CN109371035A - A kind of gene and preparation method thereof of Araneus ventricosus pyriform gland silk-fibroin - Google Patents

Abstract

The present invention relates to a kind of genes and preparation method thereof of Araneus ventricosus pyriform gland silk-fibroin, and the gene order is as shown in SEQ ID NO.1.Amplification method: the NT sequence of part pyriform gland protein gene is amplified using the method for degenerate pcr, with anchor PCR by NT sequence completion, PySp1 gene of the pair of primers for PCR amplification overall length is designed according to the CT sequence that obtained NT sequence is sequenced and obtains before, the segment that amplification obtains is subjected to flat end clone, and plasmid is extracted to the positive colony bacterium containing overall length PySp1 gene, overall length sequencing is carried out, that is, obtains the gene nucleotide series.The present invention obtain for the first time include the complete end NT, the end CT and intermediate duplicate block Araneus ventricosus pyriform gland silk-fibroin encoding gene, for further research pyriform gland silk-fibroin structure and manually prepare spider's thread fiber and lay a good foundation.

Description

A kind of gene and preparation method thereof of Araneus ventricosus pyriform gland silk-fibroin

Technical field

The invention belongs to gene and its preparation field, in particular to the gene of a kind of Araneus ventricosus pyriform gland silk-fibroin and its Preparation method.

Background technique

Araneidae spider can secrete 7 kinds of spider silks, respectively major ampullate gland silk, secondary ampulla gland silk, flagelliform gland silk, tubular gland Silk, pyriform gland silk, racemose glands silk and aggregate gland silk；Spider's thread fiber is made of different spider's thread proteins, has different materials Learn performance and biological function.Spider silk possesses excellent mechanical performance and mechanical property, is many natural or artificial materials Incomparable.The full-length gene order for obtaining spider's thread protein gene is the basis for studying spider silk performance and its mechanism, the spider's thread Protein sequence is by the non-duplicate area NT guarded relatively, and CT and very big duplicate block composition, spider's thread protein is often very big Protein, relative molecular weight is up to 200-350kDa.It obtains spider's thread full-length gene from DNA library to take time and effort, Direct PCR Obtain that overall length spider's thread gene is convenient and efficient, but the uncertainty due to lacking related aim sequence segment and long range PCR result, The spider's thread protein gene of only a small number of overall lengths is cloned out, seriously constrains the further research to spider silk.

Pyriform gland silk belongs to fixed silk as secreted by pyriform gland, and pyriform gland silk is answering for stemness silk fiber and wet glue in fact Zoarium, the connection being mainly used between the spider's thread and solid support.First PySp1 full-length gene was just reported out in 2017 Come comprising 21 repetitive units, each repetitive unit includes 234 amino acid residues, wherein by small comprising a large amount of PX and QQ Module.

Spider silk smooth in appearance is dainty, silk is sliding glittering, tough and tensile soft, and has biocompatibility and preferable Memorability Can, there is great potential using value, the spider's thread egg manually prepared as the native protein class high molecular material haveing excellent performance It is white that different forms can be prepared by different methods, such as fiber, film, microballoon, capsule be applied to as biomaterial Military, space flight and field of biomedicine, and its protein ingredient determines its degradability, the strategy for meeting sustainable development is wanted It asks.

Summary of the invention

Technical problem to be solved by the invention is to provide a kind of gene of Araneus ventricosus pyriform gland silk-fibroin and its preparations Method, overcomes the defect of the Araneus ventricosus pyriform gland Silk gene blank of the prior art, and this method has cloned whole gene It include the complete end NT, duplicate block and the complete end CT, for encoding Araneus ventricosus pyriform gland silk-fibroin；And to environment It is pollution-free；Preparation method simple process, mild condition is easily operated, establishes for spider albumen is mass produced in genetic engineering Gene basis.

A kind of gene of Araneus ventricosus pyriform gland silk-fibroin of the invention, the gene order is as shown in SEQ ID NO.1.

The full length gene size is 11,931bp, encodes 3976 amino acid, particular sequence such as SEQ ID NO.1 institute altogether Show, molecular weight of albumen 400.0kDa.

The end NT size is 450bp in the full-length gene, encodes 150 amino acid, particular sequence such as SEQ ID NO.2 institute Show.

The area N-linker size is 768bp, encodes 256 amino acid altogether, particular sequence is as shown in SEQ ID NO.3.

Duplicate block size is 10,116bp, encodes 3372 amino acid altogether, particular sequence is as shown in SEQ ID NO.4.

The area C-linker size is 327bp, encodes 109 amino acid altogether, particular sequence is as shown in SEQ ID NO.5

The end CT size is 267bp, encodes 89 amino acid altogether, particular sequence is as shown in SEQ ID NO.6.

A kind of preparation method of the gene of Araneus ventricosus pyriform gland silk-fibroin of the present invention, comprising:

(1) according to the PySp1 protein gene of spider pyriform gland silk, in the end NT degenerate primer, 2 are designed specifically in duplicate block Property primer, expands to obtain the end the NT gene order of part by degenerate pcr；

(2) 2 specific primers and 1 anchor primer are separately designed according to step (1) sequence obtained, passes through anchoring PCR is by the end NT polishing；

(3) obtained according to step (2) includes complete NT terminal sequence, in the end NT 5 ' tip designs, 1 forward primer, The end CT 3 ' tip designs, 1 reverse primer, for expanding overall length PySp1 gene；

(4) after carrying out Ago-Gel gel extraction to PCR product obtained by step (3), blunt end cloning is carried out with carrier Reaction is attached product recycling later；

(5) flat end clone is carried out to the connection product of step (4), using thermal shock conversion method, it is thin is transferred to DH5 α competence It being converted in born of the same parents, the bacterium solution after conversion is coated on LB solid medium by recovery time 1h, after being incubated overnight, picking list Clone carries out PCR detection；

(6) plasmid of positive monoclonal obtained in step (5) is completely sequenced, finally obtains Araneus ventricosus pears shape Gland silk-fibroin full-length gene.

The preferred embodiment of above-mentioned preparation method is as follows:

For expanding the primer at the part end NT: positive degenerate primer in the step (1): ATGTTYCARATGAGTTCAATG；

As shown in SEQ ID NO.7.

Inverted repeat area specific primer: CTGTTGTGTTTGAGCGGATTG.As shown in SEQ ID NO.8.The step (1) condition that degenerate pcr expands in are as follows: 95 DEG C of initial denaturations 5min, 95 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 30s, Circulation 30 times.

The specific primer at the end completion NT is used in the step (2):

Primer 1:TTTCAATAGCTGCCGCCTGC；As shown in SEQ ID NO.9.

Primer 2: GTGGCAATAGCTTGGGTTACGC；As shown in SEQ ID NO.10.

Anchor primer: ACTCCTGTGGAACCATCGGACGGGGGG.As shown in SEQ ID NO.11.

Anchor PCR condition in the step (2) are as follows: in first round PCR, carry out single primer expansion using specific primer 1 Increase, condition: 95 DEG C of initial denaturations 5min, 95 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 72 DEG C of extension 30s are recycled 30 times；Later by PCR Product carries out Ago-Gel gel extraction, is carried out adding poly- dCTP reaction with TdT enzyme, the second wheel PCR is carried out after product recycling； It is expanded using specific primer 2 and anchor primer, condition is same as above.

For expanding the primer of overall length PySp1 gene in the step (3):

Forward primer: ATGTCTTGGACCCTGGGGCTTCAGTTTTTATTC；As shown in SEQ ID NO.12

Reverse primer: CTATCCAAGTGCTGCAAGTACGAC.As shown in SEQ ID NO.13.

1 pair of primer is located at the 5 ' ends and 3 ' ends of PySp gene, to obtain complete PySp1 gene.

Enzyme in the step (3) for expanding overall length PySp1 gene is Phanta exo+ polymerase.

Amplification condition in the step (3) are as follows: 98 DEG C of initial denaturation 1min, 98 DEG C of denaturation 5s, 60 DEG C of annealing 15s, 72 DEG C are prolonged 10min is stretched, is recycled 30 times.

Carrier in the step (4) are as follows: pEASY-Blunt Zero Cloning Vector.

Blunt end cloning reacts in the step (4) are as follows: 16 DEG C of reactions are overnight.

The volume ratio of connection product and DH5 α competent cell is 1:10 in the step (5)；LB solid medium contains Ampicillin Amp and kanamycins Kan, the concentration of ampicillin and kanamycins in the medium are 100 μ g/mL.

A kind of application of the gene of Araneus ventricosus pyriform gland silk-fibroin of the invention is such as used as fibrous material.

The function of the gene of Araneus ventricosus pyriform gland silk-fibroin are as follows: pyriform gland silk-fibroin is a kind of spider being just found recently Silk-fibroin, it is the major protein of composition attachment disk.Attachment disk is used to connect the intersection of net, attachment drags silk to arrive other media Surface, and it is used to capture and mobile prey.Scanning electron microscopy study discovery: in attachment disk, minor diameter albumen Fiber is embedded in the binding as glue, forms the network for being similar to tree root shape, and there are also major diameters to drag silk for the inside, is one The kind extremely strong material of stickiness, plays an important role in spider vital movement.

Beneficial effect

The present invention passes through the end the NT sub-sequence that degenerate pcr amplifies PySp1 first, is then mended using anchor PCR Entirely, it expands to obtain complete PySp1 gene and carries out flat end clone using long range PCR method later, obtain containing overall length The clone of PySp1 gene；

It includes the complete end NT, duplicate block and the complete end CT that the present invention, which has cloned whole gene, for encoding greatly Abdomen epeira pyriform gland silk-fibroin；And no pollution to the environment；

The mini spider's thread protein that building has natural Araneus ventricosus pyriform gland silk-fibroin main feature module is attempted for the first time The complete repeat region (Av.PySpRp) of one of gene, (Av.PySpNT-PySpRp-PySpCT) and PySp, but it is mini Expression quantity of the PySp spider's thread protein Gene A v.PySpNT-PySpRp-PySpCT in E.coli BL21 (DE3) bacterial strain is extremely low, The simple complete duplicate block PySp (Av.PySpRp) gene can be obtained in E.coli BL21 (DE3) bacterial strain with normal expression Araneus ventricosus pyriform gland silk-fibroin, the albumen can be spun into nano fibrous membrane by electrostatic spinning；

Preparation method simple process of the present invention, mild condition is easily operated, for spider egg is mass produced in genetic engineering It is white to have established gene basis.

Detailed description of the invention

Fig. 1 is the amino acid sequence of Araneus ventricosus PySp1 albumen of the present invention.

Fig. 2 is the hydrophobicity plot of Araneus ventricosus PySp1 albumen of the present invention；It is wherein complete PySp1 albumen on Fig. 2 Hydrophobicity profile is followed successively by the hydrophobicity profile at the end NT, N-linker, repeat 8, the end CT under Fig. 2 from left to right.

Fig. 3 is the secondary structure prediction figure of Araneus ventricosus PySp1 albumen of the present invention；Wherein Fig. 3 is followed successively by NT from left to right The secondary structure prediction figure at end, the end repeat8, CT.

Fig. 4 is the sequence alignment figure of Araneus ventricosus PySp1 albumen of the present invention；Wherein, Fig. 4 a is the NT of 5 kinds of PySp1 albumen Terminal sequence comparison chart, Fig. 4 b are the repetitive unit sequence alignment figure of 4 kinds of PySp1 albumen, and Fig. 4 c is the end CT of 7 kinds of PySp1 albumen Sequence alignment figure.

Fig. 5 is the SEM photograph (1000 ×) of pure PySp azelon film.

Specific embodiment

Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.In addition, it should also be understood that, after reading the content taught by the present invention, those skilled in the art Member can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited Range.

Embodiment 1

(1) according to the conserved sequence at the end NT of the PySp1 of other spiders, 1 degeneracy is respectively designed at the end NT and the end CT respectively Primer: the end NT degenerate primer: ATGTTYCARATGAGTTCAATG, and 1 specific reverse primers is designed in duplicate block, specifically Property reverse primer: CTGTTGTGTTTGAGCGGATTG；Then PCR is carried out, condition is 95 DEG C of initial denaturation 5min, 95 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 30s are recycled 30 times.

(2) 2 specific primers and 1 anchor primer are respectively designed at the end NT and the end CT respectively according to sequence obtained For the end completion NT and the end CT.

NT end-specificity primer 1:TTTCAATAGCTGCCGCCTGC；

NT end-specificity primer 2: GTGGCAATAGCTTGGGTTACGC；

: anchor primer: ACTCCTGTGGAACC ATCGGACGGGGGG；

Anchor PCR condition are as follows: in first round PCR, carry out single primer amplification using specific primer 1, condition: 95 DEG C pre- It is denaturalized 5min, 95 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 72 DEG C of extension 30s, is recycled 30 times.PCR product is subjected to agarose later Gel gel extraction, is carried out with TdT enzyme plus C reaction, product carry out the second wheel PCR after recycling.Use specific primer 2 and anchor Determine primer to be expanded, condition is same as above.

