CN110846336B - Multifunctional fusion enzyme XAET, multifunctional fusion enzyme site-directed integration eukaryotic specific expression vector and construction method thereof - Google Patents

Abstract

The invention discloses a multifunctional fusion enzyme XAET and a multifunctional fusion enzyme site-directed integration eukaryotic specific expression vector and a construction method thereof, wherein the multifunctional fusion enzyme can express activities of xylanase, phytase, diglucanse and double cellulose, the activities are used for preparing corresponding transgenic animals in the later period, the animals secrete the enzymes by themselves to digest antinutritional factors such as xylan, phytic acid, glucan and cellulose in feed, and the effects of improving the feed utilization rate and reducing pollution emission are achieved. Meanwhile, the problem of mutual interference when rigid peptide mediates the co-expression of more than two fusion proteins is solved; the method solves the problems of low polygene co-expression efficiency and difficult gene position determination of the 2A connecting peptide and the problem of interference of 2A polypeptide residues on upstream enzyme protein in polygene co-expression, and achieves the aim of enhancing the polygene high-efficiency co-expression efficiency. In addition, the problem of poor effect of single-source cellulase gene expression and cellulose hydrolysis can be solved.

Description

A multifunctional fusion enzyme XAET and multifunctional fusion enzyme site-directed integration of eukaryotic specific expression Express vehicle and its construction method

technical field

The invention relates to the field of biotechnology, in particular to a site-specific integration vector for the specific expression of a multifunctional fusion enzyme XAET and a multifunctional fusion enzyme parotid gland tissue and a construction method thereof.

Background technique

Non-starch polysaccharide (NSP) is a polymer composed of several monosaccharides connected by glycosidic bonds, including most polysaccharide molecules except α-glucan. NSPs were originally classified according to the methods used to extract and isolate polysaccharides. The insoluble matter left after the cell wall is extracted by a series of alkalis is called cellulose, and the matter dissolved in the alkali solution is called hemicellulose. Considering the chemical structure and biological function of non-starch polysaccharides, it was found that the classification based on their solubility is inaccurate. Generally, non-starch polysaccharides are generally divided into three categories, namely cellulose, non-fibrous polysaccharides (hemicellulose polymers) and pectin polysaccharides. Among them, non-fibrous polysaccharides include xylan, β-glucan, mannan, galactan, etc. According to the difference in water solubility, non-starch polysaccharides can be divided into soluble non-starch polysaccharides (SNSP) and insoluble non-starch polysaccharides (INSP). , lignin, and protein combination, so it is soluble in water and is called soluble non-starch polysaccharide.

Non-starch polysaccharide (NSP) is the main component of feed dietary fiber. These fibers surround feed nutrients in the cell wall, and some fibers can dissolve in water and produce viscous substances. These sticky substances inhibit the normal digestive function of the animal and prevent the animal from absorbing nutrients. If these NSPs are removed, nutrients can be released from the cell wall, thereby improving the utilization rate of metabolizable energy and protein. Both corn and wheat contain large amounts of NSP, and many vegetable protein sources, such as soybean meal, also contain NSP. Adding enzyme preparations to the feed can remove these NSPs, such as starch and protein surrounded by cell structures in soybean meal can be released, thereby improving the metabolic energy of soybean meal and the utilization rate of protein.

Adding enzyme preparations to feed can solve the problem of digestion and absorption of NSP, but enzyme preparations are easily inactivated by high temperature during the feed pelleting and extruding process, which also increases the cost of feed, and also makes the storage conditions of feed more stringent, increasing the Transportation and feeding costs. Therefore, a kind of other solution is badly needed. In recent years, successive studies have shown that enzyme genes that can express cellulose or non-cellulosic polysaccharides such as xylanase and glucanase are transferred into animals, and by obtaining transgenic animals expressing this gene, the Animals secrete and express corresponding enzymes to digest NSP in the feed, so as to improve the digestion and absorption of feed nutrients. Therefore, it is a new way to solve the anti-nutritional problem of feed by using transgenic breeding technology to breed new animal varieties that express the digestive enzymes that pigs lack. In 2001, Canadian scientist Golovan et al. (2001) first used the Escherichia coli acid-resistant phytase appA gene to be integrated into pigs to improve the digestibility of feed phosphorus and significantly reduce fecal phosphorus emissions by 75%. In 2013, this project also used the upstream regulatory region of the porcine parotid gland secretion protein to successfully generate transgenic mice for cellulase (Huang et al., 2013) and transgenic mice for mannanase (Li Zicong et al., 2013), zhang 2018 etc. used 2A polypeptide to fuse xylanase-glucanase-phytase into pig gene pigs to improve pig production performance and reduce phosphorus and nitrogen emissions. It is also disclosed in the publication number CN106086068A that the fusion expression of pectinase-xylanase-phytase-cellulase has been realized by using 2A connecting peptide, but the expression efficiency is not high. However, these existing technologies have the following problems: single gene transfer, high expression level, but few types of expressed enzymes, elimination of individual feed nutritional factors has limited improvement in animal performance, and has little economic value. 2A-mediated multi-gene transfer still has the problem of low co-expression efficiency, inconsistent expression of the first and last genes of the multi-gene fusion enzyme, difficult to determine the gene position, and some gene sequences will interfere with the cleavage of 2A, resulting in loss of function of the expression product .

Contents of the invention

The present invention discloses a multifunctional fusion enzyme XAET and a multifunctional fusion enzyme site-specific integration eukaryotic expression vector and its construction method. The multifunctional fusion enzyme can express xylanase, phytase, diglucanase and Double cellulase activity, which is used to prepare corresponding transgenic animals in the later stage, the animals will secrete these enzymes by themselves to digest xylan, phytic acid, dextran, cellulose and other anti-nutritional factors in the feed, so as to improve Feed utilization, the efficacy of reducing pollution emissions. At the same time, solve the problem of mutual interference when rigid peptides mediate the co-expression of two or more fusion proteins; solve the problems of low efficiency of multi-gene co-expression of 2A-linked peptides, difficulty in determining gene positions, and the impact of 2A polypeptide residues on upstream enzyme proteins in multi-gene co-expression The problem of interference is eliminated, and the purpose of enhancing the efficient co-expression efficiency of multiple genes is achieved. In addition, the problem of poor cellulase gene expression and hydrolysis of cellulose from a single source can also be solved.

According to one aspect of the present disclosure, a multifunctional fusion enzyme is provided, which can simultaneously express xylanase, phytase, diglucanase and dicellulase.

In some embodiments, the multifunctional fusion enzyme gene consists of xylanase gene-A3-phytase gene-furin-P2A-cellulase gene-A3'-cellulase gene. When two or more proteins are co-expressed, it is difficult to achieve co-expression of all genes by using only flexible peptides or rigid peptides. The present invention uses A3 and 2A in combination to solve the problem of mutual interference when rigid peptides mediate the co-expression of two or more fusion proteins. Problem: Solve the problem of low efficiency of multi-gene co-expression of 2A-linked peptides and the interference of 2A polypeptide residues on upstream enzyme proteins during multi-gene co-expression, so as to achieve the effect of multi-gene co-expression efficiently. At the same time, compared with the simple 2A linking peptide, the present invention uses A3 and furin P2A to construct XAET to significantly improve the activity of the four enzymes under different pH conditions, eliminate the influence of 2A polypeptide on upstream genes, and avoid the inhibition of 2A reactivity by the spatial structure of some proteins At the same time, it also avoids the tedious test process of optimizing different protein sequences, and the enzyme expression level is higher than that of 2A. Rigid peptides can only be used in the construction of two fusion enzymes. The present invention combines 2A and A3 skillfully, which not only retains the advantages of A3, but also enhances the multi-gene co-expression ability of the fusion enzyme. P2A with the highest self-cleavage efficiency is used at the junction of the second and third genes of the fusion enzyme, and the furin enzyme recognition motif RVKR is added to its N-terminus. RVKR can efficiently cut at the organelle Golgi apparatus, leaving only 2 amino acid residues Base, this design has higher cutting efficiency than the general 2A sequence, and has the least impact on the expression product. Therefore, the XAET gene sequence obtained through the design of this disclosure can efficiently express xylanase, phytase, diglucanase and dicellulase, and solve the problem of co-expression of the four genes. It provides a basis for improving gene transfer and enzyme production efficiency for transgenic animals and enzyme fermentation industrial production. At the same time, as disclosed in the patent publication No. CN106086068A, when 2A is used to connect two gene sequences, the connection sequence of the two genes will affect the expression and function of the genes, and the enzyme activity of the two genes is not as good as that of a single gene. High activity; in multi-gene connection, if only 2A connecting peptide is used, this position effect will be more obvious, and the impact on the enzyme activity of each gene will be more prominent. However, the present disclosure adopts the combination of A3 and 2A connecting peptides to construct the above-mentioned multifunctional enzyme gene, which can overcome the position effect, and the enzyme activity expressed by the xylanase and phytase genes is equivalent to that of a single gene. However, since the cellulase is co-expressed with two double genes, the expressed cellulase activity is higher than that of the single-gene enzyme.

In some embodiments, the gene sequence of A3 in the multifunctional fusion enzyme gene is shown in SEQ ID No:5.

In some embodiments, the gene sequence of A3' in the multifunctional fusion enzyme gene is shown in SEQ ID No:6.

In some embodiments, the multifunctional fusion enzyme gene XAET sequence is shown in SEQ ID No:11. Due to the efficient self-cleavage function of the 2A polypeptide, it can better realize the co-expression of the two proteins before and after, and is known as the most reliable linker for protein fusion expression. However, the function of the partzyme protein was significantly impaired after fusing the C-terminal polypeptide of 2A, and the expression level of the latter protein was reduced. In the co-expression of multiple genes, the sequence of protein combinations is strict, and it is necessary to cumbersomely verify the arrangement and combination to achieve low-level co-expression (this phenomenon has been shown in the patent publication No. CN106086068A), which is not conducive to the construction of multifunctional fusion enzymes. The cleavage efficiency of 2A sequence is related to the upstream polypeptide motif. Some protein amino acid motifs will seriously affect the 2A cleavage activity, resulting in the incomplete cleavage of the two enzymes before and after 2A mediation, resulting in abnormal folding of the secondary structure, and may be blocked by upstream genes. The signal peptide migrates to the wrongly targeted organelles and cannot be processed and secreted correctly, resulting in loss of function and failure of expression. In the present disclosure, the combination of A3 and 2A linking peptides is used to construct a multifunctional fusion enzyme for fusion expression of four genes, which can overcome the respective shortcomings of 2A and A3 linking peptides, so that the multifunctional fusion enzyme can efficiently express four kinds of genes. Enzyme activity can lay the foundation for obtaining transgenic animals expressing the four enzymes.

In some embodiments, the multifunctional fusion enzyme has an amino acid sequence as shown in SEQ ID No:12. The most important indicator of the connecting peptide is the length of the amino acid chain. GGGGS connecting peptides of different lengths have different effects on the expression and activity of the fusion protein, and it does not mean that the longer the connecting peptide, the higher the expression. Fusion enzymes have strict requirements on flexible peptides, and flexible peptides that are too long or too short are not conducive to the activity of fusion enzymes. Proteins with large molecular weight and complex structure require a larger folding space, and the linking peptide should also be lengthened, but too long peptide chains may increase antigenicity and are also easily broken by enzymatic hydrolysis. The steric hindrance effect caused by too short connecting peptide will affect the correct folding of the protein and increase the probability of aggregate formation. Rigid linker peptides are often used to fix the distance between functional proteins at both ends to ensure the integrity of functional domains due to the stability of the secondary structure and the inability to stretch and bend. Therefore, the designed rigid linker peptide A3/A3' and self-cleaved P2A act together to connect 4 genes to achieve co-expression of the 4 genes. Enzyme, phytase, dextranase and cellulase can be highly expressed.

According to another aspect of the present disclosure, there is provided a multifunctional fusion enzyme site-directed integration eukaryotic specific expression vector, the expression vector comprising the multifunctional fusion enzyme according to claim 5 or 6.

In some embodiments, the multifunctional fusion enzyme site-specifically integrates the gene sequence of the eukaryotic specific expression vector as shown in SEQ ID No:13.

In some embodiments, a method for constructing a multifunctional fusion enzyme site-directed integration of a eukaryotic-specific expression vector is provided, which includes the following steps:

Screening and optimization of candidate target genes;

Design of connecting peptide;

The target gene is connected with the connecting peptide;

Synthesis of multifunctional fusion enzyme gene XAET;

Construct the CEP112 site-directed transfer XAET gene expression vector.

In some embodiments, constructing a CEP112 site-directed XAET gene expression vector comprises the following steps:

Replace the BEXA cistron in the CEP112-LA340RA3219 vector with the XAET polycistroton to construct a new vector Cep112-mPSP-XAET;

Linearize Cep112-mPSP-XAET with PacI and sexAI, then use inf-npsp primers to amplify the npsp upstream regulatory region and replace the existing mpsp sequence;

The expression vector Cep112-npsp-XAET was constructed for site-specific transfer of CEP112 to XAET.

According to another aspect of the present disclosure, the application of a multifunctional fusion enzyme XAET is provided, and the multifunctional fusion enzyme XAET can be used to prepare food-saving and environmentally friendly transgenic animals, such as transgenic pigs, transgenic cattle, transgenic sheep, etc.

Beneficial effects of the present disclosure:

1) The present invention designs and constructs pxynB-A3-APPA-furin-P2A-pegⅡ-A3'-TeEGI (XAET) fusion enzyme, which can co-express xylanase, phytase, double glucanase and double cellulase Four enzyme activities, better glucanase and cellulase activity than monocellulase gene. Including the hydrolytic enzymes needed to eliminate the main anti-nutritional factors in the feed is of great value in improving the conversion rate of high-fiber feed. The design of the present invention improves the efficiency of gene expression, and if it is used in the industrial production of transgenic animals and enzyme fermentation, it can significantly improve the production efficiency of gene transfer and enzyme, and has important economic value;

2) Compared with the simple 2A linking peptide, the XAET obtained by the present invention significantly improves the activity of the four enzymes under different pH conditions, eliminates the influence of the 2A polypeptide on the upstream gene, and avoids the problem that the spatial structure of some proteins inhibits the 2A reactivity. Avoid the tedious test process of optimizing different protein sequences;

3) Rigid peptides can only be used in the construction of two fusion enzymes. The present invention combines 2A and A3 skillfully, which not only retains the advantages of A3, but also enhances the multi-gene co-expression ability of the fusion enzyme;

4) P2A with the highest self-cleavage efficiency is used at the junction of the second and third genes of the fusion enzyme, and the furin enzyme recognition motif RVKR is added to its N-terminus. RVKR can efficiently cut in the organelle Golgi apparatus, leaving only 4 Amino acid residues, this design has higher cutting efficiency than the general 2A sequence, and has the least impact on the expression product;

5) Carrying the XAET gene is carried by an efficient site-specific integration vector at the pig CEP112 site, which can efficiently prepare transgenic pigs, quickly obtain transgenic families with consistent integration positions, and cultivate new varieties of transgenic pigs.

