A DNA molecule coding for a food protein, such as ovalbumin or casein, modified so that the codons for phenylalanine have been omitted or replaced by codons for one or more other metabolisable amino acids. Also a modified edible protein coded for by such a DNA molecule. Such modified proteins are useful in the nutrition of patients suffering from phenylketonuria.

Description

13 parts

›This application is a 371 of PCT/GB94/01046 filed…

This application is a 371 of PCT/GB94/01046 filed on May 16, 1994, which claims benefit of GB9310472 filed on May 20, 1993.

This invention relates to an edible protein which has been modified so that it is phenylalanine free, to DNA coding for it, and to a method of producing it. Such a protein is a useful nutrient in the treatment of diseases which are associated with difficulty in metabolising phenylalanine. A particular example of such a disease is phenylketonuria (PKU).

PKU is a genetically acquired disease that occurs in a relatively fixed proportion of new births in a human population. A defect in the enzyme carrying out the pterin-dependent hydroxylation of phenylalanine to tyrosine prevents the body from metabolizing the amino acid phenylalanine. This amino acid occurs in varying proportions in all proteins in foodstuffs and is, in the correct amount, essential for human protein synthesis, and therefore for the growth and maintenance of the body. Patients with PKU cannot remove excess phenylalanine from the blood and tissues and the failure to achieve this control over phenylalanine levels leads to grave neurological damage, especially in the growing child.

PKU patients are at present fed with a synthetic diet which contains a metabolically-correct amount of phenylalanine along with a mixture of the other amino acids needed for growth. Such a diet is unpalatable and is presented in liquid form only and therefore has difficulty in achieving patient compliance.

An object of this invention is to provide an edible protein which when pure contains no phenylalanine and which can form the basis for a diet containing the optimal nutritional phenylalanine content for PKU patients. This object may be achieved by taking the gene from a known nutritional protein and modifying it so that the codons coding for phenylalanine are deleted or are replaced by codons coding for another amino acid.

An alternative approach is to synthesise by chemical means DNA coding for a phenylalanine-free polypeptide, starting either from fragments of genes coding for existing proteins, or from the nucleotides themselves.

According to one aspect of the invention we provide a DNA molecule coding for a food protein, modified in that the codons coding for phenylalanine have been deleted or replaced by codons coding for one or more other amino acids.

According to another aspect of the invention we provide an edible polypeptide which comprises a food protein modified in that the phenylalanine residues have been omitted or have been replaced by one or more other amino acids also occurring in protein.

We further provide a nutrient material comprising an edible polypeptide as defined above and other edible substances.

The food protein is preferably a common food protein such as ovalbumin or caesin.

We also provide a nutrient material comprising an edible protein or modified food protein as hereinbefore defined, and other edible substances.

The protein according to the invention is phenylalanine free when pure, but the diet of the patient must contain some phenylalanine, i.e. the amount required for metabolism, but with substantially no excess.

An obvious approach would be to add an appropriate proportion of normal food proteins, which contain phenylalanine, to a pure phenylalanine-free protein according to the invention.

On the other hand, proteins are notoriously difficult to purify to a high level. If only partially purified, the phenylalanine-free protein will be accompanied by other protein products of the host organism containing their normal amounts of phenylalanine. Thus, if the modified protein is only partly purified (which is much easier than complete purification), a protein mixture containing overall a reduced proportion of phenylalanine will be obtained. By controlling the degree of purification, a protein mixture containing a metabolically-appropriate proportion of phenylalanine can be produced. This invention also provides such a mixture.

Although codons for phenylalanine may simply be deleted from the gene for a food protein, in order to preserve as far as possible the tertiary structure of the protein the codons coding for phenylalanine are preferably replaced by codons coding for another amino acid, preferably those having the most similar properties, e.g. tyrosine.

We also provide an expression vector into which has been incorporated DNA for an edible protein or modified food protein as described herein. The expression vector is preferably a Saccharomyces cerevisiae expression vector because this yeast has a long history as a human foodstuff and is amenable to genetic manipulation. Other yeasts, e.g. Pischia pastoris , may also be used.

We further provide a host, for example a yeast such as S. cerevisiae or Pichia pastoris , transformed by such an expression vector.

Ovalbumin and caesin have been selected as preferred food proteins to be modified in accordance with this invention because they are naturally-occurring proteins which are commonly used as human foodstuffs, are widely acceptable, and also because the modified proteins are likely to behave in a similar manner to the native proteins when cooked or subjected to other food processing steps. A wide variety of other food proteins may, however, also be chosen.

Preferably, apart from omitting or substituting codons coding for phenylalanine, the DNA molecule coding for the edible protein is modified as necessary to ensure that the codon for each amino acid is the codon of preference for the selected host, e.g. S.cerevisiae.

DNA sequences and polypeptides embodying the invention will now be described in more detail in non-limiting manner, with reference to the Figures and Examples. FIGS. 1 to 11 relate to modified chick ovalbumin and FIGS. 12 to 24 relate to modified bovine casein.

FIG. 1 shows the sequence of cDNA (SEQ ID NO:38) for unmodified chick ovalbumin (a copy of the Genbank entry).

FIG. 2 (SEQ ID NO:39) shows the primary sequence (386 amino acids) of the coding region (coordinates 66-1223) of the cDNA for chick ovalbumin (GenBank accession number V00383). The locations of the twenty phenylalanine residues are shown in bold face.

›FIG. 3 (SEQ ID NO:1) shows a polyypeptide…

FIG. 3 (SEQ ID NO:1) shows a polyypeptide corresponding to that of FIG. 2, but from which all phenylalanine residues have been deleted.

FIG. 4 (SEQ ID NO:2) shows the amino acid sequence of a polypeptide corresponding to FIG. 2, in which the phenylalanine residues have been replaced by tyrosine residues.

FIG. 5 (SEQ ID NO:3) shows the DNA sequence (1098 bp) of a synthetic gene encoding the derivative of chick ovalbumin lacking phenylalanine (oval-f) produced using the optimal pattern of codon usage for S. cerevisiae .

FIG. 6 (SEQ ID NO:4) shows the DNA sequence (1158 bp) of a synthetic gene encoding the derivative of chick ovalbumin in which the phenylalanines are replaced by tyrosines (Y oval-f) produced using the optimal pattern of codon usage for S. cerevisiae .

FIGS. 7 to 10 (SEQ ID NO:5-8) show, respectively, the nucleotide sequence for the constructs pl+oval−f+3end, pl+h6oval−f+3end, pl+Yoval−f+3end, and pl+h6Yoval−f+3end.

FIGS. 11A and 11B (SEQ ID NO:5) show the nucleotide sequence of the double stranded synthetic pl+oval−f+3end gene constructed from overlapping oligonucleotides. The end points of the oligonucleotides are shown by the ♦ character.

FIG. 12 shows a copy of the GenBank entry for bovine alpha-S1-casein mRNA (SEQ ID NO:40) obtained using the FETCH program of the UWGCG suite of software.

FIG. 13 shows the primary amino acid sequence (SEQ ID NO:42) of the mature alpha-S1-casein specified by the coding region (coordinates 109-705) of the alpha-s 1 -casein mRNA (EMBL accession number M33123). The locations of the eight phenylalanine residues are shown in bold face.

FIG. 14 (SEQ ID NO:9) shows a modified protein corresponding to that of FIG. 13, but from which all phenylalanine residues have been deleted and an N-terminal methionine residue has been added.

FIG. 15 (SEQ ID NO:10) shows a DNA sequence (582 nucleotides) coding for the modified protein of FIG. 14, the derivative of mature bovine alpha-s 1 -casein lacking phenylalanine residues and possessing an N-terminal methionine residue produced using the optimal pattern of codon usage for S. cerevisiae. Two stop codons have also been added at the end of the casein coding sequence.

FIG. 16 (SEQ ID NO:11) shows the DNA sequence (1056 necleotides) of a synthetic gene encoding the derivative of mature bovine alpha-s 1 -casein lacking phenylalanine residues and pocessing an N-terminal methionine residue, produced using the optimal pattern of codon usage for S. cerevisaise. Two stop codons have also been added at the end of the casein coding sequence. The non-translated 3′ region of bovine alpha-s 1 -casein mRNA had been added and the construct is bounded by polylinkers.

FIG. 17 (SEQ ID NO:26-37) shows the nucleotide sequences of bovine casein gene blocks A and B, from which the whole gene was subsequently assembled (Block A: PCR primers=SEQ ID NOS:26 and 31; casein 1=SEQ ID NO:27; casein 2=SEQ ID NO:28; casein 3=SEQ ID NO:29; casein 4=SEQ ID NO:30. Block B: PCR primers=SEQ ID NOS:32 and 37; casein 5=SEQ ID NO:33; casein 6=SEQ ID NO:34; casein 7=SEQ ID NO:35; casein 8=SEQ ID NO:36.).

FIG. 18 a (SEQ ID NO:12-13) and 18 b (SEQ ID NO:14-15) show respectively the predicted and actual DNA and protein sequences of block A. The TTG triplet deletion in the “actual” sequence obtained is indicated (*).

FIGS. 19 a (SEQ ID NO:16-17) and 19 b (SEQ ID NO:18-19) show respectively the predicted and actual DNA and protein sequences of block B. The single base change in the sequence is indicated (*).

FIG. 20 (SEQ ID NO:20-25) shows the combined DNA and protein sequences of blocks A and B. The mutagenic N-terminal (to create a unique PstI restriction site) and C-terminal oligonucleotide primers for PCR amplification are indicated (*).

FIG. 21 (SEQ ID NO:22-23) shows the complete DNA and protein sequences of the synthetic casein.

›Examples8

›EXAMPLE 1

The gene and downstream non-translated DNA sequence for chick ovalbumin were based on the nucleotide sequence of the complementary cDNA for chick ovalbumin deposited by O'Hare et al in the GenBank database with the accession number V00383. The vector pEMBLyex4 (see Cesareni, G and Murray, J. A. H. (1987) In ‘Genetic Engineering’ (Ed. Setlow, J. K.) Volume 9 Plenum Publishing Corporation, New York, pp135-154) was chosen for expression, as it can be used to direct the expression of genes which lack their own promoter. The vector harbours a hybrid promoter consisting of the upstream activator sequence of the GAL1 promoter and the 5′ non-translated leader of the CYC1 gene, up to position −4. The plasmid contains a translation initiation codon ATG downstream from the GAL1-CYC1 promoter. The codon ATG is followed by a unique HindIII site and is preceded by unique cloning sites for BamHI, PstI, SmaI and XbaI. In addition to yeast selectable markers and origin of replication it carries ampicillin resistance and a functional E. coli origin. The complete nucleotide sequence of the vector is known.

The sequence of the cDNA for chick ovalbumin is shown in FIG. 1 and a translation of the ovalbumin coding region is shown in FIG. 2 . The amino acid sequence of a polypeptide (oval-f) derived from chick ovalbumin, but lacking any phenylalanine residues, is shown in FIG. 3 . To optimize expression of this gene when expressed in S. cerevisiae the polypeptide sequence was ‘backtranslated’ using the most preferred pattern of codon usage for S. cerevisiae (FIG. 5 ). A derivative of chick ovalbumin was also designed in which the phenylalanine residues are replaced by tyrosine residues in order to attempt to produce a protein which has as near as possible the tertiary structure of chick ovalbumin. The amino acid sequence of the polypeptide (Yoval-f) and its corresponding gene produced as for the oval-f gene are shown in FIGS. 4 and 6 respectively. To further facilitate expression, cloning procedures and protein purification the following modifications were made to the basic gene.