(3) 1 pair of specific primer of design is used to expand overall length PySp genetic fragment after,

Forward primer: ATGTCTTGGACCCTGGGGCTTCAGTTTTTATTC；

Reverse primer: CTATCCAAGTGCTGCAAGTACGAC；

PCR condition is 98 DEG C of initial denaturations 1min, 98 DEG C of denaturation 5s, 60 DEG C of annealing 15s, 72 DEG C of extension 10min, circulation 30 It is secondary.Ago-Gel gel extraction later.(Phanta exo+ polymerase adds in PCR reaction solution)

(4) recovery product blunt end cloning is carried out with carrier pEASY-Blunt Zero Cloning vector to react, Condition are as follows: 16 DEG C of reactions are overnight.

(5) DH5 α competent cell, volume ratio 1:10 are transferred to after, the recovery time is 1h.Bacterium solution is coated on later On the solid medium of ampicillin and kanamycins, concentration is 100 μ g/mL, 37 DEG C of culture 12h.Then to bacterium colony into Row screening, sequencing.

It is soft using the online softwares such as SignalP4.1, Expasy tools, PSIPRED and Geneious, DNA star Part analyzes the gene, and SignalP4.1 is used to find the signal peptide of gene, and Expasy tools is used to dredge PySp1 Aqueous to be analyzed, PSIPRED predicts the secondary structure of PySp1, and Geneious is used to carry out Multiple Sequence Alignment, DNA Star is used to analyze the base composition of PySp1 gene and amino acid composition.

It is as follows to analyze result:

SignalP4.1 and DNA star analyzes result:

Fig. 1 is the structural domain and sequence map of PySp1 albumen, from figure 1 it appears that entire PySp1 gene divides For five regions, the end NT, N-linker, duplicate block, the end C-linker and CT.Wherein by 16 poles in sequence in duplicate block Its similar repetitive unit composition, and occupy 85% or more of entire protein sequence.Wherein preceding 15 repetitive unit sizes are 213 amino acid, the 16th repetitive unit are 177 amino acid.It can be found that the signal peptide of albumen is present in the end NT in Fig. 1 In (runic label), and there are 2 Cys residues.

The amino acid sequence of Araneus ventricosus PySp1 albumen of the present invention, while as shown in SEQ ID NO.16.

Expasy tools analyzes result: Fig. 2 is hydrophobicity plot and the end NT of entire PySp1 albumen, Repeat Unit 8 (R8), the hydrophobicity profile at the end CT.From fig. 2 it can be seen that water repellent region and hydrophilic region are in entire albumen Change repeatedly in sequence, entire protein molecular shows as hydrophobicity.The average hydrophilicity value at the end CT is greater than the end NT, but the end NT is thin Aqueous peak value is greater than the end CT.

PSIPRED analyzes result: Fig. 3 is PySp Protein secondary structure prognostic chart, from figure 3, it can be seen that containing in the end NT There are 5 αhelix, first Cys residue is between the 1st and the 2nd spiral, and second Cys residue is in the 4th spiral shell In rotation.It can be found that containing only 4 helical structures in the end CT in Fig. 3.There are 8 helical structures in Fig. 3 in repetitive unit.

Geneious analyzes result: Fig. 4 is the comparison diagram of PySp albumen.It can be seen that difference from Fig. 4 a, Fig. 4 b, Fig. 4 c There is higher similarity at the end NT of PySp between species, repetitive unit and the end CT.

Embodiment 2

1. according to the duplicate block PySp primers

Primer 1:TATTCTGCAGCATCAACTGCC；As shown in SEQ ID NO.14

Primer 2:AGCGTTTGAGCTTTGTTGAGATTG；As shown in SEQ ID NO.15.

Amplification condition are as follows: 95 DEG C of initial denaturations 5,95 DEG C of denaturation 30s, 57 DEG C of annealing 30s, 72 DEG C of extension 1min are recycled 30 times.

The amplification duplicate block overall length PySp genetic fragment obtains PCR product.Above-mentioned experiment is prepared into obtained Insert Fragment It is attached with pEHS carrier, thaw following reagent on ice chest, and is loaded on ice in the following order, and linked system is as follows:

Insert Fragment 7L

PEHS carrier 1L

T4DNA Ligase 1L

10×T4Ligation Buffer 1L

Total 10L

It is placed in PCR pipe, mixes, connect 6h at room temperature.Connection product it is heat-shock transformed enter E.coli DH5 α competence it is thin Born of the same parents.

It extracts plasmid and is transferred to BL21 bacterial strain, carry out protein induced expression:

(1) after protein induced expression 5h, using low-temperature and high-speed centrifuge 4000rpm, 15min, thalline were collected by centrifugation, is used in combination The Tris-HCl (pH 8.0) of 20mM is dissolved with the ratio of 30ml/L bacterium solution；

(2) the obtained mixed liquor of dissolution using mechanical biomixer is subjected to clasmatosis, after bacterial cell disruption 12000rpm, 4 DEG C, 10min collects inclusion body；

(3) inclusion body that centrifugation obtains all is dissolved in the urea of 8M, according to the ratio of 70mL/L, mixed liquor is put on Preservative film is in 30~60min of incubation on electromagnetic heating blender；

(4) mixed liquor after being incubated for, 12000rpm are centrifuged 30min under the conditions of 25 DEG C；

(5) the case where after being centrifuged, collecting supernatant, taking out cracking of the part using SDS-PAGE detection protein expression；

(6) bag filter for cutting one section of appropriate length, it is with deionized water that bag filter interior flushing is clean, for use；

(7) one end of bag filter is clamped with dialysis clamp, a small amount of deionization can be first added before pouring into protein liquid thereto Water is to check leakproofness；

(8) processed urea-containing protein solution is poured slowly into bag filter, finally, the other end by bag filter is same Sample is clamped with dialysis clamp；

(9) bag filter is put into the big container equipped with ultrapure water, is put into rotor and is placed on magnetic stirrer and stirs, About 2h replaces the dialyzate in a container, and entire dialysis procedure carries out about 48h.

After dialysis, the protein liquid in bag filter is all poured into 500mL freeze-drying cup, is frozen with freeze drier It is dry, until occurring the albumen of powdery in cup.Albumen powder is taken to carry out Static Spinning:

(1) 8% spinning solution is made in the hexafluoroisopropanol (HFIP) that 3mL is added in the albumen for taking 0.24g to extract；

(2) a clean needle tubing separately is taken, uniformly mixed spinning solution is poured into needle tubing, and the syringe needle handled well is inserted It is good, it is fixed in injection rod；

(3) the square cardboard for preparing an about 17cm*17cm, is wrapped with aluminium-foil paper, keeps one-sided smooth complete；

(4) parameter of injection rod, 5cc, 0.80mL/h, 4.00mL are set；

(5) it when having drop outflow at syringe needle, connects high voltage power supply (16kV), starts electrostatic spinning, it is observed that Under the action of electric field force, the drop that pillow goes out becomes conical (Taylor cone) from ball-type；

(6) after electrostatic spinning, power supply is closed, used syringe needle, which is removed, to be replaced in recycle in hexafluoroisopropanol makes With it is stand-by that the nano fibrous membrane spun is put in storage at dry.

The film spun is taken pictures under scanning electron microscope (SEM), referring to attached drawing 5.

It is possible thereby to illustrate, it is the spider's thread that it is feasible for, which carrying out electrospun nanofibers film using pure PySp albumen, Fiber applications provide the foundation material.

SEQUENCE LISTING

<110>Donghua University

<120>a kind of gene and preparation method thereof of Araneus ventricosus pyriform gland silk-fibroin