Description of drawings

Figure 1 is a schematic diagram of the structure of pxynB-A3-APPA-furin-P2A-pegII-A3'-teEGI (XAET);

Fig. 2 is xylanase (xynB)-phytase (appA) bicistronic optimal combination and expression result: A. xylanase-phytase bicistronic optimal combination design schematic diagram; B. xynB pH range; C. pH range of appA; D. pH stability of xynB (39.℃, 2h); pH stability of appA (39.℃, 2h);

Figure 3 is the optimal combination and expression results of glucanase (egⅡ)-cellulase (TeEGⅠ) bicistronic combination: A.egⅡ-TeEGⅠ bicistronic optimal combination design schematic diagram; B. pH range of glucanase ; The pH range of C. cellulase; the pH stability of D. glucanase; the pH stability of E.appA cellulase;

Figure 4 shows the difference in enzyme activity between the fusion enzyme and the single gene expression product of the enzyme: A. Comparison of cellulase activity under different pH conditions; B: Cellulase tolerance to different pH environments (39 degrees, treated for 2 hours); C : fusion enzyme and single gene expression Q-PCR (TeEGI gene), D: fusion enzyme and single gene expression Q-PCR (egII);

Figure 5 is the detection result of XAET expression in pK15 cells;

Figure 6 is a plasmid map of Cep112-npsp-XAET.

detailed description

1. Screening of target genes

The xylanase gene xynB from Aspergillus niger (Guo et al., 2013), the phytase gene appA from Escherichia coli, and the cellulase gene egⅡ from Trichoderma reesei (Akbarzadeh et al. , 2014), the cellulase gene TeEGⅠ from crickets (Kim et al., 2008), after the signal peptide was predicted by SignalP 4.1 Server, its own signal peptide was removed, and then optimized according to the pig codon preference, and the The signal peptide (signal peptide, sp) sequence of the parotid secretory protein (PSP) derived from pig or bovine is added to the N-terminal of the amino acid sequence of the candidate gene after codon optimization, such as pigPSP-SP-xynB, pigPSP- SP-appA, pigPSP-SP-egⅡ, and pigPSP-SP-teEGⅠ are abbreviated as pSPxyn, pSPappA, pSPeg2, pSPTeEG respectively, and the mature peptide genes after codon optimization are respectively named pxyn (the gene sequence is shown in SEQ ID No: 1) , pappA (the gene sequence is shown in SEQ ID No: 2), pegII (the gene sequence is shown in SEQ ID No: 3), pTEGI (the gene sequence is shown in SEQ ID No: 4).

2. Construction of polycistronic gene sequences of xylanase (xynB)-phytase (appA), cellulase (egⅡ)-cellulase (TeEGⅠ)

Using the A3 rigid peptide, connect xylanase (xynB)-phytase (appA), cellulase (eg II)-cellulase (TeEGⅠ) respectively to construct a bifunctional fusion enzyme, and remove the stop codon of the upstream gene of A3 , while removing the A3 C-terminal downstream gene signal peptide.

The A3 sequence after optimized mutation is as follows:

A3 (SEQ ID No: 5): GAGGCTGCCGCCAAAGAAGCTGCCGCCAAGGAGGCTGCCGCC AAG

A3' (SEQ ID No: 6): GAGGCCGCCGCCAAGGAGGCCGCCGCCAAGGAGGCCGCCGCC AAG

The optimized A3-linked bifunctional enzyme gene sequence is xynB-A3-APPA and egII-A3'-TeEGI. The cistrons of the fusion-designed multifunctional enzymes were optimized by porcine codons to remove rare codons and select codons with high frequency of use by porcine cells.

The A3 repeat sequence in the polycistronic and the signal peptide of the porcine parotid protein were re-optimized to reduce the influence of the repetitive sequence on the stability of the polycistronic structure, and the optimized sequences were artificially synthesized. The optimized xynB-A3-APPA gene sequence is shown in SEQ ID No:7, the amino acid sequence is shown in SEQ ID No:8; the optimized egII-A3'-TeEGI gene sequence is shown in SEQ ID No:9, The amino acid sequence is shown in SEQ ID No:10.

Using furin enzyme recognition gene sequence and efficient self-cleaving P2A sequence, pxynB-A3-APPA and pegII-A3'-TeEGI were connected to construct a multifunctional cistron pxynB-A3-APPA-furin-P2A-pegII-A3'- TeEGI (XAET) (gene sequence shown in SEQ ID No: 11, amino acid sequence shown in SEQ ID No: 12, gene structure map shown in Figure 1) cloned into pcDNA3.1 (+) eukaryotic expression vector polyclonal On site BamHI/EcoRI.

Use P2A to link xylanase (xynB)-phytase (appA), cellulase (egII)-cellulase (TeEGI) to construct bifunctional enzymes pxynB-p2A-pAPPA, pegII-p2A-pTeEG, Cloned into the multiple cloning site of pcDNA3.1 (+) eukaryotic expression vector, as a control group.

3. Xylanase (xynB)-phytase (appA)-cellulase (egⅡ)-cellulase (TeEGⅠ) eukaryotic expression vector construction

The optimized multifunctional enzyme cistron XAET was inserted into the eukaryotic expression vector pcDNA3.1 multiple cloning site BamHI/EcoRI, identified by enzyme digestion and sequencing, and the multifunctional enzyme cistron XAET eukaryotic expression vector pCD-XAET was constructed success.

4. In vitro expression and functional verification of xylanase (xynB)-phytase (appA)-cellulase (egⅡ)-cellulase (TeEGⅠ) multifunctional enzyme

The pCD-XAET eukaryotic expression vector was transiently transfected into the porcine kidney pK15 cell line according to the instructions of the transfection kit LipofectamineTM LTX + PLUSTMReagent (invitrogen), for 48-72 hours, the cell supernatant was collected as a crude enzyme solution to measure the enzyme activity, and the enzyme Viability and its pH tolerance. Enzyme activity determination methods and definitions refer to cellulase "NYT/912-2004", dextranase "NYT/911-2004", xylanase "GBT/23874-2009", phytase "GBT/18634- 2009".

1. Expression analysis of xylanase (xynB) and phytase (appA) fusion enzyme

Xylanase (xynB) and phytase (appA) were fused with furin-P2A and A3 respectively, and expressed in porcine pK15 cells. The results showed that the A3-linked fusion enzyme successfully expressed xylanase and phytic acid Enzyme bifunctional enzyme, and its expression xylanase and phytase have higher biological activity at pH2.0-pH6.5, wherein the xylanase activity of pxynB-A3-appA, at pH2.0 -5.0 is higher than pxynB-p2A-pappA, slightly lower than the latter after pH5.0, but the xylanase expressed by pxynB-A3-appA is significantly more resistant to pH2.0-pH7.0 than pxynB-p2A -pappA. The activity and tolerance of the phytase expressed by pxynB-A3-appA were significantly better than those of pxynB-p2A-pappA in different pH buffers, the results are shown in Figure 2.

2. Cellulase (egⅡ)-cellulase (TeEGⅠ) fusion enzyme expression analysis

The complex structure and low solubility of cellulose are the main reasons for limiting its hydrolysis. The activity of cellulase genes found so far is generally low. In order to improve the activity of cellulase, this study fused the egⅡ gene from Reesei’s Trichoderma and the TeEGⅠ gene from cricket through A3 and 2A. The results showed that the glucanase and fiber The activity and tolerance of the enzyme in different pH buffers were better than those of pegⅡ-p2A-pTeEGⅠ, and the enzyme activity was significantly improved. The results are shown in Figure 3.

Compared with single-enzyme gene expression, the expression of cellulase by fusion enzyme pegⅡ-A3'-TeEGⅠ has been improved. According to the quantitative results, the expression level of single gene is generally more than 2 times higher than that of fusion enzyme (plasmid size and transfection efficiency Inversely proportional), while the cellulase activity expressed by the fusion enzyme of this design is higher than that of the single gene, the results are shown in Figure 4.

3. Expression detection of polycistronic XAET in pK15 cells

The polycistronic XAET eukaryotic expression vector pCD-XAET was introduced into PK15 cells by electroporation and liposome chemical transfection respectively, and the cell supernatant culture fluid was collected after 48 hours to measure its expression. The results showed that polycistronic XAET Anti-XAET all successfully expressed xylanase, phytase and cellulase, the results are shown in Figure 5.

5. Site-directed integration into CEP112 site transxylanase (xynB)-phytase (appA)-cellulase (eg Ⅱ)-cellulase (TeEGⅠ) (XAET) gene expression vector construction

Firstly, replace the BEXA cistron in the previous research vector CEP112-LA340RA3219 (derived from No. "201711477805.5" "A Construction Method for Site-Directed Integration of Exogenous DNA Transgenic Pig", Publication No. "108285906A" patent) with the XAET polycistronic , constructed a new vector Cep112-mPSP-XAET, based on it, linearized Cep112-mPSP-XAET with PacI and sexAI, then amplified the npsp upstream regulatory region with inf-npsp primers in Table 1, and replaced the existing sequence, npsp (-11.5kb～-5.7kb) extended the regulatory region sequence by 395bp on the basis of the original mpsp (-11.1kb～-5.7kb), and constructed the Cep112-npsp-XAET vector (sequence shown in SEQ ID No: 12). Enzyme digestion verification showed that the size of the cut band was in line with the expectation. After sequencing verification, it was confirmed that a transgenic vector capable of specifically expressing the four functional enzymes of XAET in porcine salivary glands was successfully obtained. The plasmid map is shown in Figure 6.

The obtained Cep112-npsp-XAET vector is an efficient site-specific integration vector for the pig CEP112 site, which can efficiently prepare transgenic pigs, quickly obtain transgenic families with consistent integration positions, and lay a good foundation for the subsequent rapid cultivation of new transgenic pig varieties with consistent genetic background.

6. Acquisition of pigs transfected with multifunctional fusion enzyme XAET gene

The successfully constructed Cep112-npsp-XAET vector was transfected into a porcine fibroblast cell line to obtain a positive cell line expressing XAET polycistrons. XAET genetic pigs.

The obtained XAET transgenic pigs were identified for gene and sequencing level, and the saliva of the positive pigs was collected and tested. It was found that the XAET transgenic pigs could efficiently express phytase, xylanase, glucanase and cellulose Enzymes, and the activities of xylanase and phytase were equivalent to those of transgenic pig enzymes, and the activity of cellulase was higher than that of transgenic pig enzymes.

What have been described above are only some embodiments of the present invention. For those skilled in the art, without departing from the inventive concept of the present invention, several modifications and improvements can be made, and these all belong to the protection scope of the present invention.

sequence listing

<110> Wen's Food Group Co., Ltd.; South China Agricultural University

<120> A multifunctional fusion enzyme XAET and multifunctional fusion enzyme site-directed integration eukaryotic specific expression vector and its construction method

<130> 2019.11.21

<160> 13

<170> SIPOSequenceListing 1.0

<210> 1

<211> 627

<212>DNA

<213> artificial synthesis ()

<400> 1

atgtttcaac tttggaaact tgttttcttg tgcggtctgc tcattgggac ctcagcatct 60

agcacacctt caagcacagg cgaaaacaat gggttctatt actccttctg gaccgacggg 120

ggcggcgatg tcacctacac aaacggagac gccggagcct acaccgtgga gtggagcaac 180

gtggggaact tcgtggggagg aaagggatgg aacccaggat ccgcccagga tatcacctac 240

tccggcacct ttacaccaag cggcaacgga tacctgtccg tgtacggatg gaccacagac 300

cctctgatcg agtactacat cgtggaaagc tacggcgatt acaaccccgg atccgggggc 360

acctacaaag ggaccgtgac atccgacggc agcgtgtacg atatctacac cgctacaagg 420

accaacgctg ccagcatcca gggcacagcc accttcacac agtactggtc cgtgcgccag 480

aacaagcggg tgggagggac cgtgaccaca agcaaccact ttaacgcctg ggccaaactg 540

ggaatgaacc tggggacaca caactaccag attgtcgcca ccgaaggcta ccagtcctca 600

ggctcatcct ccattacagt ccagtga 627

<210> 2

<211> 1293

<212>DNA

<213> artificial synthesis ()

<400> 2

atgttccaac tgtggaagct ggtcttcctg tgtggtctgc tgattggcac ctctgcttcc 60

cagagcgaac ccgaactgaa actggaaagc gtcgtcatcg tctcccgcca cggagtccgc 120

gcccctacaa aagccaccca gctcatgcag gacgtgaccc ccgatgcctg gcctacatgg 180

ccagtcaagc tgggatggct cacccctagg ggaggagagc tgatcgccta cctcggacac 240

tatcagaggc agagactggt ggctgacgga ctgctcgcta agaaaggatg cccacagtcc 300

ggacaggtgg ctatcattgc tgacgtggat gagcgcaccc ggaagacagg agaagccttc 360

gccgctggac tggctccaga ttgcgctatc accgtgcaca cacaggccga caccagctcc 420

cccgatcctc tgtttaaccc cctcaaaacc ggcgtgtgcc agctggacaa cgccaatgtc 480

accgatgcta tcctgtctag ggccggaggc agcattgctg acttcaccgg ccatagacag 540

acagcctttc gcgagctgga acgggtgctc aacttccctc agagcaatct gtgcctcaag 600

cgcgagaaac aggacgaatc ttgtagcctg acccaggccc tcccatccga gctgaaggtg 660

tctgctgata acgtcagcct gaccggagcc gtgtccctcg cttctatgct gacagagatc 720

ttcctgctcc agcaggctca gggaatgcca gaaccaggat ggggccgcat taccgactcc 780

caccagtgga acacactgct ctctctgcat aatgcccagt tttacctgct ccagaggacc 840

ccagaggtgg ctaggtctag agctacaccc ctgctcgacc tcatcaagac cgccctgaca 900

cctcaccccc ctcagaaaca ggcttatggg gtgaccctgc caacaagcgt cctgttcatt 960

gccggacatg ataccaacct ggccaatctc gggggagctc tggaactcaa ctggaccctg 1020

cccggccagc ctgacaatac accacccggc ggggagctgg tgttcgaaag gtggcgccgg 1080

ctgagcgata actcccagtg gatccaggtg agcctggtct ttcagaccct gcagcagatg 1140

agagacaaga cccccctgtc cctcaacaca cctccaggag aggtcaaact gaccctcgcc 1200

ggctgcgagg aacgcaatgc tcaggggatg tgctctctcg ccggattcac ccagattgtc 1260

aacgaagccc gcattccagc ctgctccctg tga 1293

<210> 3

<211> 1251

<212>DNA

<213> artificial synthesis ()