1. Addition of a sequence corresponding to the 3′ end of the mRNA from the end of the coding region to the poly A site, in order to enhance expression.

2. Addition of an extra TAA stop codon at the end of the gene, in order to ensure that no translation would take place beyond the normal coding region.

3. In order to assist in vitro manipulation, addition at either end of the synthetic gene of polylinkers which contained restriction sites for PstI, BamHI, SmaI, EcoRI and HindIII. The synthetic genes do not contain sites for these restriction enzymes. The polylinkers have the following sequence:

5′ CTGCAGGATCCCGGGAATTCAAGCTT 3′ (SEQ ID NO:43)

[PstI] [SmaI] [HindIII] [BamHI] [EcoRI]

4. In some versions of the synthetic gene a sequence corresponding to 6 histidine residues was added immediately downstream of the initiating methionine, in order to facilitate purification of the protein by a form of affinity chromatography.

Thus 4 basic variations on the original synthetic gene were obtained, with the following structures:

The synthetic gene is constructed via the synthesis of oligonucleotides each approximately 100 nucleotides long and designed in such a way that they overlap each other and will self-assemble by complementary base pairing into a contiguous structure which can be ligated via the appropriate sticky ends, generated by restriction endonuclease digestion into pEMBLyex4 or an appropriate E. coli vector such as pBR322 or pUC19. The sequences of the oligonucleotides and their arrangement is shown in FIG. 11 . The end points of the individual oligonucleotides are marked by the character.

›EXAMPLE 2

This example utilises bovine alpha-s1-casein. In this illustration only one synthetic gene was designed, but the general approach used in Example 1 can be applied to produce the other three genes analogous to those of Example 1, (i.e. those genes containing tyrosine replacements for phenylalanine and/or a run of six histidine residues immediately downstream of the N-terminal methionine).

The sequence of the mRNA for bovine alpha-s1-casein is shown in FIG. 12 and a translation of the region coding for the mature polypeptide is shown in FIG. 13 . The modified form of the protein lacking phenylalanine residues and with an added N-terminal methionine (to permit translation) is shown in FIG. 14. A DNA sequence corresponding to this modified polypeptide produced using the most preferred pattern of codon usage for S. cerevisiae is shown in FIG. 15 . Finally, the nucleotide sequence of the complete synthetic gene with the 3′ untranslated region from the bovine alpha-s1-casein mRNA added on as well as the polylinkers (described in section A) is shown in FIG. 16 . It should be noted that this particular synthetic gene has an internal EcoR1 site, as well as those present in the polylinkers and therefore EcoR1 should not be used in any in vitro manipulations of this gene during insertion into a vector.

›EXAMPLE 3 · 1 of 6

This example concerns the construction of a bovine casein gene modified in that the codons for phenylalamine are replaced by codons for tyrosine.

Synthetic Gene Design

Eight C.100′mer oligonucleotides were designed, synthesised, and purified. These oligonucleotides (casein 1-8. see FIG. 16) formed the basis of two self-priming block assemblies in which the two blocks (designated A and B) overlapped by about 100 bp.

Following an initial round of PCR-mediated extension of the self-primed oligonucleotides as separate Blocks (A & B), a second round of PCR amplification using terminal flanking c. 20′ mer primers (AL1 & AR1,BL1 & BR2; see FIG. 17 generated the two independent c.380 bp gene blocks A and B.

As mentioned above the design of the casein 1-8 c. 100′ mer oligonucleotides was such that the encoded gene contained no phenylalanine codons, all these being substituted with tyrosine codons. A further feature was the incorporation of a number of unique restriction sites to facilitate in the final assembly of the whole gene from components of the two overlapping gene blocks. This duplication facilitates correction of erroneous PCR-mediated DNA synthesis.

Gene Block Synthesis

Using the 2-step PCR stategy described above both casein gene blocks A and B were amplified as discrete c. 380 bp products using Stratagene's native pfu DNA polymerase. Little success was achieved with the cloned enzyme. This particular enzyme was used because of its apparently superior fidelity properties.

Cloning and Sequencing of the Gene Blocks

Both blocks A & B were successfully cloned into Invitrogen's PCRII-TA cloning vector. Plasmid DNA was prepared from numerous isolates and these subjected to DNA sequence analysis using both universal and reverse sequencing primers. For the majority of clones full c. 380 bp reads were obtained. All these sequences were computer aligned against the “desired” sequence and against each other. Representative sample alignments for block A and B are shown in FIGS. 18 a and b , and 19 a and b , respectively.

PCRII-TA clones A100 and B69 were chosen as primary DNA sources. Two mutagenic c. 60′ mer mutagenic oligo nucleotides, casein 9 and casein 10 , were synthesised, purified, and used to amplify a “corrected” c.200 bp HpaI/HindIII C-terminus. This product was cloned into PCRII-TA vector and the sequence of several clones analysed using universal and reverse sequence primers. No perfect sequences were obtained but one clone (C20) which had only one base change, a G to T conversion resulting in a single amino acid change of trp to leu, was chosen.

The strategy taken was to assemble the gene sequence in pMTL22 by cloning the c. 200 bp hpa/HindIII C-terminal fragment of clone C20 next to the remainder of the gene derived from cloning of the c.265 bp BamHI/AatII of clone A100 and the c. 270 bp AatII/KpnI of clone B69. This has been achieved and the final nucleotide sequence verified yielding the “casein” gene sequence with a TTG triplet deletion at nt. pos. 258 and a G to T base change at nt. pos.

Cloning of the Casein Sequence into pMTL8133

The casein gene sequence was sub-cloned from the pMTL22 construct above into the “in house” E.coli /yeast expression vector pMTL8133. This vector is based on chloramphenicol resistance and has a hybrid PGK::REP 2 promoter element which has been shown to elicit high expression levels of other heterologous genes in both E.coli and Saccharomyces cerevisiae . The casein sequence was cloned as a PstI(flush-ended)/HindIII fragment into SspI/HindIII cleaved pMTL8133, such that it is correctly juxtaposed to the 5′-UTR sequence for elevated expression in yeast. The correct sequence at the cloning junction was verified by sequence analysis.

The modified gene has been clone into the E.coli /yeast expression vector pMTL8133 which has previously been shown to elicit expression of heterologous genes in both Escherichia coli and Sacharomyces cerevisiae.

Casein Expression Studies

E.coli strain INV alpha F′ (endA1, recA1, hsdR17(r−k, m+k), supE44, λ-, thi-1, qyrA, relA1, φ80 lacZ∝ΔM15Δ(lacZYA-argF), deoR+, F genotype) has been transformed with the pMTL8133-casein recombinant plasmid and cultured in the presence of chloramphenicol (30 μg ml −1 ) to maintain selection for the plasmid. Sonic extracts have been prepared from this culture and subjected to polyacrylamide gel electrophoresis alongside native bovine alpha casein (purchased from Sigma). Blotting of this gel onto nitrocellulose membrane followed by probing of the membrane sequentially with rabbit anti-casein and peroxidase-conjugated goat anti-rabbit antibody has revealed the presence of a polypeptide equal in size to the bovine alpha casein control. This polypeptide has a predicted molecular weight of 22 kDa. This protein product is detectable by means of antibody probing. No product is visible in coomassie blue stained polyacrylamide gels.

The pMTL8133-casein recombinant plasmid is used to transform a yeast (e.g. S.cerevisiae ) in order to obtain expression of the modified casein encoded thereby.

If necessary or desirable, the base change at nt. pos. 512 can be corrected using a two-step strategy as follows. Firstly, the major part of the casein gene, 510 bp PstII/KpnI (nt. pos. 15 to 530) fragment is sub-cloned into PstI/KpnI cleaved pMTL20 with conomitant loss of AatII and NcoI polylinker sites. This enables the substitution of the c. 100 bp AatI/NcoI fragment containing the TTG triplet deletion with a correct sequence derived from the annealing of two complementary c. 100 bp oligonucleotides (nt. pos. 178 to 279). Such a clone is used for the second step involving mutagenic PCR using oligonucleotide primers AL2 and casein 15 whereby the base change at nt. pos. 512 is corrected.