<130> 1

<160> 16

<170> PatentIn version 3.3

<210> 1

<211> 11931

<212> DNA

<213>artificial sequence

<400> 1

atgtcttgga ccctggggct tcagttttta ttcacatttt gcttggtgct cttcgcatgg 60

aggggagatg ctctttcaga cccaaaatct tgggttcagg acgaagcgac ggccgtttat 120

ttcttggagc gcaccttgga gtatctggga gaatgcggcg ttctgaaaat tgaccaggag 180

agagatgcca tcgacaccat gtttcagatg agttcaatgt ttcaacagaa tgcaaaaccc 240

agcaagataa agcactctat atcttccaag ttggccgaca tagtcataga aggtctggag 300

ggagacagcg acccgtccta caagatcgac tgcgtaaccc aagctattgc caccgctttc 360

gaagcaacaa caggtgttcg agatgaaaat ttcattgagt ctgtcattga acttatagta 420

ttaatattta acaatgatat cgaagagaag atggaagaag aaatcgaaga gactgctgca 480

agtcagcagg cggcagctat tgaaaaccaa atttcgacaa tagaatcgaa ctttggatcg 540

gcacagactc agtcatcagt gcagaacgat atacagcagc aaagcactgt agatatttct 600

tcttcggtca ccaatcagat tcagtctcag cagtatgcag acagtcaatc ttccgcgcaa 660

attcagtcac agcaaagtgt cgctagccaa caacaagatc ttacttctca actaacgact 720

tctagcgctc agcaacaata tgaaacttcc caagcttcta tcgcacagca gcaatatgaa 780

acttcccaag cttctatcgc gcagcagcaa tatgaagctt ctcaacagca ggcttctatc 840

gagcagcaac aatatgaagc ttcgcaagct tctatcgcgc aacagcaata tgaagcttct 900

caacagcagg cttctatcga gcagcaacaa tatgaagctt cgcaagcttc tatcgcgcaa 960

cagcaatatg aagcttctca acagcaagct tctatcgcgc agcaacaata tcaaagttcg 1020

cagcaacagg cttctataaa tcagcaacaa tatgaagctt ctcagcagca atcttccagt 1080

tctgaacagc agtacacaac ttccgaacag caaattcagc agtccgagaa tgcatatacc 1140

gatcagtcaa atattcaaca atccgctcaa acacaacagt acagcacttc acaacaagaa 1200

caagaatcct ctgcaagcta ttctacagcc tcaattgccg gttcaagtgt gtcgcaatct 1260

caggcgactg tctcaagcgc acctgattat tttaacacgc aaactttaag tagcagcctg 1320

tcttcttctc tgcaatcact cagtgcactc aattcgatag cgagtggtca actgagctcc 1380

tcgaatgccg cttctatttt agcgagtgca gtggcacagt cacttggagt gtcccaagcg 1440

tcggttcaaa atagcataag ccaacagttg agaagcgtag ggcccggatc ttccacgtcc 1500

tctgtcgctc aagcaatagc aaatggagtg gctaacgcag ttggagcatc aggaactgga 1560

gttgcaggac aagaacaatc tatttcacaa tccatatata cttcaatttc cactgctctt 1620

tctcaactgg cagcaccggc tccagcacct gcaccgagac ttgctcctag accactacca 1680

gccccaattc aagccccaag accagcaccc gcaccacaac ctgcaccggt ttacgcacca 1740

gccccagtcg tttcacaagt tcaggcaact tcttcctctc aagcctcggc tcaacagagt 1800

gccttcgcac agtcccaaca atcttcagtt gttcaatctc aacaaagttc aaacgcttat 1860

tctgcagcat caactgccgg ttcaagtgtg tcgcaatctc aggcgattgt ctcaagcgct 1920

cctgtttatt ttaacacgca aactttaagt agcagcctgt cttcttctct gcaatcactc 1980

agtgcactca attcgttagc gagtggtcaa ctgagctcct ggaatgccgc ttctattata 2040

gcgagtgcgg tagcaccgtc acttggagtg tcccaagcgt cggttcaaaa tagtataagc 2100

caacagttga gaagcgtagg gcccggatct tccacgtcct ctgtcgctca agcaatagca 2160

aatggagtgg ctaacgcagt tggagcatca ggaacaggag ttgcaggaca agaacaatct 2220

atttcacaat ccatatatac ttcagtttcc actgctctat ctcaactggc agcaccggct 2280

ccagcacctg cacctagacc agctcctcga ccactaccag ccccaattca agccccaaga 2340

ccagcacccg caccacaacc tgcaccggtt tacgcaccag ccccagtcgt ttcacaagtt 2400

caggcaactt cttcctctca agcctcggct caacagagtg ccttcgcaca gtcccaacaa 2460

tcttcagttg ttcaatctca acaaagctca aacgcttatt ctgcagcttc atctgtaggc 2520

tcaagttttt cgcagtctca ggggactgtc ccaagcgctc ctgtttattt taacacgcaa 2580

actttaagta gcagcctgtc ttcttctctg caatcactca gtgcactcaa ttcgatagcg 2640

agtggtcaac tgagctcctc gaatgccgct tctattatag cgagtgcagt ggcacggtca 2700

cttggagtgt cccaagcgtc ggttcaaaat agtataagcc aacagttgag aagcgtaggg 2760

cccggatctt ccacgtcctc tgtcgctcaa gcaatagcaa atggagtggc taacgcagtt 2820

ggagcatcag gaactggagt tgcaggacaa gaacaatcta tttcacaatc catatatact 2880

tcaatttcca ctgctctttc tcaactggca gcaccggctc cagcacctgc accgagactt 2940

gctcctagac cactaccagc cccaattcaa gccccaagac cagcacccgc accacaacct 3000

gcaccggttt acgcaccagc cccagtcgtt tcacaagttc aggcaacttc ttcctctcaa 3060

gcttcggctc aacagagtgc cttcgcacag tcccagcaat cttcagttgc acaatctcaa 3120

caaagctcaa acgttgattc tgcagcttca tctgtaggct caagtttttc gcagtctcag 3180

gggactgtcc caagcgctcc tgtttatttt aacacgcaaa ctttaagtag cagcctgtct 3240

tcttctctgc aatcactcag tgcactcaat tcgatagcga gtggtcaact gagctcatcg 3300

tatgccgatt ctattttagc gagtgcagtg gctcggtctc ttggagtttc ccaagcgtcg 3360

gttctaaata gtataagcca acagttgaga agcgtagggc ccggatcttc cacgtcctct 3420

gtcgctcaag caatagcaaa tggagtggct aacgcagttg gagcatcagg aacaggagtt 3480

gcaggacaag aacaatctat ttcacaatcc atatatactt cagtttccac tgctctttct 3540

caactggcag caccggctcc agcacctgca ccgagacttg ctcctagacc actaccagcc 3600

ccaattcaag ccccaagacc agcacccgca ccacaacctg caccggttta cgcaccagcc 3660

ccagtcgttt cacaagttca ggcaacttct tcctctcaag cctcggctca acagagtgcc 3720

ttcgcacagt cccaacaatc ttcagttgtt caatctcaac aaagttcaaa cgcttattct 3780

gcagcatcaa ctgccggttc aagtgtgtcg caatctcagg cgattgtctc aagcgctcct 3840

gattatttta acacgcaaac tttaagtagc agcctgtctt cttctcagca atcactcagt 3900

gcactcaatt cgatagcgag tggtcaactg agctcctcga atgccgcttc tattttagcg 3960

agtgcagtgg cacggtcact tgtagtgtcc cacgcgtcgg ttcaaaatag tataagccaa 4020

cagttgagaa gcgtagggcc cggatcttcc acgtcctctg tcgctcaagc aatagcaaat 4080

ggagtggcta acgcagttgg agcatcagga actggagttg caggacaaga acaatctatt 4140

tcacaatcca tatatacttc aatttccact gctctttctc aactggcagc accggctcca 4200

gcacctgcac cgagacttgc tcctagacca ctaccagccc caattcaagc cccaagacca 4260

gcacccgcac cacaacctgc accggtttac gcaccagccc cagtcgtttc acaagttcag 4320

gcaacttctt cctctcaagc ctcggctcaa cagagtgcct tcgcacagtc ccaacaatct 4380

tcagttgttc aatctcaaca aagttcaaac gcttattctg cagcatcaac tgccggttca 4440

agtgtgtcgc aatctcaggc gattgtctca agcgctcctg attattttaa cacgcaaact 4500

ttaagtagca gcctgtcttc ttctcagcaa tcactcagtg cactcaattc gatagcgagt 4560

ggtcaactga gctcctcgaa tgccgcttct attttagcga gtgcagtggc acggtcactt 4620

ggagtgtccc aagcgtcggt tcaaaatagt ataagccaac agttgagaag cgtagggccc 4680

ggatcttcca cgtcctctgt cgctcaagca atagcaaatg gagtggctaa cgcagttgga 4740

gcatcaggaa ctggagttgc aggacaagaa caatctattt cacaatccat atatacttca 4800

atttccactg ctctttctca actggcagca ccggctccag cacctgcacc gagacttgct 4860

cctagaccac taccagcccc aattcaagcc ccaagaccag cacccgcacc acaacctgca 4920

ccggtttacg caccagcccc agtcgtttca caagttcagg caacttcttc ctctcaagcc 4980

tcggctcaac agagtgcctt cgcacagtcc caacaatctt cagttgttca atctcaacaa 5040

agttcaaacg cttattctgc agcatcaact gccggttcaa gtgtgtcgca atctcaggcg 5100

attgtctcaa gcgctcctga ttattttaac acgcaaactt taagtagcag cctgtcttct 5160

tctcagcaat cactcagtgc actcaattcg atagcgagtg gtcaactgag ctcctcgaat 5220

gccgcttcta ttttagcgag tgcagtggca cggtcacttg gagtgtccca agcgtcggtt 5280

caaaatagta taagccaaca gttgagaagc gtagggcccg gatcttccac gtcctctgtc 5340

gctcaagcaa tagcaaatgg agtggctaac gcagttggag catcaggaac tggagttgca 5400

ggacaagaac aatctatttc acaatccata tatacttcaa tttccactgc tctttctcaa 5460

ctggcagcac cggctccagc acctgcaccg agacttgctc ctagaccact accagcccca 5520

attcaagccc caagaccagc acccgcacca caacctgcac cggtttacgc accagcccca 5580

gtcgtttcac aagttcaggc aacttcttcc tctcaagcct cggctcaaca gagtgccttc 5640

gcacagtccc aacaatcttc agttgttcaa tctcaacaaa gttcaaacgc ttattctgca 5700

gcatcaactg ccggttcaag tgtgtcgcaa tctcaggcga ttgtctcaag cgctcctgat 5760

tattttaaca cgcaaacttt aagtagcagc ctgtcttctt ctcagcaatc actcagtgca 5820

ctcaattcga tagcgagtgg tcaactgagc tcctcgaatg ccgcttctat tttagcgagt 5880

gcagtggcac ggtcacttgg agtgtcccaa gcgtcggttc aaaatagtat aagccaacag 5940

ttgagaagcg tagggcccgg atcttccacg tcctctgtcg ctcaagcaat agcaaatgga 6000

gtggctaacg cagttggagc atcaggaact ggagttgcag gacaagaaca atctatttca 6060

caatccatat atacttcaat ttccactgct ctttctcaac tggcagcacc ggctccagca 6120

cctgcaccga gacttgctcc tagaccacta ccagccccaa ttcaagcccc aagaccagca 6180

cccgcaccac aacctgcacc ggtttacgca ccagccccag tcgtttcaca agttcaggca 6240

acttcttcct ctcaagcctc ggctcaacag agtgccttcg cacagtccca acaatcttca 6300

gttgttcaat ctcaacaaag ttcaaacgct tattctgcag catcaactgc cggttcaagt 6360

gtgtcgcaat ctcaggcgat tgtctcaagc gctcctgatt attttaacac gcaaacttta 6420

agtagcagcc tgtcttcttc tcagcaatca ctcagtgcac tcaattcgat agcgagtggt 6480

caactgagct cctcgaatgc cgcttctatt ttagcgagtg cagtggcacg gtcacttgga 6540

gtgtcccaag cgtcggttca aaatagtata agccaacagt tgagaagcgt agggcccgga 6600

tcttccacgt cctctgtcgc tcaagcaata gcaaatggag tggctaacgc agttggagca 6660

tcaggaactg gagttgcagg acaagaacaa tctatttcac aatccatata tacttcaatt 6720

tccactgctc tttctcaact ggcagcaccg gctccagcac ctgcaccgag acttgctcct 6780

agaccactac cagccccaat tcaagcccca agaccagcac ccgcaccaca acctgcaccg 6840

gtttacgcac cagccccagt cgtttcacaa gttcaggcaa cttcttcctc tcaagcctcg 6900

gctcaacaga gtgccttcgc acagtcccaa caatcttcag ttgttcaatc tcaacaaagt 6960

tcaaacgctt attctgcagc atcaactgcc ggttcaagtg tgtcgcaatc tcaggcgatt 7020

gtctcaagcg ctcctgatta ttttaacacg caaactttaa gtagcagcct gtcttcttct 7080

cagcaatcac tcagtgcact caattcgata gcgagtggtc aactgagctc ctcgaatgcc 7140

gcttctattt tagcgagtgc agtggcacgg tcacttggag tgtcccaagc gtcggttcaa 7200

aatagtataa gccaacagtt gagaagcgta gggcccggat cttccacgtc ctctgtcgct 7260