<400> 3

atgttccagc tgtggaagct ggtgttcctg tgcggcctgc tgatcggcac cagcgcccag 60

cagaccgtgt ggggccagtg tggcggaatt ggatggtccg ggcccacaaa ttgcgctcca 120

ggatccgcct gtagcaccct gaacccttac tatgctcagt gcatccccgg agccaccaca 180

attaccacat ccaccaggcc accaagcgga ccaaccaaa ccacaagagc cacctccaca 240

tccagctcta cacctcccac ctcatccgga gtgagattcg ctggggtcaa catcgccggg 300

ttcgactttg gctgcaccac agatgggaca tgtgtgacct ctaaggtcta cccacctctg 360

aaaaatttta ccggatccaa caattatcca gacgggatcg gccagatgca gcacttcgtg 420

aacgaagatg gaatgaccat ttttcggctc cctgtggggt ggcagtacct ggtcaacaat 480

aacctcgggg gcaacctgga ctcaacctcc atcagcaagt atgatcagct ggtgcaggga 540

tgcctgagcc tcggagctta ctgtatcgtc gacattcaca attatgccag gtggaacgga 600

gggatcattg gccagggcgg acctacaaat gcccagttca cctctctctg gtcacagctg 660

gcttccaaat acgcctctca gtcacgagtg tggtttggca tcatgaacga gccccatgac 720

gtgaatatta acacatgggc cgctaccgtc caggaagtgg tcacagccat ccgcaatgct 780

ggcgccacct ctcagttcat ttcactgcca ggaaacgact ggcagtccgc tggagccttt 840

atctccgatg gaagcgctgc tgctctgtcc caggtgacca atccagacgg cagcaccaca 900

aacctcatct tcgatgtcca caagtacctg gactctgata actcagggac ccatgccgag 960

tgcaccacaa ataacatcga cggcgctttt agccactcg ccacctggct gcgccagaat 1020

aaccggcagg ccatcctgac cgaaacaggg ggcggaaacg tgcagtcctg catccaggac 1080

atgtgtcagc agattcagta cctcaatcag aacagcgatg tgtacctggg atatgtcgga 1140

tggggagctg gatccttcga cagcacctac gtgctgaccg agacacccac cagctctggc 1200

aactcttgga cagatacctc actcgtgtca tcctgtctgg cccgaaaatg a 1251

<210> 4

<211> 1365

<212>DNA

<213> artificial synthesis ()

<400> 4

atggttcagc tttggaaact tgttctcttg tgcggcctgc tcgccgggac ctcagcgtct 60

ggcagctacg actacgccga cgtgatcaag aagtccctgc tgttctacca ggctcagcgc 120

agcggccggc tgagcggcat ggaccccctg gtgagctgga ggaaggactc cgccctgaac 180

gacagaggaa acaacggaga ggacctgacc ggaggatact acgacgctgg cgacttcgtg 240

aagttcggct tccccatggc cctacaccatc accctgctga gctggggcgt gatcgactac 300

gagaacacct acagctccat cggcgccctg tccgccgccc gcgccgccat caagtggggc 360

accgactact tcatcaaggc ccacgtgagc gccaacgagc tgtacggaca ggtcggaaac 420

ggaggagctg accactcctg gtggggcagg cccgaggaca tgaacatgga ccggcccgcc 480

tacaagatcg acacctcccg gccaggcagc gacctggccg ccgagaccgc cgccgccatg 540

gccgccgcca gcatcgtgtt caagaacgcc gactccaact acgccaacac cctgctgagg 600

cacgccaagg agctgtacaa cttcgccgac aactacaggg gcaagtacag cgactccatc 660

agcgacgccg ccgccttcta caactcctac agctacgagg acgagctggt gtggggagct 720

atctggctgt ggagggctac caacgaccag aactacctga acaaggccac ccagtactac 780

aaccagtaca gcatccagta caagaactcc cccctgagct gggacgacaa gtccaccgga 840

gctagcgccc tgctggctaa gctgaccgga ggcgaccagt acaagtccgc cgtgcagagc 900

ttctgcgacg gcttctacta caaccagcag aagaccccca agggcctgat ctggtactcc 960

gactggggca gcctgaggca gtccatgaac gccgtgtggg tgtgcctcca ggccgccgac 1020

gctggagtga agaccggaga gtaccgcagc ctggccaaga agcagctgga ctacgctctg 1080

ggcgacgccg gccggtcctt cgtggtgggc ttcggcaaca accccccag ccacgagcag 1140

cacagggctg cttcctgccc agacgctcct gccgcctgcg actggaacac ctacaacggc 1200

ggccagtcca actaccacgt gctgtacggc gccctggtgg gaggaccaga cgccaacgac 1260

tactacaacg acgtgagaag cgactacgtg cacaacgagg tggcctgcga ctacaacgcc 1320

ggcttccaga acgtgctggt gtccctgaag gccaacggct actga 1365

<210> 5

<211> 45

<212>DNA

<213> artificial synthesis ()

<400> 5

gaggctgccg ccaaagaagc tgccgccaag gaggctgccg ccaag 45

<210> 6

<211> 45

<212>DNA

<213> artificial synthesis ()

<400> 6

gaggccgccg ccaaggaggc cgccgccaag gaggccgccg ccaag 45

<210> 7

<211> 1980

<212>DNA

<213> artificial synthesis ()

<400> 7

atgttccagc tgtggaagct ggtgttcctg tgcggactgc tgatcggcac cagcgcctcc 60

agcaccccct ccagcaccgg agagaacaac ggcttctact actccttctg gaccgacgga 120

ggaggcgacg tgacctacac caacggcgac gccggagctt acaccgtgga gtggagcaac 180

gtggggcaact tcgtgggagg caagggatgg aacccaggct ccgcccagga catcacctac 240

tccggcacct tcaccccaag cggcaacggc tacctgtccg tgtacggctg gaccaccgac 300

cccctgatcg agtactacat cgtggagagc tacggcgact acaacccagg ctccggaggc 360

acctacaagg gcaccgtgac cagcgacggc tccgtgtacg acatctacac cgctaccagg 420

accaacgctg ccagcatcca gggcaccgcc accttcaccc agtactggtc cgtgaggcag 480

aacaagagag tgggcggcac cgtgaccacc agcaaccact tcaacgcctg ggccaagctg 540

ggcatgaacc tgggcaccca caactaccag atcgtggcta ccgagggcta ccagtccagc 600

ggctccagct ccatcaccgt gcaggaggct gccgccaaag aagctgccgc caaggaggct 660

gccgccaagc agtccgagcc agagctgaag ctggagagcg tggtcatcgt gtcccgccac 720

ggcgtgcgcg ctccaaccaa ggccaccag ctgatgcagg acgtgacccc agacgcttgg 780

ccaacctggc cagtgaagct gggatggctg acccccaggg gcggagagct gatcgcctac 840

ctgggccact accagaggca gagactggtg gctgacggac tgctggccaa gaagggatgc 900

ccacagagcg gacaggtggc tatcatcgct gacgtggacg agcgcacccg gaagaccgga 960

gaggccttcg ccgccggcct ggccccagac tgcgctatca ccgtgcacac ccaggctgac 1020

accagctccc ccgacccact gttcaaccca ctgaagaccg gcgtgtgcca gctggacaac 1080

gccaacgtga ccgacgctat cctgagccgc gccggaggct ccatcgctga cttcaccgga 1140

cacaggcaga ccgccttcag ggagctggag agagtgctga acttccccca gtccaacctg 1200

tgcctgaagc gggagaagca ggacgagagc tgctccctga cccaggccct gccaagcgag 1260

ctgaaggtgt ccgccgacaa cgtgagcctg accggagccg tgagcctggc ctccatgctg 1320

accgagatct tcctgctcca gcaggctcag ggaatgccag agccaggatg gggaaggatc 1380

accgacagcc accacgtggaa caccctgctg tccctgcaca acgcccagtt ctacctgctc 1440

cagcggaccc cagaggtggc taggagcaga gccaccccac tgctggacct gatcaagacc 1500

gccctgaccc cacacccacc acagaagcag gcctacggcg tgaccctgcc aacctccgtg 1560

ctgttcatcg ccggccacga caccaacctg gctaacctgg gaggcgccct ggagctgaac 1620

tggaccctgc caggacagcc agacaacacc ccaccaggag gagagctggt gttcgagagg 1680

tggcgccggc tgagcgacaa ctcccagtgg attcaggtgt ccctggtgtt ccagaccctc 1740

cagcagatga gagacaagac cccactgtcc ctgaacaccc caccaggaga ggtgaagctg 1800

accctggccg gatgcgagga gaggaacgct cagggaatgt gcagcctggc cggcttcacc 1860

cagatcgtga acgaggctag aatccccgcc tgctccctga gggtgaagag gggcagcgga 1920

gctaccaact tctccctgct gaagcaggct ggcgacgtgg aggagaaccc aggaccatga 1980

<210> 8

<211> 659

<212> PRT

<213> artificial synthesis ()

<400> 8

Met Phe Gln Leu Trp Lys Leu Val Phe Leu Cys Gly Leu Leu Ile Gly

1 5 10 15

Thr Ser Ala Ser Ser Thr Pro Ser Ser Thr Gly Glu Asn Asn Gly Phe

20 25 30

Tyr Tyr Ser Phe Trp Thr Asp Gly Gly Gly Asp Val Thr Tyr Thr Asn

35 40 45

Gly Asp Ala Gly Ala Tyr Thr Val Glu Trp Ser Asn Val Gly Asn Phe

50 55 60

Val Gly Gly Lys Gly Trp Asn Pro Gly Ser Ala Gln Asp Ile Thr Tyr

65 70 75 80

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

85 90 95

Trp Thr Thr Asp Pro Leu Ile Glu Tyr Tyr Ile Val Glu Ser Tyr Gly

100 105 110

Asp Tyr Asn Pro Gly Ser Gly Gly Thr Tyr Lys Gly Thr Val Thr Ser

115 120 125

Asp Gly Ser Val Tyr Asp Ile Tyr Thr Ala Thr Arg Thr Asn Ala Ala

130 135 140

Ser Ile Gln Gly Thr Ala Thr Phe Thr Gln Tyr Trp Ser Val Arg Gln

145 150 155 160

Asn Lys Arg Val Gly Gly Thr Val Thr Thr Ser Asn His Phe Asn Ala

165 170 175

Trp Ala Lys Leu Gly Met Asn Leu Gly Thr His Asn Tyr Gln Ile Val

180 185 190

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

195 200 205

Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Gln

210 215 220

Ser Glu Pro Glu Leu Lys Leu Glu Ser Val Val Ile Val Ser Arg His

225 230 235 240

Gly Val Arg Ala Pro Thr Lys Ala Thr Gln Leu Met Gln Asp Val Thr

245 250 255

Pro Asp Ala Trp Pro Thr Trp Pro Val Lys Leu Gly Trp Leu Thr Pro

260 265 270

Arg Gly Gly Glu Leu Ile Ala Tyr Leu Gly His Tyr Gln Arg Gln Arg

275 280 285

Leu Val Ala Asp Gly Leu Leu Ala Lys Lys Gly Cys Pro Gln Ser Gly

290 295 300

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

305 310 315 320

Glu Ala Phe Ala Ala Gly Leu Ala Pro Asp Cys Ala Ile Thr Val His

325 330 335

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

340 345 350

Thr Gly Val Cys Gln Leu Asp Asn Ala Asn Val Thr Asp Ala Ile Leu

355 360 365

Ser Arg Ala Gly Gly Ser Ile Ala Asp Phe Thr Gly His Arg Gln Thr

370 375 380

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

385 390 395 400

Cys Leu Lys Arg Glu Lys Gln Asp Glu Ser Cys Ser Leu Thr Gln Ala

405 410 415

Leu Pro Ser Glu Leu Lys Val Ser Ala Asp Asn Val Ser Leu Thr Gly

420 425 430

Ala Val Ser Leu Ala Ser Met Leu Thr Glu Ile Phe Leu Leu Gln Gln

435 440 445

Ala Gln Gly Met Pro Glu Pro Gly Trp Gly Arg Ile Thr Asp Ser His

450 455 460

Gln Trp Asn Thr Leu Leu Ser Leu His Asn Ala Gln Phe Tyr Leu Leu

465 470 475 480

Gln Arg Thr Pro Glu Val Ala Arg Ser Arg Ala Thr Pro Leu Leu Asp

485 490 495

Leu Ile Lys Thr Ala Leu Thr Pro His Pro Pro Gln Lys Gln Ala Tyr

500 505 510

Gly Val Thr Leu Pro Thr Ser Val Leu Phe Ile Ala Gly His Asp Thr

515 520 525

Asn Leu Ala Asn Leu Gly Gly Ala Leu Glu Leu Asn Trp Thr Leu Pro

530 535 540

Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Glu Leu Val Phe Glu Arg

545 550 555 560

Trp Arg Arg Leu Ser Asp Asn Ser Gln Trp Ile Gln Val Ser Leu Val

565 570 575

Phe Gln Thr Leu Gln Gln Met Arg Asp Lys Thr Pro Leu Ser Leu Asn

580 585 590

Thr Pro Pro Gly Glu Val Lys Leu Thr Leu Ala Gly Cys Glu Glu Arg

595 600 605

Asn Ala Gln Gly Met Cys Ser Leu Ala Gly Phe Thr Gln Ile Val Asn

610 615 620

Glu Ala Arg Ile Pro Ala Cys Ser Leu Arg Val Lys Arg Gly Ser Gly

625 630 635 640

Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn

645 650 655

Pro Gly Pro

<210> 9

<211> 2598

<212>DNA

<213> artificial synthesis ()