Met Gly Ser Ile Gly Ala Ala Ser Met Glu Cys Asp Val Lys Glu Leu

1 5 10 15

Lys Val His His Ala Asn Glu Asn Ile Tyr Cys Pro Ile Ala Ile Met

20 25 30

Ser Ala Leu Ala Met Val Tyr Leu Gly Ala Lys Asp Ser Thr Arg Thr

35 40 45

Gln Ile Asn Lys Val Val Arg Asp Lys Leu Pro Gly Gly Asp Ser Ile

›EXAMPLE 3 · 2 of 6

50 55 60

Glu Ala Gln Cys Gly Thr Ser Val Asn Val His Ser Ser Leu Arg Asp

65 70 75 80

Ile Leu Asn Gln Ile Thr Lys Pro Asn Asp Val Tyr Ser Ser Leu Ala

85 90 95

Ser Arg Leu Tyr Ala Glu Glu Arg Tyr Pro Ile Leu Pro Glu Tyr Leu

100 105 110

Gln Cys Val Lys Glu Leu Tyr Arg Gly Gly Leu Glu Pro Ile Asn Gln

115 120 125

Thr Ala Ala Asp Gln Ala Arg Glu Leu Ile Asn Ser Trp Val Glu Ser

130 135 140

Gln Thr Asn Gly Ile Ile Arg Asn Val Leu Gln Pro Ser Ser Val Asp

145 150 155 160

Ser Gln Thr Ala Met Val Leu Val Asn Ala Ile Val Lys Gly Leu Trp

165 170 175

Glu Lys Thr Lys Asp Glu Asp Thr Gln Ala Met Pro Arg Val Thr Glu

180 185 190

Gln Glu Ser Lys Pro Val Gln Met Met Tyr Gln Ile Gly Leu Arg Val

195 200 205

Ala Ser Met Ala Ser Glu Lys Met Lys Ile Leu Glu Leu Pro Ala Ser

210 215 220

Gly Thr Met Ser Met Leu Val Leu Leu Pro Asp Glu Val Ser Gly Leu

225 230 235 240

Glu Gln Leu Glu Ser Ile Ile Asn Glu Lys Leu Thr Glu Trp Thr Ser

245 250 255

Ser Asn Val Met Glu Glu Arg Lys Ile Lys Val Tyr Leu Pro Arg Met

260 265 270

Lys Met Glu Glu Lys Tyr Asn Leu Thr Ser Val Leu Met Ala Met Gly

275 280 285

Ile Thr Asp Val Ser Ser Ser Ala Asn Leu Ser Gly Ile Ser Ser Ala

290 295 300

Glu Ser Leu Lys Ile Ser Gln Ala Val His Ala Ala His Ala Glu Ile

305 310 315 320

Asn Glu Ala Gly Arg Glu Val Val Gly Ser Ala Glu Ala Gly Val Asp

325 330 335

Ala Ala Ser Val Ser Glu Glu Arg Ala Asp His Pro Leu Cys Ile Lys

340 345 350

His Ile Ala Thr Asn Ala Val Leu Gly Arg Cys Val Ser Pro

355 360 365

Met Gly Ser Ile Gly Ala Ala Ser Met Glu Tyr Cys Tyr Asp Val Tyr

1 5 10 15

Lys Glu Leu Lys Val His His Ala Asn Glu Asn Ile Tyr Tyr Cys Pro

20 25 30

Ile Ala Ile Met Ser Ala Leu Ala Met Val Tyr Leu Gly Ala Lys Asp

35 40 45

Ser Thr Arg Thr Gln Ile Asn Lys Val Val Arg Tyr Asp Lys Leu Pro

50 55 60

Gly Tyr Gly Asp Ser Ile Glu Ala Gln Cys Gly Thr Ser Val Asn Val

65 70 75 80

His Ser Ser Leu Arg Asp Ile Leu Asn Gln Ile Thr Lys Pro Asn Asp

85 90 95

Val Tyr Ser Tyr Ser Leu Ala Ser Arg Leu Tyr Ala Glu Glu Arg Tyr

100 105 110

Pro Ile Leu Pro Glu Tyr Leu Gln Cys Val Lys Glu Leu Tyr Arg Gly

115 120 125

Gly Leu Glu Pro Ile Asn Tyr Gln Thr Ala Ala Asp Gln Ala Arg Glu

130 135 140

Leu Ile Asn Ser Trp Val Glu Ser Gln Thr Asn Gly Ile Ile Arg Asn

145 150 155 160

Val Leu Gln Pro Ser Ser Val Asp Ser Gln Thr Ala Met Val Leu Val

165 170 175

Asn Ala Ile Val Tyr Lys Gly Leu Trp Glu Lys Thr Tyr Lys Asp Glu

180 185 190

Asp Thr Gln Ala Met Pro Tyr Arg Val Thr Glu Gln Glu Ser Lys Pro

195 200 205

Val Gln Met Met Tyr Gln Ile Gly Leu Tyr Arg Val Ala Ser Met Ala

210 215 220

Ser Glu Lys Met Lys Ile Leu Glu Leu Pro Tyr Ala Ser Gly Thr Met

225 230 235 240

Ser Met Leu Val Leu Leu Pro Asp Glu Val Ser Gly Leu Glu Gln Leu

245 250 255

Glu Ser Ile Ile Asn Tyr Glu Lys Leu Thr Glu Trp Thr Ser Ser Asn

260 265 270

Val Met Glu Glu Arg Lys Ile Lys Val Tyr Leu Pro Arg Met Lys Met

275 280 285

Glu Glu Lys Tyr Asn Leu Thr Ser Val Leu Met Ala Met Gly Ile Thr

290 295 300

Asp Val Tyr Ser Ser Ser Ala Asn Leu Ser Gly Ile Ser Ser Ala Glu

305 310 315 320

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

325 330 335

Glu Ala Gly Arg Glu Val Val Gly Ser Ala Glu Ala Gly Val Asp Ala

340 345 350

Ala Ser Val Ser Glu Glu Tyr Arg Ala Asp His Pro Tyr Leu Tyr Cys

355 360 365

Ile Lys His Ile Ala Thr Asn Ala Val Leu Tyr Tyr Gly Arg Cys Val

370 375 380

Ser Pro

385

ATGGGTTCTA TTGGTGCTGC TTCTATGGAA TGTGATGTTA AAGAATTGAA AGTTCATCAT 60

GCTAATGAAA ATATTTATTG TCCAATTGCT ATTATGTCTG CTTTGGCTAT GGTTTATTTG 120

GGTGCTAAAG ATTCTACTAG AACTCAAATT AATAAAGTTG TTAGAGATAA ATTGCCAGGT 180

GGTGATTCTA TTGAAGCTCA ATGTGGTACT TCTGTTAATG TTCATTCTTC TTTGAGAGAT 240

ATTTTGAATC AAATTACTAA ACCAAATGAT GTTTATTCTT CTTTGGCTTC TAGATTGTAT 300

GCTGAAGAAA GATATCCAAT TTTGCCAGAA TATTTGCAAT GTGTTAAAGA ATTGTATAGA 360

GGTGGTTTGG AACCAATTAA TCAAACTGCT GCTGATCAAG CTAGAGAATT GATTAATTCT 420

TGGGTTGAAT CTCAAACTAA TGGTATTATT AGAAATGTTT TGCAACCATC TTCTGTTGAT 480

TCTCAAACTG CTATGGTTTT GGTTAATGCT ATTGTTAAAG GTTTGTGGGA AAAAACTAAA 540

GATGAAGATA CTCAAGCTAT GCCAAGAGTT ACTGAACAAG AATCTAAACC AGTTCAAATG 600

ATGTATCAAA TTGGTTTGAG AGTTGCTTCT ATGGCTTCTG AAAAAATGAA AATTTTGGAA 660

TTGCCAGCTT CTGGTACTAT GTCTATGTTG GTTTTGTTGC CAGATGAAGT TTCTGGTTTG 720

GAACAATTGG AATCTATTAT TAATGAAAAA TTGACTGAAT GGACTTCTTC TAATGTTATG 780

GAAGAAAGAA AAATTAAAGT TTATTTGCCA AGAATGAAAA TGGAAGAAAA ATATAATTTG 840

ACTTCTGTTT TGATGGCTAT GGGTATTACT GATGTTTCTT CTTCTGCTAA TTTGTCTGGT 900

ATTTCTTCTG CTGAATCTTT GAAAATTTCT CAAGCTGTTC ATGCTGCTCA TGCTGAAATT 960

AATGAAGCTG GTAGAGAAGT TGTTGGTTCT GCTGAAGCTG GTGTTGATGC TGCTTCTGTT 1020

TCTGAAGAAA GAGCTGATCA TCCATTGTGT ATTAAACATA TTGCTACTAA TGCTGTTTTG 1080

GGTAGATGTG TTTCTCCA 1098

ATGGGTTCTA TTGGTGCTGC TTCTATGGAA TATTGTTATG ATGTTTATAA AGAATTGAAA 60

GTTCATCATG CTAATGAAAA TATTTATTAT TGTCCAATTG CTATTATGTC TGCTTTGGCT 120

ATGGTTTATT TGGGTGCTAA AGATTCTACT AGAACTCAAA TTAATAAAGT TGTTAGATAT 180

GATAAATTGC CAGGTTATGG TGATTCTATT GAAGCTCAAT GTGGTACTTC TGTTAATGTT 240

CATTCTTCTT TGAGAGATAT TTTGAATCAA ATTACTAAAC CAAATGATGT TTATTCTTAT 300

TCTTTGGCTT CTAGATTGTA TGCTGAAGAA AGATATCCAA TTTTGCCAGA ATATTTGCAA 360

TGTGTTAAAG AATTGTATAG AGGTGGTTTG GAACCAATTA ATTATCAAAC TGCTGCTGAT 420

CAAGCTAGAG AATTGATTAA TTCTTGGGTT GAATCTCAAA CTAATGGTAT TATTAGAAAT 480

GTTTTGCAAC CATCTTCTGT TGATTCTCAA ACTGCTATGG TTTTGGTTAA TGCTATTGTT 540

TATAAAGGTT TGTGGGAAAA AACTTATAAA GATGAAGATA CTCAAGCTAT GCCATATAGA 600

GTTACTGAAC AAGAATCTAA ACCAGTTCAA ATGATGTATC AAATTGGTTT GTATAGAGTT 660

GCTTCTATGG CTTCTGAAAA AATGAAAATT TTGGAATTGC CATATGCTTC TGGTACTATG 720

TCTATGTTGG TTTTGTTGCC AGATGAAGTT TCTGGTTTGG AACAATTGGA ATCTATTATT 780

AATTATGAAA AATTGACTGA ATGGACTTCT TCTAATGTTA TGGAAGAAAG AAAAATTAAA 840

GTTTATTTGC CAAGAATGAA AATGGAAGAA AAATATAATT TGACTTCTGT TTTGATGGCT 900

ATGGGTATTA CTGATGTTTA TTCTTCTTCT GCTAATTTGT CTGGTATTTC TTCTGCTGAA 960

TCTTTGAAAA TTTCTCAAGC TGTTCATGCT GCTCATGCTG AAATTAATGA AGCTGGTAGA 1020

GAAGTTGTTG GTTCTGCTGA AGCTGGTGTT GATGCTGCTT CTGTTTCTGA AGAATATAGA 1080

GCTGATCATC CATATTTGTA TTGTATTAAA CATATTGCTA CTAATGCTGT TTTGTATTAT 1140

GGTAGATGTG TTTCTCCA 1158

CTGCAGGATC CCGGGAATTC AAGCTTATGG GTTCTATTGG TGCTGCTTCT ATGGAATGTG 60

ATGTTAAAGA ATTGAAAGTT CATCATGCTA ATGAAAATAT TTATTGTCCA ATTGCTATTA 120

TGTCTGCTTT GGCTATGGTT TATTTGGGTG CTAAAGATTC TACTAGAACT CAAATTAATA 180

›EXAMPLE 3 · 3 of 6

