 18

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

405

GGAGAGATTT TTAAGAAT 18

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

406

AAAAATCTCA CCCCATGT 18

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

407

GGTGAGATTT TTAAGAAT 18

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

408

AAAAATCTCT ACCCATGT 18

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

409

GTAGAGATTT TTAAGAAT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

410

CTGCCCCTGG CCACGGCCGC AGCTACG 27

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

411

ATGGATTGGA TGTCGCGTAG CTGC 24

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

412

CTGCCCCTGG CCACGGCCGC AGGTACG 27

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

413

ATGGATTGGA TGTCGCGTAC CTGC 24

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

414

CTGCCCCTGG CCACGGCCGC AATCACG 27

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

415

ATGGATTGGA TGTCGCGTGA TTGC 24

21 base pairs

nucleic acid

single

linear

DNA (synthetic)

416

GCTCATCCAA TCCATATCAA G 21

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

417

ATGGATTGGA TGAGCCGTGG GTGC 24

21 base pairs

nucleic acid

single

linear

DNA (synthetic)

418

CAGCATCCAA TCCATATCAA G 21

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

419

ATGGATTGGA TGCTGCGTGG GTGC 24

21 base pairs

nucleic acid

single

linear

DNA (synthetic)

420

CACCATCCAA TCCATATCAA G 21

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

421

ATGGATTGGA TGGTGCGTGG GTGC 24

21 base pairs

nucleic acid

single

linear

DNA (synthetic)

422

AAACATCCAA TCCATATCAA G 21

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

423

ATGGATTGGA TGTTTCGTGG GTGC 24

21 base pairs

nucleic acid

single

linear

DNA (synthetic)

424

CGAGCTCCAA TCCATATCAA G 21

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

425

ATGGATTGGA GCTCGCGTGG GTGC 24

21 base pairs

nucleic acid

single

linear

DNA (synthetic)

426

CGAAACCCAA TCCATATCAA G 21

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

427

ATGGATTGGG TTTCGCGTGG GTGC 24

21 base pairs

nucleic acid

single

linear

DNA (synthetic)

428

CGAGACCCAA TCCATATCAA G 21

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

429

ATGGATTGGG TCTCGCGTGG GTGC 24

21 base pairs

nucleic acid

single

linear

DNA (synthetic)

430

CGAATCCCAA TCCATATCAA G 21

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

431

ATGGATTGGG ATTCGCGTGG GTGC 24

21 base pairs

nucleic acid

single

linear

DNA (synthetic)

432

CGAAAACCAA TCCATATCAA G 21

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

433

ATGGATTGGT TTTCGCGTGG GTGC 24

21 base pairs

nucleic acid

single

linear

DNA (synthetic)

434

CGAATGCCAA TCCATATCAA G 21

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

435

ATGGATTGGC ATTCGCGTGG GTGC 24

21 base pairs

nucleic acid

single

linear

DNA (synthetic)

436

CGATTCCCAA TCCATATCAA G 21

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

437

ATGGATTGGG AATCGCGTGG GTGC 24

21 base pairs

nucleic acid

single

linear

DNA (synthetic)

438

CGATCCCCAA TCCATATCAA G 21

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

439

ATGGATTGGG GATCGCGTGG GTGC 24

21 base pairs

nucleic acid

single

linear

DNA (synthetic)

440

CGATGGCCAA TCCATATCAA G 21

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

441

ATGGATTGGC CATCGCGTGG GTGC 24

21 base pairs

nucleic acid

single

linear

DNA (synthetic)

442

CGATACCCAA TCCATATCAA G 21

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

443

ATGGATTGGG TATCGCGTGG GTGC 24

34 base pairs

nucleic acid

single

linear

DNA (synthetic)

444

CATCCAATCC AAATCAAGGA CGGTGACTGG AATG 34

44 base pairs

nucleic acid

single

linear

DNA (synthetic)

445

AATTCATTCC AGTCACCGTC CTTGATTTGG ATTGGATGTC GCGT 44

34 base pairs

nucleic acid

single

linear

DNA (synthetic)

446

CATCCAATCG AAATCAAGGA CGGTGACTGG AATG 34

44 base pairs

nucleic acid

single

linear

DNA (synthetic)

447

AATTCATTCC AGTCACCGTC CTTGATTTCG ATTGGATGTC GCGT 44

34 base pairs

nucleic acid

single

linear

DNA (synthetic)

448

CATCCAATCA TGATCAAGGA CGGTGACTGG AATG 34

44 base pairs

nucleic acid

single

linear

DNA (synthetic)

449

AATTCATTCC AGTCACCGTC CTTGATCATG ATTGGATGTC GCGT 44

34 base pairs

nucleic acid

single

linear

DNA (synthetic)