<400> 9

atgtttcagc tctggaagct cgtgtttctc tgcggactcc tcatcgggac ctcagcccag 60

cagaccgtgt ggggacagtg tggcggaatc ggctggtccg gcccaaccaa ctgcgcccca 120

ggcagcgcct gctccaccct gaacccctac tacgcccagt gcatcccagg cgccaccacc 180

atcaccacca gcaccaggcc cccatccggc cccaccacca ccaccagagc cacctccacc 240

agcagctcca ccccaccac ctccagcggc gtgagattcg ccggcgtgaa catcgccggc 300

ttcgacttcg gctgcaccac cgacggcacc tgcgtgacca gcaaggtgta ccccccactg 360

aagaacttca ccggcagcaa caactaccca gacggcatcg gccagatgca gcacttcgtg 420

aacgaggacg gcatgaccat cttccggctg cccgtgggct ggcagtacct ggtgaacaac 480

aacctgggcg gcaacctgga cagcacctcc atcagcaagt acgaccagct ggtgcagggc 540

tgcctgagcc tgggcgccta ctgcatcgtg gacatccaca actacgccag atggaacggc 600

ggcatcatcg gccagggcgg ccccaccaac gccccagttca ccagcctgtg gtcccagctg 660

gcctccaagt acgccagcca gtccagagtg tggttcggca tcatgaacga gccacacgac 720

gtgaacatca acacctgggc cgccaccgtg caggaggtgg tgaccgccat cagaaacgcc 780

ggcgccacct cccagttcat ctccctgcca ggcaacgact ggcagagcgc cggcgccttc 840

atctccgacg gcagcgccgc cgccctgagc caggtgacca accccgacgg cagcaccacc 900

aacctgatct tcgacgtgca caagtacctg gactccgaca acagcggcac ccacgccgag 960

tgcaccacca acaacatcga cggcgccttc tccccactgg ccacctggct gagacagaac 1020

aacagacagg ccatcctgac cgagaccggc ggcggcaacg tgcagtcctg catccaggac 1080

atgtgccagc agatccagta cctgaaccag aacagcgacg tgtacctggg ctacgtgggc 1140

tggggcgccg gctccttcga cagcacctac gtgctgaccg agaccccac ctcctccggc 1200

aacagctgga ccgacacctc cctggtgtcc agctgcctgg ccagaaagga ggccgccgcc 1260

aaggaggccg ccgccaagga ggccgccgcc aagggcagct acgactacgc cgacgtgatc 1320

aagaagagcc tgctgttcta ccaggcccag cggtccggca ggctgagcgg catggaccca 1380

ctggtgtcct ggagaaagga cagcgccctg aacgacaggg gcaacaacgg cgaggacctg 1440

accggcggct actacgacgc cggcgacttc gtgaagttcg gcttcccaat ggcctacacc 1500

atcaccctgc tgagctgggg cgtgatcgac tacgagaaca cctactccag catcggcgcc 1560

ctgagcgccg ccagagccgc catcaagtgg ggcaccgact acttcatcaa ggcccacgtg 1620

tccgccaacg agctgtacgg ccaggtgggc aacggcggcg ccgaccacag ctggtggggc 1680

agacccgagg acatgaacat ggacaggcca gcctacaaga tcgacacctc cagaccaggc 1740

tccgacctgg ccgccgagac cgccgccgcc atggccgccg cctccatcgt gttcaagaac 1800

gccgactcca actacgccaa caccctgctg agacacgcca aggagctgta caacttcgcc 1860

gacaactaca ggggcaagta ctccgactcc atcagcgacg ccgccgcctt ctacaacagc 1920

tacagctacg aggacgagct ggtgtggggc gccatctggc tgtggagagc caccaacgac 1980

cagaactacc tgaacaaggc cacccagtac tacaaccagt actccatcca gtacaagaac 2040

agcccactgt cctgggacga caagagcacc ggcgcctccg ccctgctggc caagctgacc 2100

ggcggcgacc agtacaagag cgccgtgcag agcttctgcg acggcttcta ctacaaccag 2160

cagaagaccc caaagggcct gatctggtac tccgactggg gctccctgag acagtccatg 2220

aacgccgtgt gggtgtgcct gcaagccgcc gacgccggcg tgaagaccgg cgagtacaga 2280

tccctggcca agaagcagct ggactacgcc ctgggcgacg ccggccggag cttcgtggtg 2340

ggcttcggca acaacccacc ctcccacgag cagcacagggg ccgccagctg cccagacgcc 2400

cccgccgcct gcgactggaa cacctacaac ggcggccagt ccaactacca cgtgctgtac 2460

ggcgccctgg tgggcggccc cgacgccaac gactactaca acgacgtgag atccgactac 2520

gtgcacaacg aggtggcctg cgactacaac gctggatttc agaatgtcct cgtgtcactc 2580

aaggctaatg gctactga 2598

<210> 10

<211> 865

<212> PRT

<213> artificial synthesis ()

<400> 10

Met Phe Gln Leu Trp Lys Leu Val Phe Leu Cys Gly Leu Leu Ile Gly

1 5 10 15

Thr Ser Ala Gln Gln Thr Val Trp Gly Gln Cys Gly Gly Ile Gly Trp

20 25 30

Ser Gly Pro Thr Asn Cys Ala Pro Gly Ser Ala Cys Ser Thr Leu Asn

35 40 45

Pro Tyr Tyr Ala Gln Cys Ile Pro Gly Ala Thr Thr Ile Thr Thr Ser

50 55 60

Thr Arg Pro Pro Ser Gly Pro Thr Thr Thr Thr Thr Arg Ala Thr Ser Thr

65 70 75 80

Ser Ser Ser Thr Pro Pro Thr Ser Ser Gly Val Arg Phe Ala Gly Val

85 90 95

Asn Ile Ala Gly Phe Asp Phe Gly Cys Thr Thr Asp Gly Thr Cys Val

100 105 110

Thr Ser Lys Val Tyr Pro Pro Leu Lys Asn Phe Thr Gly Ser Asn Asn

115 120 125

Tyr Pro Asp Gly Ile Gly Gln Met Gln His Phe Val Asn Glu Asp Gly

130 135 140

Met Thr Ile Phe Arg Leu Pro Val Gly Trp Gln Tyr Leu Val Asn Asn

145 150 155 160

Asn Leu Gly Gly Asn Leu Asp Ser Thr Ser Ile Ser Lys Tyr Asp Gln

165 170 175

Leu Val Gln Gly Cys Leu Ser Leu Gly Ala Tyr Cys Ile Val Asp Ile

180 185 190

His Asn Tyr Ala Arg Trp Asn Gly Gly Ile Ile Gly Gln Gly Gly Pro

195 200 205

Thr Asn Ala Gln Phe Thr Ser Leu Trp Ser Gln Leu Ala Ser Lys Tyr

210 215 220

Ala Ser Gln Ser Arg Val Trp Phe Gly Ile Met Asn Glu Pro His Asp

225 230 235 240

Val Asn Ile Asn Thr Trp Ala Ala Thr Val Gln Glu Val Val Thr Ala

245 250 255

Ile Arg Asn Ala Gly Ala Thr Ser Gln Phe Ile Ser Leu Pro Gly Asn

260 265 270

Asp Trp Gln Ser Ala Gly Ala Phe Ile Ser Asp Gly Ser Ala Ala Ala

275 280 285

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

290 295 300

Asp Val His Lys Tyr Leu Asp Ser Asp Asn Ser Gly Thr His Ala Glu

305 310 315 320

Cys Thr Thr Asn Asn Ile Asp Gly Ala Phe Ser Pro Leu Ala Thr Trp

325 330 335

Leu Arg Gln Asn Asn Arg Gln Ala Ile Leu Thr Glu Thr Gly Gly Gly

340 345 350

Asn Val Gln Ser Cys Ile Gln Asp Met Cys Gln Gln Ile Gln Tyr Leu

355 360 365

Asn Gln Asn Ser Asp Val Tyr Leu Gly Tyr Val Gly Trp Gly Ala Gly

370 375 380

Ser Phe Asp Ser Thr Tyr Val Leu Thr Glu Thr Pro Thr Ser Ser Ser Gly

385 390 395 400

Asn Ser Trp Thr Asp Thr Ser Leu Val Ser Ser Cys Leu Ala Arg Lys

405 410 415

Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Gly

420 425 430

Ser Tyr Asp Tyr Ala Asp Val Ile Lys Lys Ser Leu Leu Phe Tyr Gln

435 440 445

Ala Gln Arg Ser Gly Arg Leu Ser Gly Met Asp Pro Leu Val Ser Trp

450 455 460

Arg Lys Asp Ser Ala Leu Asn Asp Arg Gly Asn Asn Gly Glu Asp Leu

465 470 475 480

Thr Gly Gly Tyr Tyr Asp Ala Gly Asp Phe Val Lys Phe Gly Phe Pro

485 490 495

Met Ala Tyr Thr Ile Thr Leu Leu Ser Trp Gly Val Ile Asp Tyr Glu

500 505 510

Asn Thr Tyr Ser Ser Ile Gly Ala Leu Ser Ala Ala Arg Ala Ala Ile

515 520 525

Lys Trp Gly Thr Asp Tyr Phe Ile Lys Ala His Val Ser Ala Asn Glu

530 535 540

Leu Tyr Gly Gln Val Gly Asn Gly Gly Ala Asp His Ser Trp Trp Gly

545 550 555 560

Arg Pro Glu Asp Met Asn Met Asp Arg Pro Ala Tyr Lys Ile Asp Thr

565 570 575

Ser Arg Pro Gly Ser Asp Leu Ala Ala Glu Thr Ala Ala Ala Met Ala

580 585 590

Ala Ala Ser Ile Val Phe Lys Asn Ala Asp Ser Asn Tyr Ala Asn Thr

595 600 605

Leu Leu Arg His Ala Lys Glu Leu Tyr Asn Phe Ala Asp Asn Tyr Arg

610 615 620

Gly Lys Tyr Ser Asp Ser Ile Ser Asp Ala Ala Ala Phe Tyr Asn Ser

625 630 635 640

Tyr Ser Tyr Glu Asp Glu Leu Val Trp Gly Ala Ile Trp Leu Trp Arg

645 650 655

Ala Thr Asn Asp Gln Asn Tyr Leu Asn Lys Ala Thr Gln Tyr Tyr Asn

660 665 670

Gln Tyr Ser Ile Gln Tyr Lys Asn Ser Pro Leu Ser Trp Asp Asp Lys

675 680 685

Ser Thr Gly Ala Ser Ala Leu Leu Ala Lys Leu Thr Gly Gly Asp Gln

690 695 700

Tyr Lys Ser Ala Val Gln Ser Phe Cys Asp Gly Phe Tyr Tyr Asn Gln

705 710 715 720

Gln Lys Thr Pro Lys Gly Leu Ile Trp Tyr Ser Asp Trp Gly Ser Leu

725 730 735

Arg Gln Ser Met Asn Ala Val Trp Val Cys Leu Gln Ala Ala Asp Ala

740 745 750

Gly Val Lys Thr Gly Glu Tyr Arg Ser Leu Ala Lys Lys Gln Leu Asp

755 760 765

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

770 775 780

Asn Pro Pro Ser His Glu Gln His Arg Ala Ala Ser Cys Pro Asp Ala

785 790 795 800

Pro Ala Ala Cys Asp Trp Asn Thr Tyr Asn Gly Gly Gln Ser Asn Tyr

805 810 815

His Val Leu Tyr Gly Ala Leu Val Gly Gly Pro Asp Ala Asn Asp Tyr

820 825 830

Tyr Asn Asp Val Arg Ser Asp Tyr Val His Asn Glu Val Ala Cys Asp

835 840 845

Tyr Asn Ala Gly Phe Gln Asn Val Leu Val Ser Leu Lys Ala Asn Gly

850 855 860

Tyr

865

<210> 11

<211> 4581

<212>DNA

<213> artificial synthesis ()

