CN114836461B - Recombinant plasmid expressing collagenase, yeast strain, fermentation medium and fermentation culture method thereof - Google Patents

Abstract

The invention discloses a recombinant plasmid for expressing collagenase, a yeast strain and its fermentation medium, fermentation culture method and use. The recombinant plasmid inserts different modules of the expression cassette into the expression plasmid, including the signal peptide αfactor, ColG or ColH leader. Peptides and functional regions; then the recombinant plasmid is transformed into a yeast strain to obtain Saccharomyces cerevisiae secreting collagenase. The present invention constructs an engineering strain of Saccharomyces cerevisiae that secretes and produces collagenase, and improves the ability of Saccharomyces cerevisiae to secrete collagenase by optimizing the components of the yeast culture medium and optimizing the signal peptide component in the collagenase sequence. The obtained recombinant collagenase can effectively degrade gelatin and collagen, which has important practical significance for the application of collagenase and the effective utilization of collagen resources.

Description

Recombinant plasmid, yeast strain and fermentation medium and fermentation medium expressing collagenase Ferment culture method

Technical Field

The invention belongs to the field of biotechnology, and specifically relates to a recombinant plasmid expressing collagenase, a yeast strain and its fermentation medium, fermentation culture method and use.

Background technique

In the processing and production of livestock, poultry and aquatic products, the output of by-products is huge. The horns, hooves, skins, bones and scales of these animals are rich in collagen resources.

Enzymatic extraction is an important means to improve the processing utilization of these by-products. Compared with other non-biological processes such as acid and alkali treatment, it can better obtain high-quality biologically active extracts and is more environmentally friendly. In view of the high strength of collagen's special triple helical structure, it is difficult for ordinary proteases to directly hydrolyze it, and chemical methods or high-temperature assisted hydrolysis are usually required.

Currently, enzymes commonly used to prepare collagen oligopeptides in industrial production mainly include papain, alkaline protease, trypsin, etc., but their specificity for collagen is poor.

Collagenase is a specific metalloproteinase for collagen. It can hydrolyze collagen under physiological conditions, making it easier to obtain bioactive collagen peptides. It can effectively improve the degree and quality of collagen hydrolysis under mild treatment conditions. .

Collagenase derived from Clostridium histolytica is the most widely studied and is currently recognized as the standard enzyme for identification and analysis of new collagenases. It is also a commercially available microbial-derived collagenase. However, collagenase derived from Clostridium histolytica is mainly purified from the fermentation supernatant of Clostridium histolytica. The composition of the fermentation supernatant is complex, the protein purification steps are cumbersome, and the activity of collagenase preparations between batches big different. In addition, Clostridium histolytica is a pathogenic bacterium, and the collagenase it secretes has certain limitations in its application in areas with high safety requirements such as food.

Contents of the invention

The purpose of the present invention is to provide a recombinant plasmid expressing collagenase, a yeast strain and its fermentation medium, fermentation culture method and use, so as to solve the shortcomings of existing collagenase sources that need to be expanded. By introducing the collagenase gene into Saccharomyces cerevisiae cells and optimizing the composition of the yeast fermentation medium, the secretion and expression of collagenase in a food-grade safe host can be achieved. Furthermore, by optimizing the signal peptide element in the collagenase sequence, the secretion level of collagenase was improved. The present invention focuses on the optimization of culture medium and signal peptide components, and obtains a Saccharomyces cerevisiae strain that efficiently secretes and expresses collagenase. The obtained recombinant collagenase has the ability to hydrolyze gelatin and collagen under physiological conditions. The fermentation supernatant containing the recombinant collagenase The liquid can be used directly for substrate processing.

The purpose of the present invention is achieved through the following technical solutions:

A recombinant plasmid for expressing collagenase is to insert the leader sequence (leader) of αfactor, the leader peptide sequence (pro peptide) and the enzyme functional region sequence (Chain) of Clostridium histolytica collagenase ColG into the expression plasmid, or It is to insert the leader sequence (leader) and spacer sequence (spacer) of αfactor, the leader peptide sequence (pro peptide) and enzyme functional region sequence (Chain) of Clostridium histolytica collagenase ColH into the expression plasmid;

The leader sequence (leader) of the α factor is shown in SEQ ID No. 1;

The leader peptide sequence and enzyme functional region sequence (pro peptide-Chain) of ColG are shown in SEQ ID No.2;

The leader sequence and spacer sequence (leader-spacer) of the αfactor are shown in SEQ ID No. 3;

The leader peptide sequence and enzyme functional region sequence (pro peptide-Chain) of ColH are shown in SEQ ID No. 4;

The collagenase expression plasmid constructed by the present invention is not limited to using only the native signal peptide of collagenase to mediate collagenase secretion, but by comparing the collagenase secretion yield under the combination of different modules (signal peptide, leader peptide, functional region) of the expression cassette, The optimized module combination was obtained to achieve efficient secretion in Saccharomyces cerevisiae cells.

The expression plasmids are CPOTud, p426GPD, p426TEF, p426ADH, p426CYC, p416GPD, p4216TEF, p4126ADH, p416CYC, p425GPD, p425TEF, p425ADH, p425CYC, p415GPD, p4215TEF, p4125ADH, p41 5CYC, p424GPD, p424TEF, p424ADH, p424CYC, p414GPD , p4214TEF, p4124ADH, p414CYC, p423GPD, p423TEF, p423ADH, p423CYC, p413GPD, p4213TEF, p4123ADH, p413CYC, pSP-GM1, pRS303, pRS304, pRS305, pRS306, pRS413, pRS414 , pRS415, pRS416, pRS423, pRS424, pRS425 or pRS426 .

A Saccharomyces cerevisiae that secretes collagenase is obtained by transforming the above-mentioned recombinant plasmid into a yeast strain;

The starting yeast strain is Saccharomyces cerevisiae B184M, CEN.PK 530.1C, CEN.PK 113.5D, BY4742, BY4741, CEN.PK2-1D, CEN.PK2-1C or IMX581.

A fermentation medium suitable for the above-mentioned Saccharomyces cerevisiae is to add Ca ²⁺ and/or Zn ²⁺ to the YPD medium;

The Ca ²⁺ concentration is 10-25 mM. At this concentration, collagenase is secreted in a relatively high amount. Considering that a higher metal ion concentration may potentially cause precipitation of the fermentation broth, 10 mM is preferred.

The concentration of Zn ²⁺ is 0.4-1mM, preferably 0.6mM.

The above-mentioned fermentation culture method of Saccharomyces cerevisiae is to inoculate the Saccharomyces cerevisiae into the above-mentioned fermentation medium and culture it;

The recombinant collagenase can be collected by culturing, preferably at 30-37°C for at least 96 hours.

The recombinant collagenase fermentation product of Saccharomyces cerevisiae of the present invention can effectively degrade gelatin and collagen. Therefore, the above-mentioned recombinant plasmid and Saccharomyces cerevisiae can be used to produce collagenase and degrade gelatin and collagen.

Compared with the existing technology, the present invention has the following advantages and effects:

The present invention constructs an engineering strain of Saccharomyces cerevisiae that secretes and produces collagenase, and improves the ability of Saccharomyces cerevisiae to secrete collagenase by optimizing the components of the yeast culture medium and optimizing the signal peptide component in the collagenase sequence. The obtained recombinant collagenase can effectively degrade gelatin and collagen, which has important practical significance for the application of collagenase and the effective utilization of collagen resources.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a map of collagenase expression plasmids pCP_G01 and pCP_H01.

Figure 2 shows the enzyme activity and OD ₆₀₀ values under different Ca ²⁺ ion concentrations.

Figure 3 shows the enzyme activity and OD ₆₀₀ values under different Zn ²⁺ ion concentrations.

Figure 4 shows the enzyme activity and OD ₆₀₀ values under different Zn ²⁺ ion concentrations.

Figure 5 is a diagram of the plasmid construction framework.

Figure 6 shows the enzyme activities and OD ₆₀₀ values of different strains.

Figure 7 shows the results of plate hydrolysis experiments of different strains.

Figure 8 shows the enzyme activities and OD ₆₀₀ values of different strains.

Figure 9 shows the results of plate hydrolysis experiments of different strains.

Figure 10 shows the degradation effect of collagenase on gelatin.

Figure 11 shows the degradation effect of collagenase on collagen.

Figure 12 shows the morphology of collagen after collagenase treatment for 24 hours.

Figure 13 shows the production of collagenase secreted by the strain.

Detailed ways

The present invention will be described in further detail below with reference to the examples and drawings, but the implementation of the present invention is not limited thereto.

The materials, reagents, etc. used in the following examples can all be obtained from commercial sources unless otherwise specified;

The experimental methods for which specific conditions are not specified in the following examples are generally carried out according to conventional conditions, such as those described in "Molecular Cloning Experiment Guide" (Beijing: Science Press, 2017) and "Yeast Genetics Methods Experiment Guide" (Beijing: Science Press, 2016).

In order to better understand the content of the present invention, Saccharomyces cerevisiae B184M (PNAS, 2015, 112: E4689-E4696) was used as the starting strain, and the plasmid CPOTud (Biotechnology and Bioengineering, 2012, 109 (5): 1259-1268) was used as the starting strain. ) is an expression plasmid, and specific examples will be further described.

The culture media involved in the following examples are as follows:

LB medium: 10 g/L peptone, 5 g/L yeast extract, 10 g/L NaCl;

LB/Amp medium: 10g/L peptone, 5g/L yeast extract, 10g/L NaCl. After sterilization, cool to about 50°C, add 100μg/mL ampicillin (filter sterilization);

YPD medium: 20g/L peptone, 10g/L yeast extract, 20g/L glucose (added after separate sterilization);

Modified YPD medium: 20g/L peptone, 10g/L yeast extract, 20g/L glucose (added after separate sterilization), 10mM CaCl ₂ and 0.6mM ZnCl ₂ ;

YPE culture medium: 20g/L peptone, 10g/L yeast extract, 0.5g/L glucose (added after separate sterilization), 10ml absolute ethanol/L (filter sterilization);

For solid culture medium, 20g/L agar powder is added to the corresponding liquid culture medium.