450

CATCCAATCT TCATCAAGGA CGGTGACTGG AATG 34

44 base pairs

nucleic acid

single

linear

DNA (synthetic)

451

AATTCATTCC AGTCACCGTC CTTGATGAAG ATTGGATGTC GCGT 44

34 base pairs

nucleic acid

single

linear

DNA (synthetic)

452

CATCCAATCT CCATCAAGGA CGGTGACTGG AATG 34

44 base pairs

nucleic acid

single

linear

DNA (synthetic)

453

AATTCATTCC AGTCACCGTC CTTGATGGAG ATTGGATGTC GCGT 44

34 base pairs

nucleic acid

single

linear

DNA (synthetic)

454

CATCCAATCG TAATCAAGGA CGGTGACTGG AATG 34

44 base pairs

nucleic acid

single

linear

DNA (synthetic)

455

AATTCATTCC AGTCACCGTC CTTGATTACG ATTGGATGTC GCGT 44

21 base pairs

nucleic acid

single

linear

DNA (synthetic)

456

CGACATCCAA TCCGTATCAA G 21

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

457

ACGGATTGGA TGTCGCGTGG GTGC 24

21 base pairs

nucleic acid

single

linear

DNA (synthetic)

458

CGACATCCAA TCAAAATCAA G 21

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

459

TTTGATTGGA TGTCGCGTGG GTGC 24

21 base pairs

nucleic acid

single

linear

DNA (synthetic)

460

CGACATCCAA TCTACATCAA G 21

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

461

GTAGATTGGA TGTCGCGTGG GTGC 24

16 base pairs

nucleic acid

single

linear

DNA (synthetic)

462

GCTGGTGACT GGAATG 16

26 base pairs

nucleic acid

single

linear

DNA (synthetic)

463

AATTCATTCC AGTCACCAGC CTTGAT 26

16 base pairs

nucleic acid

single

linear

DNA (synthetic)

464

AACGGTGACT GGAATG 16

26 base pairs

nucleic acid

single

linear

DNA (synthetic)

465

AATTCATTCC AGTCACCGTT CTTGAT 26

16 base pairs

nucleic acid

single

linear

DNA (synthetic)

466

GAAGGTGACT GGAATG 16

26 base pairs

nucleic acid

single

linear

DNA (synthetic)

467

AATTCATTCC AGTCACCTTC CTTGAT 26

16 base pairs

nucleic acid

single

linear

DNA (synthetic)

468

GGTGGTGACT GGAATG 16

26 base pairs

nucleic acid

single

linear

DNA (synthetic)

469

AATTCATTCC AGTCACCACC CTTGAT 26

16 base pairs

nucleic acid

single

linear

DNA (synthetic)

470

ATCGGTGACT GGAATG 16

26 base pairs

nucleic acid

single

linear

DNA (synthetic)

471

AATTCATTCC AGTCACCGAT CTTGAT 26

16 base pairs

nucleic acid

single

linear

DNA (synthetic)

472

CTGGGTGACT GGAATG 16

26 base pairs

nucleic acid

single

linear

DNA (synthetic)

473

AATTCATTCC AGTCACCCAG CTTGAT 26

16 base pairs

nucleic acid

single

linear

DNA (synthetic)

474

TTCGGTGACT GGAATG 16

26 base pairs

nucleic acid

single

linear

DNA (synthetic)

475

AATTCATTCC AGTCACCGAA CTTGAT 26

16 base pairs

nucleic acid

single

linear

DNA (synthetic)

476

TCCGGTGACT GGAATG 16

26 base pairs

nucleic acid

single

linear

DNA (synthetic)

477

AATTCATTCC AGTCACCGGA CTTGAT 26

32 base pairs

nucleic acid

single

linear

DNA (synthetic)

478

AATTCGCTAG GAAACTGACG TTCTATCTGA AA 32

37 base pairs

nucleic acid

single

linear

DNA (synthetic)

479

CTCAAGGGTT TTCAGATAGA ACGTCAGTTT CCTAGCG 37

32 base pairs

nucleic acid

single

linear

DNA (synthetic)

480

AATTCCAGAG GAAACTGACG TTCTATCTGA AA 32

37 base pairs

nucleic acid

single

linear

DNA (synthetic)

481

CTCAAGGGTT TTCAGATAGA ACGTCAGTTT CCTCTGG 37

32 base pairs

nucleic acid

single

linear

DNA (synthetic)

482

AATTCCACAG GAAACTGACG TTCTATCTGA AA 32

37 base pairs

nucleic acid

single

linear

DNA (synthetic)

483

CTCAAGGGTT TTCAGATAGA ACGTCAGTTT CCTGTGG 37

32 base pairs

nucleic acid

single

linear

DNA (synthetic)