caagcaatag caaatggagt ggctaacgca gttggagcat caggaacagg agttgcagga 7320

caagaacaat ctatttcaca atccatagat acttcaattt ccactgctct ttctcaactg 7380

gcagcaccgg ctccagcacc tgcaccgaga cttgctccta gaccactacc agccccaatt 7440

caagccccaa gaccagcacc cgcaccacaa cctgcaccgg tttacgcacc agccccagtc 7500

gtttcacaag ttcaggcaac ttcttcctct caagcctcgg ctcaacagag tgccttcgca 7560

cagtcccaac aatcttcagt tgttcaatct caacaaagtt caaacgctta ttctgcagca 7620

tcaactgccg gttcaagtgt gtcgcaatct caggcgattg tctcaagcgc tcctgattat 7680

tttaacacgc aaactttaag tagcagcctg tcttcttctc agcaatcact cagtgcactc 7740

aattcgatag cgagtggtca actgagctcc tcgaatgccg cttctatttt agcgagtgca 7800

gtggcacggt cacttggagt gtcccaagcg tcggttcaaa atagtataag ccaacagttg 7860

agaagcgtag ggcccggatc ttccacgtcc tctgtcgctc aagcaatagc aaatggagtg 7920

gctaacgcag ttggagcatc aggaactgga gttgcaggac aagaacaatc tatttcacaa 7980

tccatatata cttcaatttc cactgctctt tctcaactgg cagcaccggc tccagcacct 8040

gcaccgagac ttgctcctag accactacca gccccaattc aagccccaag accagcaccc 8100

gcaccacaac ctgcaccggt ttacgcacca gccccagtcg tttcacaagt tcaggcaact 8160

tcttcctctc aagcctcggc tcaacagagt gccttcgcac agtcccaaca atcttcagtt 8220

gttcaatctc aacaaagttc aaacgcttat tctgcagcat caactgccgg ttcaagtgtg 8280

tcgcaatctc aggcgattgt ctcaagcgct cctgtttatt ttaacacgca aactttaagt 8340

agcagcctgt cttcttctct gcagtcactc agtgcactca attcgatagc gagtggtcaa 8400

ctgagctcct cgaatgccgc ttctattata gcgagtgcag tggcacggtc acttggagtg 8460

tcccaagcgt cggttcaaaa tagtataagc caacagttga gaagcgtagg gcccggatct 8520

tccacgtcct ctgtcgatca agcaatagca aatgtagagg ctaacgcaga tggatcatca 8580

ggaacaggag ttgcaggaca agaacaatct atttcacaat ccatatatac ttcaatttcc 8640

actgctcttt ctcaactggc agcaccggct ccagcacctg caccgagact tgctcctaga 8700

ccactaccag ccccaattca agccccaaga ccagcacccg caccacaacc tgcaccggtt 8760

tacgcaccag ccccagtcgt ttcacaagtt caggcaactt cttcctctca agcctcggct 8820

caacagagtg ccttcgcaca gtcccaacaa tcttcagttg ttcaatctca acaaagttca 8880

aacgcttatt ctgcagcatc aactgccggt tcaagtgtgt cgcaatctca ggcgattgtc 8940

tcaagcgctc ctgattattt taacacgcaa actttaagta gcagcctgtc ttcttctcag 9000

caatcactca gtgcactcaa ttcgatagcg agtggtcaac tgagctcctc gaatgccgct 9060

tctattttag cgagtgcagt ggcacggtca cttggagtgt cccaagcgtc ggttcaaaat 9120

agtataagcc aacagttgag aagcgtaggg cccggatctt ccacgtcctc tgtcgctcaa 9180

gcaatagcaa atggagtggc taacgcagtt ggagcatcag gaactggagt tgcaggacaa 9240

gaacaatcta tttcacaatc catatatact tcaatttcca ctgctctttc tcaactggca 9300

gcaccggctc cagcacctgc accgagactt gctcctagac cactaccagc cccaattcaa 9360

gccccaagac cagcacccgc accacaacct gcaccggttt acgcaccagc cccagtcgtt 9420

tcacaagttc aggcaacttc ttcttctcaa gcctcggctc aacagagtgc cttcgcacag 9480

tcccaacaat cttcagttgt tcaatctcaa caaagttcaa acgcttattc tgcagcatca 9540

actgccggtt caagtgtgtc gcaatctcag gcgattgtct caagcgctcc tgattatttt 9600

aacacgcaaa ctttaagtag cagcctgtct tcttctcagc aatcactcag tgcactcaat 9660

tcgatagcga gtggtcaact gagctcctcg aatgccgctt ctattttagc gagtgcagtg 9720

gcacggtcac ttggagtgtc ccaagcgtcg gttcaaaata gtataagcca acagttgaga 9780

agcgtagggc ccggatcttc cacgtcctct gtcgctcaag caatagcaaa tggagtggct 9840

aacgcagttg gagcatcagg aactggagtt gcaggacaag aacaatctat ttcacaatcc 9900

atatatactt caatttccac tgctctttct caactggcag caccggctcc agcacctgca 9960

ccgagacttg ctcctagacc actaccagcc ccaattcaag ccccaagacc agcacccgca 10020

ccacaacctg caccggttta cgcaccagcc ccagtcgttt cacaagttca ggcaacttct 10080

tcctctcaag cctcggctca acagagtgcc ttcgcacagt cccaacaatc ttcagttgtt 10140

caatctcaac aaagttcaaa cgcttattct gcagcatcaa ctgccggttc aagtgtgtcg 10200

caatctcagg cgattgtctc aagcgctcct gattatttta acacgcaaac tttaagtagc 10260

agcctgtctt cttctcagca atcactcagt gcactcaatt cgatagcgag tggtcaactg 10320

agctcctcga atgccgcttc tattttagcg agtgcagtgg cacggtcact tggagtgtcc 10380

caagcgtcgg ttcaaaatag tataagccaa cagttgagaa gcgtagggcc cggatcttcc 10440

acgtcctctg tcgctcaagc aatagcaaat ggagtggcta acgcagttgg agcatcagga 10500

actggagttg caggacaaga acaatctatt tcacaatcca tatatacttc aatttccact 10560

gctctttctc aactggcagc accggctcca gcacctgcac ctagaccagc tcctcgacca 10620

ctaccagccc caattcaagc cccaagacca gcacccgcac cacaacctgc accggtttac 10680

gcaccagccc cagtcgtttc acaagttcag gcaacttctt cctctcaagc ctcggctcaa 10740

cagagtgcct tcgcacagtc ccaacaatct tcagttgttc aatctcaaca aagttcaaac 10800

gcttattctg cagcatcaac tgccggttca agtgtgtcgc aatctcaggc gattgtctca 10860

agcgctcctg tttattttaa cacgcaaact ttaagtagca gcctgtcttc ttctctgcaa 10920

tcactcagtg cactcaattc gatagcgagt ggtcaactga gctcctcgaa tgccgcttct 10980

attatagcga gtgcagtggc acggtcactt ggagtgtccc aagcgtcggt tcaaaatagt 11040

ataagccaac agttgagaag cgtagggccc ggatcttcca cgtcctctgt cgctcaagca 11100

atagcaaatg gagtggctaa cgcagttgga gcatcaggaa caggagttgc aggacaagaa 11160

caatctattt cacaatccat atatacttca atttccactg ctctttctca actggcagca 11220

acggctccag cacctgcacc gagaccagct cctcgaccac taccagcccc aattcaagcc 11280

ccaagaccag cacccgcacc acaacctgcg ccagtttacg cacccgcgcc agtgtacgct 11340

ccagcgccag tgtacgctcc agcgccaatc gtttcccaga ttcaggaaac ttcttcttct 11400

caagcctcgg cgcaacagaa ttatttggca cagtcccagc aatcttccgt tgctcaatct 11460

caacaaagtt cgaacgctta ttcggcagta tcagctgctg attcctcatt ttcaagcgga 11520

gcttcatcag gatttggctc ttctctgtct tcttcctctg gcttttctgc cccagccagc 11580

cccgcgccgt attctccctc agtttccgca agttcttcga ttggagcctc gtccggatct 11640

gcgttggccg cttctgccgc ccagcaactc atgtctcccg cagcgaacca aaggattgcg 11700

gctctgtcca attctctgag ggctgctgta tccggaggac aagtcaacta tggcgccctc 11760

tccaactcgc ttgccagcgc agcgagtcag atacagagtt catccggctt gtccaagagt 11820

gaagttttag tcgaggctct cctggaaacg ttgtttgctc tgctgagctc tctcacgtct 11880

ggagcgtcta cggctgattt cgcacaggtc gtacttgcag cacttggata g 11931

<210> 2

<211> 450

<212> DNA

<213>artificial sequence

<400> 2

atgtcttgga ccctggggct tcagttttta ttcacatttt gcttggtgct cttcgcatgg 60

aggggagatg ctctttcaga cccaaaatct tgggttcagg acgaagcgac ggccgtttat 120

ttcttggagc gcaccttgga gtatctggga gaatgcggcg ttctgaaaat tgaccaggag 180

agagatgcca tcgacaccat gtttcagatg agttcaatgt ttcaacagaa tgcaaaaccc 240

agcaagataa agcactctat atcttccaag ttggccgaca tagtcataga aggtctggag 300

ggagacagcg acccgtccta caagatcgac tgcgtaaccc aagctattgc caccgctttc 360

gaagcaacaa caggtgttcg agatgaaaat ttcattgagt ctgtcattga acttatagta 420

ttaatattta acaatgatat cgaagagaag 450

<210> 3

<211> 768

<212> DNA

<213>artificial sequence

<400> 3

atggaagaag aaatcgaaga gactgctgca agtcagcagg cggcagctat tgaaaaccaa 60

atttcgacaa tagaatcgaa ctttggatcg gcacagactc agtcatcagt gcagaacgat 120

atacagcagc aaagcactgt agatatttct tcttcggtca ccaatcagat tcagtctcag 180

cagtatgcag acagtcaatc ttccgcgcaa attcagtcac agcaaagtgt cgctagccaa 240

caacaagatc ttacttctca actaacgact tctagcgctc agcaacaata tgaaacttcc 300

caagcttcta tcgcacagca gcaatatgaa acttcccaag cttctatcgc gcagcagcaa 360

tatgaagctt ctcaacagca ggcttctatc gagcagcaac aatatgaagc ttcgcaagct 420

tctatcgcgc aacagcaata tgaagcttct caacagcagg cttctatcga gcagcaacaa 480

tatgaagctt cgcaagcttc tatcgcgcaa cagcaatatg aagcttctca acagcaagct 540

tctatcgcgc agcaacaata tcaaagttcg cagcaacagg cttctataaa tcagcaacaa 600

tatgaagctt ctcagcagca atcttccagt tctgaacagc agtacacaac ttccgaacag 660

caaattcagc agtccgagaa tgcatatacc gatcagtcaa atattcaaca atccgctcaa 720

acacaacagt acagcacttc acaacaagaa caagaatcct ctgcaagc 768

<210> 4

<211> 10116

<212> DNA

<213>artificial sequence

<400> 4

tattctacag cctcaattgc cggttcaagt gtgtcgcaat ctcaggcgac tgtctcaagc 60

gcacctgatt attttaacac gcaaacttta agtagcagcc tgtcttcttc tctgcaatca 120

ctcagtgcac tcaattcgat agcgagtggt caactgagct cctcgaatgc cgcttctatt 180

ttagcgagtg cagtggcaca gtcacttgga gtgtcccaag cgtcggttca aaatagcata 240

agccaacagt tgagaagcgt agggcccgga tcttccacgt cctctgtcgc tcaagcaata 300

gcaaatggag tggctaacgc agttggagca tcaggaactg gagttgcagg acaagaacaa 360

tctatttcac aatccatata tacttcaatt tccactgctc tttctcaact ggcagcaccg 420

gctccagcac ctgcaccgag acttgctcct agaccactac cagccccaat tcaagcccca 480

agaccagcac ccgcaccaca acctgcaccg gtttacgcac cagccccagt cgtttcacaa 540

gttcaggcaa cttcttcctc tcaagcctcg gctcaacaga gtgccttcgc acagtcccaa 600

caatcttcag ttgttcaatc tcaacaaagt tcaaacgctt attctgcagc atcaactgcc 660

ggttcaagtg tgtcgcaatc tcaggcgatt gtctcaagcg ctcctgttta ttttaacacg 720

caaactttaa gtagcagcct gtcttcttct ctgcaatcac tcagtgcact caattcgtta 780

gcgagtggtc aactgagctc ctggaatgcc gcttctatta tagcgagtgc ggtagcaccg 840

tcacttggag tgtcccaagc gtcggttcaa aatagtataa gccaacagtt gagaagcgta 900

gggcccggat cttccacgtc ctctgtcgct caagcaatag caaatggagt ggctaacgca 960

gttggagcat caggaacagg agttgcagga caagaacaat ctatttcaca atccatatat 1020

acttcagttt ccactgctct atctcaactg gcagcaccgg ctccagcacc tgcacctaga 1080

ccagctcctc gaccactacc agccccaatt caagccccaa gaccagcacc cgcaccacaa 1140

cctgcaccgg tttacgcacc agccccagtc gtttcacaag ttcaggcaac ttcttcctct 1200

caagcctcgg ctcaacagag tgccttcgca cagtcccaac aatcttcagt tgttcaatct 1260

caacaaagct caaacgctta ttctgcagct tcatctgtag gctcaagttt ttcgcagtct 1320