In the following examples, the relevant enzyme activity detection of collagenase was carried out according to the following method:

(1) After the yeast strain (BG01, BH01) containing the collagenase expression plasmid is activated on the YPD plate, use a 1 μL inoculation loop to pick a single colony and transfer it to 2.5 mL medium at 30°C, and culture at 200 rpm for 96 hours.

(2) Determination of biomass. Dilute the bacterial solution to an appropriate multiple and measure the absorbance value at a wavelength of 600nm, which is the OD ₆₀₀ value.

(3) The collagenase activity was determined by the ninhydrin method (Anal Biochem, 2013, 437: 46-48). The cultured bacterial solution was centrifuged to obtain the fermentation supernatant. 20 μL of the fermentation supernatant was taken, and 480 μL of 2 mg/mL gelatin solution prepared with reaction buffer (50 mM Tris·Cl, 5 mM CaCl ₂ , 1 μM ZnCl ₂ , pH = 7.5) was added. The reaction was carried out at 37°C for 30 min, and 500 μL of stop solution (12% (w/v) PEG6000, 25 mM EDTA) was added to terminate the reaction. Take 10 μL of the reaction solution, add 90 μL of deionized water, add 500 μL of ninhydrin colorimetric solution (mix equal volumes of 200 mM citric acid buffer containing 0.16% SnCl ₂ , pH=5.0 and 50 g/L ninhydrin-DMSO solution, prepare and use immediately), heat in a 80°C water bath for 10 min, add 600 μL of deionized water after cooling and mix evenly, and measure the absorbance at 570 nm. Under the conditions of 37°C and pH=7.5, the amount of collagen produced by hydrolyzing collagen per minute by 100 μL of enzyme solution is equivalent to 1 μg of glycine, which is 1 enzyme activity unit U.

(4) Plate transparent circle method to qualitatively characterize enzyme activity. Add an additional 1% skimmed milk powder to the solid medium. After culturing the strain on the plate at 30°C for 4 days, transfer it to 37°C and culture it for 2-3 days. Check the size of the hydrolysis circle formed around the colony and take photos.

The primer sequences involved in the following examples are shown in Table 1:

Table 1: Primer sequences

#Underline marks the enzyme cutting site

Example 1 Acquisition of Clostridium histolyticum collagenase encoding gene

According to the amino acid sequences of Clostridium histolytica collagenase ColG (Q9X721) and ColH (Q46085) published in the UniProt database, the DNA coding sequence was synthesized by Nanjing GenScript Biotechnology Co., Ltd. after codon optimization for Saccharomyces cerevisiae to obtain Plasmids pUC57-Mini_G (SEQ ID No. 5) and pUC57-Mini_H (SEQ ID No. 6) (containing pre peptide-pro peptide-Chain).

Example 2 Construction of Saccharomyces cerevisiae engineering strain expressing collagenase ColG and ColH

(1) CPOTud is digested with Kpn I and Nhe I and the vector is recovered;

(2) Use the primer pair AF/ARproG, AF/ARproH, and use pAlphaAmyCPOT (Biotechnology and Bioengineering, 2012, 109:1259-1268) as the template to amplify the signal peptide with G and H homology arms (αfactorleader+spacer) (SEQ ID No.3); use the primer pair proGF/GR, proHF/HR, and use pUC57-Mini_G and pUC57-Mini_H as templates to amplify the ColG and ColH fragments (SEQ ID No.2 and SEQ ID No.4); The corresponding fragments (ColG or ColH and the corresponding signal peptide) were amplified and fused by PCR using corresponding primers AF/GR and AF/HR to obtain insert fragments G01 and H01.

(3) After the fused fragments were digested with Kpn I and Nhe I, they were connected to the vector using T4 DNA Ligase to obtain plasmids pCP_G01 (SEQ ID No. 7) and pCP_H01 (SEQ ID No. 8).

(4) The ligation product is transformed into E. coli competent cells using conventional procedures. After the coated plate was cultured at 37°C overnight, bacterial colonies were picked and placed in a PCR system containing identification primers G1/G2 or H1/H2 for colony PCR identification.

(5) Identify the correct clone and inoculate it into LB/Amp medium, culture it at 37°C, 250rpm for 12-16 hours, extract and digest the plasmid, identify it and sequence it to confirm the plasmid pCP_G01 (SEQ ID No. 7) and pCP_H01 (SEQ ID No. 8) ) build successfully. The collagenase ColG expression cassette contained in plasmid pCP_G01 is: αfactor leader+spacer-colG pro peptide-colG chain; the collagenase ColH expression cassette contained in plasmid pCP_H01 is: αfactor leader+spacer-colH pro peptide-colH chain (see Figure 1 for the map) .

(6) The obtained plasmids pCP_G01, pCP_H01 and the control empty plasmid CPOTud were transformed into Saccharomyces cerevisiae B184M using the conventional lithium acetate transformation method to obtain strains BG01, BH01 and B0.

Example 3 Optimization of culture conditions for collagenase-producing bacteria

Since ColG and ColH are metalloproteases, they require Ca ²⁺ and Zn ²⁺ as metal cofactors.

The strain BG01 was used as a representative for medium optimization. The conventional YPD medium was supplemented with Ca ²⁺ (CaCl ₂ ) at concentrations of 2.5mM, 5mM, 10mM, 15mM, 20mM, and 25mM and Zn ²⁺ (ZnCl ₂ ) at concentrations of 0.1mM, 0.2mM, 0.4mM, 0.6mM, 0.8mM, and 1mM, respectively, and then fermented for 96h and the enzyme activity was determined. The results are shown in Figures 2 and 3. The single factor experiment showed that the addition of Ca ²⁺ or Zn ²⁺ significantly increased the production of collagenase. Among them, Ca ²⁺ between 10mM and 25mM can achieve a higher enzyme activity. In order to avoid the potential precipitation effect caused by a higher concentration of Ca ²⁺ , 10mM Ca ²⁺ is preferably used as the added concentration of metal ions. For Zn ²⁺ , 0.6mM Zn ²⁺ is selected as the added concentration of metal ions.

Based on the optimization results of ColG, we also added 10mM Ca ²⁺ to YPD medium, supplemented with 0.2mM, 0.4mM, 0.6mM, 0.8mM, and 1mM Zn ²⁺ (ZnCl ₂ ), and inoculated strain BH01 for 96h fermentation to measure enzyme activity. The results are shown in Figure 4, and the enzyme activity was the highest at 0.6mM Zn ²⁺ concentration.

In summary, 10mM Ca ²⁺ and 0.6mM Zn ²⁺ are the optimal metal ion addition concentrations.

Example 4 Optimization of component adaptation assembly of Saccharomyces cerevisiae expression cassette

The construction framework of a series of plasmids optimized for component adaptation assembly is shown in Figure 5. The construction process is the same as in Example 2. Different fragments are obtained by amplification and fusion using corresponding primer pairs.

Use the primer pair AF/ARWproG and use pAlphaAmyCPOT as the template to amplify the signal peptide with G homology arm (αfactor leader) (SEQ ID No. 1); use the primer pair WproGF/GR and use pUC57-Mini_G as the template to amplify The ColG fragment was obtained; the corresponding fragment (ColG and the corresponding signal peptide) was amplified and fused by PCR using the corresponding primer AF/GR to obtain the inserted fragment G02.

The primer pair PproG/GR was used to amplify ColG with αfactor pre homology arm using pUC57-Mini_G as the template, and the resulting fragment was amplified using the primer pair AP/GR to obtain the inserted fragment G03.

Use the primer pair AF/ARG and use pAlphaAmyCPOT as the template to amplify the signal peptide with G homology arm (αfactor leader+spacer); use the primer pair AGF/GR and use pUC57-Mini_G as the template to amplify the ColG fragment; The corresponding fragment (ColG and the corresponding signal peptide) was amplified and fused by PCR using the corresponding primer AF/GR to obtain the inserted fragment G04.

Using primer pair AF/ARWG, pAlphaAmyCPOT was used as a template to amplify the signal peptide with G homology arm (α factor leader + spacer); using primer pair WGF/GR, pUC57-Mini_G was used as a template to amplify the ColG fragment; the corresponding fragments (ColG and the corresponding signal peptide) were amplified and fused using corresponding primers AF/GR by PCR to obtain the inserted fragment G05.

The primer pair PG/GR was used to amplify ColG with αfactor pre homology arm using pUC57-Mini_G as the template, and the resulting fragment was amplified using the primer pair AP/GR to obtain the insert fragment G06.

Using the primer pair GF/GR, the insert G07 was amplified using pUC57-Mini_G as the template.

Use the primer pair AF/ARWproH and use pAlphaAmyCPOT as the template to amplify the signal peptide with H homology arm (αfactor leader); use the primer pair WproHF/HR and use pUC57-Mini_H as the template to amplify the ColH fragment; the corresponding fragment (ColH and the corresponding signal peptide) were amplified and fused by PCR using the corresponding primers AF/HR to obtain the insert H02.

Use the primer pair AF/ARH and use pAlphaAmyCPOT as the template to amplify the signal peptide with H homology arm (αfactor leader+spacer); use the primer pair AHF/HR and use pUC57-Mini_H as the template to amplify the ColH fragment; The corresponding fragment (ColH and the corresponding signal peptide) was amplified and fused by PCR using the corresponding primer AF/HR to obtain the insert fragment H03.

Using primer pair AF/ARWH, pAlphaAmyCPOT was used as a template to amplify the signal peptide with H homology arm (α factor leader + spacer); using primer pair WHF/HR, pUC57-Mini_H was used as a template to amplify the ColH fragment; the corresponding fragments (ColH and the corresponding signal peptide) were amplified and fused using corresponding primers AF/HR by PCR to obtain the inserted fragment H04.