<400> 11

gccaccatgt tccagctgtg gaagctggtg ttcctgtgcg gactgctgat cggcaccagc 60

gcctccagca ccccctccag caccggagag aacaacggct tctactactc cttctggacc 120

gacggaggag gcgacgtgac ctacaccaac ggcgacgccg gagcttacac cgtggagtgg 180

agcaacgtgg gcaacttcgt gggaggcaag ggatggaacc caggctccgc ccaggacatc 240

acctactccg gcaccttcac cccaagcggc aacggctacc tgtccgtgta cggctggacc 300

accgaccccc tgatcgagta ctacatcgtg gagagctacg gcgactacaa cccaggctcc 360

ggaggcacct acaagggcac cgtgaccagc gacggctccg tgtacgacat ctacaccgct 420

accaggacca acgctgccag catccagggc accgccacct tcacccagta ctggtccgtg 480

aggcagaaca agagagtggg cggcaccgtg accaccagca accacttcaa cgcctgggcc 540

aagctgggca tgaacctggg cacccacaac taccagatcg tggctaccga gggctaccag 600

tccagcggct ccagctccat caccgtgcag gaggctgccg ccaaagaagc tgccgccaag 660

gaggctgccg ccaagcagtc cgagccagag ctgaagctgg agagcgtggt catcgtgtcc 720

cgccacggcg tgcgcgctcc aaccaaggcc acccagctga tgcaggacgt gaccccagac 780

gcttggccaa cctggccagt gaagctggga tggctgaccc ccaggggcgg agagctgatc 840

gcctacctgg gccactacca gaggcagaga ctggtggctg acggactgct ggccaagaag 900

ggatgcccac agagcggaca ggtggctatc atcgctgacg tggacgagcg cacccggaag 960

accggagagg ccttcgccgc cggcctggcc ccagactgcg ctatcaccgt gcacacccag 1020

gctgacacca gctcccccga cccactgttc aacccactga agaccggcgt gtgccagctg 1080

gacaacgcca acgtgaccga cgctatcctg agccgcgccg gaggctccat cgctgacttc 1140

accggacaca ggcagaccgc cttcagggag ctggagagag tgctgaactt cccccagtcc 1200

aacctgtgcc tgaagcggga gaagcaggac gagagctgct ccctgaccca ggccctgcca 1260

agcgagctga aggtgtccgc cgacaacgtg agcctgaccg gagccgtgag cctggcctcc 1320

atgctgaccg agatcttcct gctccagcag gctcagggaa tgccagagcc aggatgggga 1380

aggatcaccg acagccacca gtggaacacc ctgctgtccc tgcacaacgc ccagttctac 1440

ctgctccagc ggaccccaga ggtggctagg agcagagcca ccccactgct ggacctgatc 1500

aagaccgccc tgaccccaca cccaccacag aagcaggcct acggcgtgac cctgccaacc 1560

tccgtgctgt tcatcgccgg ccacgacacc aacctggcta acctgggagg cgccctggag 1620

ctgaactgga ccctgccagg acagccagac aacacccac caggaggaga gctggtgttc 1680

gagaggtggc gccggctgag cgacaactcc cagtggattc aggtgtccct ggtgttccag 1740

accctccagc agatgagaga caagacccca ctgtccctga acaccccacc aggagaggtg 1800

aagctgaccc tggccggatg cgaggagagg aacgctcagg gaatgtgcag cctggccggc 1860

ttcacccaga tcgtgaacga ggctagaatc cccgcctgct ccctgagggt gaagaggggc 1920

agcggagcta ccaacttctc cctgctgaag caggctggcg acgtggagga gaacccagga 1980

ccaatgtttc agctctggaa gctcgtgttt ctctgcggac tcctcatcgg gacctcagcc 2040

cagcagaccg tgtggggaca gtgtggcgga atcggctggt ccggcccaac caactgcgcc 2100

ccaggcagcg cctgctccac cctgaaccccc tactacgccc agtgcatccc aggcgccacc 2160

accatcacca ccagcaccag gcccccatcc ggccccacca ccaccaccag agccacctcc 2220

accagcagct ccaccccacc cacctccagc ggcgtgagat tcgccggcgt gaacatcgcc 2280

ggcttcgact tcggctgcac caccgacggc acctgcgtga ccagcaaggt gtacccccca 2340

ctgaagaact tcaccggcag caacaactac ccagacggca tcggccagat gcagcacttc 2400

gtgaacgagg acggcatgac catcttccgg ctgcccgtgg gctggcagta cctggtgaac 2460

aacaacctgg gcggcaacct ggacagcacc tccatcagca agtacgacca gctggtgcag 2520

ggctgcctga gcctgggcgc ctactgcatc gtggacatcc acaactacgc cagatggaac 2580

ggcggcatca tcggccaggg cggccccacc aacgcccagt tcaccagcct gtggtcccag 2640

ctggcctcca agtacgccag ccagtccaga gtgtggttcg gcatcatgaa cgagccacac 2700

gacgtgaaca tcaacacctg ggccgccacc gtgcaggagg tggtgaccgc catcagaaac 2760

gccggcgcca cctcccagtt catctccctg ccaggcaacg actggcagag cgccggcgcc 2820

ttcatctccg acggcagcgc cgccgccctg agccaggtga ccaaccccga cggcagcacc 2880

accaacctga tcttcgacgt gcacaagtac ctggactccg acaacagcgg cacccacgcc 2940

gagtgcacca ccaacaacat cgacggcgcc ttctccccac tggccacctg gctgagacag 3000

aacaacagac aggccatcct gaccgagacc ggcggcggca acgtgcagtc ctgcatccag 3060

gacatgtgcc agcagatcca gtacctgaac cagaacagcg acgtgtacct gggctacgtg 3120

ggctggggcg ccggctcctt cgacagcacc tacgtgctga ccgagacccc cacctcctcc 3180

ggcaacagct ggaccgacac ctccctggtg tccagctgcc tggccagaaa ggaggccgcc 3240

gccaaggagg ccgccgccaa ggaggccgcc gccaagggca gctacgacta cgccgacgtg 3300

atcaagaaga gcctgctgtt ctaccaggcc cagcggtccg gcaggctgag cggcatggac 3360

ccactggtgt cctggagaaa ggacagcgcc ctgaacgaca ggggcaacaa cggcgaggac 3420

ctgaccggcg gctactacga cgccggcgac ttcgtgaagt tcggcttccc aatggcctac 3480

accatcaccc tgctgagctg gggcgtgatc gactacgaga acacctactc cagcatcggc 3540

gccctgagcg ccgccagagc cgccatcaag tggggcaccg actacttcat caaggcccac 3600

gtgtccgcca acgagctgta cggccaggtg ggcaacggcg gcgccgacca cagctggtgg 3660

ggcagacccg aggacatgaa catggacagg ccagcctaca agatcgacac ctccagacca 3720

ggctccgacc tggccgccga gaccgccgcc gccatggccg ccgcctccat cgtgttcaag 3780

aacgccgact ccaactacgc caacaccctg ctgagacacg ccaaggagct gtacaacttc 3840

gccgacaact acaggggcaa gtactccgac tccatcagcg acgccgccgc cttctacaac 3900

agctacagct acgaggacga gctggtgtgg ggcgccatct ggctgtggag agccaccaac 3960

gaccagaact acctgaacaa ggccaccag tactacaacc agtactccat ccagtacaag 4020

aacagcccac tgtcctggga cgacaagagc accggcgcct ccgccctgct ggccaagctg 4080

accggcggcg accagtacaa gagcgccgtg cagagcttct gcgacggctt ctactacaac 4140

cagcagaaga ccccaaaggg cctgatctgg tactccgact ggggctccct gagacagtcc 4200

atgaacgccg tgtgggtgtg cctgcaagcc gccgacgccg gcgtgaagac cggcgagtac 4260

agatccctgg ccaagaagca gctggactac gccctgggcg acgccggccg gagcttcgtg 4320

gtgggcttcg gcaacaaccc accctcccac gagcagcaca gggccgccag ctgcccagac 4380

gcccccgccg cctgcgactg gaacacctac aacggcggcc agtccaacta ccacgtgctg 4440

tacggcgccc tggtgggcgg ccccgacgcc aacgactact acaacgacgt gagatccgac 4500

tacgtgcaca acgaggtggc ctgcgactac aacgctggat ttcagaatgt cctcgtgtca 4560

ctcaaggcta atggctactg a 4581

<210> 12

<211> 1524

<212> PRT

<213> artificial synthesis ()

<400> 12

Met Phe Gln Leu Trp Lys Leu Val Phe Leu Cys Gly Leu Leu Ile Gly

1 5 10 15

Thr Ser Ala Ser Ser Thr Pro Ser Ser Thr Gly Glu Asn Asn Gly Phe

20 25 30

Tyr Tyr Ser Phe Trp Thr Asp Gly Gly Gly Asp Val Thr Tyr Thr Asn

35 40 45

Gly Asp Ala Gly Ala Tyr Thr Val Glu Trp Ser Asn Val Gly Asn Phe

50 55 60

Val Gly Gly Lys Gly Trp Asn Pro Gly Ser Ala Gln Asp Ile Thr Tyr

65 70 75 80

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

85 90 95

Trp Thr Thr Asp Pro Leu Ile Glu Tyr Tyr Ile Val Glu Ser Tyr Gly

100 105 110

Asp Tyr Asn Pro Gly Ser Gly Gly Thr Tyr Lys Gly Thr Val Thr Ser

115 120 125

Asp Gly Ser Val Tyr Asp Ile Tyr Thr Ala Thr Arg Thr Asn Ala Ala

130 135 140

Ser Ile Gln Gly Thr Ala Thr Phe Thr Gln Tyr Trp Ser Val Arg Gln

145 150 155 160

Asn Lys Arg Val Gly Gly Thr Val Thr Thr Ser Asn His Phe Asn Ala

165 170 175

Trp Ala Lys Leu Gly Met Asn Leu Gly Thr His Asn Tyr Gln Ile Val

180 185 190

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

195 200 205

Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Gln

210 215 220

Ser Glu Pro Glu Leu Lys Leu Glu Ser Val Val Ile Val Ser Arg His

225 230 235 240

Gly Val Arg Ala Pro Thr Lys Ala Thr Gln Leu Met Gln Asp Val Thr

245 250 255

Pro Asp Ala Trp Pro Thr Trp Pro Val Lys Leu Gly Trp Leu Thr Pro

260 265 270

Arg Gly Gly Glu Leu Ile Ala Tyr Leu Gly His Tyr Gln Arg Gln Arg

275 280 285

Leu Val Ala Asp Gly Leu Leu Ala Lys Lys Gly Cys Pro Gln Ser Gly

290 295 300

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

305 310 315 320

Glu Ala Phe Ala Ala Gly Leu Ala Pro Asp Cys Ala Ile Thr Val His

325 330 335

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

340 345 350

Thr Gly Val Cys Gln Leu Asp Asn Ala Asn Val Thr Asp Ala Ile Leu

355 360 365

Ser Arg Ala Gly Gly Ser Ile Ala Asp Phe Thr Gly His Arg Gln Thr

370 375 380

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

385 390 395 400

Cys Leu Lys Arg Glu Lys Gln Asp Glu Ser Cys Ser Leu Thr Gln Ala

405 410 415

Leu Pro Ser Glu Leu Lys Val Ser Ala Asp Asn Val Ser Leu Thr Gly

420 425 430

Ala Val Ser Leu Ala Ser Met Leu Thr Glu Ile Phe Leu Leu Gln Gln

435 440 445

Ala Gln Gly Met Pro Glu Pro Gly Trp Gly Arg Ile Thr Asp Ser His

450 455 460

Gln Trp Asn Thr Leu Leu Ser Leu His Asn Ala Gln Phe Tyr Leu Leu

465 470 475 480

Gln Arg Thr Pro Glu Val Ala Arg Ser Arg Ala Thr Pro Leu Leu Asp

485 490 495

Leu Ile Lys Thr Ala Leu Thr Pro His Pro Pro Gln Lys Gln Ala Tyr

500 505 510

Gly Val Thr Leu Pro Thr Ser Val Leu Phe Ile Ala Gly His Asp Thr

515 520 525

Asn Leu Ala Asn Leu Gly Gly Ala Leu Glu Leu Asn Trp Thr Leu Pro

530 535 540

Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Glu Leu Val Phe Glu Arg

545 550 555 560

Trp Arg Arg Leu Ser Asp Asn Ser Gln Trp Ile Gln Val Ser Leu Val

565 570 575

Phe Gln Thr Leu Gln Gln Met Arg Asp Lys Thr Pro Leu Ser Leu Asn

580 585 590

Thr Pro Pro Gly Glu Val Lys Leu Thr Leu Ala Gly Cys Glu Glu Arg

595 600 605

Asn Ala Gln Gly Met Cys Ser Leu Ala Gly Phe Thr Gln Ile Val Asn

610 615 620

Glu Ala Arg Ile Pro Ala Cys Ser Leu Arg Val Lys Arg Gly Ser Gly

625 630 635 640

Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn

645 650 655

Pro Gly Pro Met Phe Gln Leu Trp Lys Leu Val Phe Leu Cys Gly Leu

660 665 670

Leu Ile Gly Thr Ser Ala Gln Gln Thr Val Trp Gly Gln Cys Gly Gly

675 680 685

Ile Gly Trp Ser Gly Pro Thr Asn Cys Ala Pro Gly Ser Ala Cys Ser

690 695 700

Thr Leu Asn Pro Tyr Tyr Ala Gln Cys Ile Pro Gly Ala Thr Thr Ile

705 710 715 720

Thr Thr Ser Thr Arg Pro Pro Ser Gly Pro Thr Thr Thr Thr Thr Arg Ala

725 730 735

Thr Ser Thr Ser Ser Ser Thr Pro Pro Thr Ser Ser Gly Val Arg Phe

740 745 750

Ala Gly Val Asn Ile Ala Gly Phe Asp Phe Gly Cys Thr Thr Asp Gly

755 760 765

Thr Cys Val Thr Ser Lys Val Tyr Pro Pro Leu Lys Asn Phe Thr Gly

770 775 780

Ser Asn Asn Tyr Pro Asp Gly Ile Gly Gln Met Gln His Phe Val Asn

785 790 795 800

Glu Asp Gly Met Thr Ile Phe Arg Leu Pro Val Gly Trp Gln Tyr Leu

805 810 815

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

820 825 830

Tyr Asp Gln Leu Val Gln Gly Cys Leu Ser Leu Gly Ala Tyr Cys Ile

835 840 845

Val Asp Ile His Asn Tyr Ala Arg Trp Asn Gly Gly Ile Ile Gly Gln

850 855 860

Gly Gly Pro Thr Asn Ala Gln Phe Thr Ser Leu Trp Ser Gln Leu Ala

865 870 875 880

Ser Lys Tyr Ala Ser Gln Ser Arg Val Trp Phe Gly Ile Met Asn Glu

885 890 895

Pro His Asp Val Asn Ile Asn Thr Trp Ala Ala Thr Val Gln Glu Val

900 905 910

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

915 920 925

Pro Gly Asn Asp Trp Gln Ser Ala Gly Ala Phe Ile Ser Asp Gly Ser

930 935 940

Ala Ala Ala Leu Ser Gln Val Thr Asn Pro Asp Gly Ser Thr Thr Asn

945 950 955 960

Leu Ile Phe Asp Val His Lys Tyr Leu Asp Ser Asp Asn Ser Gly Thr

965 970 975

His Ala Glu Cys Thr Thr Asn Asn Ile Asp Gly Ala Phe Ser Pro Leu

980 985 990

Ala Thr Trp Leu Arg Gln Asn Asn Arg Gln Ala Ile Leu Thr Glu Thr

995 1000 1005

Gly Gly Gly Asn Val Gln Ser Cys Ile Gln Asp Met Cys Gln Gln Ile

1010 1015 1020

Gln Tyr Leu Asn Gln Asn Ser Asp Val Tyr Leu Gly Tyr Val Gly Trp

1025 1030 1035 1040

Gly Ala Gly Ser Phe Asp Ser Thr Tyr Val Leu Thr Glu Thr Pro Thr

1045 1050 1055

Ser Ser Gly Asn Ser Trp Thr Asp Thr Ser Leu Val Ser Ser Cys Leu

1060 1065 1070

Ala Arg Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala

1075 1080 1085

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

1090 1095 1100

Phe Tyr Gln Ala Gln Arg Ser Gly Arg Leu Ser Gly Met Asp Pro Leu

1105 1110 1115 1120

Val Ser Trp Arg Lys Asp Ser Ala Leu Asn Asp Arg Gly Asn Asn Gly

1125 1130 1135

Glu Asp Leu Thr Gly Gly Tyr Tyr Asp Ala Gly Asp Phe Val Lys Phe

1140 1145 1150

Gly Phe Pro Met Ala Tyr Thr Ile Thr Leu Leu Ser Trp Gly Val Ile

1155 1160 1165

Asp Tyr Glu Asn Thr Tyr Ser Ser Ile Gly Ala Leu Ser Ala Ala Arg

1170 1175 1180

Ala Ala Ile Lys Trp Gly Thr Asp Tyr Phe Ile Lys Ala His Val Ser

1185 1190 1195 1200

Ala Asn Glu Leu Tyr Gly Gln Val Gly Asn Gly Gly Ala Asp His Ser

1205 1210 1215

Trp Trp Gly Arg Pro Glu Asp Met Asn Met Asp Arg Pro Ala Tyr Lys

1220 1225 1230

Ile Asp Thr Ser Arg Pro Gly Ser Asp Leu Ala Ala Glu Thr Ala Ala

1235 1240 1245

Ala Met Ala Ala Ala Ser Ile Val Phe Lys Asn Ala Asp Ser Asn Tyr

1250 1255 1260

Ala Asn Thr Leu Leu Arg His Ala Lys Glu Leu Tyr Asn Phe Ala Asp

1265 1270 1275 1280

Asn Tyr Arg Gly Lys Tyr Ser Asp Ser Ile Ser Asp Ala Ala Ala Phe

1285 1290 1295

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

1300 1305 1310

Leu Trp Arg Ala Thr Asn Asp Gln Asn Tyr Leu Asn Lys Ala Thr Gln

1315 1320 1325

Tyr Tyr Asn Gln Tyr Ser Ile Gln Tyr Lys Asn Ser Pro Leu Ser Trp

1330 1335 1340

Asp Asp Lys Ser Thr Gly Ala Ser Ala Leu Leu Ala Lys Leu Thr Gly

1345 1350 1355 1360

Gly Asp Gln Tyr Lys Ser Ala Val Gln Ser Phe Cys Asp Gly Phe Tyr

1365 1370 1375

Tyr Asn Gln Gln Lys Thr Pro Lys Gly Leu Ile Trp Tyr Ser Asp Trp

1380 1385 1390

Gly Ser Leu Arg Gln Ser Met Asn Ala Val Trp Val Cys Leu Gln Ala

1395 1400 1405

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

1410 1415 1420

Gln Leu Asp Tyr Ala Leu Gly Asp Ala Gly Arg Ser Phe Val Val Gly

1425 1430 1435 1440

Phe Gly Asn Asn Pro Pro Ser His Glu Gln His Arg Ala Ala Ser Cys

1445 1450 1455

Pro Asp Ala Pro Ala Ala Cys Asp Trp Asn Thr Tyr Asn Gly Gly Gln

1460 1465 1470

Ser Asn Tyr His Val Leu Tyr Gly Ala Leu Val Gly Gly Pro Asp Ala

1475 1480 1485

Asn Asp Tyr Tyr Asn Asp Val Arg Ser Asp Tyr Val His Asn Glu Val

1490 1495 1500

Ala Cys Asp Tyr Asn Ala Gly Phe Gln Asn Val Leu Val Ser Leu Lys

1505 1510 1515 1520

Ala Asn Gly Tyr

<210> 13

<211> 26848

<212>DNA

<213> artificial synthesis ()