AAGTTGTTAG AGATAAATTG CCAGGTGGTG ATTCTATTGA AGCTCAATGT GGTACTTCTG 240

TTAATGTTCA TTCTTCTTTG AGAGATATTT TGAATCAAAT TACTAAACCA AATGATGTTT 300

ATTCTTCTTT GGCTTCTAGA TTGTATGCTG AAGAAAGATA TCCAATTTTG CCAGAATATT 360

TGCAATGTGT TAAAGAATTG TATAGAGGTG GTTTGGAACC AATTAATCAA ACTGCTGCTG 420

ATCAAGCTAG AGAATTGATT AATTCTTGGG TTGAATCTCA AACTAATGGT ATTATTAGAA 480

ATGTTTTGCA ACCATCTTCT GTTGATTCTC AAACTGCTAT GGTTTTGGTT AATGCTATTG 540

TTAAAGGTTT GTGGGAAAAA ACTAAAGATG AAGATACTCA AGCTATGCCA AGAGTTACTG 600

AACAAGAATC TAAACCAGTT CAAATGATGT ATCAAATTGG TTTGAGAGTT GCTTCTATGG 660

CTTCTGAAAA AATGAAAATT TTGGAATTGC CAGCTTCTGG TACTATGTCT ATGTTGGTTT 720

TGTTGCCAGA TGAAGTTTCT GGTTTGGAAC AATTGGAATC TATTATTAAT GAAAAATTGA 780

CTGAATGGAC TTCTTCTAAT GTTATGGAAG AAAGAAAAAT TAAAGTTTAT TTGCCAAGAA 840

TGAAAATGGA AGAAAAATAT AATTTGACTT CTGTTTTGAT GGCTATGGGT ATTACTGATG 900

TTTCTTCTTC TGCTAATTTG TCTGGTATTT CTTCTGCTGA ATCTTTGAAA ATTTCTCAAG 960

CTGTTCATGC TGCTCATGCT GAAATTAATG AAGCTGGTAG AGAAGTTGTT GGTTCTGCTG 1020

AAGCTGGTGT TGATGCTGCT TCTGTTTCTG AAGAAAGAGC TGATCATCCA TTGTGTATTA 1080

AACATATTGC TACTAATGCT GTTTTGGGTA GATGTGTTTC TCCATAATAA AAAGAAGAAA 1140

GCTGAAAAAC TCTGTCCCTT CCAACAAGAC CCAGAGCACT GTAGTATCAG GGGTAAAATG 1200

AAAAGTATGT TCTCTGCTGC ATCCAGACTT CATAAAAGCT GGAGCTTAAT CTAGAAAAAA 1260

AATCAGAAAG AAATTACACT GTGAGAACAG GTGCAATTCA CTTTTCCTTT ACACAGAGTA 1320

ATACTGGTAA CTCATGGATG AAGGCTTAAG GGAATGAAAT TGGACTCACA GTACTGAGTC 1380

ATCACACTGA AAAATGCAAC CTGATACATC AGCAGAAGGT TTATGGGGGA AAAATGCAGC 1440

CTTCCAATTA AGCCAGATAT CTGTATGACC AAGCTGCTCC AGAATTAGTC ACTCAAAATC 1500

TCTCAGATTA AATTATCAAC TGTCACCAAC CATTCCTATG CTGACAAGGC AATTGCTTGT 1560

TCTCTGTGTT CCTGATACTA CAAGGCTCTT CCTGACTTCC TAAAGATGCA TTATAAAAAT 1620

CTTATAATTC ACATTTCTCC CTAAACTTTG ACTCAATCAT GGTATGTTGG CAAATATGGT 1680

ATATTACTAT TCAAATTGTT TTCCTTGTAC CCATATGTAA TGGGTCTTGT GAATGTGCTC 1740

TTTTGTTCCT TTAATCATAA TAAAAACATG TTTAAGCCTG CAGGATCCCG GGAATTCAAG 1800

CTT 1803

CTGCAGGATC CCGGGAATTC AAGCTTATGC ATCATCATCA TCATCATGGT TCTATTGGTG 60

CTGCTTCTAT GGAATGTGAT GTTAAAGAAT TGAAAGTTCA TCATGCTAAT GAAAATATTT 120

ATTGTCCAAT TGCTATTATG TCTGCTTTGG CTATGGTTTA TTTGGGTGCT AAAGATTCTA 180

CTAGAACTCA AATTAATAAA GTTGTTAGAG ATAAATTGCC AGGTGGTGAT TCTATTGAAG 240

CTCAATGTGG TACTTCTGTT AATGTTCATT CTTCTTTGAG AGATATTTTG AATCAAATTA 300

CTAAACCAAA TGATGTTTAT TCTTCTTTGG CTTCTAGATT GTATGCTGAA GAAAGATATC 360

CAATTTTGCC AGAATATTTG CAATGTGTTA AAGAATTGTA TAGAGGTGGT TTGGAACCAA 420

TTAATCAAAC TGCTGCTGAT CAAGCTAGAG AATTGATTAA TTCTTGGGTT GAATCTCAAA 480

CTAATGGTAT TATTAGAAAT GTTTTGCAAC CATCTTCTGT TGATTCTCAA ACTGCTATGG 540

TTTTGGTTAA TGCTATTGTT AAAGGTTTGT GGGAAAAAAC TAAAGATGAA GATACTCAAG 600

CTATGCCAAG AGTTACTGAA CAAGAATCTA AACCAGTTCA AATGATGTAT CAAATTGGTT 660

TGAGAGTTGC TTCTATGGCT TCTGAAAAAA TGAAAATTTT GGAATTGCCA GCTTCTGGTA 720

CTATGTCTAT GTTGGTTTTG TTGCCAGATG AAGTTTCTGG TTTGGAACAA TTGGAATCTA 780

TTATTAATGA AAAATTGACT GAATGGACTT CTTCTAATGT TATGGAAGAA AGAAAAATTA 840

AAGTTTATTT GCCAAGAATG AAAATGGAAG AAAAATATAA TTTGACTTCT GTTTTGATGG 900

CTATGGGTAT TACTGATGTT TCTTCTTCTG CTAATTTGTC TGGTATTTCT TCTGCTGAAT 960

CTTTGAAAAT TTCTCAAGCT GTTCATGCTG CTCATGCTGA AATTAATGAA GCTGGTAGAG 1020

AAGTTGTTGG TTCTGCTGAA GCTGGTGTTG ATGCTGCTTC TGTTTCTGAA GAAAGAGCTG 1080

ATCATCCATT GTGTATTAAA CATATTGCTA CTAATGCTGT TTTGGGTAGA TGTGTTTCTC 1140

CATAATAAAA AGAAGAAAGC TGAAAAACTC TGTCCCTTCC AACAAGACCC AGAGCACTGT 1200

AGTATCAGGG GTAAAATGAA AAGTATGTTC TCTGCTGCAT CCAGACTTCA TAAAAGCTGG 1260

AGCTTAATCT AGAAAAAAAA TCAGAAAGAA ATTACACTGT GAGAACAGGT GCAATTCACT 1320

TTTCCTTTAC ACAGAGTAAT ACTGGTAACT CATGGATGAA GGCTTAAGGG AATGAAATTG 1380

GACTCACAGT ACTGAGTCAT CACACTGAAA AATGCAACCT GATACATCAG CAGAAGGTTT 1440

ATGGGGGAAA AATGCAGCCT TCCAATTAAG CCAGATATCT GTATGACCAA GCTGCTCCAG 1500

AATTAGTCAC TCAAAATCTC TCAGATTAAA TTATCAACTG TCACCAACCA TTCCTATGCT 1560

GACAAGGCAA TTGCTTGTTC TCTGTGTTCC TGATACTACA AGGCTCTTCC TGACTTCCTA 1620

AAGATGCATT ATAAAAATCT TATAATTCAC ATTTCTCCCT AAACTTTGAC TCAATCATGG 1680

TATGTTGGCA AATATGGTAT ATTACTATTC AAATTGTTTT CCTTGTACCC ATATGTAATG 1740

GGTCTTGTGA ATGTGCTCTT TTGTTCCTTT AATCATAATA AAAACATGTT TAAGCCTGCA 1800

GGATCCCGGG AATTCAAGCT T 1821

CTGCAGGATC CCGGGAATTC AAGCTTATGG GTTCTATTGG TGCTGCTTCT ATGGAATATT 60

GTTATGATGT TTATAAAGAA TTGAAAGTTC ATCATGCTAA TGAAAATATT TATTATTGTC 120

CAATTGCTAT TATGTCTGCT TTGGCTATGG TTTATTTGGG TGCTAAAGAT TCTACTAGAA 180

CTCAAATTAA TAAAGTTGTT AGATATGATA AATTGCCAGG TTATGGTGAT TCTATTGAAG 240

CTCAATGTGG TACTTCTGTT AATGTTCATT CTTCTTTGAG AGATATTTTG AATCAAATTA 300

CTAAACCAAA TGATGTTTAT TCTTATTCTT TGGCTTCTAG ATTGTATGCT GAAGAAAGAT 360

ATCCAATTTT GCCAGAATAT TTGCAATGTG TTAAAGAATT GTATAGAGGT GGTTTGGAAC 420

CAATTAATTA TCAAACTGCT GCTGATCAAG CTAGAGAATT GATTAATTCT TGGGTTGAAT 480

CTCAAACTAA TGGTATTATT AGAAATGTTT TGCAACCATC TTCTGTTGAT TCTCAAACTG 540

CTATGGTTTT GGTTAATGCT ATTGTTTATA AAGGTTTGTG GGAAAAAACT TATAAAGATG 600

AAGATACTCA AGCTATGCCA TATAGAGTTA CTGAACAAGA ATCTAAACCA GTTCAAATGA 660

TGTATCAAAT TGGTTTGTAT AGAGTTGCTT CTATGGCTTC TGAAAAAATG AAAATTTTGG 720

AATTGCCATA TGCTTCTGGT ACTATGTCTA TGTTGGTTTT GTTGCCAGAT GAAGTTTCTG 780

GTTTGGAACA ATTGGAATCT ATTATTAATT ATGAAAAATT GACTGAATGG ACTTCTTCTA 840

ATGTTATGGA AGAAAGAAAA ATTAAAGTTT ATTTGCCAAG AATGAAAATG GAAGAAAAAT 900

ATAATTTGAC TTCTGTTTTG ATGGCTATGG GTATTACTGA TGTTTATTCT TCTTCTGCTA 960

ATTTGTCTGG TATTTCTTCT GCTGAATCTT TGAAAATTTC TCAAGCTGTT CATGCTGCTC 1020

ATGCTGAAAT TAATGAAGCT GGTAGAGAAG TTGTTGGTTC TGCTGAAGCT GGTGTTGATG 1080

CTGCTTCTGT TTCTGAAGAA TATAGAGCTG ATCATCCATA TTTGTATTGT ATTAAACATA 1140

TTGCTACTAA TGCTGTTTTG TATTATGGTA GATGTGTTTC TCCATAATAA AAAGAAGAAA 1200

GCTGAAAAAC TCTGTCCCTT CCAACAAGAC CCAGAGCACT GTAGTATCAG GGGTAAAATG 1260

AAAAGTATGT TCTCTGCTGC ATCCAGACTT CATAAAAGCT GGAGCTTAAT CTAGAAAAAA 1320

AATCAGAAAG AAATTACACT GTGAGAACAG GTGCAATTCA CTTTTCCTTT ACACAGAGTA 1380

ATACTGGTAA CTCATGGATG AAGGCTTAAG GGAATGAAAT TGGACTCACA GTACTGAGTC 1440

ATCACACTGA AAAATGCAAC CTGATACATC AGCAGAAGGT TTATGGGGGA AAAATGCAGC 1500

CTTCCAATTA AGCCAGATAT CTGTATGACC AAGCTGCTCC AGAATTAGTC ACTCAAAATC 1560

TCTCAGATTA AATTATCAAC TGTCACCAAC CATTCCTATG CTGACAAGGC AATTGCTTGT 1620

TCTCTGTGTT CCTGATACTA CAAGGCTCTT CCTGACTTCC TAAAGATGCA TTATAAAAAT 1680

CTTATAATTC ACATTTCTCC CTAAACTTTG ACTCAATCAT GGTATGTTGG CAAATATGGT 1740

›EXAMPLE 3 · 4 of 6

ATATTACTAT TCAAATTGTT TTCCTTGTAC CCATATGTAA TGGGTCTTGT GAATGTGCTC 1800

TTTTGTTCCT TTAATCATAA TAAAAACATG TTTAAGCCTG CAGGATCCCG GGAATTCAAG 1860

CTT 1863

CTGCAGGATC CCGGGAATTC AAGCTTATGC ATCATCATCA TCATCATGGT TCTATTGGTG 60

CTGCTTCTAT GGAATATTGT TATGATGTTT ATAAAGAATT GAAAGTTCAT CATGCTAATG 120

AAAATATTTA TTATTGTCCA ATTGCTATTA TGTCTGCTTT GGCTATGGTT TATTTGGGTG 180

CTAAAGATTC TACTAGAACT CAAATTAATA AAGTTGTTAG ATATGATAAA TTGCCAGGTT 240