484

AATTCTCCAG GAAACTGACG TTCTATCTGA AA 32

37 base pairs

nucleic acid

single

linear

DNA (synthetic)

485

CTCAAGGGTT TTCAGATAGA ACGTCAGTTT CCTGGAG 37

32 base pairs

nucleic acid

single

linear

DNA (synthetic)

486

AATTCCGGAG GCGTCTGACG TTCTATCTGA AA 32

37 base pairs

nucleic acid

single

linear

DNA (synthetic)

487

CTCAAGGGTT TTCAGATAGA ACGTCAGACG CCTCCGG 37

32 base pairs

nucleic acid

single

linear

DNA (synthetic)

488

AATTCCGGAG GGAACTGACG TTCTATCTGA AA 32

37 base pairs

nucleic acid

single

linear

DNA (synthetic)

489

CTCAAGGGTT TTCAGATAGA ACGTCAGTTC CCTCCGG 37

32 base pairs

nucleic acid

single

linear

DNA (synthetic)

490

AATTCCGGAG GCACCTGACG TTCTATCTGA AA 32

37 base pairs

nucleic acid

single

linear

DNA (synthetic)

491

CTCAAGGGTT TTCAGATAGA ACGTCAGGTG CCTCCGG 37

32 base pairs

nucleic acid

single

linear

DNA (synthetic)

492

AATTCCGGAG GATCCTGACG TTCTATCTGA AA 32

37 base pairs

nucleic acid

single

linear

DNA (synthetic)

493

CTCAAGGGTT TTCAGATAGA ACGTCAGGAT CCTCCGG 37

32 base pairs

nucleic acid

single

linear

DNA (synthetic)

494

AATTCCGGAG GTCCCTGACG TTCTATCTGA AA 32

37 base pairs

nucleic acid

single

linear

DNA (synthetic)

495

CTCAAGGGTT TTCAGATAGA ACGTCAGGGA CCTCCGG 37

32 base pairs

nucleic acid

single

linear

DNA (synthetic)

496

AATTCCGGAG GAAACTGACG GACTATCTGA AA 32

37 base pairs

nucleic acid

single

linear

DNA (synthetic)

497

CTCAAGGGTT TTCAGATAGT CCGTCAGTTT CCTCCGG 37

32 base pairs

nucleic acid

single

linear

DNA (synthetic)

498

AATTCCGGAG GAAACTGACG ATCTATCTGA AA 32

37 base pairs

nucleic acid

single

linear

DNA (synthetic)

499

CTCAAGGGTT TTCAGATAGA TCGTCAGTTT CCTCCGG 37

32 base pairs

nucleic acid

single

linear

DNA (synthetic)

500

AATTCCGGAG GAAACTGACG CTGTATCTGA AA 32

37 base pairs

nucleic acid

single

linear

DNA (synthetic)

501

CTCAAGGGTT TTCAGATACA GCGTCAGTTT CCTCCGG 37

32 base pairs

nucleic acid

single

linear

DNA (synthetic)

502

AATTCCGGAG GAAACTGACG AAATATCTGA AA 32

37 base pairs

nucleic acid

single

linear

DNA (synthetic)

503

CTCAAGGGTT TTCAGATATT TCGTCAGTTT CCTCCGG 37

32 base pairs

nucleic acid

single

linear

DNA (synthetic)

504

AATTCCGGAG GAAACTGACG GTTTATCTGA AA 32

37 base pairs

nucleic acid

single

linear

DNA (synthetic)

505

CTCAAGGGTT TTCAGATAAA CCGTCAGTTT CCTCCGG 37

32 base pairs

nucleic acid

single

linear

DNA (synthetic)

506

AATTCCGGAG GAAACTGACG TTCTATCTGG CT 32

37 base pairs

nucleic acid

single

linear

DNA (synthetic)

507

CTCAAGGGTA GCCAGATAGA ACGTCAGTTT CCTCCGG 37

32 base pairs

nucleic acid

single

linear

DNA (synthetic)

508

AATTCCGGAG GAAACTGACG TTCTATCTGC GT 32

37 base pairs

nucleic acid

single

linear

DNA (synthetic)

509

CTCAAGGGTA CGCAGATAGA ACGTCAGTTT CCTCCGG 37

32 base pairs

nucleic acid

single

linear

DNA (synthetic)

510

AATTCCGGAG GAAACTGACG TTCTATCTGA AC 32

37 base pairs

nucleic acid

single

linear

DNA (synthetic)

511

CTCAAGGGTG TTCAGATAGA ACGTCAGTTT CCTCCGG 37

32 base pairs

nucleic acid

single

linear

DNA (synthetic)

512

AATTCCGGAG GAAACTGACG TTCTATCTGC AG 32

37 base pairs

nucleic acid

single

linear

DNA (synthetic)