caggggactg tcccaagcgc tcctgtttat tttaacacgc aaactttaag tagcagcctg 1380

tcttcttctc tgcaatcact cagtgcactc aattcgatag cgagtggtca actgagctcc 1440

tcgaatgccg cttctattat agcgagtgca gtggcacggt cacttggagt gtcccaagcg 1500

tcggttcaaa atagtataag ccaacagttg agaagcgtag ggcccggatc ttccacgtcc 1560

tctgtcgctc aagcaatagc aaatggagtg gctaacgcag ttggagcatc aggaactgga 1620

gttgcaggac aagaacaatc tatttcacaa tccatatata cttcaatttc cactgctctt 1680

tctcaactgg cagcaccggc tccagcacct gcaccgagac ttgctcctag accactacca 1740

gccccaattc aagccccaag accagcaccc gcaccacaac ctgcaccggt ttacgcacca 1800

gccccagtcg tttcacaagt tcaggcaact tcttcctctc aagcttcggc tcaacagagt 1860

gccttcgcac agtcccagca atcttcagtt gcacaatctc aacaaagctc aaacgttgat 1920

tctgcagctt catctgtagg ctcaagtttt tcgcagtctc aggggactgt cccaagcgct 1980

cctgtttatt ttaacacgca aactttaagt agcagcctgt cttcttctct gcaatcactc 2040

agtgcactca attcgatagc gagtggtcaa ctgagctcat cgtatgccga ttctatttta 2100

gcgagtgcag tggctcggtc tcttggagtt tcccaagcgt cggttctaaa tagtataagc 2160

caacagttga gaagcgtagg gcccggatct tccacgtcct ctgtcgctca agcaatagca 2220

aatggagtgg ctaacgcagt tggagcatca ggaacaggag ttgcaggaca agaacaatct 2280

atttcacaat ccatatatac ttcagtttcc actgctcttt ctcaactggc agcaccggct 2340

ccagcacctg caccgagact tgctcctaga ccactaccag ccccaattca agccccaaga 2400

ccagcacccg caccacaacc tgcaccggtt tacgcaccag ccccagtcgt ttcacaagtt 2460

caggcaactt cttcctctca agcctcggct caacagagtg ccttcgcaca gtcccaacaa 2520

tcttcagttg ttcaatctca acaaagttca aacgcttatt ctgcagcatc aactgccggt 2580

tcaagtgtgt cgcaatctca ggcgattgtc tcaagcgctc ctgattattt taacacgcaa 2640

actttaagta gcagcctgtc ttcttctcag caatcactca gtgcactcaa ttcgatagcg 2700

agtggtcaac tgagctcctc gaatgccgct tctattttag cgagtgcagt ggcacggtca 2760

cttgtagtgt cccacgcgtc ggttcaaaat agtataagcc aacagttgag aagcgtaggg 2820

cccggatctt ccacgtcctc tgtcgctcaa gcaatagcaa atggagtggc taacgcagtt 2880

ggagcatcag gaactggagt tgcaggacaa gaacaatcta tttcacaatc catatatact 2940

tcaatttcca ctgctctttc tcaactggca gcaccggctc cagcacctgc accgagactt 3000

gctcctagac cactaccagc cccaattcaa gccccaagac cagcacccgc accacaacct 3060

gcaccggttt acgcaccagc cccagtcgtt tcacaagttc aggcaacttc ttcctctcaa 3120

gcctcggctc aacagagtgc cttcgcacag tcccaacaat cttcagttgt tcaatctcaa 3180

caaagttcaa acgcttattc tgcagcatca actgccggtt caagtgtgtc gcaatctcag 3240

gcgattgtct caagcgctcc tgattatttt aacacgcaaa ctttaagtag cagcctgtct 3300

tcttctcagc aatcactcag tgcactcaat tcgatagcga gtggtcaact gagctcctcg 3360

aatgccgctt ctattttagc gagtgcagtg gcacggtcac ttggagtgtc ccaagcgtcg 3420

gttcaaaata gtataagcca acagttgaga agcgtagggc ccggatcttc cacgtcctct 3480

gtcgctcaag caatagcaaa tggagtggct aacgcagttg gagcatcagg aactggagtt 3540

gcaggacaag aacaatctat ttcacaatcc atatatactt caatttccac tgctctttct 3600

caactggcag caccggctcc agcacctgca ccgagacttg ctcctagacc actaccagcc 3660

ccaattcaag ccccaagacc agcacccgca ccacaacctg caccggttta cgcaccagcc 3720

ccagtcgttt cacaagttca ggcaacttct tcctctcaag cctcggctca acagagtgcc 3780

ttcgcacagt cccaacaatc ttcagttgtt caatctcaac aaagttcaaa cgcttattct 3840

gcagcatcaa ctgccggttc aagtgtgtcg caatctcagg cgattgtctc aagcgctcct 3900

gattatttta acacgcaaac tttaagtagc agcctgtctt cttctcagca atcactcagt 3960

gcactcaatt cgatagcgag tggtcaactg agctcctcga atgccgcttc tattttagcg 4020

agtgcagtgg cacggtcact tggagtgtcc caagcgtcgg ttcaaaatag tataagccaa 4080

cagttgagaa gcgtagggcc cggatcttcc acgtcctctg tcgctcaagc aatagcaaat 4140

ggagtggcta acgcagttgg agcatcagga actggagttg caggacaaga acaatctatt 4200

tcacaatcca tatatacttc aatttccact gctctttctc aactggcagc accggctcca 4260

gcacctgcac cgagacttgc tcctagacca ctaccagccc caattcaagc cccaagacca 4320

gcacccgcac cacaacctgc accggtttac gcaccagccc cagtcgtttc acaagttcag 4380

gcaacttctt cctctcaagc ctcggctcaa cagagtgcct tcgcacagtc ccaacaatct 4440

tcagttgttc aatctcaaca aagttcaaac gcttattctg cagcatcaac tgccggttca 4500

agtgtgtcgc aatctcaggc gattgtctca agcgctcctg attattttaa cacgcaaact 4560

ttaagtagca gcctgtcttc ttctcagcaa tcactcagtg cactcaattc gatagcgagt 4620

ggtcaactga gctcctcgaa tgccgcttct attttagcga gtgcagtggc acggtcactt 4680

ggagtgtccc aagcgtcggt tcaaaatagt ataagccaac agttgagaag cgtagggccc 4740

ggatcttcca cgtcctctgt cgctcaagca atagcaaatg gagtggctaa cgcagttgga 4800

gcatcaggaa ctggagttgc aggacaagaa caatctattt cacaatccat atatacttca 4860

atttccactg ctctttctca actggcagca ccggctccag cacctgcacc gagacttgct 4920

cctagaccac taccagcccc aattcaagcc ccaagaccag cacccgcacc acaacctgca 4980

ccggtttacg caccagcccc agtcgtttca caagttcagg caacttcttc ctctcaagcc 5040

tcggctcaac agagtgcctt cgcacagtcc caacaatctt cagttgttca atctcaacaa 5100

agttcaaacg cttattctgc agcatcaact gccggttcaa gtgtgtcgca atctcaggcg 5160

attgtctcaa gcgctcctga ttattttaac acgcaaactt taagtagcag cctgtcttct 5220

tctcagcaat cactcagtgc actcaattcg atagcgagtg gtcaactgag ctcctcgaat 5280

gccgcttcta ttttagcgag tgcagtggca cggtcacttg gagtgtccca agcgtcggtt 5340

caaaatagta taagccaaca gttgagaagc gtagggcccg gatcttccac gtcctctgtc 5400

gctcaagcaa tagcaaatgg agtggctaac gcagttggag catcaggaac tggagttgca 5460

ggacaagaac aatctatttc acaatccata tatacttcaa tttccactgc tctttctcaa 5520

ctggcagcac cggctccagc acctgcaccg agacttgctc ctagaccact accagcccca 5580

attcaagccc caagaccagc acccgcacca caacctgcac cggtttacgc accagcccca 5640

gtcgtttcac aagttcaggc aacttcttcc tctcaagcct cggctcaaca gagtgccttc 5700

gcacagtccc aacaatcttc agttgttcaa tctcaacaaa gttcaaacgc ttattctgca 5760

gcatcaactg ccggttcaag tgtgtcgcaa tctcaggcga ttgtctcaag cgctcctgat 5820

tattttaaca cgcaaacttt aagtagcagc ctgtcttctt ctcagcaatc actcagtgca 5880

ctcaattcga tagcgagtgg tcaactgagc tcctcgaatg ccgcttctat tttagcgagt 5940

gcagtggcac ggtcacttgg agtgtcccaa gcgtcggttc aaaatagtat aagccaacag 6000

ttgagaagcg tagggcccgg atcttccacg tcctctgtcg ctcaagcaat agcaaatgga 6060

gtggctaacg cagttggagc atcaggaaca ggagttgcag gacaagaaca atctatttca 6120

caatccatag atacttcaat ttccactgct ctttctcaac tggcagcacc ggctccagca 6180

cctgcaccga gacttgctcc tagaccacta ccagccccaa ttcaagcccc aagaccagca 6240

cccgcaccac aacctgcacc ggtttacgca ccagccccag tcgtttcaca agttcaggca 6300

acttcttcct ctcaagcctc ggctcaacag agtgccttcg cacagtccca acaatcttca 6360

gttgttcaat ctcaacaaag ttcaaacgct tattctgcag catcaactgc cggttcaagt 6420

gtgtcgcaat ctcaggcgat tgtctcaagc gctcctgatt attttaacac gcaaacttta 6480

agtagcagcc tgtcttcttc tcagcaatca ctcagtgcac tcaattcgat agcgagtggt 6540

caactgagct cctcgaatgc cgcttctatt ttagcgagtg cagtggcacg gtcacttgga 6600

gtgtcccaag cgtcggttca aaatagtata agccaacagt tgagaagcgt agggcccgga 6660

tcttccacgt cctctgtcgc tcaagcaata gcaaatggag tggctaacgc agttggagca 6720

tcaggaactg gagttgcagg acaagaacaa tctatttcac aatccatata tacttcaatt 6780

tccactgctc tttctcaact ggcagcaccg gctccagcac ctgcaccgag acttgctcct 6840

agaccactac cagccccaat tcaagcccca agaccagcac ccgcaccaca acctgcaccg 6900

gtttacgcac cagccccagt cgtttcacaa gttcaggcaa cttcttcctc tcaagcctcg 6960

gctcaacaga gtgccttcgc acagtcccaa caatcttcag ttgttcaatc tcaacaaagt 7020

tcaaacgctt attctgcagc atcaactgcc ggttcaagtg tgtcgcaatc tcaggcgatt 7080

gtctcaagcg ctcctgttta ttttaacacg caaactttaa gtagcagcct gtcttcttct 7140

ctgcagtcac tcagtgcact caattcgata gcgagtggtc aactgagctc ctcgaatgcc 7200

gcttctatta tagcgagtgc agtggcacgg tcacttggag tgtcccaagc gtcggttcaa 7260

aatagtataa gccaacagtt gagaagcgta gggcccggat cttccacgtc ctctgtcgat 7320

caagcaatag caaatgtaga ggctaacgca gatggatcat caggaacagg agttgcagga 7380

caagaacaat ctatttcaca atccatatat acttcaattt ccactgctct ttctcaactg 7440

gcagcaccgg ctccagcacc tgcaccgaga cttgctccta gaccactacc agccccaatt 7500

caagccccaa gaccagcacc cgcaccacaa cctgcaccgg tttacgcacc agccccagtc 7560

gtttcacaag ttcaggcaac ttcttcctct caagcctcgg ctcaacagag tgccttcgca 7620

cagtcccaac aatcttcagt tgttcaatct caacaaagtt caaacgctta ttctgcagca 7680

tcaactgccg gttcaagtgt gtcgcaatct caggcgattg tctcaagcgc tcctgattat 7740

tttaacacgc aaactttaag tagcagcctg tcttcttctc agcaatcact cagtgcactc 7800

aattcgatag cgagtggtca actgagctcc tcgaatgccg cttctatttt agcgagtgca 7860

gtggcacggt cacttggagt gtcccaagcg tcggttcaaa atagtataag ccaacagttg 7920

agaagcgtag ggcccggatc ttccacgtcc tctgtcgctc aagcaatagc aaatggagtg 7980

gctaacgcag ttggagcatc aggaactgga gttgcaggac aagaacaatc tatttcacaa 8040

tccatatata cttcaatttc cactgctctt tctcaactgg cagcaccggc tccagcacct 8100

gcaccgagac ttgctcctag accactacca gccccaattc aagccccaag accagcaccc 8160

gcaccacaac ctgcaccggt ttacgcacca gccccagtcg tttcacaagt tcaggcaact 8220

tcttcttctc aagcctcggc tcaacagagt gccttcgcac agtcccaaca atcttcagtt 8280

gttcaatctc aacaaagttc aaacgcttat tctgcagcat caactgccgg ttcaagtgtg 8340

tcgcaatctc aggcgattgt ctcaagcgct cctgattatt ttaacacgca aactttaagt 8400

agcagcctgt cttcttctca gcaatcactc agtgcactca attcgatagc gagtggtcaa 8460

ctgagctcct cgaatgccgc ttctatttta gcgagtgcag tggcacggtc acttggagtg 8520

tcccaagcgt cggttcaaaa tagtataagc caacagttga gaagcgtagg gcccggatct 8580

tccacgtcct ctgtcgctca agcaatagca aatggagtgg ctaacgcagt tggagcatca 8640

ggaactggag ttgcaggaca agaacaatct atttcacaat ccatatatac ttcaatttcc 8700