ATGGTGATTC TATTGAAGCT CAATGTGGTA CTTCTGTTAA TGTTCATTCT TCTTTGAGAG 300

ATATTTTGAA TCAAATTACT AAACCAAATG ATGTTTATTC TTATTCTTTG GCTTCTAGAT 360

TGTATGCTGA AGAAAGATAT CCAATTTTGC CAGAATATTT GCAATGTGTT AAAGAATTGT 420

ATAGAGGTGG TTTGGAACCA ATTAATTATC AAACTGCTGC TGATCAAGCT AGAGAATTGA 480

TTAATTCTTG GGTTGAATCT CAAACTAATG GTATTATTAG AAATGTTTTG CAACCATCTT 540

CTGTTGATTC TCAAACTGCT ATGGTTTTGG TTAATGCTAT TGTTTATAAA GGTTTGTGGG 600

AAAAAACTTA TAAAGATGAA GATACTCAAG CTATGCCATA TAGAGTTACT GAACAAGAAT 660

CTAAACCAGT TCAAATGATG TATCAAATTG GTTTGTATAG AGTTGCTTCT ATGGCTTCTG 720

AAAAAATGAA AATTTTGGAA TTGCCATATG CTTCTGGTAC TATGTCTATG TTGGTTTTGT 780

TGCCAGATGA AGTTTCTGGT TTGGAACAAT TGGAATCTAT TATTAATTAT GAAAAATTGA 840

CTGAATGGAC TTCTTCTAAT GTTATGGAAG AAAGAAAAAT TAAAGTTTAT TTGCCAAGAA 900

TGAAAATGGA AGAAAAATAT AATTTGACTT CTGTTTTGAT GGCTATGGGT ATTACTGATG 960

TTTATTCTTC TTCTGCTAAT TTGTCTGGTA TTTCTTCTGC TGAATCTTTG AAAATTTCTC 1020

AAGCTGTTCA TGCTGCTCAT GCTGAAATTA ATGAAGCTGG TAGAGAAGTT GTTGGTTCTG 1080

CTGAAGCTGG TGTTGATGCT GCTTCTGTTT CTGAAGAATA TAGAGCTGAT CATCCATATT 1140

TGTATTGTAT TAAACATATT GCTACTAATG CTGTTTTGTA TTATGGTAGA TGTGTTTCTC 1200

CATAATAAAA AGAAGAAAGC TGAAAAACTC TGTCCCTTCC AACAAGACCC AGAGCACTGT 1260

AGTATCAGGG GTAAAATGAA AAGTATGTTC TCTGCTGCAT CCAGACTTCA TAAAAGCTGG 1320

AGCTTAATCT AGAAAAAAAA TCAGAAAGAA ATTACACTGT GAGAACAGGT GCAATTCACT 1380

TTTCCTTTAC ACAGAGTAAT ACTGGTAACT CATGGATGAA GGCTTAAGGG AATGAAATTG 1440

GACTCACAGT ACTGAGTCAT CACACTGAAA AATGCAACCT GATACATCAG CAGAAGGTTT 1500

ATGGGGGAAA AATGCAGCCT TCCAATTAAG CCAGATATCT GTATGACCAA GCTGCTCCAG 1560

AATTAGTCAC TCAAAATCTC TCAGATTAAA TTATCAACTG TCACCAACCA TTCCTATGCT 1620

GACAAGGCAA TTGCTTGTTC TCTGTGTTCC TGATACTACA AGGCTCTTCC TGACTTCCTA 1680

AAGATGCATT ATAAAAATCT TATAATTCAC ATTTCTCCCT AAACTTTGAC TCAATCATGG 1740

TATGTTGGCA AATATGGTAT ATTACTATTC AAATTGTTTT CCTTGTACCC ATATGTAATG 1800

GGTCTTGTGA ATGTGCTCTT TTGTTCCTTT AATCATAATA AAAACATGTT TAAGCCTGCA 1860

GGATCCCGGG AATTCAAGCT T 1881

Met Arg Pro Lys His Pro Ile Lys His Gln Gly Leu Pro Gln Glu Val

1 5 10 15

Leu Asn Glu Asn Leu Leu Arg Val Ala Pro Pro Glu Val Gly Lys Glu

20 25 30

Lys Val Asn Glu Leu Ser Lys Asp Ile Gly Ser Glu Ser Thr Glu Asp

35 40 45

Gln Ala Met Glu Asp Ile Lys Gln Met Glu Ala Glu Ser Ile Ser Ser

50 55 60

Ser Glu Glu Ile Val Pro Asn Ser Val Glu Gln Lys His Ile Gln Lys

65 70 75 80

Glu Asp Val Pro Ser Glu Arg Tyr Leu Gly Tyr Leu Glu Gln Leu Leu

85 90 95

Arg Leu Lys Lys Tyr Lys Val Pro Gln Leu Glu Ile Val Pro Asn Ser

100 105 110

Ala Glu Glu Arg Leu His Ser Met Lys Glu Gly Ile His Ala Gln Gln

115 120 125

Lys Glu Pro Met Ile Gly Val Asn Gln Glu Leu Ala Tyr Tyr Pro Glu

130 135 140

Leu Arg Gln Tyr Gln Leu Asp Ala Tyr Pro Ser Gly Ala Trp Tyr Tyr

145 150 155 160

Val Pro Leu Gly Thr Gln Tyr Thr Asp Ala Pro Ser Ser Asp Ile Pro

165 170 175

Asn Pro Ile Gly Ser Glu Asn Ser Glu Lys Thr Thr Met Pro Leu Trp

180 185 190

ATGAGACCAA AACATCCAAT TAAACATCAA GGTTTGCCAC AAGAAGTTTT GAATGAAAAT 60

TTGTTGAGAG TTGCTCCACC AGAAGTTGGT AAAGAAAAAG TTAATGAATT GTCTAAAGAT 120

ATTGGTTCTG AATCTACTGA AGATCAAGCT ATGGAAGATA TTAAACAAAT GGAAGCTGAA 180

TCTATTTCTT CTTCTGAAGA AATTGTTCCA AATTCTGTTG AACAAAAACA TATTCAAAAA 240

GAAGATGTTC CATCTGAAAG ATATTTGGGT TATTTGGAAC AATTGTTGAG ATTGAAAAAA 300

TATAAAGTTC CACAATTGGA AATTGTTCCA AATTCTGCTG AAGAAAGATT GCATTCTATG 360

AAAGAAGGTA TTCATGCTCA ACAAAAAGAA CCAATGATTG GTGTTAATCA AGAATTGGCT 420

TATTATCCAG AATTGAGACA ATATCAATTG GATGCTTATC CATCTGGTGC TTGGTATTAT 480

GTTCCATTGG GTACTCAATA TACTGATGCT CCATCTTCTG ATATTCCAAA TCCAATTGGT 540

TCTGAAAATT CTGAAAAAAC TACTATGCCA TTGTGGTGAT GA 582

CTGCAGGATC CCGGGAATTC TAGAAGCTTA TGAGACCAAA ACATCCAATT AAACATCAAG 60

GTTTGCCACA AGAAGTTTTG AATGAAAATT TGTTGAGAGT TGCTCCACCA GAAGTTGGTA 120

AAGAAAAAGT TAATGAATTG TCTAAAGATA TTGGTTCTGA ATCTACTGAA GATCAAGCTA 180

TGGAAGATAT TAAACAAATG GAAGCTGAAT CTATTTCTTC TTCTGAAGAA ATTGTTCCAA 240

ATTCTGTTGA ACAAAAACAT ATTCAAAAAG AAGATGTTCC ATCTGAAAGA TATTTGGGTT 300

ATTTGGAACA ATTGTTGAGA TTGAAAAAAT ATAAAGTTCC ACAATTGGAA ATTGTTCCAA 360

ATTCTGCTGA AGAAAGATTG CATTCTATGA AAGAAGGTAT TCATGCTCAA CAAAAAGAAC 420

CAATGATTGG TGTTAATCAA GAATTGGCTT ATTATCCAGA ATTGAGACAA TATCAATTGG 480

ATGCTTATCC ATCTGGTGCT TGGTATTATG TTCCATTGGG TACTCAATAT ACTGATGCTC 540

CATCTTCTGA TATTCCAAAT CCAATTGGTT CTGAAAATTC TGAAAAAACT ACTATGCCAT 600

TGTGGTGATG AAAGAGTCAA GTGAATTCTG AGGGACTCCA CAGTTATGGT CTTTGATGGG 660

TCTGAAAATT CCATGCTCTA CATGTCGCCT CATCTACATG TCAAACCATT CATCCAAAGG 720

CTTCAACTGC TGTTTTAGAA CAGGGCAATC TCAAACTGAG GCACTCCTTG ATGCTCTACT 780

GTATTTTAGA TAGTGTAACA TCCTTAAGTG AAATTGTCCT AACAGCTTGT TACCTAAATT 840

CCAGTAGTAT CATGCTGGTA TAAAGGCCAC TGAGTCAAAG GGAATTAAAG TCTTCATTAA 900

ATTTCTGTAT GGAAAATGTT TTAAAAGCCT TTGAATCACT TCTCCTGTAA GTGCCATCAT 960

ATCAAATAAT TGTGTGCATT AACTGAGATT TTGTCTTTCT TCTTTTCAAT AAATTACATT 1020

TTAAGGCCTG CAGGATCCCG GGAATTCTAG AAGCTT 1056

AATCAGGATC CCGGGCATAT GAGACCAAAA CATCCAATTA AACATCAAGG TTTGCCACAA 60

GAAGTTTTGA ACGAAAACTT GTTGAGATAC TACGTTGCTC CATACCCAGA AGTTTACGGT 120

AAAGAAAAAG TTAATGAATT GTCTAAAGAT ATCGGTTCTG AATCTACTGA AGATCAAGCC 180

ATGGAAGATA TTAAACAAAT GGAAGCTGAA TCTATCTCTT CTTCTGAAGA AATCGTCCCA 240

AACTCTGTTG AACAAAAACA TATTCAAAAA GAAGACGTCC CATCTGAAAG ATACTTGGGT 300

TACTTAGAAC AATTGTTGAG ATTGAAAAAA TACAAAGTTC CACAATTGGA AATCGTCCCA 360

AACTCAGCTG AAGA 374

Asn Gln Asp Pro Gly His Met Arg Pro Lys His Pro Ile Lys His Gln

1 5 10 15

Gly Leu Pro Gln Glu Val Leu Asn Glu Asn Leu Leu Arg Tyr Tyr Val

20 25 30

Ala Pro Tyr Pro Glu Val Tyr Gly Lys Glu Lys Val Asn Glu Leu Ser

35 40 45

Lys Asp Ile Gly Ser Glu Ser Thr Glu Asp Gln Ala Met Glu Asp Ile

50 55 60

Lys Gln Met Glu Ala Glu Ser Ile Ser Ser Ser Glu Glu Ile Val Pro

65 70 75 80

Asn Ser Val Glu Gln Lys His Ile Gln Lys Glu Asp Val Pro Ser Glu

85 90 95

Arg Tyr Leu Gly Tyr Leu Glu Gln Leu Leu Arg Leu Lys Lys Tyr Lys

›EXAMPLE 3 · 5 of 6

100 105 110

Val Pro Gln Leu Glu Ile Val Pro Asn Ser Ala Glu Glu

115 120 125

AATCAGGATC CCGGGCATAT GAGACCAAAA CATCCAATTA AACATCAAGG TTTGCCACAA 60

GAAGTTTTGA ACGAAAACTT GTTGAGATAC TACGTTGCTC CATACCCAGA AGTTTACGGT 120

AAAGAAAAAG TTAATGAATT GTCTAAAGAT ATCGGTTCTG AATCTACTGA AGATCAAGCC 180

ATGGAAGATA TTAAACAAAT GGAAGCTGAA TCTATCTCTT CTTCTGAAGA AATCGTCCCA 240

AACTCTGAAC AAAAACATAT TCAAAAAGAA GACGTCCCAT CTGAAAGATA CTTGGGTTAC 300

TTAGAACAAT TGTTGAGATT GAAAAAATAC AAAGTTCCAC AATTGGAAAT CGTCCCAAAC 360

TCAGCTGAAG A 371

Asn Gln Asp Pro Gly His Met Arg Pro Lys His Pro Ile Lys His Gln

1 5 10 15

Gly Leu Pro Gln Glu Val Leu Asn Glu Asn Leu Leu Arg Tyr Tyr Val

20 25 30

Ala Pro Tyr Pro Glu Val Tyr Gly Lys Glu Lys Val Asn Glu Leu Ser

35 40 45