Using the primer pair HF/HR, the insert fragment H05 was amplified using pUC57-Mini_H as the template.

Then the above G02, G03, G04, G05, G06, G07, H02, H03, H04, H05 fragments were inserted into the plasmid CPOTud through enzyme digestion and ligation, and some fragments were ligated with the plasmid using Seamless Cloning master mix (for details, please refer to Shilian Bo Research Kit B632219 instructions); the expression plasmids pCP_G02, pCP_G03, pCP_G04, pCP_G05, pCP_G06, pCP_G07, pCP_H02, pCP_H03, pCP_H04, and pCP_H05 were constructed accordingly.

The obtained plasmid and corresponding expression framework are:

pCP_G01 (containing αfactor leader+spacer-colG pro peptide-colG chain)

pCP_G02 (containing αfactor leader-colG pro peptide-colG chain)

pCP_G03 (containing αfactor pre-colG pro peptide-colG chain)

pCP_G04 (including αfactor leader+space-colG chain)

pCP_G05 (containing αfactor leader-colG chain)

pCP_G06 (containing αfactor pre-colG chain)

pCP_G07 (containing colG pre peptide-colG pro peptide-colG Chain)

pCP_H01 (containing αfactor leader+spacer-colH pro peptide-colH chain)

pCP_H02 (contains αfactor leader-colH pro peptide-colH chain)

pCP_H03 (containing α factor leader + spacer-colH chain)

pCP_H04 (containing αfactor leader-colH chain)

pCP_H05 (containing colH pre peptide-colH pro peptide-colH chain)

After the sequencing identification was correct, it was transferred into B184M to obtain the corresponding strains BG01, BG02, BG03, BG04, BG05, BG06, BG07, BH01, BH02, BH03, BH04, and BH05.

The constructed strain was cultured and the enzyme activity was tested. Figures 6 and 7 show that BG02 expresses collagenase ColG with the highest enzyme activity and the most obvious plate hydrolysis zone. It can be seen from Figures 8 and 9 that BH01 expresses collagenase ColH with the highest enzyme activity and the most obvious hydrolysis zone on the plate. The plasmid carried by strain BG02 is pCP_G02 (SEQ ID No. 9), and the expression cassette contained is αfactor leader-colG pro peptide-colG chain; the plasmid carried by strain BH01 is pCP_H01 (SEQ ID No. 8), and the expression cassette contained is αfactor leader+ spacer-colH pro peptide-colH chain.

Subsequent experiments were carried out using the above-mentioned strain BG02 and strain BH01.

Example 5 Degradation of different substrates by recombinant collagenase

(1) Degradation of gelatin. Add an additional 1% gelatin to modified YPD medium. The strains B0, BG02 and BH01 were respectively inoculated into 2.5 mL of modified YPD+gelatin culture medium, and fermented for 96 hours at 30°C and 200 rpm. After the fermentation is completed, 500 μL of the fermentation broth is centrifuged to remove bacterial cells. Take another 20 μL of fermentation supernatant, add 5 μL of 5×SDS-PAGE protein loading buffer, boil at 100°C for 10 minutes, then load the sample for electrophoresis until bromophenol blue reaches the bottom of the precast gel. After the electrophoresis is completed, place the protein gel in the staining solution for 30 minutes and take pictures with a gel imager after eluting the excess staining solution. As shown in Figure 10, the gelatin in the lanes corresponding to the blank control and control strain B0 was not decomposed and the entire lane was stained; while the gelatin in the lanes corresponding to strains BG02 and BH01 was obviously hydrolyzed by secreted collagenase.

(2) Degradation of insoluble bovine collagen. Inoculate strains B0, BG02, and BH01 into 40 mL of modified YPD medium, and ferment for 96 h at 30°C and 200 rpm. After the fermentation is completed, take 20 mL of fermentation broth and centrifuge to remove bacterial cells. The supernatant obtained from fermentation was concentrated in a 50kDa ultrafiltration centrifuge tube, and then diluted to an appropriate concentration with 50mM Tris·Cl. Add 2 mg of insoluble bovine bone collagen to it, let it stand at 37°C, observe the dissolution of the collagen, and take photos to record it. The G+H group refers to the mixture of BG02 and BH01 at 1:1. As can be seen from Figure 11, compared with the control group, the collagen fibers swelled significantly after treatment with recombinant collagenase, and the two recombinant collagenases showed a synergistic effect. Over time, collagen fibers are enzymatically broken down and disappear. The collagen fibers treated for 24 hours were naturally air-dried on the silicon wafer, adhered to the conductive adhesive, and sprayed with gold coating to observe the morphology and structure using ultra-high-resolution cold field scanning electron microscopy (SU8220, Hitachi). The results are shown in Figure 12. Compared with the tight and complete fiber structure of the control group, the sample fibers treated with recombinant collagenase ColG were in a loose state, and the surface of the sample fibers treated with recombinant collagenase ColH also underwent significant changes. . These phenomena confirmed from the microstructure that recombinant collagenase has good collagen hydrolytic activity.

Example 6 Production of recombinant collagenase by shake flask fermentation of engineered strains

After the strains B0, BG02, and BH01 were streaked and activated on the YPD plate, a single colony was picked with a 1 μL inoculating loop and transferred to 2.5 mL of modified YPD medium and cultured overnight to obtain a seed liquid. Take appropriate seed liquid and transfer it to 40 mL of fresh modified YPD medium. Adjust the initial OD ₆₀₀ to about 0.005. Carry out shake flask fermentation at 30°C, 200 rpm and 10% liquid volume. Take samples at appropriate intervals for enzyme analysis. Live detection. The production of recombinant collagenase obtained from shake flask fermentation of the engineering strain is shown in Figure 13. The production of collagenase ColG and ColH reached 6.8×10 ⁴ U/L and 5.5×10 ⁴ U/L respectively. The results show that the Saccharomyces cerevisiae engineering strain constructed in the present invention can efficiently secrete and express recombinant collagenase, can be used for processing and utilizing collagen resources, and has broad industrial application prospects.

The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, etc. may be made without departing from the spirit and principles of the present invention. All simplifications should be equivalent substitutions, and are all included in the protection scope of the present invention.

Sequence Listing

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tctactggtg ctgttggtag aaacattgaa ttttctggta aggattccaa ggacgaagat 2340