<400> 13

gcggccgccc atccatagtg tgtccttcac cctctgaagt tcatgtgcga agttggctgc 60

gtctcttctc ataaaaatga cacaaaggaa aaagtacatc agttgtaatg aagtagcatt 120

gttttatgct ccagagggcc tttgacttcc tagacctatt ttttgttttt accataatca 180

taaactttct catctgaggt gaagagtgtg gaattaacac attttgttcc tttgttaggc 240

aaagactctg aggctgaaca atcgggaggt tctatcgctt aaataagaaa agttaagata 300

attaactggc attgagcact tgtccacatt cttgtgctgt ggtcagaggt aggacacagt 360

ctcccatccc cgggttaatt aagtgcctcc aacaaagggg tactgttgcc cacatagaaa 420

gatctaaact aattaattaa tccctcaccc gcaaatcttt cagtcactaa gttagcacga 480

ttgttgaaca agttctccaa aggagagata cagatgagtg cgtatagggt ggacctggct 540

gctgaggaga cacctgcatc tgactaagaa gagccacggt gttagttgaa tggtgtggag 600

tagggtggtt ctgtgggaca gtagaaaatc gagaggcatg tgccgtttag tgaactgatg 660

gaagctaccc caaacgacag agattgtcag tcaggccaat ccgtttcgag tttgatgggc 720

agccggacag tgagacagac acacctactc agttggagga aggatgagaa caatggccag 780

cagggattga gagaccctga caggcgcaag gccctaacacacacacctaccacctcactt 840

gacaaagctg ccaaagacca aagacttgtt ctccattaga aatgacagct ggcttgaccc 900

gacagcataa taagcagagt gtactctgat tggagaactt taatgtgttt cattcagtat 960

tataaaagga cagtattaca gattttgttg tacactgctg ttacatgtgg ggcagtgtgt 1020

ctttaagtag ggtaaagtac tctttaaaaa tgggtcctag atattttttc ctttaactca 1080

agtctcttac tgtttaaatg attttattt tgtttaatat ggaggaaaaa gaagcgtaaa 1140

tggacaatat atatttagag aaagatggtt agctgtcaga aaaatatgca aatcaaaatc 1200

acaccaagac tgcagcacac ccctgtcaga tggctgtgat caagaaaata aatgacaatg 1260

agtggtggtg aagatgtact aaagggaaac acacacacac aacacacacacacacacacac 1320

acacactgga gcaaccactg tggaaatcag tatgaatggt cctcaaaaac ctgaagatag 1380

agcggggcgt ggtggcatac acttttattc ccagcactgg ggaggcagag gcaggtggat 1440

ctctgagttc caggccagcc tggtctatag cacaggttct aggacagcca gggctacaca 1500

gaaaaaccct gccttgatta aaccaaacca aaccaaacca aaccaaacca aaccaaacca 1560

aaccaaacca aaccaaacca gaccaaacca aaacactgaa gatagaactt cagtattcca 1620

ttcctagata tatacccaat ggagactaag tcagcaagac acctgcacag ccatgttcac 1680

tactacactg ttcaccacag ccaggctgtg gaaccagcct gagtgtccat gataaatgaa 1740

tggataggta actttcaagg taaatggact ctgctgtgta catgcctcac attctgttta 1800

ttcatttttc tttatgaggt gtccattcag gagtcacatg gtagttctat tttcagtctt 1860

ctgaagatac tacactggtc cccacagttt acacttttat cagcagtgaa taagggttcc 1920

tctatcctta ccatcatttg ttgtaatttt tcttgatgac cctctttctg acagggatag 1980

gatgtaatat cagtgtgagg aagtacaact tgttttctaa gtatttattg gccccttgca 2040

tttcttcttt tgaaaactgt cggttcctga catctgctca ggtattcatt ggatgttgtt 2100

tctttggtgt ttgagttctt atgaattcta gatgttaaat ccctgcctgt ggttctctcc 2160

cattctgtag gctgcctcct caccctggca attgttgtcc ttgttttgca gaaacttttg 2220

acttcatgga atctcatttg tcagttttcc ctcctctgct atagcctgag ctaatgcact 2280

ggtttttaca gagccctggt ctatgccttt atcctcctct ggcagcttcg gagtttcatt 2340

tcttacattt agatctttga tccactttga acaagttttg gagcagggtg agagatacga 2400

atctagttcc attcttccat atgtgatcct agtttacata gcatcgttgg ttgaagaggt 2460

tttattttat ttttaaataa tgtgtcataa aaaacgaggt ggttgtagca gtgtggattt 2520

gtttctttgt cctttgatct acaggtcttg ttttgtgtca gtctcatgat gttttattgc 2580

tatggctctg tcatacagtc tgaggtcagg tattgtgata taccttcagt attgctccct 2640

cagactcagg tttgctttgg ccaggagtca tcttactcag tgctcttaga gctcccccag 2700

catgtagctg ctactattct tagttgataa atcaggaaac tggggctcag agagattaac 2760

tgtcttgaac tacttctggg gaggtgaaac gtggagaacac taaactgtgt ttaccctgta 2820

ctgctccagt agctgtcggg tgctgggcta cagcaaagca cctatactat atattactca 2880

ggaggtggaa aaactcagcc tcccttgggg ttcccaagct cccaggtgtc cagtcactgc 2940

tggaaacctc atggagtctg aaaggaaggg ttgagggtac atggggcagc gatgaggagc 3000

ctggggctgg gatctcccaa acacctggat atccagatgc cactgggtca ggggggagttg 3060

ggaacagagt tgggatgtcc atggacctgt gacaaggcca gggccagggg gaggataact 3120

ctggctttac taatttgcga aagtccttag cttagcagca gttgtctggg agcacagagg 3180

ggccttctgt aagaggctca ggcagtgccg ctctgtaggc gaaggtcttc tccatgttcc 3240

ccatggtggt tcttgatgaa agagacagtc cttggctcca aactggttta ttgattgttc 3300

attgtggaaa atgggtgcac accaccttct cagggtggac cagagatcaa ataccttttg 3360

caggggaggaa tatctgggaa gggacgctta ctggctaaac cctcagggcc tctagataca 3420

tcattagcat ggagaactct gttctgggct acatgaccac aggccacatt tccacaagcc 3480

acatgtggga agtgtggcac atgttctagg ccaggaatct ggtagggagc gtggagccac 3540

ctaccatccc aggtgggtgc ctgggtgcca gggaccctga acccgctcaa ccttaccaag 3600

tttcctggca gggtccactg tcctacacag aagctggagg aggtgtgagg gttgtgtctt 3660

tgtggaatgt cccatgctgc ttggggctca gtttctccac ctgtacctca ttggtttggg 3720

tataaaaagt ggggatactt tattattctc tgactcggtc ctgaggaaaa agcatcgtgg 3780

cagtccagga accacacccct gaggttcctg cactgaaggg actccctaag tctctggagt 3840

ctctcccctt cacagagctg ccaaagtcta ggttcttttg aggataacag agccatgctt 3900

ggtaagcaga caacagcatt tgtttactca accttctttt gtcagctccc tcttcataaa 3960

caagttgaga caccatgctg gcttgaggaa gacttctaaa gccagacaac tgtgcaagga 4020

agaagaagaa ggggcaagtg gagttagcct ggatgtagcc ctcaaagtct ccagagacca 4080

gccatgaagg ctcaagtgga gggcaagacc tgcagcagcc aagcatctgg caggagagga 4140

tcctgggaac ccctctacca tgacacacat tcttcctgca ggtcacactt aataggccat 4200

ttcttatttg gatctatcat ggtgttctgt gcgagattaa tgaggtgtta tgctgcgaac 4260

agaaagttat ataaaaacaa gtcccccccc cttgtcactg ctgctaagaa tgtagcagaa 4320

attgtctcaa gtgtctctct aatcagaaac aataaaggtc tccttggatt caagccctcc 4380

agtttcctcc ttccttgctg agccttggac acccatacaa acctcctgga tgctacagct 4440

ctgggcagag actccaaggt ggggagagac tgatggtaca aaagcaaaat acttgtttgg 4500

gggtacaccc actcctctgc ctgtgtggtt cctgcagtca gtcctgcaga caggccctca 4560

gtgggtcttc catgggcaac acgcagaggg aggcaatgga tgggaatacc cacaccctgg 4620

ttagtttacc ccggccatgc tctctgctct tcatccctcc tctgccctct gccacggctt 4680

tctctgcagg aatcatatct tcatattggc ccacaggtgt tctcctcacc ctagctatga 4740

tgtttacttt agagtgacct tagcagggct ggtgggaatg agttctagaa ggctcacgga 4800

gatgctaggg aagaaacgtc ttctaactac tgaggttact aagttcctgg tggttgtctc 4860

tgcctttccc ttgttaaagt caccttgaag ttagtgcaga agaaatcaga gcccagtcac 4920

agagtaaata tggtcctgaa gatttccttt gagtgcccag aatccatgac atttcaagag 4980

ccctctttgt accttaagtc atttggggtt gtatcttctg cttgatgtat gtgtgtgtgt 5040

ttatcaaaga gtgagatggt tacataagag gtgctctaaa ggacagagag gatttgcaat 5100

tgtggcatgt gacatcctca ggccttgctc tggtgccagg aggaactgat gcagaaaaga 5160

gtaagaggtc atttcctgga ggctgtcact atagaggaga tcttacagtg cattccctcc 5220

tccaggccct gcctgaggat agacatgtgc tgactgcaac tgaaacagag gcttgggatg 5280

gagagttagg ttcacagaag ggagggtggg agatggatgc ttgctgggtt ctgggtctca 5340

tcaccagctc ctgaccacccc ggtcagccca tgtgcttatt ccatagcttt cttttgctat 5400

gtttactcag tgtggtgttt gttgggaccc agcagaagcc agtcccaggc tgacagctgt 5460

ggatacacag ggcagcatga gggtcctcag cctgaagcag tcaggctggc agaagagaaa 5520

gaccagcaca cattccttca accaactatg tcttgaaaaa caaacatatt atatcacata 5580

tattgcattt atgagacagc taaaatgtac tcgggtagca tgactccagg tggggatatc 5640

tgcaagtgcc atgagtggca gagggacagc caatgtgagg caagaaggaa ttctggctca 5700

acacagctta gctccctggt gttggttcaa actttgagag tttgaccaca agcactttat 5760

ttttgacata tttaaacaga gcacaacttt gggaaaaagt tttcttatga aaattatcac 5820

aataaagctt aaggcatgac tacattaaaa tgcctttgca aagtatatgt gccctcttcc 5880

acaagaatgg ttctattgac tgagaaataa tgttcaggat aaagatccag gaagaaaaga 5940

tcagggataa gtaaaatact aaactctttt gcaaagtaca tagacccctct ttcataacaa 6000

tgggttctat tgactgacaa gcactgctca ggagttggga aagagtctag cataagcacg 6060

atagcctgga gactctagtg aggtctagtc ttacagacag caaaaatcac caggttacaa 6120

actacattca tttccagttt tctgatcagg cacaggtatg aatcccttct gttgaagaga 6180

aaagtccatg tgtttaaaat atctggtttc tccagtgcta ttagcgagaa gacttgagcc 6240

ctatacaact cccacctgga gtgacatcct gtcttcatgg tatattacat acctagacac 6300

gctcatctca cagacttagg actttgtctt ctgatctcca tttctgatcc cacttccacc 6360

tttgccttga tagtgtcatt ttcttcactg ccttggtgac aaccatgtta tcctctgtgt 6420

atttgagtgt taccattttc agattttacc tgtatgcaag atcacacagt ctttgtcttt 6480

ctgtctggat gcatgctaat ctctacacaa caacccttcc ccgtcactca gatcttcctc 6540

cattaacaca tacatggtgc tgaagaggct agggagcttc ccttcagtgg ggagctagct 6600

ggctattggg cctttttgac tgtccaggaa ggcccccaat tgctgagaca agaacttaga 6660

ttcttcatta ttgactctaa ctcatgtatc aagcagaagc taatgaatag ttatcaacag 6720

gatcagaggt tccagtgtaa gacactttga catgaaagaa cggaggaagg acagatggat 6780

gcataaaagc aggaccactg ccccaggaag gtcctggaaa ctgatgcagg gcaaaggaca 6840

ggttataaac caaatcttag ggagtcagga agagcacaga ggagctcaac caactgacca 6900

ctgcttaggg gctaccaacc caatcctccc tgtgggaaca gctaagctat cagccaaggg 6960

taataaacag gcaggacctg tggatgacat ggagagcata gggaccctgg gtccagcctt 7020

tagcacctgc actctcagga tactccacca ttgtgtctta gagagcctag ggatactggg 7080

tccagccttt ggtaccttca ctctcagggt accccatcac tgtgtcttgg agagcctagg 7140

caccctgggt ccagccttca gtacctgcgc tctcaggaca ccccaccatt gtctcttgcc 7200

ccgtctcttc ttcctcttcc tccctttcat tgtctcttct ctgtttcttt cttgactctc 7260

ctttcccctc acaccctcac tctagttctc cccttccctc tctgcatcac cctattctct 7320

ctgtggtccc tccactttcc tttatctctc atgcttctct cctccctcaa atacttgtca 7380

cccactatac ttcaggggcc agctctagtg acaaagctgt taatagcaag actctcagat 7440

ctccaacggc tcagaggagc cagacccacc aagaactctc tccaggtcca atttcaggtt 7500

ccttcgaaag ctttcagcaa atgctcaggg aacatgccac taacaagaag atgcaaattc 7560

cagttgagag tgggaaaggc ccttgcgtag gtcccatctt ccaggccaag gtcagagggg 7620

ctctgtgtaa tccggattga cagggctcag aacaatgttt tgtttttaag gtttatttat 7680

tttaggtgtt agtgtctttg cttgcatgac cttatgtgca tcatgtgtgt gcaggttcct 7740

gatgacagta gaggagggct ttgaatccct ggggatagga agttacagga aattataagc 7800

tgctttgtgg gtcttctagc tttcccaaca gaagtgaatg ctcttcacca ctgagccatc 7860

tctctaggcc caagagacat tgctttatgg atataattgt gtgtgtgtgtcaacattgag 7920

gaaagggaaa taaaaaaaaa acttcagccg ctaaggttgt acagtttcac taattgctac 7980

ttttagttgt gataaaatgg caggtgcttc aacatttata tatacaaaaa cttccctgct 8040

ggtggttcaa ctgtgagaac tggggtaagt gggtgagttc tctttttctg tctctgtctc 8100

tgtctctctc cttccattct ttcttaaagg aaataaacat tgcagctggg ttatagctca 8160

tcaatatgga agttacagaa gtgaaaaaag gcattgcctt ggtgggtggt gttaccagct 8220

gatttttggt tgtcctgcaa ggaggtctgg ggactggctg ctctgtctct gtctgtatga 8280

gtgagggaag tctggggagc agattcccta accttcagcc tggcctggtt cctgagtgaa 8340

cccagcctct ctggtcctag tagctttttc caaacaggaa tctgagtggt gacagggaac 8400

aagtaccagc ccattgctta agtgccaggg ttagtgaggg caggaagctg ccatagctgg 8460

gattagtagt tgtattggat gtaggaagtc ctatcctggg acagctaatc cttaatgctt 8520

cactggagat tttcaatgag aaatttatcc cacggcccat atggccccat ccttttgtct 8580

ccaacagcca agtattttcc attagaggag acttcctgta cacttgatgg atgctcattt 8640

caaggtgact tggggcagtc agtacagact tgggatgacc tctgacagcc taacctctcc 8700

ccaacaaggg ccctctatgt ttgctatgta atgtaatgtc agacattgtc aggagtgtcc 8760

gcagcacagc ctgcccagtg tgagggctct cataggtttc ccactgtctt atctacacag 8820

ggataacgag gaggtaagct gcagttccca gtctcacttc acagaggaag agataaccccc 8880

atcccaggtc atgtagccag cagtggaaag aatgaggatt tgaactcagg tcttccaagt 8940

cccattgata gcatctcctc acaagtccct tgccaccctc acgatgcctt agacacttgc 9000

ctgcccttta tactaaggag atgcaggtac aaggggttta cccatgtagc agctgaggca 9060

gctggggata gataccagca gcaggcctga tgtcaccact ctaactccag catccccagt 9120

ctgtgttcct ggagtgtgaa aatccctact taacaagatt gtgcaacagt ccttggctct 9180

gtgacccata gctggaaaca ggattctcat tgatttgtgg aacatggtgg cagccagcca 9240

aaaagagggt ctgcatacag aagacagctg tggcaaggcc acagcagact ctgactacct 9300

tagcttacag aattacaagg tcataatgtc ctctgctttg gtcacctcat gttaaggaca 9360

ggccctaatg aagatggggc agaagactga aggaatggcc aaccaataac tggcccaact 9420

tgagacccat cctacaggca agcatcaatt cctgacacta ctaatgatac tctgttatgc 9480

ttgcagacag aagcctagca taactatcct ccgagaggtc cacccagcaa ctgactgaaa 9540

cagaaaaaga tatccacagg caaacagtgg atggaggtca gggactatta tgggagagct 9600

gtgggaagga ttaaaaaccc tgaaggggat aggaaccccca caggaagacc aacagagtca 9660

actaagagac ctgtgggagc tctcagagac tgagccacca accaaagagc atacacaggc 9720

cggtccgagg cacctggcac gtgtgaagca gacatgcagc tcagtctcca tgtaggtcct 9780

ccaataagcg gtagcctgac tgcagtatcc aatccctaac agggctgcac agtctggcct 9840

cagtggggga gggtgcccct aatcctgcag agacttgatg agtggagagc tatccagggg 9900

gaacccacccc tctctgagaa gggaatgggg atgggggagg gactctgtga agagggggaca 9960

aggacaaaca agaacctcaa ataggtcagg ccctaaaggc ttgctaagta gcagtggccc 10020

agctctgtcc tgttcctcag cccaaggctc agctcccacc tgtttctgtg tttttctggc 10080

ttttcatggg cctaggactt ggtggccagt tcaaacaatg gggcctgtgg aagacacaat 10140

atacaagact agggaacattc ctgttctgct gactatccac agcctgatgt aggtggaagg 10200

acccaatcac tggatttcta cccttgcgca accttgacag ctgagggcct ctcagaaacc 10260

tatttcttcc actgaaaaat gagactctca aatgaacgtc ctgacaatca tcaggcttat 10320

taaagaggtg tatctaacct gaatggcaag cagacagcag gcaaatgtct gtatcaacct 10380

ctaggaagga caagaactgc tcactgctgc cccccaggag gccatttgct gaaacagctg 10440

ctctcctgct ggtgcacagg ccctgccttc tcattgcagc tacagcccct tcctgtctga 10500

acctcctgtc aggtcactgg gaaacagatc aagatggaac aggacagctc ctgatggtaa 10560

ataaaaaaca gtggtcatgg ctattcatag gggtttatgc ttcttcagtc cacactgtga 10620

agagctgtgg gcatgaacca cagtgttcga ggtaggttg gggttctgaa attcacagtg 10680

gggtgagctc agtaaatgtg agctggaggt cactcgtgag acacacagtc ctgctgcttc 10740

tgttcccaat atcctgagga gacgacacat ctactttgtt cagaggccac agtctagttg 10800

acctgagagt taccagtttc ttattgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgt10860