actgctcttt ctcaactggc agcaccggct ccagcacctg caccgagact tgctcctaga 8760

ccactaccag ccccaattca agccccaaga ccagcacccg caccacaacc tgcaccggtt 8820

tacgcaccag ccccagtcgt ttcacaagtt caggcaactt cttcctctca agcctcggct 8880

caacagagtg ccttcgcaca gtcccaacaa tcttcagttg ttcaatctca acaaagttca 8940

aacgcttatt ctgcagcatc aactgccggt tcaagtgtgt cgcaatctca ggcgattgtc 9000

tcaagcgctc ctgattattt taacacgcaa actttaagta gcagcctgtc ttcttctcag 9060

caatcactca gtgcactcaa ttcgatagcg agtggtcaac tgagctcctc gaatgccgct 9120

tctattttag cgagtgcagt ggcacggtca cttggagtgt cccaagcgtc ggttcaaaat 9180

agtataagcc aacagttgag aagcgtaggg cccggatctt ccacgtcctc tgtcgctcaa 9240

gcaatagcaa atggagtggc taacgcagtt ggagcatcag gaactggagt tgcaggacaa 9300

gaacaatcta tttcacaatc catatatact tcaatttcca ctgctctttc tcaactggca 9360

gcaccggctc cagcacctgc acctagacca gctcctcgac cactaccagc cccaattcaa 9420

gccccaagac cagcacccgc accacaacct gcaccggttt acgcaccagc cccagtcgtt 9480

tcacaagttc aggcaacttc ttcctctcaa gcctcggctc aacagagtgc cttcgcacag 9540

tcccaacaat cttcagttgt tcaatctcaa caaagttcaa acgcttattc tgcagcatca 9600

actgccggtt caagtgtgtc gcaatctcag gcgattgtct caagcgctcc tgtttatttt 9660

aacacgcaaa ctttaagtag cagcctgtct tcttctctgc aatcactcag tgcactcaat 9720

tcgatagcga gtggtcaact gagctcctcg aatgccgctt ctattatagc gagtgcagtg 9780

gcacggtcac ttggagtgtc ccaagcgtcg gttcaaaata gtataagcca acagttgaga 9840

agcgtagggc ccggatcttc cacgtcctct gtcgctcaag caatagcaaa tggagtggct 9900

aacgcagttg gagcatcagg aacaggagtt gcaggacaag aacaatctat ttcacaatcc 9960

atatatactt caatttccac tgctctttct caactggcag caacggctcc agcacctgca 10020

ccgagaccag ctcctcgacc actaccagcc ccaattcaag ccccaagacc agcacccgca 10080

ccacaacctg cgccagttta cgcacccgcg ccagtg 10116

<210> 5

<211> 327

<212> DNA

<213>artificial sequence

<400> 5

tacgctccag cgccagtgta cgctccagcg ccaatcgttt cccagattca ggaaacttct 60

tcttctcaag cctcggcgca acagaattat ttggcacagt cccagcaatc ttccgttgct 120

caatctcaac aaagttcgaa cgcttattcg gcagtatcag ctgctgattc ctcattttca 180

agcggagctt catcaggatt tggctcttct ctgtcttctt cctctggctt ttctgcccca 240

gccagccccg cgccgtattc tccctcagtt tccgcaagtt cttcgattgg agcctcgtcc 300

ggatctgcgt tggccgcttc tgccgcc 327

<210> 6

<211> 267

<212> DNA

<213>artificial sequence

<400> 6

cagcaactca tgtctcccgc agcgaaccaa aggattgcgg ctctgtccaa ttctctgagg 60

gctgctgtat ccggaggaca agtcaactat ggcgccctct ccaactcgct tgccagcgca 120

gcgagtcaga tacagagttc atccggcttg tccaagagtg aagttttagt cgaggctctc 180

ctggaaacgt tgtttgctct gctgagctct ctcacgtctg gagcgtctac ggctgatttc 240

gcacaggtcg tacttgcagc acttgga 267

<210> 7

<211> 21

<212> DNA

<213>artificial sequence

<400> 7

atgttycara tgagttcaat g 21

<210> 8

<211> 21

<212> DNA

<213>artificial sequence

<400> 8

ctgttgtgtt tgagcggatt g 21

<210> 9

<211> 20

<212> DNA

<213>artificial sequence

<400> 9

tttcaatagc tgccgcctgc 20

<210> 10

<211> 22

<212> DNA

<213>artificial sequence

<400> 10

gtggcaatag cttgggttac gc 22

<210> 11

<211> 27

<212> DNA

<213>artificial sequence

<400> 11

actcctgtgg aaccatcgga cgggggg 27

<210> 12

<211> 33

<212> DNA

<213>artificial sequence

<400> 12

atgtcttgga ccctggggct tcagttttta ttc 33

<210> 13

<211> 24

<212> DNA

<213>artificial sequence

<400> 13

ctatccaagt gctgcaagta cgac 24

<210> 14

<211> 21

<212> DNA

<213>artificial sequence

<400> 14

tattctgcag catcaactgc c 21

<210> 15

<211> 24

<212> DNA

<213>artificial sequence

<400> 15

agcgtttgag ctttgttgag attg 24

<210> 16

<211> 3976

<212> PRT

<213>artificial sequence

<400> 16

Met Ser Trp Thr Leu Gly Leu Gln Phe Leu Phe Thr Phe Cys Leu Val

1 5 10 15

Leu Phe Ala Trp Arg Gly Asp Ala Leu Ser Asp Pro Lys Ser Trp Val

20 25 30

Gln Asp Glu Ala Thr Ala Val Tyr Phe Leu Glu Arg Thr Leu Glu Tyr

35 40 45

Leu Gly Glu Cys Gly Val Leu Lys Ile Asp Gln Glu Arg Asp Ala Ile

50 55 60

Asp Thr Met Phe Gln Met Ser Ser Met Phe Gln Gln Asn Ala Lys Pro

65 70 75 80

Ser Lys Ile Lys His Ser Ile Ser Ser Lys Leu Ala Asp Ile Val Ile

85 90 95

Glu Gly Leu Glu Gly Asp Ser Asp Pro Ser Tyr Lys Ile Asp Cys Val

100 105 110

Thr Gln Ala Ile Ala Thr Ala Phe Glu Ala Thr Thr Gly Val Arg Asp

115 120 125

Glu Asn Phe Ile Glu Ser Val Ile Glu Leu Ile Val Leu Ile Phe Asn

130 135 140

Asn Asp Ile Glu Glu Lys Met Glu Glu Glu Ile Glu Glu Thr Ala Ala

145 150 155 160

Ser Gln Gln Ala Ala Ala Ile Glu Asn Gln Ile Ser Thr Ile Glu Ser

165 170 175

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

180 185 190

Gln Gln Ser Thr Val Asp Ile Ser Ser Ser Val Thr Asn Gln Ile Gln

195 200 205

Ser Gln Gln Tyr Ala Asp Ser Gln Ser Ser Ala Gln Ile Gln Ser Gln

210 215 220

Gln Ser Val Ala Ser Gln Gln Gln Asp Leu Thr Ser Gln Leu Thr Thr

225 230 235 240

Ser Ser Ala Gln Gln Gln Tyr Glu Thr Ser Gln Ala Ser Ile Ala Gln

245 250 255

Gln Gln Tyr Glu Thr Ser Gln Ala Ser Ile Ala Gln Gln Gln Tyr Glu

260 265 270

Ala Ser Gln Gln Gln Ala Ser Ile Glu Gln Gln Gln Tyr Glu Ala Ser

275 280 285

Gln Ala Ser Ile Ala Gln Gln Gln Tyr Glu Ala Ser Gln Gln Gln Ala

290 295 300

Ser Ile Glu Gln Gln Gln Tyr Glu Ala Ser Gln Ala Ser Ile Ala Gln

305 310 315 320

Gln Gln Tyr Glu Ala Ser Gln Gln Gln Ala Ser Ile Ala Gln Gln Gln

325 330 335

Tyr Gln Ser Ser Gln Gln Gln Ala Ser Ile Asn Gln Gln Gln Tyr Glu

340 345 350

Ala Ser Gln Gln Gln Ser Ser Ser Ser Glu Gln Gln Tyr Thr Thr Ser

355 360 365

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

370 375 380

Ile Gln Gln Ser Ala Gln Thr Gln Gln Tyr Ser Thr Ser Gln Gln Glu

385 390 395 400

Gln Glu Ser Ser Ala Ser Tyr Ser Thr Ala Ser Ile Ala Gly Ser Ser

405 410 415

Val Ser Gln Ser Gln Ala Thr Val Ser Ser Ala Pro Asp Tyr Phe Asn

420 425 430

Thr Gln Thr Leu Ser Ser Ser Leu Ser Ser Ser Leu Gln Ser Leu Ser

435 440 445

Ala Leu Asn Ser Ile Ala Ser Gly Gln Leu Ser Ser Ser Asn Ala Ala

450 455 460

Ser Ile Leu Ala Ser Ala Val Ala Gln Ser Leu Gly Val Ser Gln Ala

465 470 475 480

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

485 490 495

Ser Ser Thr Ser Ser Val Ala Gln Ala Ile Ala Asn Gly Val Ala Asn

500 505 510

Ala Val Gly Ala Ser Gly Thr Gly Val Ala Gly Gln Glu Gln Ser Ile

515 520 525

Ser Gln Ser Ile Tyr Thr Ser Ile Ser Thr Ala Leu Ser Gln Leu Ala

530 535 540

Ala Pro Ala Pro Ala Pro Ala Pro Arg Leu Ala Pro Arg Pro Leu Pro

545 550 555 560

Ala Pro Ile Gln Ala Pro Arg Pro Ala Pro Ala Pro Gln Pro Ala Pro

565 570 575

Val Tyr Ala Pro Ala Pro Val Val Ser Gln Val Gln Ala Thr Ser Ser

580 585 590

Ser Gln Ala Ser Ala Gln Gln Ser Ala Phe Ala Gln Ser Gln Gln Ser

595 600 605

Ser Val Val Gln Ser Gln Gln Ser Ser Asn Ala Tyr Ser Ala Ala Ser

610 615 620

Thr Ala Gly Ser Ser Val Ser Gln Ser Gln Ala Ile Val Ser Ser Ala

625 630 635 640

Pro Val Tyr Phe Asn Thr Gln Thr Leu Ser Ser Ser Leu Ser Ser Ser

645 650 655

Leu Gln Ser Leu Ser Ala Leu Asn Ser Leu Ala Ser Gly Gln Leu Ser

660 665 670

Ser Trp Asn Ala Ala Ser Ile Ile Ala Ser Ala Val Ala Pro Ser Leu

675 680 685

Gly Val Ser Gln Ala Ser Val Gln Asn Ser Ile Ser Gln Gln Leu Arg

690 695 700

Ser Val Gly Pro Gly Ser Ser Thr Ser Ser Val Ala Gln Ala Ile Ala

705 710 715 720

Asn Gly Val Ala Asn Ala Val Gly Ala Ser Gly Thr Gly Val Ala Gly

725 730 735

Gln Glu Gln Ser Ile Ser Gln Ser Ile Tyr Thr Ser Val Ser Thr Ala

740 745 750

Leu Ser Gln Leu Ala Ala Pro Ala Pro Ala Pro Ala Pro Arg Pro Ala

755 760 765

Pro Arg Pro Leu Pro Ala Pro Ile Gln Ala Pro Arg Pro Ala Pro Ala

770 775 780

Pro Gln Pro Ala Pro Val Tyr Ala Pro Ala Pro Val Val Ser Gln Val

785 790 795 800

Gln Ala Thr Ser Ser Ser Gln Ala Ser Ala Gln Gln Ser Ala Phe Ala

805 810 815

Gln Ser Gln Gln Ser Ser Val Val Gln Ser Gln Gln Ser Ser Asn Ala

820 825 830

Tyr Ser Ala Ala Ser Ser Val Gly Ser Ser Phe Ser Gln Ser Gln Gly

835 840 845

Thr Val Pro Ser Ala Pro Val Tyr Phe Asn Thr Gln Thr Leu Ser Ser

850 855 860

Ser Leu Ser Ser Ser Leu Gln Ser Leu Ser Ala Leu Asn Ser Ile Ala

865 870 875 880

Ser Gly Gln Leu Ser Ser Ser Asn Ala Ala Ser Ile Ile Ala Ser Ala

885 890 895

Val Ala Arg Ser Leu Gly Val Ser Gln Ala Ser Val Gln Asn Ser Ile

900 905 910

Ser Gln Gln Leu Arg Ser Val Gly Pro Gly Ser Ser Thr Ser Ser Val

915 920 925

Ala Gln Ala Ile Ala Asn Gly Val Ala Asn Ala Val Gly Ala Ser Gly

930 935 940

Thr Gly Val Ala Gly Gln Glu Gln Ser Ile Ser Gln Ser Ile Tyr Thr

945 950 955 960

Ser Ile Ser Thr Ala Leu Ser Gln Leu Ala Ala Pro Ala Pro Ala Pro

965 970 975

Ala Pro Arg Leu Ala Pro Arg Pro Leu Pro Ala Pro Ile Gln Ala Pro

980 985 990

Arg Pro Ala Pro Ala Pro Gln Pro Ala Pro Val Tyr Ala Pro Ala Pro

995 1000 1005

Val Val Ser Gln Val Gln Ala Thr Ser Ser Ser Gln Ala Ser Ala

1010 1015 1020

Gln Gln Ser Ala Phe Ala Gln Ser Gln Gln Ser Ser Val Ala Gln

1025 1030 1035

Ser Gln Gln Ser Ser Asn Val Asp Ser Ala Ala Ser Ser Val Gly

1040 1045 1050

Ser Ser Phe Ser Gln Ser Gln Gly Thr Val Pro Ser Ala Pro Val

1055 1060 1065

Tyr Phe Asn Thr Gln Thr Leu Ser Ser Ser Leu Ser Ser Ser Leu

1070 1075 1080

Gln Ser Leu Ser Ala Leu Asn Ser Ile Ala Ser Gly Gln Leu Ser