513

CTCAAGGGTC TGCAGATAGA ACGTCAGTTT CCTCCGG 37

32 base pairs

nucleic acid

single

linear

DNA (synthetic)

514

AATTCCGGAG GAAACTGACG TTCTATCTGC AC 32

37 base pairs

nucleic acid

single

linear

DNA (synthetic)

515

CTCAAGGGTG TGCAGATAGA ACGTCAGTTT CCTCCGG 37

32 base pairs

nucleic acid

single

linear

DNA (synthetic)

516

AATTCCGGAG GAAACTGACG TTCTATCTGA TG 32

37 base pairs

nucleic acid

single

linear

DNA (synthetic)

517

CTCAAGGGTC ATCAGATAGA ACGTCAGTTT CCTCCGG 37

32 base pairs

nucleic acid

single

linear

DNA (synthetic)

518

AATTCCGGAG GAAACTGACG TTCTATCTGT TC 32

37 base pairs

nucleic acid

single

linear

DNA (synthetic)

519

CTCAAGGGTG AACAGATAGA ACGTCAGTTT CCTCCGG 37

32 base pairs

nucleic acid

single

linear

DNA (synthetic)

520

AATTCCGGAG GAAACTGACG TTCTATCTGT AC 32

37 base pairs

nucleic acid

single

linear

DNA (synthetic)

521

CTCAAGGGTG TACAGATAGA ACGTCAGTTT CCTCCGG 37

40 base pairs

nucleic acid

single

linear

DNA (synthetic)

522

CATGGCTAAC TGCTCTAACA TGATCGATGA AATTATAACA 40

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

523

CACTTAAAGC AGCCACCTTT GCCTTTGCTG GACTTC 36

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

524

AACAACCTCA ATGGGGAAGA CCAAGAT 27

45 base pairs

nucleic acid

single

linear

DNA (synthetic)

525

CTTTAAGTGT GTTATAATTT CATCGATCAT GTTAGAGCAG TTAGC 45

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

526

GAGGTTGTTG AAGTCCAGCA AAGGCAAAGG TGGCTG 36

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

527

ATCTTGGTCT TCCCCATT 18

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

528

ATCCTGATGG AAAATAACCT TCGAAGGCCA AACCTG 36

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

529

GAGGCATTCA ACAGGGCTGT CAAG 24

15 base pairs

nucleic acid

single

linear

DNA (synthetic)

530

AGTTTACAGA ATGCA 15

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

531

CCTTCGAAGG TTATTTTCCA TCAGGAT 27

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

532

CCTGTTGAAT GCCTCCAGGT TTGG 24

20 base pairs

nucleic acid

single

linear

DNA (synthetic)

533

TTCTGTAAAC TCTTGACAGC 20

21 base pairs

nucleic acid

single

linear

DNA (synthetic)

534

TCAGCAATTG AGAGCATTCT T 21

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

535

AAAAATCTCC TGCCATGT 18

48 base pairs

nucleic acid

single

linear

DNA (synthetic)

536

CTGCCCCTGG CCACGGCCGC ACCCACGCGA CATCCAATCC ATATCAAG 48

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

537

CTGCCCCTGG CCACGGCCGC ACCCACG 27

21 base pairs

nucleic acid

single

linear

DNA (synthetic)

538

CGACATCCAA TCCATATCAA G 21

16 base pairs

nucleic acid

single

linear

DNA (synthetic)

539

GACGGTGACT GGAATG 16

19 base pairs

nucleic acid

single

linear

DNA (synthetic)

540

GCTCTCAATT GCTGATGCA 19

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

541

CAGGAGATTT TTAAGAAT 18

48 base pairs

nucleic acid

single

linear

DNA (synthetic)

542

ATGGATTGGA TGTCGCGTGG GTGCGGCCGT GGCCAGGGGC AGACATGG 48

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

543

GGCCGTGGCC AGGGGCAGAC ATGG 24

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

544

ATGGATTGGA TGTCGCGTGG GTGC 24

26 base pairs

nucleic acid

single

linear

DNA (synthetic)

545

AATTCATTCC AGTCACCGTC CTTGAT 26

32 base pairs

nucleic acid

single

linear

DNA (synthetic)

546

AATTCCGGAG GAAACTGACG TTCTATCTGA AA 32

32 base pairs

nucleic acid

single

linear

DNA (synthetic)

547

ACCCTTGAGA ATGCGCAGGC TCAACAGTAA TA 32

37 base pairs

nucleic acid

single

linear

DNA (synthetic)

548

CTCAAGGGTT TTCAGATAGA ACGTCAGTTT CCTCCGG 37

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

549

AGCTTATTAC TGTTGAGCCT GCGCATT 27