Lys Asp Ile Gly Ser Glu Ser Thr Glu Asp Gln Ala Met Glu Asp Ile

50 55 60

Lys Gln Met Glu Ala Glu Ser Ile Ser Ser Ser Glu Glu Ile Val Pro

65 70 75 80

Asn Ser Glu Gln Lys His Ile Gln Lys Glu Asp Val Pro Ser Glu Arg

85 90 95

Tyr Leu Gly Tyr Leu Glu Gln Leu Leu Arg Leu Lys Lys Tyr Lys Val

100 105 110

Pro Gln Leu Glu Ile Val Pro Asn Ser Ala Glu Glu

115 120

CAAAAAGAAG ACGTCCCATC TGAAAGATAC TTGGGTTACT TAGAACAATT GTTGAGATTG 60

AAAAAATACA AAGTTCCACA ATTGGAAATC GTCCCAAACT CAGCTGAAGA AAGATTGCAT 120

TCTATGAAAG AAGGTATTCA TGCTCAACAA AAAGAACCAA TGATTGGTGT TAACCAAGAA 180

TTGGCTTACT ACTACCCAGA ATTGTACAGA CAATACTATC AATTGGATGC TTACCCATCT 240

GGTGCTTGGT ACTACGTTCC TTTAGGTACC CAATACACTG ATGCTCCATC TTACTCTGAT 300

ATTCCAAACC CAATCGGTTC TGAAAACTCT GAAAAAACTA CTATGCCATT GTGGTAATAA 360

GCTTCATGAT C 371

Gln Lys Glu Asp Val Pro Ser Glu Arg Tyr Leu Gly Tyr Leu Glu Gln

1 5 10 15

Leu Leu Arg Leu Lys Lys Tyr Lys Val Pro Gln Leu Glu Ile Val Pro

20 25 30

Asn Ser Ala Glu Glu Arg Leu His Ser Met Lys Glu Gly Ile His Ala

35 40 45

Gln Gln Lys Glu Pro Met Ile Gly Val Asn Gln Glu Leu Ala Tyr Tyr

50 55 60

Tyr Pro Glu Leu Tyr Arg Gln Tyr Tyr Gln Leu Asp Ala Tyr Pro Ser

65 70 75 80

Gly Ala Trp Tyr Tyr Val Pro Leu Gly Thr Gln Tyr Thr Asp Ala Pro

85 90 95

Ser Tyr Ser Asp Ile Pro Asn Pro Ile Gly Ser Glu Asn Ser Glu Lys

100 105 110

Thr Thr Met Pro Leu Trp

115

CAAAAAGAAG ACGTCCCATC TGAAAGATAC TTGGGTTACT TAGAACAATT GTTGAGATTG 60

AAAAAATACA AAGTTCCACA ATTGGAAATC GTCCCAAACT CAGCTGAAGA AAGATTGCAT 120

TCTATGAAAA GAAGGTATTC ATGCTCAACA AAAAGAACCA ATGATTGGTG TTAACCAAGA 180

ATTGGCTTAC TACTACCCAG AATTGTACAG ACAATACTAT CAATTGATGC TTACCCATCT 240

GGTGCTTTGT ACTACGTTCC TTTAGGTACC CAATACACTG ATGCTCCATC TTACTCTGAT 300

ATTCCAAACC CAATCGGTTC TGAAAACTCT GAAAAAACTA CTATGCCATT GTGGTAATAA 360

GCTTCATGAT C 371

Gln Lys Glu Asp Val Pro Ser Glu Arg Tyr Leu Gly Tyr Leu Glu Gln

1 5 10 15

Leu Leu Arg Leu Lys Lys Tyr Lys Val Pro Gln Leu Glu Ile Val Pro

20 25 30

Asn Ser Ala Glu Glu Arg Leu His Ser Met Lys Glu Gly Ile His Ala

35 40 45

Gln Gln Lys Glu Pro Met Ile Gly Val Asn Gln Glu Leu Ala Tyr Tyr

50 55 60

Tyr Pro Glu Leu Tyr Arg Gln Tyr Tyr Gln Leu Asp Ala Tyr Pro Ser

65 70 75 80

Gly Ala Leu Tyr Tyr Val Pro Leu Gly Thr Gln Tyr Thr Asp Ala Pro

85 90 95

Ser Tyr Ser Asp Ile Pro Asn Pro Ile Gly Ser Glu Asn Ser Glu Lys

100 105 110

Thr Thr Met Pro Leu Trp

115

AATCAGGATC CCGGGCATAT GAGACCAAAA CATCCAATTA AACATCAAGG TTTGCCACAA 60

GAAGTTTTGA ACGAAAACTT GTTGAGATAC TACGTTGCTC CATACCCAGA AGTTTACGGT 120

AAAGAAAAAG TTAATGAATT GTCTAAAGAT ATCGGTTCTG AATCTACTGA AGATCAAGCC 180

ATGGAAGATA TTAAACAAAT GGAAGCTGAA TCTATCTCTT CTTCTGAAGA AATCGTCCCA 240

AACTCTGAAC AAAAACATAT TCAAAAAGAA GACGTCCCAT CTGAAAGATA CTTGGGTTAC 300

TTAGAACAAT TGTTGAGATT GAAAAAATAC AAAGTTCCAC CATTGGAAAT CGTCCCAAAC 360

TCAGCTGAAG AAAGATTGCA TTCTATGAAA GAAGGTATTC ATGCTCAACA AAAAGAACCA 420

ATGATTGGTG TTAACCAAGA ATTGGCTTAC TACTACCCAG AATTGTACAG ACAATACTAT 480

CAATTGGATG CTTACCCATC TGGTGCTTTG TACTACGTTC CTTTAGGTAC CCAATACACT 540

GATGCTCCAT CTTACTCTGA TATTCCAAAC CCAATCGGTT CTGAAAACTC TGAAAAAACT 600

ACTATGCCAT TGTGGTAATA AGCTTCATGA TC 632

Asn Gln Asp Pro Gly His Asn Arg Pro Lys His Pro Ile Lys His Gln

1 5 10 15

Gly Leu Pro Gln Glu Val Leu Asn Glu Asn Leu Leu Arg Tyr Tyr Val

20 25 30

Ala Pro Tyr Pro Glu Val Tyr Gly Lys Glu Lys Val Asn Glu Leu Ser

35 40 45

Lys Asp Ile Gly Ser Glu Ser Thr Glu Asp Gln Ala Asn Glu Asp Ile

50 55 60

Lys Gln Asn Glu Ala Glu Ser Ile Ser Ser Ser Glu Glu Ile Val Pro

65 70 75 80

Asn Ser Glu Gln Lys His Ile Gln Lys Glu Asp Val Pro Ser Glu Arg

85 90 95

Tyr Leu Gly Tyr Leu Glu Gln Leu Leu Arg Leu Lys Lys Tyr Lys Val

100 105 110

Pro Gln Leu Glu Ile Val Pro Asn Ser Ala Glu Glu Arg Leu His Ser

115 120 125

Asn Lys Glu Gly Ile His Ala Gln Gln Lys Glu Pro Asn Ile Gly Val

130 135 140

Asn Gln Glu Leu Ala Tyr Tyr Tyr Pro Glu Leu Tyr Arg Gln Tyr Tyr

145 150 155 160

Gln Leu Asp Ala Tyr Pro Ser Gly Ala Leu Tyr Tyr Val Pro Leu Gly

165 170 175

Thr Gln Tyr Thr Asp Ala Pro Ser Tyr Ser Asp Ile Pro Asn Pro Ile

180 185 190

Gly Ser Glu Asn Ser Glu Lys Thr

195 200

AATCAGGATC CCGGGCTGCA GAGACCAAAA CATCCAATTA AACATCAAGG TTTGCCACAA 60

GAAGTTTTGA ACGAAAACTT GTTGAGATAC TACGTTGCTC CATACCCAGA AGTTTACGGT 120

AAAGAAAAAG TTAATGAATT GTCTAAAGAT ATCGGTTCTG AATCTACTGA AGATCAAGCC 180

ATGGAAGATA TTAAACAAAT GGAAGCTGAA TCTATCTCTT CTTCTGAAGA AATCGTCCCA 240

AACTCTGAAC AAAAACATAT TCAAAAAGAA GACGTCCCAT CTGAAAGATA CTTGGGTTAC 300

TTAGAACAAT TGTTGAGATT GAAAAAATAC AAAGTTCCAC AATTGGAAAT CGTCCCAAAC 360

TCAGCTGAAG AAAGATTGCA TTCTATGAAA GAAGGTATTC ATGCTCAACA AAAAGAACCA 420

ATGATTGGTG TTAACCACGA ATTGGCTTAC TACTACCCAG AATTGTACAG ACAATACTAT 480

CAATTGGATG CTTACCCATC TGGTGCTTTG TACTACGTTC CTTTAGGTAC CCAATACACT 540

GATGCTCCAT CTTACTCTGA TATTCCAAAC CCAATCGGTT CTGAAAACTC TGAAAAAACT 600

ACTATGCCAT TGTGGTAATA AGCTTCATGA TC 632

Asn Gln Asp Pro Gly His Asn Arg Pro Lys His Pro Ile Lys His Gln

1 5 10 15

Gly Leu Pro Gln Glu Val Leu Asn Glu Asn Leu Leu Arg Tyr Tyr Val

20 25 30

Ala Pro Tyr Pro Glu Val Tyr Gly Lys Glu Lys Val Asn Glu Leu Ser

35 40 45

Lys Asp Ile Gly Ser Glu Ser Thr Glu Asp Gln Ala Asn Glu Asp Ile

50 55 60

Lys Gln Asn Glu Ala Glu Ser Ile Ser Ser Ser Glu Glu Ile Val Pro

65 70 75 80

Asn Ser Glu Gln Lys His Ile Gln Lys Glu Asp Val Pro Ser Glu Arg

85 90 95

Tyr Leu Gly Tyr Leu Glu Gln Leu Leu Arg Leu Lys Lys Tyr Lys Val

100 105 110

Pro Gln Leu Glu Ile Val Pro Asn Ser Ala Glu Glu Arg Leu His Ser

115 120 125

Asn Lys Glu Gly Ile His Ala Gln Gln Lys Glu Pro Asn Ile Gly Val

130 135 140

›EXAMPLE 3 · 6 of 6

Asn Gln Glu Leu Ala Tyr Tyr Tyr Pro Glu Leu Tyr Arg Gln Tyr Tyr

145 150 155 160

Gln Leu Asp Ala Tyr Pro Ser Gly Ala Leu Tyr Tyr Val Pro Leu Gly

165 170 175

Thr Gln Tyr Thr Asp Ala Pro Ser Tyr Ser Asp Ile Pro Asn Pro Ile

180 185 190

Gly Ser Glu Asn Ser Glu Lys Thr

195 200

25 base pairs

nucleic acid

single

linear

›DNA

GATCCCGGGC TGCAGAGACC AAAAC 25

26 base pairs

nucleic acid

single

linear

›DNA · 1 of 2

GATCATGAAG CTTATTACCA CAATGG 26

AATCAGGATC CCGGGCATAT 20

AATCAGGATC CCGGGCATAT GAGACCAAAA CATCCAATTA AACATCAAGG TTTGCCACAA 60

GAAGTTTTGA ACGAAAACTT GTTGAGATAC TACGTTGCTC CAT 103

AATATCTTCC ATGGCTTGAT CTTCAGTAGA TTCAGAACCG ATATCTTTAG ACAATTCATT 60

AACTTTTTCT TTACCGTAAA CTTCTGGGTA TGGAGCAACG TAGTATCTC 109

ATCAAGCCAT GGAAGATATT AAACAAATGG AAGCTGAATC TATCTCTTCT TCTGAAGAAA 60

TCGTCCCAAA CTCTGTTGAA CAAAAACATA TTCAAAAAGA AGACGTCCCA TC 112

TCTTCAGCTG AGTTTGGGAC GATTTCCAAT TGTGGAACTT TGTATTTTTT CAATCTCAAC 60

AATTGTTCTA AGTAACCCAA GTATCTTTCA GATGGGACGT CTTCTTTTTG 110

TCTTCAGCTG AGTTTGGGAC 20

CAAAAAGAAG ACGTCCCATC 20