ggcaagattg tctcttatga ctgggatttc ggtgatggtg ctacctccag aggtaagaac 2400

tctgtccacg cttacaagaa ggctggtact tacaacgtca ctttgaaggt caccgatgac 2460

aagggtgcta cggctactga aagtttcact attgaaatta aaaacgaaga cactaccacc 2520

ccaatcacca aggaaatgga accaaacgat gacattaagg aagccaacgg cccgattgtc 2580

gaaggcgtca cggtcaaagg tgacttgaac ggttctgatg acgctgacac tttctacttt 2640

gatgtcaagg aagacggtga cgttaccatc gaattgccat actctggttc ctctaacttt 2700

acctggttag tctacaagga aggtgacgac caaaaccaca tcgcctccgg tatcgataaa 2760

aacaactcca aggttggtac tttcaagtcc actaagggtc gtcattacgt tttcatctac 2820

aagcacgact ctgcttccaa catttcatac tccttgaaca tcaagggttt gggtaacgaa 2880

aagttgaaag agaaggaaaa caatgactcc tctgacaagg caaccgttat tccaaatttc 2940

aataccacca tgcaaggttc tctattagggg gacgattcta gagactacta ctccttcgaa 3000

gtcaaggaag aaggtgaagt taacattgaa ttggataaaa aggatgaatt tggtgttat 3060

tggaccttgc acccagaaag caacatcaac gacagaatta cctacggtca agtcgatggt 3120

aacaaggtat ccaacaaggt caaattgaga ccaggtaagt actacttgtt ggtttacaag 3180

tacagtggtt ccggtaacta cgaattgcgg gttaacaag 3219

<210> 3

<211> 273

<212> DNA

<213> Artificial Sequence

<400> 3

atgagatttc catctatttt tactgctgtt ttgtttgctg cttcttctgc tttggctgct 60

ccagttaata ctactactga agatgaaact gctcaaattc cagctgaagc tgttatattggt 120

tattctgatt tggagggtga ctttgatgtt gctgttttgc cattttctaa ctctactaac 180

aacggtttgc tattcatcaa cactactatc gcttctatcg ctgctaaaga agaaggtgtt 240

tctttggata aaagagaaga aggtgaacca aaa 273

<210> 4

<211> 2973

<212> DNA

<213> Artificial Sequence

<400> 4

gctgttgaca agaacaatgc cactgctgcc gtccaaaacg aatccaagcg ttacactgtt 60

tcttacttaa agaccttgaa ctattacgac ttggtcgatc tattggttaa gaccgaaatc 120

gaaaacttac cagacttgtt tcaatactct tccgacgcta aggaatttta cggtaacaag 180

actagaatgt cctttatcat ggacgaaatt ggtcgtagag ctccacaata cactgaaatt 240

gaccacaagg gtataccaac cctcgttgaa gtcgtcagag ccgggttcta cttgggtttt 300

cacaacaaag aattgaatga aatcaacaag agatctttca aagaaagagt tatcccgtct 360

atcttggcta tccaaaagaa cccaaacttc aaattaggta ctgaagttca agacaagatt 420

gtctctgcta ccggtctatt ggctggtaac gaaaccgctc caccagaagt tgttaacaac 480

ttcaccccaa tcttgcaaga ttgtatcaag aacatcgaca gatacgcctt ggacgacttg 540

aaaagtaaag cattgttcaa cgttttggcc gccccaacct acgacattac tgaatacctc 600

agagctacca aggaaaagcc agaaaacact ccatggtacg gtaagatcga tggtttcatc 660

aacgaattga agaagctcgc tttgtacggt aaaattaacg acaacaactc ttggattatc 720

gataacggta tttaccacat tgctccattg ggtaaattgc actctaacaa caaaatcggt 780

atcgaaactc taactgaagt tatgaaggtc tacccatact tgtccatgca acacttgcaa 840

tccgctgacc aaatcaagag acactacgac tctaaggatg ccgaaggtaa caagatccca 900

ttagataagt tcaagaagga aggcaaggaa aagtactgcc caaagaccta cacttttgat 960

gatggtaagg ttatcatcaa ggccggtgct cgtgtcgaag aagaaaaggt caaaagatta 1020

tactgggctt ccaaggaagt caacagtcaa ttcttcagag tttatggtat tgacaagcca 1080

ttggaagaag gtaacccaga cgacatcctt accatggtta tttacaactc tccagaagaa 1140

tacaaactta actccgtctt gtacggttac gatacccaaca acggtggtat gtacattgaa 1200

ccagaaggta cttttttcac ttacgaaaga gaagctcaag aatctacgta cactttggaa 1260

gaattattca gacacgaata cacccattac ttgcaaggta gatacgctgt cccaggtcaa 1320

tggggtcgta ccaagttgta tgataacgac agattgacct ggtacgaaga aggtggtgct 1380

gaactgtttg ctggttctac tagaacttcc ggcatcttgc caagaaagtc tattgtctct 1440

aacattcaca acaccactag aaacaacaga tacaagctgt ctgacactgt tcattcgaag 1500

tacggtgcct ctttcgaatt ttacaactac gcttgtatgt tcatggacta catgtacaat 1560

aaggacatgg gtattttgaa caagttgaat gatttggcta agaacaacga cgttgacggt 1620

tacgacaatt acatcagaga tttgtcatct aattacgctt tgaacgataa gtaccaagat 1680

cacatgcaag aacgtattga caactatgaa aacttgaccg ttccattcgt tgctgacgac 1740

tacttagtta gacacgctta caagaaccca aacgaaattt actccgaaat ctccgaagtt 1800

gccaagttga aggatgctaa gtctgaagtt aagaaatctc aatacttctc aactttcact 1860

ttgcgtggta gttacaccgg aggtgcttcc aagggtaagc tggaagatca aaaagctatg 1920

aacaagttca ttgacgactc tttgaagaag ttggacacct attcctggtc tggttacaag 1980

actcttactg cttattttac caactacaag gtcgattctt ctaacagagt tacttacgat 2040

gtcgttttcc atggttatactt gccaaacgaa ggtgattcca agaacagctt gccatacggt 2100

aagattaacg gtacttacaa gggtactgag aaggaaaaga ttaagttctc ctccgaaggt 2160

tctttcgacc cagacggaaa gatagtttcc tacgaatggg acttcggtga tggtaacaag 2220

tccaacgaag agaaccctga acacagttac gataaggtcg gtacttacac tgtcaagttg 2280

aaggttaccg atgataaggg tgaaagctct gtttctacta ccactgctga aatcaaagat 2340

ttgtctgaaa acaagttgcc agtcatctat atgcacgttc ctaaatcggg tgctttaaac 2400

caaaaggttg tcttctacgg taagggtact tacgacccag acggttctat cgctggctac 2460

caatgggatt tcggtgacgg ttctgacttt tcctctgaac agaacccatc tcacgtctac 2520

accaagaaag gtgaatacac tgtgaccttg agagtcatgg attcttccgg tcaaatgtct 2580

gaaaagacca tgaaaatcaa gatcactgat ccagtatacc ctattggtac tgaaaaggaa 2640

ccaaacaact ctaaggaaac agcctccggt ccaatcgttc cgggtattcc agtctccggt 2700

actatcgaaa acacctctga tcaagactat ttctacttcg atgttattac tccaggtgaa 2760

gtcaagatcg acatcaacaa gctaggttac ggtggtgcta catgggttgt ttacgatgaa 2820

aacaataacg ctgtctccta cgctaccgat gacggtcaaa atttgtctgg taaattcaag 2880

gctgacaagc ctgggagata ctacattcat ttgtacatgt tcaatggttc ttacatgcca 2940

tacagaatta acattgaagg ttctgttggt aga 2973

<210> 5

<211> 5216

<212> DNA

<213> Artificial Sequence

<400> 5

ccaatgataa aggtaccaac aaaatgaaga agaacatctt gaagattttg atggactctt 60

actccaagga atccaaaatc caaactgtta gaagagttac ttctgtttct ttgttggctg 120

tctacttgac tatgaacacc tcaagtctag tgttggctaa gccaatcgaa aacactaacg 180

acaccagcat caagaacgtt gaaaagttga gaaacgcccc aaacgaagaa aactctaaga 240

aagttgaaga ctctaagaat gataaggttg aacacgttaa gaacattgaa gaagctaagg 300

tcgaacaagt tgctccagaa gtcaagtcta aaagtacctt gagatctgct tctatagcta 360

atacaaattc cgaaaagtac gacttcgaat atcttaatgg tttgagttac accgaattga 420

ctaacttgat caagaacatc aagtggaacc aaatcaacgg tttattcaac tactccaccg 480

gttcgcaaaa atttttcggt gacaagaaca gagtccaagc catcatcaac gctctacaag 540

aatctggtag aacttacact gccaatgaca tgaagggtat tgaaactttc accgaagttt 600

tgagagccgg tttttacttg ggctattaca acgatggtct ctcctacctg aacgaccgta 660

atttccaaga caagtgtatc ccagccatga ttgctattca aaagaaccca aacttcaagt 720

taggtactgc tgttcaagac gaagttatca catctttggg taagttgatt ggtaacgcct 780

ctgctaacgc tgaagttgtt aataactgtg tcccagtttt gaagcaattc agagaaaact 840

taaaccaata cgctccagac tacgtcaaag gtactgccgt aaatgaattg atcaagggta 900

ttgaatttga cttttccggt gctgcttacg aaaaggacgt caagaccatg ccatggtacg 960

gtaagattga cccattcatc aatgaattaa aggccttggg tctctacggt aacatcactt 1020

ctgccactga atgggcttca gatgttggta tctactactt gtctaagttc ggtttgtact 1080

caaccaatcg taacgacatt gttcaatcct tagaaaaggc tgttgatatg tacaaatatg 1140

gtaagatcgc tttcgttgct atggaaagaa tcacctggga ttacgatggt atcggttcca 1200

acgggaagaa ggtcgaccac gataagttct tggacgatgc agaaaagcat tatttgccaa 1260

agacctatac tttcgacaac ggaaccttca tcatcagagc tggagataag gtttctgaag 1320

aaaagattaa gagattgtac tgggcttccc gtgaagttaa gtctcaattc cacagagttg 1380

ttggtaacga taaggctttg gaagtgggta acgctgacga tgtcttgacc atgaaaatct 1440

tcaactctcc agaagaatac aagttcaaca ccaacattaa cggtgtctcc actgataacg 1500

gtggtttgta catcgaacca agaggtactt tctacactta cgaaagaacc ccacaacaat 1560

ctattttctc cttggaagaa ctgttcagac acgaatacac ccactacttg caagctagat 1620

acttggttga cggtttgtgg ggtcaaggtc ctttctacga aaaaaacaga ttgacttggt 1680

tcgatgaagg tactgctgaa tttttcgctg gttccaccag aacttctggt gtgttgccaa 1740

gaaagtccat cttgggttac cttgccaagg ataaggtcga tcatagatac tccttgaaga 1800

aaactttgaa ctctggttac gatgactctg actggatgtt ctacaactac ggttttgctg 1860

ttgctcacta cttatacgaa aaggacatgc caacttttat taagatgaac aaggccatct 1920

tgaacactga tgttaagtct tacgacgaaa tcattaaaaa actgtctgac gacgctaaca 1980

agaacactga ataccaaaac cacatccaag aactcgctga taagtaccaa ggtgccggta 2040

taccattggt gtctgatgac tacctgaagg accatggtta caagaaggct tccgaagttt 2100

actcagaaat ctccaaggct gcgtctttaa ctaacacttc tgttaccgct gaaaagtctc 2160

aatacttcaa cacattcact ttgaggggta cttacactgg tgaaacctct aagggtgaat 2220

ttaaggactg ggacgaaatg tctaagaagt tggacggtac tttggagtct ttagctaaga 2280

actcttggtc tggttacaag actttgaccg cttacttcac caactacaga gtcacttctg 2340

ataacaaagt ccaatatgat gttgtcttcc acggtgtttt gactgacaac gccgatattt 2400

ctaacaacaa ggctcctatt gctaaggtca ccggtccatc tactggtgct gttggtagaa 2460

acattgaatt ttctggtaag gattccaagg acgaagatgg caagattgtc tctttatgact 2520

gggatttcgg tgatggtgct acctccagag gtaagaactc tgtccacgct tacaagaagg 2580

ctggtactta caacgtcact ttgaaggtca ccgatgacaa gggtgctacg gctactgaaa 2640

gtttcactat tgaaattaaa aacgaagaca ctaccacccc aatcaccaag gaaatggaac 2700

caaacgatga cattaaggaa gccaacggcc cgattgtcga aggcgtcacg gtcaaaggtg 2760

acttgaacgg ttctgatgac gctgacactt tctactttga tgtcaaggaa gacggtgacg 2820

ttaccatcga attgccatac tctggttcct ctaactttac ctggttagtc tacaaggaag 2880

gtgacgacca aaaccacatc gcctccggta tcgataaaaa caactccaag gttggtactt 2940

tcaagtccac taagggtcgt cattacgttt tcatctacaa gcacgactct gcttccaaca 3000