1085 1090 1095

Ser Ser Tyr Ala Asp Ser Ile Leu Ala Ser Ala Val Ala Arg Ser

1100 1105 1110

Leu Gly Val Ser Gln Ala Ser Val Leu Asn Ser Ile Ser Gln Gln

1115 1120 1125

Leu Arg Ser Val Gly Pro Gly Ser Ser Thr Ser Ser Val Ala Gln

1130 1135 1140

Ala Ile Ala Asn Gly Val Ala Asn Ala Val Gly Ala Ser Gly Thr

1145 1150 1155

Gly Val Ala Gly Gln Glu Gln Ser Ile Ser Gln Ser Ile Tyr Thr

1160 1165 1170

Ser Val Ser Thr Ala Leu Ser Gln Leu Ala Ala Pro Ala Pro Ala

1175 1180 1185

Pro Ala Pro Arg Leu Ala Pro Arg Pro Leu Pro Ala Pro Ile Gln

1190 1195 1200

Ala Pro Arg Pro Ala Pro Ala Pro Gln Pro Ala Pro Val Tyr Ala

1205 1210 1215

Pro Ala Pro Val Val Ser Gln Val Gln Ala Thr Ser Ser Ser Gln

1220 1225 1230

Ala Ser Ala Gln Gln Ser Ala Phe Ala Gln Ser Gln Gln Ser Ser

1235 1240 1245

Val Val Gln Ser Gln Gln Ser Ser Asn Ala Tyr Ser Ala Ala Ser

1250 1255 1260

Thr Ala Gly Ser Ser Val Ser Gln Ser Gln Ala Ile Val Ser Ser

1265 1270 1275

Ala Pro Asp Tyr Phe Asn Thr Gln Thr Leu Ser Ser Ser Leu Ser

1280 1285 1290

Ser Ser Gln Gln Ser Leu Ser Ala Leu Asn Ser Ile Ala Ser Gly

1295 1300 1305

Gln Leu Ser Ser Ser Asn Ala Ala Ser Ile Leu Ala Ser Ala Val

1310 1315 1320

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

1325 1330 1335

Ser Gln Gln Leu Arg Ser Val Gly Pro Gly Ser Ser Thr Ser Ser

1340 1345 1350

Val Ala Gln Ala Ile Ala Asn Gly Val Ala Asn Ala Val Gly Ala

1355 1360 1365

Ser Gly Thr Gly Val Ala Gly Gln Glu Gln Ser Ile Ser Gln Ser

1370 1375 1380

Ile Tyr Thr Ser Ile Ser Thr Ala Leu Ser Gln Leu Ala Ala Pro

1385 1390 1395

Ala Pro Ala Pro Ala Pro Arg Leu Ala Pro Arg Pro Leu Pro Ala

1400 1405 1410

Pro Ile Gln Ala Pro Arg Pro Ala Pro Ala Pro Gln Pro Ala Pro

1415 1420 1425

Val Tyr Ala Pro Ala Pro Val Val Ser Gln Val Gln Ala Thr Ser

1430 1435 1440

Ser Ser Gln Ala Ser Ala Gln Gln Ser Ala Phe Ala Gln Ser Gln

1445 1450 1455

Gln Ser Ser Val Val Gln Ser Gln Gln Ser Ser Asn Ala Tyr Ser

1460 1465 1470

Ala Ala Ser Thr Ala Gly Ser Ser Val Ser Gln Ser Gln Ala Ile

1475 1480 1485

Val Ser Ser Ala Pro Asp Tyr Phe Asn Thr Gln Thr Leu Ser Ser

1490 1495 1500

Ser Leu Ser Ser Ser Gln Gln Ser Leu Ser Ala Leu Asn Ser Ile

1505 1510 1515

Ala Ser Gly Gln Leu Ser Ser Ser Asn Ala Ala Ser Ile Leu Ala

1520 1525 1530

Ser Ala Val Ala Arg Ser Leu Gly Val Ser Gln Ala Ser Val Gln

1535 1540 1545

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

1550 1555 1560

Thr Ser Ser Val Ala Gln Ala Ile Ala Asn Gly Val Ala Asn Ala

1565 1570 1575

Val Gly Ala Ser Gly Thr Gly Val Ala Gly Gln Glu Gln Ser Ile

1580 1585 1590

Ser Gln Ser Ile Tyr Thr Ser Ile Ser Thr Ala Leu Ser Gln Leu

1595 1600 1605

Ala Ala Pro Ala Pro Ala Pro Ala Pro Arg Leu Ala Pro Arg Pro

1610 1615 1620

Leu Pro Ala Pro Ile Gln Ala Pro Arg Pro Ala Pro Ala Pro Gln

1625 1630 1635

Pro Ala Pro Val Tyr Ala Pro Ala Pro Val Val Ser Gln Val Gln

1640 1645 1650

Ala Thr Ser Ser Ser Gln Ala Ser Ala Gln Gln Ser Ala Phe Ala

1655 1660 1665

Gln Ser Gln Gln Ser Ser Val Val Gln Ser Gln Gln Ser Ser Asn

1670 1675 1680

Ala Tyr Ser Ala Ala Ser Thr Ala Gly Ser Ser Val Ser Gln Ser

1685 1690 1695

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

1700 1705 1710

Leu Ser Ser Ser Leu Ser Ser Ser Gln Gln Ser Leu Ser Ala Leu

1715 1720 1725

Asn Ser Ile Ala Ser Gly Gln Leu Ser Ser Ser Asn Ala Ala Ser

1730 1735 1740

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

1745 1750 1755

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

1760 1765 1770

Gly Ser Ser Thr Ser Ser Val Ala Gln Ala Ile Ala Asn Gly Val

1775 1780 1785

Ala Asn Ala Val Gly Ala Ser Gly Thr Gly Val Ala Gly Gln Glu

1790 1795 1800

Gln Ser Ile Ser Gln Ser Ile Tyr Thr Ser Ile Ser Thr Ala Leu

1805 1810 1815

Ser Gln Leu Ala Ala Pro Ala Pro Ala Pro Ala Pro Arg Leu Ala

1820 1825 1830

Pro Arg Pro Leu Pro Ala Pro Ile Gln Ala Pro Arg Pro Ala Pro

1835 1840 1845

Ala Pro Gln Pro Ala Pro Val Tyr Ala Pro Ala Pro Val Val Ser

1850 1855 1860

Gln Val Gln Ala Thr Ser Ser Ser Gln Ala Ser Ala Gln Gln Ser

1865 1870 1875

Ala Phe Ala Gln Ser Gln Gln Ser Ser Val Val Gln Ser Gln Gln

1880 1885 1890

Ser Ser Asn Ala Tyr Ser Ala Ala Ser Thr Ala Gly Ser Ser Val

1895 1900 1905

Ser Gln Ser Gln Ala Ile Val Ser Ser Ala Pro Asp Tyr Phe Asn

1910 1915 1920

Thr Gln Thr Leu Ser Ser Ser Leu Ser Ser Ser Gln Gln Ser Leu

1925 1930 1935

Ser Ala Leu Asn Ser Ile Ala Ser Gly Gln Leu Ser Ser Ser Asn

1940 1945 1950

Ala Ala Ser Ile Leu Ala Ser Ala Val Ala Arg Ser Leu Gly Val

1955 1960 1965

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

1970 1975 1980

Val Gly Pro Gly Ser Ser Thr Ser Ser Val Ala Gln Ala Ile Ala

1985 1990 1995

Asn Gly Val Ala Asn Ala Val Gly Ala Ser Gly Thr Gly Val Ala

2000 2005 2010

Gly Gln Glu Gln Ser Ile Ser Gln Ser Ile Tyr Thr Ser Ile Ser

2015 2020 2025

Thr Ala Leu Ser Gln Leu Ala Ala Pro Ala Pro Ala Pro Ala Pro

2030 2035 2040

Arg Leu Ala Pro Arg Pro Leu Pro Ala Pro Ile Gln Ala Pro Arg

2045 2050 2055

Pro Ala Pro Ala Pro Gln Pro Ala Pro Val Tyr Ala Pro Ala Pro

2060 2065 2070

Val Val Ser Gln Val Gln Ala Thr Ser Ser Ser Gln Ala Ser Ala

2075 2080 2085

Gln Gln Ser Ala Phe Ala Gln Ser Gln Gln Ser Ser Val Val Gln

2090 2095 2100

Ser Gln Gln Ser Ser Asn Ala Tyr Ser Ala Ala Ser Thr Ala Gly

2105 2110 2115

Ser Ser Val Ser Gln Ser Gln Ala Ile Val Ser Ser Ala Pro Asp

2120 2125 2130

Tyr Phe Asn Thr Gln Thr Leu Ser Ser Ser Leu Ser Ser Ser Gln

2135 2140 2145

Gln Ser Leu Ser Ala Leu Asn Ser Ile Ala Ser Gly Gln Leu Ser

2150 2155 2160

Ser Ser Asn Ala Ala Ser Ile Leu Ala Ser Ala Val Ala Arg Ser

2165 2170 2175

Leu Gly Val Ser Gln Ala Ser Val Gln Asn Ser Ile Ser Gln Gln

2180 2185 2190

Leu Arg Ser Val Gly Pro Gly Ser Ser Thr Ser Ser Val Ala Gln

2195 2200 2205

Ala Ile Ala Asn Gly Val Ala Asn Ala Val Gly Ala Ser Gly Thr

2210 2215 2220

Gly Val Ala Gly Gln Glu Gln Ser Ile Ser Gln Ser Ile Tyr Thr

2225 2230 2235

Ser Ile Ser Thr Ala Leu Ser Gln Leu Ala Ala Pro Ala Pro Ala

2240 2245 2250

Pro Ala Pro Arg Leu Ala Pro Arg Pro Leu Pro Ala Pro Ile Gln

2255 2260 2265

Ala Pro Arg Pro Ala Pro Ala Pro Gln Pro Ala Pro Val Tyr Ala

2270 2275 2280

Pro Ala Pro Val Val Ser Gln Val Gln Ala Thr Ser Ser Ser Gln

2285 2290 2295

Ala Ser Ala Gln Gln Ser Ala Phe Ala Gln Ser Gln Gln Ser Ser

2300 2305 2310

Val Val Gln Ser Gln Gln Ser Ser Asn Ala Tyr Ser Ala Ala Ser

2315 2320 2325

Thr Ala Gly Ser Ser Val Ser Gln Ser Gln Ala Ile Val Ser Ser

2330 2335 2340

Ala Pro Asp Tyr Phe Asn Thr Gln Thr Leu Ser Ser Ser Leu Ser

2345 2350 2355

Ser Ser Gln Gln Ser Leu Ser Ala Leu Asn Ser Ile Ala Ser Gly

2360 2365 2370

Gln Leu Ser Ser Ser Asn Ala Ala Ser Ile Leu Ala Ser Ala Val

2375 2380 2385

Ala Arg Ser Leu Gly Val Ser Gln Ala Ser Val Gln Asn Ser Ile

2390 2395 2400

Ser Gln Gln Leu Arg Ser Val Gly Pro Gly Ser Ser Thr Ser Ser

2405 2410 2415

Val Ala Gln Ala Ile Ala Asn Gly Val Ala Asn Ala Val Gly Ala

2420 2425 2430

Ser Gly Thr Gly Val Ala Gly Gln Glu Gln Ser Ile Ser Gln Ser

2435 2440 2445

Ile Asp Thr Ser Ile Ser Thr Ala Leu Ser Gln Leu Ala Ala Pro

2450 2455 2460

Ala Pro Ala Pro Ala Pro Arg Leu Ala Pro Arg Pro Leu Pro Ala

2465 2470 2475

Pro Ile Gln Ala Pro Arg Pro Ala Pro Ala Pro Gln Pro Ala Pro

2480 2485 2490

Val Tyr Ala Pro Ala Pro Val Val Ser Gln Val Gln Ala Thr Ser

2495 2500 2505

Ser Ser Gln Ala Ser Ala Gln Gln Ser Ala Phe Ala Gln Ser Gln

2510 2515 2520

Gln Ser Ser Val Val Gln Ser Gln Gln Ser Ser Asn Ala Tyr Ser

2525 2530 2535

Ala Ala Ser Thr Ala Gly Ser Ser Val Ser Gln Ser Gln Ala Ile

2540 2545 2550

Val Ser Ser Ala Pro Asp Tyr Phe Asn Thr Gln Thr Leu Ser Ser

2555 2560 2565

Ser Leu Ser Ser Ser Gln Gln Ser Leu Ser Ala Leu Asn Ser Ile

2570 2575 2580

Ala Ser Gly Gln Leu Ser Ser Ser Asn Ala Ala Ser Ile Leu Ala

2585 2590 2595

Ser Ala Val Ala Arg Ser Leu Gly Val Ser Gln Ala Ser Val Gln

2600 2605 2610

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

2615 2620 2625

Thr Ser Ser Val Ala Gln Ala Ile Ala Asn Gly Val Ala Asn Ala

2630 2635 2640

Val Gly Ala Ser Gly Thr Gly Val Ala Gly Gln Glu Gln Ser Ile

2645 2650 2655

Ser Gln Ser Ile Tyr Thr Ser Ile Ser Thr Ala Leu Ser Gln Leu

2660 2665 2670

Ala Ala Pro Ala Pro Ala Pro Ala Pro Arg Leu Ala Pro Arg Pro

2675 2680 2685

Leu Pro Ala Pro Ile Gln Ala Pro Arg Pro Ala Pro Ala Pro Gln

2690 2695 2700

Pro Ala Pro Val Tyr Ala Pro Ala Pro Val Val Ser Gln Val Gln

2705 2710 2715

Ala Thr Ser Ser Ser Gln Ala Ser Ala Gln Gln Ser Ala Phe Ala

2720 2725 2730

Gln Ser Gln Gln Ser Ser Val Val Gln Ser Gln Gln Ser Ser Asn

2735 2740 2745

Ala Tyr Ser Ala Ala Ser Thr Ala Gly Ser Ser Val Ser Gln Ser

2750 2755 2760

Gln Ala Ile Val Ser Ser Ala Pro Val Tyr Phe Asn Thr Gln Thr

2765 2770 2775

Leu Ser Ser Ser Leu Ser Ser Ser Leu Gln Ser Leu Ser Ala Leu

2780 2785 2790

Asn Ser Ile Ala Ser Gly Gln Leu Ser Ser Ser Asn Ala Ala Ser

2795 2800 2805

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

2810 2815 2820