CAAAAAGAAG ACGTCCCATC TGAAAGATAC TTGGGTTACT TAGAACAATT GTTGAGATTG 60

AAAAAATACA AAGTTCCACA ATTGGAAATC GTCCCAAACT CAGCTGAAGA 110

TCTGGGTAGT AGTAAGCCAA TTCTTGGTTA ACACCAATCA TTGGTTCTTT TTGTTGAGCA 60

TGAATACCTT CTTTCATAGA ATGCAATCTT TCTTCAGCTG AGTTTGGAAC 110

TTGGCTTACT ACTACCCAGA ATTGTACAGA CAATACTATC AATTGGATGC TTACCCATCT 60

GGTGCTTGGT ACTACGTTCC TTTAGGTACC CAATACACTG 100

GACTTGATCA TGAAGCTTAT TACCACAATG GCATAGTAGT TTTTTCAGAG TTTTCAGAAC 60

CGATTGGGTT TGGAATATCA GAGTAAGATG GAGCATCAGT GTATTGGGTA CCTAAA 116

GATCATGAAG CTTATTACCA CAATGGCATA GTAGTTTTTT CAGAGTTTTC AGAACCGATT 60

G 61

GACATACAGC TAGAAAGCTG TATTGCCTTT AGCACTCAAG CTCAAAAGAC AACTCAGAGT 60

TCACC ATG GGC TCC ATC GGC GCA GCA AGC ATG GAA TTT TGT TTT GAT 107

Met Gly Ser Ile Gly Ala Ala Ser Met Glu Phe Cys Phe Asp

1 5 10

GTA TTC AAG GAG CTC AAA GTC CAC CAT GCC AAT GAG AAC ATC TTC TAC 155

Val Phe Lys Glu Leu Lys Val His His Ala Asn Glu Asn Ile Phe Tyr

15 20 25 30

TGC CCC ATT GCC ATC ATG TCA GCT CTA GCC ATG GTA TAC CTG GGT GCA 203

Cys Pro Ile Ala Ile Met Ser Ala Leu Ala Met Val Tyr Leu Gly Ala

35 40 45

AAA GAC AGC ACC AGG ACA CAG ATA AAT AAG GTT GTT CGC TTT GAT AAA 251

Lys Asp Ser Thr Arg Thr Gln Ile Asn Lys Val Val Arg Phe Asp Lys

50 55 60

CTT CCA GGA TTC GGA GAC AGT ATT GAA GCT CAG TGT GGC ACA TCT GTA 299

Leu Pro Gly Phe Gly Asp Ser Ile Glu Ala Gln Cys Gly Thr Ser Val

65 70 75

AAC GTT CAC TCT TCA CTT AGA GAC ATC CTC AAC CAA ATC ACC AAA CCA 347

Asn Val His Ser Ser Leu Arg Asp Ile Leu Asn Gln Ile Thr Lys Pro

80 85 90

AAT GAT GTT TAT TCG TTC AGC CTT GCC AGT AGA CTT TAT GCT GAA GAG 395

Asn Asp Val Tyr Ser Phe Ser Leu Ala Ser Arg Leu Tyr Ala Glu Glu

95 100 105 110

AGA TAC CCA ATC CTG CCA GAA TAC TTG CAG TGT GTG AAG GAA CTG TAT 443

Arg Tyr Pro Ile Leu Pro Glu Tyr Leu Gln Cys Val Lys Glu Leu Tyr

115 120 125

AGA GGA GGC TTG GAA CCT ATC AAC TTT CAA ACA GCT GCA GAT CAA GCC 491

Arg Gly Gly Leu Glu Pro Ile Asn Phe Gln Thr Ala Ala Asp Gln Ala

130 135 140

AGA GAG CTC ATC AAT TCC TGG GTA GAA AGT CAG ACA AAT GGA ATT ATC 539

Arg Glu Leu Ile Asn Ser Trp Val Glu Ser Gln Thr Asn Gly Ile Ile

145 150 155

AGA AAT GTC CTT CAG CCA AGC TCC GTG GAT TCT CAA ACT GCA ATG GTT 587

Arg Asn Val Leu Gln Pro Ser Ser Val Asp Ser Gln Thr Ala Met Val

160 165 170

CTG GTT AAT GCC ATT GTC TTC AAA GGA CTG TGG GAG AAA ACA TTT AAG 635

Leu Val Asn Ala Ile Val Phe Lys Gly Leu Trp Glu Lys Thr Phe Lys

175 180 185 190

GAT GAA GAC ACA CAA GCA ATG CCT TTC AGA GTG ACT GAG CAA GAA AGC 683

Asp Glu Asp Thr Gln Ala Met Pro Phe Arg Val Thr Glu Gln Glu Ser

195 200 205

AAA CCT GTG CAG ATG ATG TAC CAG ATT GGT TTA TTT AGA GTG GCA TCA 731

Lys Pro Val Gln Met Met Tyr Gln Ile Gly Leu Phe Arg Val Ala Ser

210 215 220

ATG GCT TCT GAG AAA ATG AAG ATC CTG GAG CTT CCA TTT GCC AGT GGG 779

Met Ala Ser Glu Lys Met Lys Ile Leu Glu Leu Pro Phe Ala Ser Gly

225 230 235

ACA ATG AGC ATG TTG GTG CTG TTG CCT GAT GAA GTC TCA GGC CTT GAG 827

Thr Met Ser Met Leu Val Leu Leu Pro Asp Glu Val Ser Gly Leu Glu

240 245 250

CAG CTT GAG AGT ATA ATC AAC TTT GAA AAA CTG ACT GAA TGG ACC AGT 875

Gln Leu Glu Ser Ile Ile Asn Phe Glu Lys Leu Thr Glu Trp Thr Ser

255 260 265 270

TCT AAT GTT ATG GAA GAG AGG AAG ATC AAA GTG TAC TTA CCT CGC ATG 923

Ser Asn Val Met Glu Glu Arg Lys Ile Lys Val Tyr Leu Pro Arg Met

275 280 285

AAG ATG GAG GAA AAA TAC AAC CTC ACA TCT GTC TTA ATG GCT ATG GGC 971

Lys Met Glu Glu Lys Tyr Asn Leu Thr Ser Val Leu Met Ala Met Gly

290 295 300

ATT ACT GAC GTG TTT AGC TCT TCA GCC AAT CTG TCT GGC ATC TCC TCA 1019

Ile Thr Asp Val Phe Ser Ser Ser Ala Asn Leu Ser Gly Ile Ser Ser

305 310 315

GCA GAG AGC CTG AAG ATA TCT CAA GCT GTC CAT GCA GCA CAT GCA GAA 1067

Ala Glu Ser Leu Lys Ile Ser Gln Ala Val His Ala Ala His Ala Glu

320 325 330

ATC AAT GAA GCA GGC AGA GAG GTG GTA GGG TCA GCA GAG GCT GGA GTG 1115

Ile Asn Glu Ala Gly Arg Glu Val Val Gly Ser Ala Glu Ala Gly Val

335 340 345 350

GAT GCT GCA AGC GTC TCT GAA GAA TTT AGG GCT GAC CAT CCA TTC CTC 1163

Asp Ala Ala Ser Val Ser Glu Glu Phe Arg Ala Asp His Pro Phe Leu

355 360 365

TTC TGT ATC AAG CAC ATC GCA ACC AAC GCC GTT CTC TTC TTT GGC AGA 1211

Phe Cys Ile Lys His Ile Ala Thr Asn Ala Val Leu Phe Phe Gly Arg

370 375 380

TGT GTT TCC CCT TAAAAAGAAG AAAGCTGAAA AACTCTGTCC CTTCCAACAA 1263

Cys Val Ser Pro

385

GACCCAGAGC ACTGTAGTAT CAGGGGTAAA ATGAAAAGTA TGTTCTCTGC TGCATCCAGA 1323

CTTCATAAAA GCTGGAGCTT AATCTAGAAA AAAAATCAGA AAGAAATTAC ACTGTGAGAA 1383

CAGGTGCAAT TCACTTTTCC TTTACACAGA GTAATACTGG TAACTCATGG ATGAAGGCTT 1443

AAGGGAATGA AATTGGACTC ACAGTACTGA GTCATCACAC TGAAAAATGC AACCTGATAC 1503

ATCAGCAGAA GGTTTATGGG GGAAAAATGC AGCCTTCCAA TTAAGCCAGA TATCTGTATG 1563

ACCAAGCTGC TCCAGAATTA GTCACTCAAA ATCTCTCAGA TTAAATTATC AACTGTCACC 1623

AACCATTCCT ATGCTGACAA GGCAATTGCT TGTTCTCTGT GTTCCTGATA CTACAAGGCT 1683

CTTCCTGACT TCCTAAAGAT GCATTATAAA AATCTTATAA TTCACATTTC TCCCTAAACT 1743

TTGACTCAAT CATGGTATGT TGGCAAATAT GGTATATTAC TATTCAAATT GTTTTCCTTG 1803

TACCCATATG TAATGGGTCT TGTGAATGTG CTCTTTTGTT CCTTTAATCA TAATAAAAAC 1863

ATGTTTAAGC 1873

386 amino acids

amino acid

linear

protein

Met Gly Ser Ile Gly Ala Ala Ser Met Glu Phe Cys Phe Asp Val Phe

1 5 10 15

Lys Glu Leu Lys Val His His Ala Asn Glu Asn Ile Phe Tyr Cys Pro

20 25 30

Ile Ala Ile Met Ser Ala Leu Ala Met Val Tyr Leu Gly Ala Lys Asp

35 40 45

Ser Thr Arg Thr Gln Ile Asn Lys Val Val Arg Phe Asp Lys Leu Pro

50 55 60

Gly Phe Gly Asp Ser Ile Glu Ala Gln Cys Gly Thr Ser Val Asn Val

65 70 75 80

His Ser Ser Leu Arg Asp Ile Leu Asn Gln Ile Thr Lys Pro Asn Asp

85 90 95

Val Tyr Ser Phe Ser Leu Ala Ser Arg Leu Tyr Ala Glu Glu Arg Tyr

100 105 110

Pro Ile Leu Pro Glu Tyr Leu Gln Cys Val Lys Glu Leu Tyr Arg Gly

›DNA · 2 of 2

115 120 125

Gly Leu Glu Pro Ile Asn Phe Gln Thr Ala Ala Asp Gln Ala Arg Glu

130 135 140

Leu Ile Asn Ser Trp Val Glu Ser Gln Thr Asn Gly Ile Ile Arg Asn

145 150 155 160

Val Leu Gln Pro Ser Ser Val Asp Ser Gln Thr Ala Met Val Leu Val

165 170 175

Asn Ala Ile Val Phe Lys Gly Leu Trp Glu Lys Thr Phe Lys Asp Glu

180 185 190

Asp Thr Gln Ala Met Pro Phe Arg Val Thr Glu Gln Glu Ser Lys Pro

195 200 205

Val Gln Met Met Tyr Gln Ile Gly Leu Phe Arg Val Ala Ser Met Ala

210 215 220

Ser Glu Lys Met Lys Ile Leu Glu Leu Pro Phe Ala Ser Gly Thr Met

225 230 235 240

Ser Met Leu Val Leu Leu Pro Asp Glu Val Ser Gly Leu Glu Gln Leu

245 250 255

Glu Ser Ile Ile Asn Phe Glu Lys Leu Thr Glu Trp Thr Ser Ser Asn

260 265 270

Val Met Glu Glu Arg Lys Ile Lys Val Tyr Leu Pro Arg Met Lys Met

275 280 285

Glu Glu Lys Tyr Asn Leu Thr Ser Val Leu Met Ala Met Gly Ile Thr

290 295 300

Asp Val Phe Ser Ser Ser Ala Asn Leu Ser Gly Ile Ser Ser Ala Glu