tttcatactc cttgaacatc aagggtttgg gtaacgaaaa gttgaaagag aaggaaaaca 3060

atgactcctc tgacaaggca accgttattc caaatttcaa taccaccatg caaggttctc 3120

tattagggga cgattctaga gactactact ccttcgaagt caaggaagaa ggtgaagtta 3180

acattgaatt ggataaaaag gatgaatttg gtgttatacttg gaccttgcac ccagaaagca 3240

acatcaacga cagaattacc tacggtcaag tcgatggtaa caaggtatcc aacaaggtca 3300

aattgagacc aggtaagtac tacttgttgg tttacaagta cagtggttcc ggtaactacg 3360

aattgcgggt taacaagtaa gctagcttta tcggaaagaa catgtgagca aaaggccagc 3420

aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc 3480

ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat 3540

aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc 3600

cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct 3660

cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg 3720

aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc 3780

cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga 3840

ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa 3900

gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta 3960

gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc 4020

agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg 4080

acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga 4140

tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg 4200

agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct 4260

gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg 4320

agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc 4380

cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa 4440

ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc 4500

cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt 4560

cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc 4620

ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt 4680

tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc 4740

catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt 4800

gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata 4860

gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga 4920

tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag 4980

catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa 5040

aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt 5100

attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga 5160

aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtc 5216

<210> 6

<211> 4925

<212> DNA

<213> Artificial Sequence

<400> 6

ccaatgataa aggtaccaac aaaatgaaga gaaaatgttt gtctaagaga ttgatgttgg 60

ctatcaccat ggctaccatc ttcaccgtca attctacatt gccaatttac gctgctgttg 120

acaagaacaa tgccactgct gccgtccaaa acgaatccaa gcgttacact gtttcttact 180

taaagacctt gaactattac gacttggtcg atctattggt taagaccgaa atcgaaaact 240

taccagactt gtttcaatac tcttccgacg ctaaggaatt ttacggtaac aagactagaa 300

tgtcctttat catggacgaa attggtcgta gagctccaca atacactgaa attgaccaca 360

agggtatacc aaccctcgtt gaagtcgtca gagccgggtt ctacttgggt tttcacaaca 420

aagaattgaa tgaaatcaac aagagatctt tcaaagaaag agttatcccg tctatcttgg 480

ctatccaaaa gaacccaaac ttcaaattag gtactgaagt tcaagacaag attgtctctg 540

ctaccggtct attggctggt aacgaaaccg ctccaccaga agttgttaac aacttcaccc 600

caatcttgca agattgtatc aagaacatcg acagatacgc cttggacgac ttgaaaagta 660

aagcattgtt caacgttttg gccgccccaa cctacgacat tactgaatac ctcagagcta 720

ccaaggaaaa gccagaaaac actccatggt acggtaagat cgatggtttc atcaacgaat 780

tgaagaagct cgctttgtac ggtaaaatta acgacaacaa ctcttggatt atcgataacg 840

gtatttacca cattgctcca ttgggtaaat tgcactctaa caacaaaatc ggtatcgaaa 900

ctctaactga agttatgaag gtctacccat acttgtccat gcaacacttg caatccgctg 960

accaaatcaa gagacactac gactctaagg atgccgaagg taacaagatc ccattagata 1020

agttcaagaa ggaaggcaag gaaaagtact gcccaaagac ctacactttt gatgatggta 1080

aggttatcat caaggccggt gctcgtgtcg aagaagaaaa ggtcaaaaga ttatactggg 1140

cttccaagga agtcaacagt caattcttca gagtttatgg tattgacaag ccattggaag 1200

aaggtaaccc agacgacatc cttaccatgg ttatttacaa ctctccagaa gaatacaaac 1260

ttaactccgt cttgtacggt tacgatacca acaacggtgg tatgtacatt gaaccagaag 1320

gtactttttt cacttacgaa agagaagctc aagaatctac gtacactttg gaagaattat 1380

tcagacacga atacacccat tacttgcaag gtagatacgc tgtcccaggt caatggggtc 1440

gtaccaagtt gtatgataac gacagattga cctggtacga agaaggtggt gctgaactgt 1500

ttgctggttc tactagaact tccggcatct tgccaagaaa gtctattgtc tctaacattc 1560

acaacaccac tagaaacaac agatacaagc tgtctgacac tgttcattcg aagtacggtg 1620

cctctttcga attttacaac tacgcttgta tgttcatgga ctacatgtac aataaggaca 1680

tgggtatttt gaacaagttg aatgatttgg ctaagaacaa cgacgttgac ggttacgaca 1740

attacatcag agatttgtca tctaattacg ctttgaacga taagtaccaa gatcacatgc 1800

aagaacgtat tgacaactat gaaaacttga ccgttccatt cgttgctgac gactacttag 1860

ttagacacgc ttacaagaac ccaaacgaaa tttactccga aatctccgaa gttgccaagt 1920

tgaaggatgc taagtctgaa gttaagaaat ctcaatactt ctcaactttc actttgcgtg 1980

gtagttacac cggaggtgct tccaagggta agctggaaga tcaaaaagct atgaacaagt 2040

tcattgacga ctctttgaag aagttggaca cctattcctg gtctggttac aagactctta 2100

ctgcttattt taccaactac aaggtcgatt cttctaacag agttacttac gatgtcgttt 2160

tccatggtta cttgccaaac gaaggtgatt ccaagaacag cttgccatac ggtaagatta 2220

acggtactta caagggtact gagaaggaaa agattaagtt ctcctccgaa ggttctttcg 2280

acccagacgg aaagatagtt tcctacgaat gggacttcgg tgatggtaac aagtccaacg 2340

aagagaaccc tgaacacagt tacgataagg tcggtactta cactgtcaag ttgaaggtta 2400

ccgatgataa gggtgaaagc tctgtttcta ctaccactgc tgaaatcaaa gatttgtctg 2460

aaaacaagtt gccagtcatc tatatgcacg ttcctaaatc gggtgcttta aaccaaaagg 2520

ttgtcttcta cggtaagggt acttacgacc cagacggttc tatcgctggc taccaatggg 2580

atttcggtga cggttctgac ttttcctctg aacagaaccc atctcacgtc tacaccaaga 2640

aaggtgaata cactgtgacc ttgagagtca tggattcttc cggtcaaatg tctgaaaaga 2700

ccatgaaaat caagatcact gatccagtat accctattgg tactgaaaag gaaccaaaca 2760

actctaagga aacagcctcc ggtccaatcg ttccgggtat tccagtctcc ggtactatcg 2820

aaaacacctc tgatcaagac tatttctact tcgatgttat tactccaggt gaagtcaaga 2880

tcgacatcaa caagctaggt tacggtggtg ctacatgggt tgtttacgat gaaaacaata 2940

acgctgtctc ctacgctacc gatgacggtc aaaatttgtc tggtaaattc aaggctgaca 3000

agcctgggag atactacatt catttgtaca tgttcaatgg ttcttacatg ccatacagaa 3060

ttaacattga aggttctgtt ggtagataag ctagctttat cggaaagaac atgtgagcaa 3120

aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 3180

tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 3240

caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 3300

cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 3360

ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 3420

gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 3480

agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 3540

gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 3600

acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 3660

gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 3720

gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 3780

cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat 3840

caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa 3900

gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct 3960

cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta 4020

cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct 4080

caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg 4140

gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa 4200

gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt 4260

cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta 4320

catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca 4380

gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta 4440

ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct 4500

gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg 4560

cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac 4620

tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact 4680

gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa 4740

atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt 4800

ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat 4860

gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg 4920

acgtc 4925

<210> 7

<211> 11468

<212> DNA

<213> Artificial Sequence

<400> 7

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accataccac agcttttcaa ttcaattcat catttttttt ttattctttt ttttgatttc 240

ggtttctttg aaattttttt gattcggtaa tctccgaaca gaaggaagaa cgaaggaagg 300

agcacagact tagattggta tatatacgca tatgtagtgt tgaagaaaca tgaaattgcc 360

cagtattctt aacccaactg cacagaacaa aaacctgcag gaaacgaaga taaatctaaa 420

aaactgtatt ataagtaaat gcatgtatac taaactcaca aattagagct tcaatttaat 480

tatatcagtt attaccctat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac 540

cgcatcagga aattgtaaac gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa 600

tcagctcattttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat 660

agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg 720

tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac 780

catcacccta atcaagttttttggggtcga ggtgccgtaa agcactaaat cggaacccta 840

aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag 900

ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg 960

taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtccatt cgccattcag 1020

gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctgga 1080

taaaggcgcg ccgtcgacaa tatagaaatt tcttagtttt aatgattgaa tgaatccatt 1140

tcgaacacgt caatgtactt ggacaagctt tgtagaacat gaagtacttg agcaatggac 1200

gcccaactgg aatttacttt agcgacacta ttccaagacc agcaactcaa agaagtaaat 1260

tctttttttc acaatagttg tttttttgat gatgtacgtc ccagcagcac ctaatggtat 1320

ttttatgagt attggcagtt tgccttatta attaagcaaa ccggtcatat attttaaaaa 1380

ataggaactt tgtagtcctt attaatgctg aataaaaaaa taggaagaga aatcagcaag 1440

tataaacagt taaatgtttt ttaactataa tggttgcatt caagaggaca tgtaaattgc 1500

gtactcggta caagttggta aaaacgttaa agcactccta cgagatttag tcagtcctgg 1560

cattaataaa caaaaagacc tgtatctata tttcatgaac tgggtaaaat caccacacaa 1620

aaaagaataa acaaatataa tgaactactt ttggtcttgg actcgcagtt tatagtttgt 1680

taaaattgac aaacacacat gttctgatta acttacttcc gcttcaaagt actgaaaaca 1740

tcatacgaac tggtataaga aggaagatag caaataaaaa taaatcaaat attgcttgta 1800

aaaaattagg atataaaaaa atctctattc attaaagaca aaaagctaaa ggaataaaaa 1860

agtcattcgc tttacgccaa ataaaacgtc tatgcacgtt tttaaaggct gtgaacatta 1920

acaatgttgt ggaattcagg cttgagagaa gcaccgccaa ccaagaaacc gtcaatatcg 1980

tggaacttga ggaactcctt gcagttacca ccgttaacgg aaccaccgta gatgacacgg 2040

agaccctcgg caacagatgc accaagcttg ttggtagccc acttgcggat ctcagcgtga 2100

acctcttgag cttgctcagg ggtggcagtc ttaccagtac caatggccca gacaggctca 2160

taagcaatga caatcttgga ccagttctgg accttgtcag cgatggcgtt caattgacga 2220

acaacaacgt tgatggtctc gttagcctca cgctcggcca aagtctcacc aatgcaggca 2280

acgacagtaa gaccttgttc aagggcaaac ttggtcttgt cggcaacgaa ctcgtcagac 2340

tccttgaaga tggtacgacg ctcggagtga ccagtcaaag tgtaggtaat accagcatca 2400

atcaaagatt gagcactgtt ctcaccagtg taggcaccgt tcttcttgtc gaagacgttt 2460

tgggcaccaa cgccaatatc cttcttgact tgttggcggg tggtgatgag gtacatgttt 2520

tgagggaaga tgacagtttc gacatcacca acgttaagct tggtggtgtt caaaccctca 2580

ataatagtct tcatggactc caaagagcca ttcatcttaa agttaccacc gacaaagaat 2640

ttacgtgcca ttttgatcct agttctacta ttgaaatgta gttggggaga gacgagagtt 2700

ggcccgtccg ctcattatat ataacgtagc ggacagtcac acagttaagg ggaattaccg 2760

agcttcggca atttaccccg tcgatagcaa ccgttggcat ggatccggcc ggccagatct 2820

acgtatggtc atttcttctt cagattccct catggagaaa gtgcggcaga tgtatatgac 2880

agagtcgcca gtttccaaga gactttatattc aggcacttcc atgataggca agagagaaga 2940

cccagagatg ttgttgtcct agttacacat ggtatttatt ccagagtatt cctgatgaaa 3000

tggtttagat ggacatacga agagtttgaa tcgtttacca atgttcctaa cgggagcgta 3060

atggtgatgg aactggacga atccatcaat agatacgtcc tgaggaccgt gctacccaaa 3120

tggactgatt gtgagggaga cctaactaca tagtgtttaa agattacgga tatttaactt 3180

acttagaata atgccatttttttgagttat aataatccta cgttagtgtg agcgggattt 3240

aaactgtgag gaccttaata cattcagaca cttctgcggt atcaccctac ttatccctt 3300

cgagattata tctaggaacc catcaggttg gtggaagatt acccgttcta agacttttca 3360

gcttcctcta ttgatgttac acctggacac cccttttctg gcatccagtt tttaatcttc 3420

agtggcatgt gagattctcc gaaattaatt aaagcaatca cacaattctc tcggatacca 3480

cctcggttga aactgacagg tggtttgtta cgcatgctaa tgcaaaggag cctatatacc 3540

tttggctcgg ctgctgtaac agggaatata aagggcagca taatttagga gtttagtgaa 3600

cttgcaacat ttactatttt cccttcttac gtaaatattt ttctttttaa ttctaaatca 3660

atctttttca attttttgtt tgtattcttt tcttgcttaa atctataact acaaaaaaca 3720

catacataaa ctaaaaggta ccaacaaaat gagatttcca tctattttta ctgctgtttt 3780

gtttgctgct tcttctgctt tggctgctcc agttaatact actactgaag atgaaactgc 3840

tcaaattcca gctgaagctg ttattggtta ttctgatttg gagggtgact ttgatgttgc 3900

tgttttgcca ttttctaact ctactaacaa cggtttgcta ttcatcaaca ctactatcgc 3960

ttctatcgct gctaaagaag aaggtgtttc tttggataaa agagaagaag gtgaaccaaa 4020

aaagccaatc gaaaacacta acgacaccag catcaagaac gttgaaaagt tgagaaacgc 4080

cccaaacgaa gaaaactcta agaaagttga agactctaag aatgataagg ttgaacacgt 4140

taagaacatt gaagaagcta aggtcgaaca agttgctcca gaagtcaagt ctaaaagtac 4200

cttgagatct gcttctatag ctaatacaaa ttccgaaaag tacgacttcg aatatcttaa 4260

tggtttgagt tacaccgaat tgactaactt gatcaagaac atcaagtgga accaaatcaa 4320

cggtttattc aactactcca ccggttcgca aaaatttttc ggtgacaaga acagagtcca 4380

agccatcatc aacgctctac aagaatctgg tagaacttac actgccaatg acatgaaggg 4440

tattgaaact ttcaccgaag ttttgagagc cggtttttac ttgggctatt acaacgatgg 4500

tctctcctac ctgaacgacc gtaatttcca agacaagtgt atcccagcca tgattgctat 4560

tcaaaagaac ccaaacttca agttaggtac tgctgttcaa gacgaagtta tcacatcttt 4620

gggtaagttg attggtaacg cctctgctaa cgctgaagtt gttaataact gtgtcccagt 4680

tttgaagcaa ttcagagaaa acttaaacca atacgctcca gactacgtca aaggtactgc 4740

cgtaaatgaa ttgatcaagg gtattgaatt tgacttttcc ggtgctgctt acgaaaagga 4800

cgtcaagacc atgccatggt acggtaagat tgacccattc atcaatgaat taaaggcctt 4860

gggtctctac ggtaacatca cttctgccac tgaatgggct tcagatgttg gtatctacta 4920

cttgtctaag ttcggtttgt actcaaccaa tcgtaacgac attgttcaat ccttagaaaa 4980

ggctgttgat atgtacaaat atggtaagat cgctttcgtt gctatggaaa gaatcacctg 5040

ggattacgat ggtatcggtt ccaacgggaa gaaggtcgac cacgataagt tcttggacga 5100

tgcagaaaag cattatttgc caaagaccta tactttcgac aacggaacct tcatcatcag 5160

agctggagat aaggtttctg aagaaaagat taagagattg tactgggctt cccgtgaagt 5220

taagtctcaa ttccacagag ttgttggtaa cgataaggct ttggaagtgg gtaacgctga 5280

cgatgtcttg accatgaaaa tcttcaactc tccagaagaa tacaagttca acaccaacat 5340

taacggtgtc tccactgata acggtggttt gtacatcgaa ccaagaggta ctttctacac 5400

ttacgaaaga accccacaac aatctatttt ctccttggaa gaactgttca gacacgaata 5460

cacccactac ttgcaagcta gatacttggt tgacggtttg tggggtcaag gtcctttcta 5520

cgaaaaaaac agattgactt ggttcgatga aggtactgct gaatttttcg ctggttccac 5580

cagaacttct ggtgtgttgc caagaaagtc catcttgggt taccttgcca aggataaggt 5640

cgatcataga tactccttga agaaaacttt gaactctggt tacgatgact ctgactggat 5700

gttctacaac tacggttttg ctgttgctca ctacttatac gaaaaggaca tgccaacttt 5760

tattaagatg aacaaggcca tcttgaacac tgatgttaag tcttacgacg aaatcattaa 5820

aaaactgtct gacgacgcta acaagaacac tgaataccaa aaccacatcc aagaactcgc 5880

tgataagtac caaggtgccg gtataccatt ggtgtctgat gactacctga aggaccatgg 5940

ttacaagaag gcttccgaag tttactcaga aatctccaag gctgcgtctt taactaacac 6000

ttctgttacc gctgaaaagt ctcaatactt caacacattc actttgaggg gtacttacac 6060

tggtgaaacc tctaagggtg aatttaagga ctgggacgaa atgtctaaga agttggacgg 6120

tactttggag tctttagcta agaactcttg gtctggttac aagactttga ccgcttactt 6180

caccaactac agagtcactt ctgataacaa agtccaatat gatgttgtct tccacggtgt 6240