gtgttgttcg tgtgtgagtg caggtgcaca tatgatagcg tacacgttga ggtcagagga 10920

taactatcag gcgttgtccc ctcctacttt tcctcggact ctggagaaca aacatgggtc 10980

cttattccag gggagcaagt gcctgttggc tgacacatct tgctcacata cattttacct 11040

agacaatgga gcctccatca gagtattact ttagctcctc accgatggca atgcaccacc 11100

tctctaccca cataggagtt gggtctccac accccccac acccccttca ccaaaacgtt 11160

ttcagttatact ttatctggta aagttcatca gagaatgaag ccagttattaa gaacatggaa 11220

tcatttggga acctggatct agcaataccc caccctagat ggagttgctg agttttcacc 11280

tcagattata attcccccct agcttctatg gtttatctg aaaccagggg aactcgattc 11340

ctccctttgg accacagaca tcctggcttg tgaattcaca tgtcatctac tgctaatcca 11400

ttggtagtat gtggctcaca gagaacacact acagtcatgg ccaatgtcaa ggtagcaag 11460

atgtgaatca ttcccccagt cctgctgttt tcatgactaa ccctcctcag cacagtgacc 11520

atgaacctac ttttcccctc cttttatttt tagaattgct ggaattttct attttgagaa 11580

ataatagcct tggggcagca ttaaacaaaa tcatctagaa agctggttta aaatacagat 11640

ggttgagtca gtgaaagagt gaggaatgtc attattggcc cctcacagag gctggctcac 11700

tccagcagag gtggttgaag ctcttggaca cgggtcaggt gcataggaag ggtggtctgg 11760

gacacctgag aaccacaatt gaacaaacag aagctgctgg cttttttttt tttaaatgag 11820

ttctcaaaaa atgactgggc tagcttaggc aaatacttcg agccaaccca acagaacatt 11880

cttccattga ttcattctgg atcttctttc tagacaatac tgaactgacc ccttgttggc 11940

agtctcaagt ttgacaacat agggctttga acttggcaca aggtccatca ctgtcaccca 12000

agcatcctgg gtgacctttg ggttggaata tcttggctaa ccttagatat tttctttgga 12060

gtatctttag aacatccagg aaatagggct tgattctcat cctgggacca caatataagt 12120

caccctagaa tcccaggaga tcgtgcagag aaacaaggat ctctctcgtg tgcatccttc 12180

ttcaaagcag tgagtagtga ctccactaaa ctgagttccc atctgagagt ccacaggagg 12240

ctttggggca agaagcagag ggaaggcact gtttgtgttg gtaaagtttt gactctaaca 12300

aatttgaaga catagatgac atgtgtcag actaacaaca acctagactc atgtgggttc 12360

tgtttaggga tcagatttta ttcatcaatg acttgtctta gtgtataggag aaaggcttcc 12420

tactggagtg taggctcaat aatgacagaa gagatagcta tttcccctag ggactgtgct 12480

gctccaagtt tggtggagaa aggcagtgggg gaacctagat gtgctctctg gggaggggggt 12540

ctgaagctgg cttcatagaa ggtgtgaagt tttgctgaaa catctaaaca gaattatagc 12600

ttaggaaagt gagcaggcaa ggcagggaat gtgttgcata tgtatatgta catgaatata 12660

ttatgttata gatacacaca catttgaacc tcatttgcag atgacagaaa ataggttat 12720

ttgcctctct taactgctaa gcacaatgac ttccagttcc atccatttcc tgaaatgcca 12780

caatttcatt tttcattgtg gctgaataaa attccattgc agactgggcc ctacttcatc 12840

cactcctgag ggcaggcata tcccctggct ccatttctta cctattgtga agagaagtgc 12900

aactgtcttg ttgaaaggca agcgtgagag aggcaggcac taattgtggg tttttgtttc 12960

ttcttcctgc tatgactctc catttgtcag ggcgcgccgc caccatgttc cagctgtgga 13020

agctggtgtt cctgtgcgga ctgctgatcg gcaccagcgc ctccagcacc ccctccagca 13080

ccggagagaa caacggcttc tactactcct tctggaccga cggaggaggc gacgtgacct 13140

acaccaacgg cgacgccgga gcttacaccg tggagtggag caacgtgggc aacttcgtgg 13200

gaggcaaggg atggaaccca ggctccgccc aggacatcac ctactccggc accttcaccc 13260

caagcggcaa cggctacctg tccgtgtacg gctggaccac cgaccccctg atcgagtact 13320

acatcgtgga gagctacggc gactacaacc caggctccgg aggcacctac aagggcaccg 13380

tgaccagcga cggctccgtg tacgacatct acaccgctac caggaccaac gctgccagca 13440

tccagggcac cgccaccttc accccagtact ggtccgtgag gcagaacaag agagtgggcg 13500

gcaccgtgac caccagcaac cacttcaacg cctgggccaa gctgggcatg aacctgggca 13560

cccacaacta ccagatcgtg gctaccgagg gctaccagtc cagcggctcc agctccatca 13620

ccgtgcagga ggctgccgcc aaagaagctg ccgccaagga ggctgccgcc aagcagtccg 13680

agccagagct gaagctggag agcgtggtca tcgtgtcccg ccacggcgtg cgcgctccaa 13740

ccaaggccac ccagctgatg caggacgtga ccccagacgc ttggccaacc tggccagtga 13800

agctgggatg gctgaccccc aggggcggag agctgatcgc ctacctgggc cactaccaga 13860

ggcagagact ggtggctgac ggactgctgg ccaagaaggg atgcccacag agcggacagg 13920

tggctatcat cgctgacgtg gacgagcgca cccggaagac cggagaggcc ttcgccgccg 13980

gcctggcccc agactgcgct atcaccgtgc acacccaggc tgacaccagc tcccccgacc 14040

cactgttcaa cccactgaag accggcgtgt gccagctgga caacgccaac gtgaccgacg 14100

ctatcctgag ccgcgccgga ggctccatcg ctgacttcac cggacacagg cagaccgcct 14160

tcagggagct ggagagagtg ctgaacttcc cccagtccaa cctgtgcctg aagcgggaga 14220

agcaggacga gagctgctcc ctgacccagg ccctgccaag cgagctgaag gtgtccgccg 14280

acaacgtgag cctgaccgga gccgtgagcc tggcctccat gctgaccgag atcttcctgc 14340

tccagcaggc tcagggaatg ccagagccag gatggggaag gatcaccgac agccaccagt 14400

ggaacaccct gctgtccctg cacaacgccc agttctacct gctccagcgg accccagagg 14460

tggctaggag cagagccacc ccactgctgg acctgatcaa gaccgccctg accccacacc 14520

caccacagaa gcaggcctac ggcgtgaccc tgccaacctc cgtgctgttc atcgccggcc 14580

acgacaccaa cctggctaac ctgggaggcg ccctggagct gaactggacc ctgccaggac 14640

agccagacaa caccccacca ggaggagagc tggtgttcga gaggtggcgc cggctgagcg 14700

acaactccca gtggattcag gtgtccctgg tgttccagac cctccagcag atgagagaca 14760

agaccccact gtccctgaac accccaccag gagaggtgaa gctgaccctg gccggatgcg 14820

aggagaggaa cgctcaggga atgtgcagcc tggccggctt cacccagatc gtgaacgagg 14880

ctagaatccc cgcctgctcc ctgagggtga agaggggcag cggagctacc aacttctccc 14940

tgctgaagca ggctggcgac gtggaggaga acccaggacc aatgtttcag ctctggaagc 15000

tcgtgtttct ctgcggactc ctcatcggga cctcagccca gcagaccgtg tggggacagt 15060

gtggcggaat cggctggtcc ggcccaacca actgcgcccc aggcagcgcc tgctccaccc 15120

tgaaccccta ctacgcccag tgcatcccag gcgccaccac catcaccacc agcaccaggc 15180

ccccatccgg cccccaccacc accacccagag ccacctccac cagcagctcc accccacccca 15240

cctccagcgg cgtgagattc gccggcgtga acatcgccgg cttcgacttc ggctgcacca 15300

ccgacggcac ctgcgtgacc agcaaggtgt accccccact gaagaacttc accggcagca 15360

acaactaccc agacggcatc ggccagatgc agcacttcgt gaacgaggac ggcatgacca 15420

tcttccggct gcccgtgggc tggcagtacc tggtgaacaa caacctgggc ggcaacctgg 15480

acagcacctc catcagcaag tacgaccagc tggtgcaggg ctgcctgagc ctgggcgcct 15540

actgcatcgt ggacatccac aactacgcca gatggaacgg cggcatcatc ggccagggcg 15600

gccccaccaa cgcccagttc accagcctgt ggtcccagct ggcctccaag tacgccagcc 15660

agtccagagt gtggttcggc atcatgaacg agccacacga cgtgaacatc aacacctggg 15720

ccgccaccgt gcaggaggtg gtgaccgcca tcagaaacgc cggcgccacc tcccagttca 15780

tctccctgcc aggcaacgac tggcagagcg ccggcgcctt catctccgac ggcagcgccg 15840

ccgccctgag ccaggtgacc aaccccgacg gcagcaccac caacctgatc ttcgacgtgc 15900

acaagtacct ggactccgac aacagcggca cccacgccga gtgcaccacc aacaacatcg 15960

acggcgcctt ctccccactg gccacctggc tgagacagaa caacagacag gccatcctga 16020

ccgagaccgg cggcggcaac gtgcagtcct gcatccagga catgtgccag cagatccagt 16080

acctgaacca gaacagcgac gtgtacctgg gctacgtggg ctggggcgcc ggctccttcg 16140

acagcaccta cgtgctgacc gagaccccca cctcctccgg caacagctgg accgacacct 16200

ccctggtgtc cagctgcctg gccagaaagg aggccgccgc caaggaggcc gccgccaagg 16260

aggccgccgc caagggcagc tacgactacg ccgacgtgat caagaagagc ctgctgttct 16320

accaggccca gcggtccggc aggctgagcg gcatggaccc actggtgtcc tggagaaagg 16380

acagcgccct gaacgacagg ggcaacaacg gcgaggacct gaccggcggc tactacgacg 16440

ccggcgactt cgtgaagttc ggcttcccaa tggcctacac catcaccctg ctgagctggg 16500

gcgtgatcga ctacgagaac acctactcca gcatcggcgc cctgagcgcc gccagagccg 16560

ccatcaagtg gggcaccgac tacttcatca aggccacgt gtccgccaac gagctgtacg 16620

gccaggtggg caacggcggc gccgaccaca gctggtgggg cagacccgag gacatgaaca 16680

tggacaggcc agcctacaag atcgacacct ccagaccagg ctccgacctg gccgccgaga 16740

ccgccgccgc catggccgcc gcctccatcg tgttcaagaa cgccgactcc aactacgcca 16800

acaccctgct gagacacgcc aaggagctgt acaacttcgc cgacaactac aggggcaagt 16860

actccgactc catcagcgac gccgccgcct tctacaacag ctacagctac gaggacgagc 16920

tggtgtgggg cgccatctgg ctgtggagag ccaccaacga ccagaactac ctgaacaagg 16980

ccaccccagta ctacaaccag tactccatcc agtacaagaa cagcccactg tcctgggacg 17040

acaagagcac cggcgcctcc gccctgctgg ccaagctgac cggcggcgac cagtacaaga 17100

gcgccgtgca gagcttctgc gacggcttct actacaacca gcagaagacc ccaaagggcc 17160

tgatctggta ctccgactgg ggctccctga gacagtccat gaacgccgtg tgggtgtgcc 17220

tgcaagccgc cgacgccggc gtgaagaccg gcgagtacag atccctggcc aagaagcagc 17280

tggactacgc cctgggcgac gccggccgga gcttcgtggt gggcttcggc aacaacccac 17340

cctcccacga gcagcacagg gccgccagct gcccagacgc ccccgccgcc tgcgactgga 17400

acacctacaa cggcggccag tccaactacc acgtgctgta cggcgccctg gtgggcggcc 17460

ccgacgccaa cgactactac aacgacgtga gatccgacta cgtgcacaac gaggtggcct 17520

gcgactacaa cgctggattt cagaatgtcc tcgtgtcact caaggctaat ggctactgag 17580

ggcgcgccga tcaattctct agagctcgct gatcagcctc gactgtgcct tctagttgcc 17640

agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca 17700

ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta 17760

ttctgggggg tggggtgggg caggacagca aggggggagga ttgggaagac aatagcaggc 17820

atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggtttaaac 17880

tcgattataa cttcgtatag catacattat acgaagttat gatcgatatg aagaatctgc 17940

ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 18000

gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 18060

tggagttccg cgttacataa ccttacggtaa atggcccgcc tggctgaccg cccaacgacc 18120

cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 18180

attgacgtca atgggtggag tattacggt aaactgccca cttggcagta catcaagtgt 18240

atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 18300

atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 18360

tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 18420

actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 18480

aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 18540

gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 18600

ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 18660

gtttaaactt aagcttggta ccgagctcgg atccactagt ccagtgtggt ggaattcgcc 18720

accatgggat cggccattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg 18780

gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg 18840

ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc 18900

ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct 18960

tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa 19020

gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg 19080

gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa 19140

gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat 19200

gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg 19260

cgcatgcccg acggcgatga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc 19320

atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac 19380

cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg 19440

gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc 19500

tatcgccttc ttgacgagtt cttcgagggc agaggaagtc tgctaacatg cggtgacgtc 19560