305 310 315 320

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

325 330 335

Glu Ala Gly Arg Glu Val Val Gly Ser Ala Glu Ala Gly Val Asp Ala

340 345 350

Ala Ser Val Ser Glu Glu Phe Arg Ala Asp His Pro Phe Leu Phe Cys

355 360 365

Ile Lys His Ile Ala Thr Asn Ala Val Leu Phe Phe Gly Arg Cys Val

370 375 380

Ser Pro

385

TCACTTCGAC CATCAACCCA GCTTGCTGTT CTTCCCAGTC TTGGGTTCAA GATCTTGACA 60

ACC ATG AAA CTT CTC ATC CTT ACC TGT CTT GTG GCT GTT GCT CTT GCC 108

Met Lys Leu Leu Ile Leu Thr Cys Leu Val Ala Val Ala Leu Ala

-15 -10 -5

AGG CCC AAA CAT CCT ATC AAG CAC CAA GGA CTC CCT CAA GAA GTC CTC 156

Arg Pro Lys His Pro Ile Lys His Gln Gly Leu Pro Gln Glu Val Leu

1 5 10 15

AAT GAA AAT TTA CTC AGG TTT TTT GTG GCA CCT TTT CCA GAA GTG TTT 204

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

20 25 30

GGA AAG GAG AAG GTC AAT GAA CTG AGC AAG GAT ATT GGG AGT GAA TCA 252

Gly Lys Glu Lys Val Asn Glu Leu Ser Lys Asp Ile Gly Ser Glu Ser

35 40 45

ACT GAG GAT CAA GCC ATG GAA GAT ATT AAG CAA ATG GAA GCT GAA AGC 300

Thr Glu Asp Gln Ala Met Glu Asp Ile Lys Gln Met Glu Ala Glu Ser

50 55 60

ATT TCG TCA AGT GAG GAA ATT GTT CCC AAT AGT GTT GAG CAG AAG CAC 348

Ile Ser Ser Ser Glu Glu Ile Val Pro Asn Ser Val Glu Gln Lys His

65 70 75 80

ATT CAA AAG GAA GAT GTG CCC TCT GAG CGT TAC CTG GGT TAT CTG GAA 396

Ile Gln Lys Glu Asp Val Pro Ser Glu Arg Tyr Leu Gly Tyr Leu Glu

85 90 95

CAG CTT CTC AGA CTG AAA AAA TAC AAA GTA CCC CAG CTG GAA ATT GTT 444

Gln Leu Leu Arg Leu Lys Lys Tyr Lys Val Pro Gln Leu Glu Ile Val

100 105 110

CCC AAT AGT GCT GAG GAA CGA CTT CAC AGT ATG AAA GAG GGA ATC CAT 492

Pro Asn Ser Ala Glu Glu Arg Leu His Ser Met Lys Glu Gly Ile His

115 120 125

GCC CAA CAG AAA GAA CCT ATG ATA GGA GTG AAT CAG GAA CTG GCC TAC 540

Ala Gln Gln Lys Glu Pro Met Ile Gly Val Asn Gln Glu Leu Ala Tyr

130 135 140

TTC TAC CCT GAG CTT TTC AGA CAA TTC TAC CAG CTG GAT GCC TAT CCA 588

Phe Tyr Pro Glu Leu Phe Arg Gln Phe Tyr Gln Leu Asp Ala Tyr Pro

145 150 155 160

TCT GGT GCT TGG TAT TAC GTT CCA CTA GGC ACA CAA TAC ACT GAT GCC 636

Ser Gly Ala Trp Tyr Tyr Val Pro Leu Gly Thr Gln Tyr Thr Asp Ala

165 170 175

CCA TCA TTC TCT GAC ATC CCT AAT CCC ATT GGC TCT GAG AAC AGT GAA 684

Pro Ser Phe Ser Asp Ile Pro Asn Pro Ile Gly Ser Glu Asn Ser Glu

180 185 190

AAG ACT ACT ATG CCA CTG TGG TGAAGAGTCA AGTGAATTCT GAGGGACTCC 735

Lys Thr Thr Met Pro Leu Trp

195

ACAGTTATGG TCTTTGATGG GTCTGAAAAT TCCATGCTCT ACATGTCGCC TCATCTACAT 795

GTCAAACCAT TCATCCAAAG GCTTCAACTG CTGTTTTAGA ACAGGGCAAT CTCAAACTGA 855

GGCACTCCTT GATGCTCTAC TGTATTTTAG ATAGTGTAAC ATCCTTAAGT GAAATTGTCC 915

TAACAGCTTG TTACCTAAAT TCCAGTAGTA TCATGCTGGT ATAAAGGCCA CTGAGTCAAA 975

GGGAATTAAA GTCTTCATTA AATTTCTGTA TGGAAAATGT TTTAAAAGCC TTTGAATCAC 1035

TTCTCCTGTA AGTGCCATCA TATCAAATAA TTGTGTGCAT TAACTGAGAT TTTGTCTTTC 1095

TTCTTTTCAA TAAATTACAT TTTAAGGC 1123

214 amino acids

amino acid

linear

protein

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

-15 -10 -5 1

Pro Lys His Pro Ile Lys His Gln Gly Leu Pro Gln Glu Val Leu Asn

5 10 15

Glu Asn Leu Leu Arg Phe Phe Val Ala Pro Phe Pro Glu Val Phe Gly

20 25 30

Lys Glu Lys Val Asn Glu Leu Ser Lys Asp Ile Gly Ser Glu Ser Thr

35 40 45

Glu Asp Gln Ala Met Glu Asp Ile Lys Gln Met Glu Ala Glu Ser Ile

50 55 60 65

Ser Ser Ser Glu Glu Ile Val Pro Asn Ser Val Glu Gln Lys His Ile

70 75 80

Gln Lys Glu Asp Val Pro Ser Glu Arg Tyr Leu Gly Tyr Leu Glu Gln

85 90 95

Leu Leu Arg Leu Lys Lys Tyr Lys Val Pro Gln Leu Glu Ile Val Pro

100 105 110

Asn Ser Ala Glu Glu Arg Leu His Ser Met Lys Glu Gly Ile His Ala

115 120 125

Gln Gln Lys Glu Pro Met Ile Gly Val Asn Gln Glu Leu Ala Tyr Phe

130 135 140 145

Tyr Pro Glu Leu Phe Arg Gln Phe Tyr Gln Leu Asp Ala Tyr Pro Ser

150 155 160

Gly Ala Trp Tyr Tyr Val Pro Leu Gly Thr Gln Tyr Thr Asp Ala Pro

165 170 175

Ser Phe Ser Asp Ile Pro Asn Pro Ile Gly Ser Glu Asn Ser Glu Lys

180 185 190

Thr Thr Met Pro Leu Trp

195

Arg Pro Lys His Pro Ile Lys His Gln Gly Leu Pro Gln Glu Val Leu

1 5 10 15

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

20 25 30

Gly Lys Glu Lys Val Asn Glu Leu Ser Lys Asp Ile Gly Ser Glu Ser

35 40 45

Thr Glu Asp Gln Ala Met Glu Asp Ile Lys Gln Met Glu Ala Glu Ser

50 55 60

Ile Ser Ser Ser Glu Glu Ile Val Pro Asn Ser Val Glu Gln Lys His

65 70 75 80

Ile Gln Lys Glu Asp Val Pro Ser Glu Arg Tyr Leu Gly Tyr Leu Glu

85 90 95

Gln Leu Leu Arg Leu Lys Lys Tyr Lys Val Pro Gln Leu Glu Ile Val

100 105 110

Pro Asn Ser Ala Glu Glu Arg Leu His Ser Met Lys Glu Gly Ile His

115 120 125

Ala Gln Gln Lys Glu Pro Met Ile Gly Val Asn Gln Glu Leu Ala Tyr

130 135 140

Phe Tyr Pro Glu Leu Phe Arg Gln Phe Tyr Gln Leu Asp Ala Tyr Pro

145 150 155 160

Ser Gly Ala Trp Tyr Tyr Val Pro Leu Gly Thr Gln Tyr Thr Asp Ala

165 170 175

Pro Ser Phe Ser Asp Ile Pro Asn Pro Ile Gly Ser Glu Asn Ser Glu

180 185 190

Lys Thr Thr Met Pro Leu Trp

195

CTGCAGGATC CCGGGAATTC AAGCTT 26

›Tables in the description — 10

374 base pairs	nucleic acid	single	linear
DNA
-	1..374	/note= “predicted sequence of bovine	casein block A”
12

371 base pairs	nucleic acid	single	linear
DNA
-	1..371	/note= “actual sequence obtained for	bovine casein block A”
14

371 base pairs	nucleic acid	single	linear
DNA
-	1..371	/note= “predicted sequence of bovine	casein block B”
16

371 base pairs	nucleic acid	single	linear
DNA
-	1..371	/note= “actual sequence obtained for	bovine casein block B”
18

632 base pairs	nucleic acid	single	linear
DNA
-	1..632	/note= “combined sequence of bovine	casein blocks A and B”
20

632 base pairs	nucleic acid	single	linear
DNA
-	1..632	/note= “final sequence of synthetic	bovine casein gene”
22

20 base pairs	nucleic acid	single	linear
DNA
-	1..20	/note “5′-terminal oligonucleotide PCR	primer for Block A”
26

20 base pairs	nucleic acid	single	linear
DNA
-	1..20	/note “3′-terminal oligonucleotide PCR	primer for Block A”
31

20 base pairs	nucleic acid	single	linear
DNA
-	1..20	/note “5′-terminal oligonucleotide PCR	primer for Block B”
32

61 base pairs	nucleic acid	single	linear
DNA
-	1..61	/note “3′-terminal oligonucleotide PCR	primer for Block B”
37

2 of 13 part labels are ours — the grant heads the rest

Claims as granted

15 claims

Classifications

15 codes

IPC · International Patent Classification

Section C — Chemistry; metallurgy

C07K14/47
C12N5/02
C07K14/77
C12N15/12

USPC · US Patent Classification

435/69.1514/2536/23.5530/360530/367435/252.33435/252.3536/23.1435/254.21435/320.1530/350

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Karen Cochrane Carlson

art unit 1653 · TC 1600

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