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

2825 2830 2835

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

2840 2845 2850

Ala Asn Ala Asp Gly Ser Ser Gly Thr Gly Val Ala Gly Gln Glu

2855 2860 2865

Gln Ser Ile Ser Gln Ser Ile Tyr Thr Ser Ile Ser Thr Ala Leu

2870 2875 2880

Ser Gln Leu Ala Ala Pro Ala Pro Ala Pro Ala Pro Arg Leu Ala

2885 2890 2895

Pro Arg Pro Leu Pro Ala Pro Ile Gln Ala Pro Arg Pro Ala Pro

2900 2905 2910

Ala Pro Gln Pro Ala Pro Val Tyr Ala Pro Ala Pro Val Val Ser

2915 2920 2925

Gln Val Gln Ala Thr Ser Ser Ser Gln Ala Ser Ala Gln Gln Ser

2930 2935 2940

Ala Phe Ala Gln Ser Gln Gln Ser Ser Val Val Gln Ser Gln Gln

2945 2950 2955

Ser Ser Asn Ala Tyr Ser Ala Ala Ser Thr Ala Gly Ser Ser Val

2960 2965 2970

Ser Gln Ser Gln Ala Ile Val Ser Ser Ala Pro Asp Tyr Phe Asn

2975 2980 2985

Thr Gln Thr Leu Ser Ser Ser Leu Ser Ser Ser Gln Gln Ser Leu

2990 2995 3000

Ser Ala Leu Asn Ser Ile Ala Ser Gly Gln Leu Ser Ser Ser Asn

3005 3010 3015

Ala Ala Ser Ile Leu Ala Ser Ala Val Ala Arg Ser Leu Gly Val

3020 3025 3030

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

3035 3040 3045

Val Gly Pro Gly Ser Ser Thr Ser Ser Val Ala Gln Ala Ile Ala

3050 3055 3060

Asn Gly Val Ala Asn Ala Val Gly Ala Ser Gly Thr Gly Val Ala

3065 3070 3075

Gly Gln Glu Gln Ser Ile Ser Gln Ser Ile Tyr Thr Ser Ile Ser

3080 3085 3090

Thr Ala Leu Ser Gln Leu Ala Ala Pro Ala Pro Ala Pro Ala Pro

3095 3100 3105

Arg Leu Ala Pro Arg Pro Leu Pro Ala Pro Ile Gln Ala Pro Arg

3110 3115 3120

Pro Ala Pro Ala Pro Gln Pro Ala Pro Val Tyr Ala Pro Ala Pro

3125 3130 3135

Val Val Ser Gln Val Gln Ala Thr Ser Ser Ser Gln Ala Ser Ala

3140 3145 3150

Gln Gln Ser Ala Phe Ala Gln Ser Gln Gln Ser Ser Val Val Gln

3155 3160 3165

Ser Gln Gln Ser Ser Asn Ala Tyr Ser Ala Ala Ser Thr Ala Gly

3170 3175 3180

Ser Ser Val Ser Gln Ser Gln Ala Ile Val Ser Ser Ala Pro Asp

3185 3190 3195

Tyr Phe Asn Thr Gln Thr Leu Ser Ser Ser Leu Ser Ser Ser Gln

3200 3205 3210

Gln Ser Leu Ser Ala Leu Asn Ser Ile Ala Ser Gly Gln Leu Ser

3215 3220 3225

Ser Ser Asn Ala Ala Ser Ile Leu Ala Ser Ala Val Ala Arg Ser

3230 3235 3240

Leu Gly Val Ser Gln Ala Ser Val Gln Asn Ser Ile Ser Gln Gln

3245 3250 3255

Leu Arg Ser Val Gly Pro Gly Ser Ser Thr Ser Ser Val Ala Gln

3260 3265 3270

Ala Ile Ala Asn Gly Val Ala Asn Ala Val Gly Ala Ser Gly Thr

3275 3280 3285

Gly Val Ala Gly Gln Glu Gln Ser Ile Ser Gln Ser Ile Tyr Thr

3290 3295 3300

Ser Ile Ser Thr Ala Leu Ser Gln Leu Ala Ala Pro Ala Pro Ala

3305 3310 3315

Pro Ala Pro Arg Leu Ala Pro Arg Pro Leu Pro Ala Pro Ile Gln

3320 3325 3330

Ala Pro Arg Pro Ala Pro Ala Pro Gln Pro Ala Pro Val Tyr Ala

3335 3340 3345

Pro Ala Pro Val Val Ser Gln Val Gln Ala Thr Ser Ser Ser Gln

3350 3355 3360

Ala Ser Ala Gln Gln Ser Ala Phe Ala Gln Ser Gln Gln Ser Ser

3365 3370 3375

Val Val Gln Ser Gln Gln Ser Ser Asn Ala Tyr Ser Ala Ala Ser

3380 3385 3390

Thr Ala Gly Ser Ser Val Ser Gln Ser Gln Ala Ile Val Ser Ser

3395 3400 3405

Ala Pro Asp Tyr Phe Asn Thr Gln Thr Leu Ser Ser Ser Leu Ser

3410 3415 3420

Ser Ser Gln Gln Ser Leu Ser Ala Leu Asn Ser Ile Ala Ser Gly

3425 3430 3435

Gln Leu Ser Ser Ser Asn Ala Ala Ser Ile Leu Ala Ser Ala Val

3440 3445 3450

Ala Arg Ser Leu Gly Val Ser Gln Ala Ser Val Gln Asn Ser Ile

3455 3460 3465

Ser Gln Gln Leu Arg Ser Val Gly Pro Gly Ser Ser Thr Ser Ser

3470 3475 3480

Val Ala Gln Ala Ile Ala Asn Gly Val Ala Asn Ala Val Gly Ala

3485 3490 3495

Ser Gly Thr Gly Val Ala Gly Gln Glu Gln Ser Ile Ser Gln Ser

3500 3505 3510

Ile Tyr Thr Ser Ile Ser Thr Ala Leu Ser Gln Leu Ala Ala Pro

3515 3520 3525

Ala Pro Ala Pro Ala Pro Arg Pro Ala Pro Arg Pro Leu Pro Ala

3530 3535 3540

Pro Ile Gln Ala Pro Arg Pro Ala Pro Ala Pro Gln Pro Ala Pro

3545 3550 3555

Val Tyr Ala Pro Ala Pro Val Val Ser Gln Val Gln Ala Thr Ser

3560 3565 3570

Ser Ser Gln Ala Ser Ala Gln Gln Ser Ala Phe Ala Gln Ser Gln

3575 3580 3585

Gln Ser Ser Val Val Gln Ser Gln Gln Ser Ser Asn Ala Tyr Ser

3590 3595 3600

Ala Ala Ser Thr Ala Gly Ser Ser Val Ser Gln Ser Gln Ala Ile

3605 3610 3615

Val Ser Ser Ala Pro Val Tyr Phe Asn Thr Gln Thr Leu Ser Ser

3620 3625 3630

Ser Leu Ser Ser Ser Leu Gln Ser Leu Ser Ala Leu Asn Ser Ile

3635 3640 3645

Ala Ser Gly Gln Leu Ser Ser Ser Asn Ala Ala Ser Ile Ile Ala

3650 3655 3660

Ser Ala Val Ala Arg Ser Leu Gly Val Ser Gln Ala Ser Val Gln

3665 3670 3675

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

3680 3685 3690

Thr Ser Ser Val Ala Gln Ala Ile Ala Asn Gly Val Ala Asn Ala

3695 3700 3705

Val Gly Ala Ser Gly Thr Gly Val Ala Gly Gln Glu Gln Ser Ile

3710 3715 3720

Ser Gln Ser Ile Tyr Thr Ser Ile Ser Thr Ala Leu Ser Gln Leu

3725 3730 3735

Ala Ala Thr Ala Pro Ala Pro Ala Pro Arg Pro Ala Pro Arg Pro

3740 3745 3750

Leu Pro Ala Pro Ile Gln Ala Pro Arg Pro Ala Pro Ala Pro Gln

3755 3760 3765

Pro Ala Pro Val Tyr Ala Pro Ala Pro Val Tyr Ala Pro Ala Pro

3770 3775 3780

Val Tyr Ala Pro Ala Pro Ile Val Ser Gln Ile Gln Glu Thr Ser

3785 3790 3795

Ser Ser Gln Ala Ser Ala Gln Gln Asn Tyr Leu Ala Gln Ser Gln

3800 3805 3810

Gln Ser Ser Val Ala Gln Ser Gln Gln Ser Ser Asn Ala Tyr Ser

3815 3820 3825

Ala Val Ser Ala Ala Asp Ser Ser Phe Ser Ser Gly Ala Ser Ser

3830 3835 3840

Gly Phe Gly Ser Ser Leu Ser Ser Ser Ser Gly Phe Ser Ala Pro

3845 3850 3855

Ala Ser Pro Ala Pro Tyr Ser Pro Ser Val Ser Ala Ser Ser Ser

3860 3865 3870

Ile Gly Ala Ser Ser Gly Ser Ala Leu Ala Ala Ser Ala Ala Gln

3875 3880 3885

Gln Leu Met Ser Pro Ala Ala Asn Gln Arg Ile Ala Ala Leu Ser

3890 3895 3900

Asn Ser Leu Arg Ala Ala Val Ser Gly Gly Gln Val Asn Tyr Gly

3905 3910 3915

Ala Leu Ser Asn Ser Leu Ala Ser Ala Ala Ser Gln Ile Gln Ser

3920 3925 3930

Ser Ser Gly Leu Ser Lys Ser Glu Val Leu Val Glu Ala Leu Leu

3935 3940 3945

Glu Thr Leu Phe Ala Leu Leu Ser Ser Leu Thr Ser Gly Ala Ser

3950 3955 3960

Thr Ala Asp Phe Ala Gln Val Val Leu Ala Ala Leu Gly

3965 3970 3975

Claims (8)

1. a kind of gene of the Araneus ventricosus pyriform gland silk-fibroin as shown in SEQ ID NO.1.

2. a kind of preparation method of the gene of Araneus ventricosus pyriform gland silk-fibroin, comprising:

(1) according to the PySp1 protein gene of spider pyriform gland silk, in the end NT degenerate primer, specific primer is designed in duplicate block, It expands to obtain the end the NT gene order of part by degenerate pcr；

(2) specific primer and anchor primer are separately designed according to step (1) sequence obtained, by anchor PCR by the end NT Polishing；

(3) obtained according to step (2) includes complete NT terminal sequence, in the end NT 5 ' tip designs, 1 forward primer, at the end CT 3 ' the reverse primers of tip designs 1, for expanding overall length PySp1 gene；

(4) after carrying out Ago-Gel gel extraction to PCR product obtained by step (3), blunt end cloning is carried out with carrier and is reacted, It is attached product recycling later；

(5) flat end clone is carried out to the connection product of step (4), is transferred in DH5 α competent cell and is converted, recovered, it will Bacterium solution after conversion is coated on LB solid medium, and after being incubated overnight, picking monoclonal carries out PCR detection；

(6) plasmid of positive monoclonal obtained in step (5) is completely sequenced, finally obtains Araneus ventricosus pyriform gland silk Full length protein gene.

3. preparation method according to claim 2, which is characterized in that for expanding the part end NT in the step (1) Primer: positive degenerate primer: ATGTTYCARATGAGTTCAATG；

Inverted repeat area specific primer: CTGTTGTGTTTGAGCGGATTG.

4. preparation method according to claim 2, which is characterized in that for the special of the end completion NT in the step (2) Property primer:

Primer 1:TTTCAATAGCTGCCGCCTGC；

Primer 2: GTGGCAATAGCTTGGGTTACGC；

Anchor primer: ACTCCTGTGGAACCATCGGACGGGGGG.

5. the preparation method according to claim 4, which is characterized in that anchor PCR condition in the step (2) are as follows: In one wheel PCR, single primer amplification is carried out using specific primer 1；PCR product is subjected to Ago-Gel gel extraction later, It is carried out adding poly- dCTP reaction with TdT enzyme, the second wheel PCR is carried out after product recycling；Use specific primer 2 and anchor primer It is expanded.

6. preparation method according to claim 2, which is characterized in that for expanding overall length PySp1 base in the step (3) The primer of cause: forward primer: ATGTCTTGGACCCTGGGGCTTCAGTTTTTATTC；

Reverse primer: CTATCCAAGTGCTGCAAGTACGAC.

7. preparation method according to claim 2, which is characterized in that for expanding overall length PySp1 base in the step (3) The enzyme of cause is Phanta high fidelity enzyme.

8. a kind of application of the gene of Araneus ventricosus pyriform gland silk-fibroin.