gaggagaatc ctggcccaat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc 19620

atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc 19680

gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg 19740

cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc 19800

taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc 19860

caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag 19920

ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac 19980

ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg 20040

gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac 20100

ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg 20160

ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag 20220

aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg 20280

gacgagctgt acaagtaatc tagagggccc gtttaaaccc gctgatcagc ctcgactgtg 20340

ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa 20400

ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt 20460

aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa 20520

gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaaga tctataactt 20580

cgtatagcat acattatacg aagttatgga tctgtcgacc atagtgtgtc cttcacacat 20640

cacggttaca attaggcagt gctgactcta aatcaagaga cctcattaca tgttcctgac 20700

tctttttttt ctcacttttt tttccatttttttattactc aaatgaattt atcacatctg 20760

tagttgtgca atgatcaaaa caatctgatt tcacaggatt tccacatttt ttattttgtc 20820

ttttcactca agtgtctttg caggcttcca tccctttatc ctcaagaatc attttcgggc 20880

tctaagaaat cttttttgta ctttttctat ttattttcag agcctcttgt tttcctttta 20940

tagatgtaat aaaacttcct ctcttcaagt ttatgaatta gaatttttta gtgccattcc 21000

ccaggttatc tcatttcttc aggttttttt cagttatctt ttaatttctc ttttatgttt 21060

ctcactcttg gcttttgtgc ttgatgattc tttctcttta ttcatattga caaaagatga 21120

atattatatt agttgcctgt tgctattata acaaatcaca acaaattttg tggcttaaaa 21180

caacagaaat ttattatctc aggttgtga aggtgaccat tccaaaatta gtcttagaat 21240

actaaaatca ataacatgac tctgtacgtc aactatactc caatacatat ttttttaatt 21300

gagaaaaaaa aaaaagaaaa tcaaaaccaa cttgtcagca gggatttcct tccagaggct 21360

ccagggaagg atctgcttgc ttgcctttcc cagcttccag aagccactta attcctgc 21420

tcatggccct gcacgcatcg ctcttcctcg tgctgctttc atcctgaccc tggcccttct 21480

cctctcttac aaggaccttt gaggttgcac cagccccaca ggtgcaccca ggatgctctc 21540

ccatctcagg atccgttatc acacccgtgttgccacgtaa ggtaacatgt tcaccggtgc 21600

cgagggttag gatgtggggca tctttggggca gaggggcgtt cttcaactta ccgcagacag 21660

ggtttctaga cagctgattt gctgtttaaa tagagtttct ttcctaatat gtctctccct 21720

gaggagaaag tcaaaatgag ttctggggtg ggaatgacac acggcctgga ggcgatatta 21780

gaatccaggc cccttaaata tgacagtgag gagggcttta ttctgggttt gtagaatcca 21840

cacttgaagt cttggtttcc cctggattgt tcattcacat ttattttag gggtgagcct 21900

tctgtttttc atctcgtttg cccacagtct gcaggttggg atggggctcg cctcctctat 21960

acacagacct ttaaggagct tgtttttatc ttcacttctc accaggctct ttgccatctt 22020

tgcggtatgt gaggccagaa cttctctaga gggctggata gacagaggcg ccagctctgc 22080

gaacgcctcc ctatagcta ggctttcaca gcttgtttta tccccaaaca tctcagtatt 22140

ctcttacctc ctttccatcc tgcagaagtg gactgagagc tgttctcagt tggtgaatgt 22200

ctcccaaaaa tgactattcc tgttctacac ctgactgtgt tatacagcaa tccgttttta 22260

ttttactcaa gattgtttct tagctgttca gtattggtta aaggtcacta aaaagcagaa 22320

ttcttaatgt attgtaataa tcacttaaga tataaaaata tttgtgcata taatgactaa 22380

atgctgcatt caaggaatga atcttggtta aaactttttg ccaatctgta tctgataaca 22440

aaataatttg aaacatatta cattttaaac gaatggccct taaaatttga atgaaggata 22500

actagacatt ttaatagaag tgcagcatga tactttcttt gcaatttcac attataaaat 22560

aatgcaatta cgaagcatat cattaggaac ttaattgtgc tcagtgttgt tgtggctcag 22620

gttattctgg aaagagagcc tgataacata tgagtactta ttggggaggg aattccagga 22680

atttgaggta agtgaatgga gataaggaaa cagagaaggg aaaagccaat aaggggagcc 22740

ttattgatgg agttactgct gagagtgaag ggggtctcca tcccactgag gaccctgaat 22800

gatccttcag gacataatca tggaatcgtc ccatcagaga atggtagcct gagtattta 22860

gccacacaag tccagcccct tttattgagg gtgctcctaa aggacatctg accctgctct 22920

tcctgctcct gcacttcctg tctgcccctg cacttcctgc ctgctcctgc acttcctgtc 22980

ttcccctgca cttcctgccc ctgaacttcc tgcctgctcc tgcacttcct gtcttcccct 23040

gcacttcctg cccctgcact tccttgctcc tacacttccc gccttctttt gtacttcttg 23100

ctgctcctgc acttccttcc tgactctgca cttcctgcct gctcctgtac ttccctcctt 23160

cgtttgcact tcctgtctgc tcctgcactt ccttctccta cactccctgc ctgctcctgc 23220

tcttcctgct cctgcatttc ctgctttttc cttttcctgc tcctgcatct cctacctgct 23280

cctggagttc cttcctgctc ctacacttcc tgtctgctcc tgcacttctg gcttcacctg 23340

ctcttggact gaatgacctt ccctagcttt aaagaaagct tgaggtggaa aaactaagcc 23400

gtcccacagc ccagttgagg gggaatcagg tatgagttgc ctgtctcagc tgggttgcaa 23460

tcagatggat caaaaagatg tggcaggatg ccagaagcat ctagaattga atggaaacag 23520

tgaaagtgga tcagaaatag agatgcatct ttctctacac agtagtcttc cctccataac 23580

tgcattaaaa cagcgttcaa agatttgaat catgtttata taaaacatac ccaaagaagc 23640

cccccaaaat tagactacat ggtttactt tttcatctct tacaaggccc ccttaaaatg 23700

acaaaaatca atcccaaagg cgtaagtctg ccacaacaaa ggaaacacaa aggtggccgt 23760

cagcaggcaa gacttggaca tcttcctgga agcaatgctg agatggccag cttctcgcta 23820

cacaggaagc cagttatagc cttagaaaga gctcgaggat ctgcaatccc gcggccatgg 23880

cggccggggag catgcgacgt cgggcccaat tcgccctata gtgagtcgta ttacaattca 23940

ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc 24000

cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc 24060

ccttcccaac agttgcgcag cctgaatggc gaatggacgc gccctgtagc ggcgcattaa 24120

gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc 24180

ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag 24240

ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca 24300

aaaaacttga ttagggtgat ggctcacgta gtgggccatc gccctgatag acggattttc 24360

gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa 24420

cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct 24480

attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa 24540

cgcttacaat ttcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg 24600

catcaggtgg cacttttcgg ggaaatgtgc gcggaaccccc tatttgttta tttttctaaa 24660

tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt 24720

gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg 24780

cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag 24840

atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg 24900

agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg 24960

gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt 25020

ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga 25080

cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac 25140

ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc 25200

atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc 25260

gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac 25320

tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag 25380

gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg 25440

gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta 25500

tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg 25560

ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata 25620

tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt 25680

ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcatcagacc 25740

ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct 25800

tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa 25860

ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag 25920

tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc 25980

tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg 26040

actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca 26100

cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat 26160

gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg 26220

tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc 26280

ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc 26340

ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc 26400

cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg 26460

cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga 26520

gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc 26580

attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa 26640

ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc 26700

gtatgttgtg tggaattgtg aggcggataac aatttcacac aggaaacagc tatgaccatg 26760

attacgccaa gctatttagg tgacactata gaatactcaa gctatgcatc caacgcgttg 26820

ggagctctcc catatggtcg acctgcag 26848

Claims (7)

1. The multifunctional fusion enzyme XAET is characterized in that the multifunctional fusion enzyme gene is composed of xylanase gene-A3-phytase gene-furin-P2A-cellulase gene-A3' -cellulase genome, and the amino acid sequence of the multifunctional fusion enzyme XAET is shown in SEQ ID No. 12.

2. A multifunctional fusion enzyme XAET gene is characterized in that the sequence of the multifunctional fusion enzyme XAET gene is shown as SEQ ID No. 11.

3. A multifunctional fusion enzyme site-directed integration eukaryotic specific expression vector comprising the multifunctional fusion enzyme XAET of claim 1.

4. The multifunctional fusion enzyme site-specific integrated eukaryotic specific expression vector of claim 3, wherein the vector gene sequence is shown in SEQ ID No. 13.

5. A method for constructing a site-directed integrated eukaryotic specific expression vector comprising the multifunctional fusion enzyme XAET of claim 1, comprising the steps of:

screening and optimizing candidate target genes;

designing a connecting peptide;

the target gene is connected with the connecting peptide;

synthesizing a multifunctional fusion enzyme gene XAET;

constructing a CEP112 site-directed XAET gene expression vector.

6. The method for constructing site-specific integrated eukaryotic specific expression vector of claim 5, wherein the construction of CEP112 site-specific XAET gene expression vector comprises the following steps:

replacing a BEXA cistron in a CEP112-LA340RA3219 vector with an XAET polycistron to construct a new vector Cep112-mPSP-XAET;

the Cep112-mPSP-XAET is linearized by PacI and sexAI, then the inf-npsp primer is used for amplifying the npsp upstream regulatory region and replacing the existing mPSP sequence;

constructing a CEP112 site-directed XAET gene transfer vector Cep112-npsp-XAET.

7. Use of the multifunctional fusion enzyme XAET according to claim 1, wherein the multifunctional fusion enzyme XAET is used for the preparation of a grain-saving environmentally friendly transgenic animal.

Images