tttgactgac aacgccgata tttctaacaa caaggctcct attgctaagg tcaccggtcc 6300

atctactggt gctgttggta gaaacattga attttctggt aaggattcca aggacgaaga 6360

tggcaagatt gtctcttatg actgggattt cggtgatggt gctacctcca gaggtaagaa 6420

ctctgtccac gcttacaaga aggctggtac ttacaacgtc actttgaagg tcaccgatga 6480

caagggtgct acggctactg aaagtttcac tattgaaatt aaaaacgaag acactaccac 6540

cccaatcacc aaggaaatgg aaccaaacga tgacattaag gaagccaacg gcccgattgt 6600

cgaaggcgtc acggtcaaag gtgacttgaa cggttctgat gacgctgaca ctttctactt 6660

tgatgtcaag gaagacggtg acgttaccat cgaattgcca tactctggtt cctctaactt 6720

tacctggtta gtctacaagg aaggtgacga ccaaaaccac atcgcctccg gtatcgataa 6780

aaacaactcc aaggttggta ctttcaagtc cactaagggt cgtcattacg ttttcatcta 6840

caagcacgac tctgcttcca acatttcata ctccttgaac atcaagggtt tgggtaacga 6900

aaagttgaaa gagaaggaaa acaatgactc ctctgacaag gcaaccgtta ttccaaattt 6960

caataccacc atgcaaggtt ctctattagg ggacgattct agagactact actccttcga 7020

agtcaaggaa gaaggtgaag ttaacattga attggataaa aaggatgaat ttggtgttac 7080

ttggaccttg cacccagaaa gcaacatcaa cgacagaatt acctacggtc aagtcgatgg 7140

taacaaggta tccaacaagg tcaaattgag accaggtaag tactacttgt tggtttacaa 7200

gtacagtggt tccggtaact acgaattgcg ggttaacaag caccaccacc accaccacta 7260

agctagcctc gagtctagaa actaagatta atataattat ataaaaatat tatcttcttt 7320

tctttatatc tagtgttatg taaaataaat tgatgactac ggaaagcttt tttatattgt 7380

ttctttttca ttctgagcca cttaaatttc gtgaatgttc ttataaggga cggtagattt 7440

acaagtgata caacaaaaag caaggcgctt tttctaataa aaagaagaaa agcatttaac 7500

aattgaacac ctctatatca acgaagaata ttactttgtc tctaaatcct tgtaaaatgt 7560

gtacgatctc tatatgggtt actcagaagt gtaccgaaga ctgcattgaa agtttatgtt 7620

ttttcactgc aagcgtcatt ttcgctttga gaagatgttc ttattcaaat ttcaactgtt 7680

atatagaaga gcaaaaaatt gccaaaaaaa acaacattta ttcatttaaa atataaaatt 7740

tgggcttcta tattttaata ttgcttttca attactgtta ttaaatgtaa gtactgcgtc 7800

tatgaaaata tatgcaaatg ctaagaaaaa tcctaaaaat ttgaatatga gatattcctc 7860

agtatttctt tttcatcctt tcttctgcgg ctctagccct ttgttctctc atcaatctgc 7920

gtctctgttc atcggtcaaa gaattcaaat tttgttgctg aattgaagga ataacgcgtg 7980

tacgcatgta acattatact gaaaaccttg cttgagaagg ttttgggacg ctcgaagatc 8040

ctccggatcg tttcgccggc gtttatccag ctgcattaat gaatcggcca acgcgcgggg 8100

agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg 8160

gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca 8220

gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac 8280

cgtaaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac 8340

aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg 8400

tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac 8460

ctgtccgcctttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat 8520

ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag 8580

cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac 8640

ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt 8700

gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt 8760

atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc 8820

aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga 8880

aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac 8940

gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc 9000

cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct 9060

gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca 9120

tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct 9180

ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca 9240

ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc 9300

atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg 9360

cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct 9420

tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa 9480

aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta 9540

tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc 9600

ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg 9660

agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa 9720

gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg 9780

agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc 9840

accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg 9900

gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat 9960

cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata 10020

ggggttccgc gcacatttcc ccgaaaagtg ccacctgaac gaagcatctg tgcttcattt 10080

tgtagaacaa aaatgcaacg cgagagcgct aatttttcaa acaaagaatc tgagctgcat 10140

ttttacagaa cagaaatgca acgcgaaagc gctattttac caacgaagaa tctgtgcttc 10200

atttttgtaa aacaaaaatg caacgcgaga gcgctaattt ttcaaacaaa gaatctgagc 10260

tgcattttta cagaacagaa atgcaacgcg agagcgctat tttaccaaca aagaatctat 10320

acttcttttt tgttctacaa aaatgcatcc cgagagcgct atttttctaa caaagcatct 10380

tagattactt tttttctcct ttgtgcgctc tataatgcag tctcttgata actttttgca 10440

ctgtaggtcc gttaaggtta gaagaaggct actttggtgt ctattttctc ttccataaaa 10500

aaagcctgac tccacttccc gcgtttactg attactagcg aagctgcggg tgcatttttt 10560

caagataaag gcatccccga ttatattcta taccgatgtg gattgcgcat actttgtgaa 10620

cagaaagtga tagcgttgat gattcttcat tggtcagaaa attatgaacg gtttcttcta 10680

ttttgtctct atatactacg tataggaaat gtttacattt tcgtattgtt ttcgattcac 10740

tctatgaata gttcttacta caattttttt gtctaaagag taatactaga gataaacata 10800

aaaaatgtag aggtcgagtt tagatgcaag ttcaaggagc gaaaggtgga tgggtaggtt 10860

atataggggat atagcacaga gatatatagc aaagagatac ttttgagcaa tgtttgtgga 10920

agcggtattc gcaatatttt agtagctcgt tacagtccgg tgcgtttttg gttttttgaa 10980

agtgcgtctt cagagcgctt ttggttttca aaagcgctct gaagttccta tactttctag 11040

agaataggaa cttcggaata ggaacttcaa agcgtttccg aaaacgagcg cttccgaaaa 11100

tgcaacgcga gctgcgcaca tacagctcac tgttcacgtc gcacctatat ctgcgtgttg 11160

cctgtatata tatatacatg agaagaacgg catagtgcgt gtttatgctt aaatgcgtac 11220

ttatatgcgt ctatttatgt aggatgaaag gtagtctagt acctcctgtg atattatccc 11280

attccatgcg gggtatcgta tgcttccttc agcactaccc tttagctgtt ctatatgctg 11340

ccactcctca attggattag tctcatcctt caatgctatc atttcctttg atattggatc 11400

atactaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc 11460

ctttcgtc 11468

<210> 8

<211> 11222

<212> DNA

<213> Artificial Sequence

<400> 8

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accataccac agcttttcaa ttcaattcat catttttttt ttattctttt ttttgatttc 240

ggtttctttg aaattttttt gattcggtaa tctccgaaca gaaggaagaa cgaaggaagg 300

agcacagact tagattggta tatatacgca tatgtagtgt tgaagaaaca tgaaattgcc 360

cagtattctt aacccaactg cacagaacaa aaacctgcag gaaacgaaga taaatctaaa 420

aaactgtatt ataagtaaat gcatgtatac taaactcaca aattagagct tcaatttaat 480

tatatcagtt attaccctat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac 540

cgcatcagga aattgtaaac gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa 600

tcagctcattttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat 660

agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg 720

tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac 780

catcacccta atcaagttttttggggtcga ggtgccgtaa agcactaaat cggaacccta 840

aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag 900

ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg 960

taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtccatt cgccattcag 1020

gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctgga 1080

taaaggcgcg ccgtcgacaa tatagaaatt tcttagtttt aatgattgaa tgaatccatt 1140

tcgaacacgt caatgtactt ggacaagctt tgtagaacat gaagtacttg agcaatggac 1200

gcccaactgg aatttacttt agcgacacta ttccaagacc agcaactcaa agaagtaaat 1260

tctttttttc acaatagttg tttttttgat gatgtacgtc ccagcagcac ctaatggtat 1320

ttttatgagt attggcagtt tgccttatta attaagcaaa ccggtcatat attttaaaaa 1380

ataggaactt tgtagtcctt attaatgctg aataaaaaaa taggaagaga aatcagcaag 1440

tataaacagt taaatgtttt ttaactataa tggttgcatt caagaggaca tgtaaattgc 1500

gtactcggta caagttggta aaaacgttaa agcactccta cgagatttag tcagtcctgg 1560

cattaataaa caaaaagacc tgtatctata tttcatgaac tgggtaaaat caccacacaa 1620

aaaagaataa acaaatataa tgaactactt ttggtcttgg actcgcagtt tatagtttgt 1680

taaaattgac aaacacacat gttctgatta acttacttcc gcttcaaagt actgaaaaca 1740

tcatacgaac tggtataaga aggaagatag caaataaaaa taaatcaaat attgcttgta 1800

aaaaattagg atataaaaaa atctctattc attaaagaca aaaagctaaa ggaataaaaa 1860

agtcattcgc tttacgccaa ataaaacgtc tatgcacgtt tttaaaggct gtgaacatta 1920

acaatgttgt ggaattcagg cttgagagaa gcaccgccaa ccaagaaacc gtcaatatcg 1980

tggaacttga ggaactcctt gcagttacca ccgttaacgg aaccaccgta gatgacacgg 2040

agaccctcgg caacagatgc accaagcttg ttggtagccc acttgcggat ctcagcgtga 2100

acctcttgag cttgctcagg ggtggcagtc ttaccagtac caatggccca gacaggctca 2160

taagcaatga caatcttgga ccagttctgg accttgtcag cgatggcgtt caattgacga 2220

acaacaacgt tgatggtctc gttagcctca cgctcggcca aagtctcacc aatgcaggca 2280

acgacagtaa gaccttgttc aagggcaaac ttggtcttgt cggcaacgaa ctcgtcagac 2340

tccttgaaga tggtacgacg ctcggagtga ccagtcaaag tgtaggtaat accagcatca 2400

atcaaagatt gagcactgtt ctcaccagtg taggcaccgt tcttcttgtc gaagacgttt 2460

tgggcaccaa cgccaatatc cttcttgact tgttggcggg tggtgatgag gtacatgttt 2520

tgagggaaga tgacagtttc gacatcacca acgttaagct tggtggtgtt caaaccctca 2580

ataatagtct tcatggactc caaagagcca ttcatcttaa agttaccacc gacaaagaat 2640

ttacgtgcca ttttgatcct agttctacta ttgaaatgta gttggggaga gacgagagtt 2700

ggcccgtccg ctcattatat ataacgtagc ggacagtcac acagttaagg ggaattaccg 2760

agcttcggca atttaccccg tcgatagcaa ccgttggcat ggatccggcc ggccagatct 2820

acgtatggtc atttcttctt cagattccct catggagaaa gtgcggcaga tgtatatgac 2880

agagtcgcca gtttccaaga gactttatattc aggcacttcc atgataggca agagagaaga 2940

cccagagatg ttgttgtcct agttacacat ggtatttatt ccagagtatt cctgatgaaa 3000

tggtttagat ggacatacga agagtttgaa tcgtttacca atgttcctaa cgggagcgta 3060

atggtgatgg aactggacga atccatcaat agatacgtcc tgaggaccgt gctacccaaa 3120

tggactgatt gtgagggaga cctaactaca tagtgtttaa agattacgga tatttaactt 3180

acttagaata atgccatttttttgagttat aataatccta cgttagtgtg agcgggattt 3240

aaactgtgag gaccttaata cattcagaca cttctgcggt atcaccctac ttatccctt 3300

cgagattata tctaggaacc catcaggttg gtggaagatt acccgttcta agacttttca 3360

gcttcctcta ttgatgttac acctggacac cccttttctg gcatccagtt tttaatcttc 3420

agtggcatgt gagattctcc gaaattaatt aaagcaatca cacaattctc tcggatacca 3480

cctcggttga aactgacagg tggtttgtta cgcatgctaa tgcaaaggag cctatatacc 3540

tttggctcgg ctgctgtaac agggaatata aagggcagca taatttagga gtttagtgaa 3600

cttgcaacat ttactatttt cccttcttac gtaaatattt ttctttttaa ttctaaatca 3660

atctttttca attttttgtt tgtattcttt tcttgcttaa atctataact acaaaaaaca 3720

catacataaa ctaaaaggta ccaacaaaat gagatttcca tctattttta ctgctgtttt 3780

gtttgctgct tcttctgctt tggctgctcc agttaatact actactgaag atgaaactgc 3840

tcaaattcca gctgaagctg ttattggtta ttctgatttg gagggtgact ttgatgttgc 3900

tgttttgcca ttttctaact ctactaacaa cggtttgcta ttcatcaaca ctactatcgc 3960

ttctatcgct gctaaagaag aaggtgtttc tttggataaa agagaagaag gtgaaccaaa 4020

agctgttgac aagaacaatg ccactgctgc cgtccaaaac gaatccaagc gttacactgt 4080

ttcttactta aagaccttga actattacga cttggtcgat ctattggtta agaccgaaat 4140

cgaaaactta ccagacttgt ttcaatactc ttccgacgct aaggaatttt acggtaacaa 4200

gactagaatg tcctttatca tggacgaaat tggtcgtaga gctccacaat acactgaaat 4260

tgaccacaag ggtataccaa ccctcgttga agtcgtcaga gccgggttct acttgggttt 4320

tcacaacaaa gaattgaatg aaatcaacaa gagatctttc aaagaaagag ttatcccgtc 4380

tatcttggct atccaaaaga acccaaactt caaattaggt actgaagttc aagacaagat 4440

tgtctctgct accggtctat tggctggtaa cgaaaccgct ccaccagaag ttgttaacaa 4500

cttcacccca atcttgcaag attgtatcaa gaacatcgac agatacgcct tggacgactt 4560

gaaaagtaaa gcattgttca acgttttggc cgccccaacc tacgacatta ctgaatacct 4620

cagagctacc aaggaaaagc cagaaaacac tccatggtac ggtaagatcg atggtttcat 4680

caacgaattg aagaagctcg ctttgtacgg taaaattaac gacaacaact cttggattat 4740

cgataacggt atttaccaca ttgctccatt gggtaaattg cactctaaca acaaaatcgg 4800

tatcgaaact ctaactgaag ttatgaaggt ctacccatac ttgtccatgc aacacttgca 4860

atccgctgac caaatcaaga gacactacga ctctaaggat gccgaaggta acaagatccc 4920

attagataag ttcaagaagg aaggcaagga aaagtactgc ccaaagacct acacttttga 4980

tgatggtaag gttatcatca aggccggtgc tcgtgtcgaa gaagaaaagg tcaaaagatt 5040

atactgggct tccaaggaag tcaacagtca attcttcaga gtttatggta ttgacaagcc 5100

attggaagaa ggtaacccag acgacatcct taccatggtt atttacaact ctccagaaga 5160

atacaaactt aactccgtct tgtacggtta cgataccaac aacggtggta tgtacattga 5220

accagaaggt acttttttca cttacgaaag agaagctcaa gaatctacgt acactttgga 5280

agaattattc agacacgaat acacccatta cttgcaaggt agatacgctg tcccaggtca 5340

atggggtcgt accaagttgt atgataacga cagattgacc tggtacgaag aaggtggtgc 5400

tgaactgttt gctggttcta ctagaacttc cggcatcttg ccaagaaagt ctattgtctc 5460

taacattcac aacaccacta gaaacaacag atacaagctg tctgacactg ttcattcgaa 5520

gtacggtgcc tctttcgaat tttacaacta cgcttgtatg ttcatggact acatgtacaa 5580

taaggacatg ggtattttga acaagttgaa tgatttggct aagaacaacg acgttgacgg 5640

ttacgacaat tacatcagag atttgtcatc taattacgct ttgaacgata agtaccaaga 5700

tcacatgcaa gaacgtattg acaactatga aaacttgacc gttccattcg ttgctgacga 5760

ctacttagtt agacacgctt acaagaaccc aaacgaaatt tactccgaaa tctccgaagt 5820

tgccaagttg aaggatgcta agtctgaagt taagaaatct caatacttct caactttcac 5880

tttgcgtggt agttacaccg gaggtgcttc caagggtaag ctggaagatc aaaaagctat 5940

gaacaagttc attgacgact ctttgaagaa gttggacacc tattcctggt ctggttacaa 6000

gactcttact gcttatttta ccaactacaa ggtcgattct tctaacagag ttacttacga 6060

tgtcgttttc catggttatact tgccaaacga aggtgattcc aagaacagct tgccatacgg 6120

taagattaac ggtacttaca agggtactga gaaggaaaag attaagttct cctccgaagg 6180

ttctttcgac ccagacggaa agatagtttc ctacgaatgg gacttcggtg atggtaacaa 6240

gtccaacgaa gagaaccctg aacacagtta cgataaggtc ggtacttaca ctgtcaagtt 6300

gaaggttacc gatgataagg gtgaaagctc tgtttctact accactgctg aaatcaaaga 6360

tttgtctgaa aacaagttgc cagtcatcta tatgcacgtt cctaaatcgg gtgctttaaa 6420

ccaaaaggtt gtcttctacg gtaagggtac ttacgaccca gacggttcta tcgctggcta 6480

ccaatgggat ttcggtgacg gttctgactt ttcctctgaa cagaacccat ctcacgtcta 6540

caccaagaaa ggtgaataca ctgtgacctt gagagtcatg gattcttccg gtcaaatgtc 6600

tgaaaagacc atgaaaatca agatcactga tccagtatac cctattggta ctgaaaagga 6660

accaaacaac tctaaggaaa cagcctccgg tccaatcgtt ccgggtattc cagtctccgg 6720

tactatcgaa aacacctctg atcaagacta tttctacttc gatgttatta ctccaggtga 6780

agtcaagatc gacatcaaca agctaggtta cggtggtgct acatgggttg tttacgatga 6840

aaacaataac gctgtctcct acgctaccga tgacggtcaa aatttgtctg gtaaattcaa 6900

ggctgacaag cctgggagat actacattca tttgtacatg ttcaatggtt cttacatgcc 6960

atacagaatt aacattgaag gttctgttgg tagacacccac caccaccacc actaagctag 7020

cctcgagtct agaaactaag attaatataa ttatataaaa atattatctt cttttcttta 7080

tatctagtgt tatgtaaaat aaattgatga ctacggaaag cttttttata ttgtttcttt 7140

ttcattctga gccacttaaa tttcgtgaat gttcttataa gggacggtag atttacaagt 7200

gatacaacaa aaagcaaggc gctttttcta ataaaaagaa gaaaagcatt taacaattga 7260

acacctctat atcaacgaag aatattactt tgtctctaaa tccttgtaaa atgtgtacga 7320

tctctatatg ggttactcag aagtgtaccg aagactgcat tgaaagttta tgttttttca 7380

ctgcaagcgt cattttcgct ttgagaagat gttcttattc aaatttcaac tgttatatag 7440

aagagcaaaa aattgccaaa aaaaacaaca tttattcatt taaaatataa aatttgggct 7500

tctatatttt aatattgctt ttcaattact gttattaaat gtaagtactg cgtctatgaa 7560

aatatatgca aatgctaaga aaaatcctaa aaatttgaat atgagatatt cctcagtatt 7620

tctttttcat cctttcttct gcggctctag ccctttgttc tctcatcaat ctgcgtctct 7680

gttcatcggt caaagaattc aaattttgtt gctgaattga aggaataacg cgtgtacgca 7740

tgtaacatta tactgaaaac cttgcttgag aaggttttgg gacgctcgaa gatcctccgg 7800

atcgtttcgc cggcgtttat ccagctgcat taatgaatcg gccaacgcgc ggggagaggc 7860

ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt 7920

cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca 7980

ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa 8040

aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat 8100

cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc 8160

cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc 8220

gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt 8280

tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac 8340

cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg 8400

ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca 8460

gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc 8520

gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa 8580

accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa 8640

ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac 8700

tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta 8760

aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt 8820

taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata 8880

gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc 8940

agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac 9000

cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag 9060

tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac 9120

gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc 9180

agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg 9240

gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc 9300

atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct 9360

gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc 9420

tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc 9480

atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc 9540

agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc 9600

gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca 9660

cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt 9720

tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt 9780

ccgcgcacat ttccccgaaa agtgccacct gaacgaagca tctgtgcttc attttgtaga 9840

acaaaaatgc aacgcgagag cgctaatttt tcaaacaaag aatctgagct gcatttttac 9900

agaacagaaa tgcaacgcga aagcgctatt ttaccaacga agaatctgtg cttcattttt 9960

gtaaaacaaa aatgcaacgc gagagcgcta atttttcaaa caaagaatct gagctgcatt 10020

tttacagaac agaaatgcaa cgcgagagcg ctattttacc aacaaagaat ctatacttct 10080

tttttgttct acaaaaatgc atcccgagag cgctattttt ctaacaaagc atcttagatt 10140

actttttttc tcctttgtgc gctctataat gcagtctctt gataactttt tgcactgtag 10200

gtccgttaag gttagaagaa ggctactttg gtgtctattt tctcttccat aaaaaaagcc 10260

tgactccact tcccgcgttt actgattact agcgaagctg cgggtgcatt ttttcaagat 10320

aaaggcatcc ccgattatat tctataccga tgtggattgc gcatactttg tgaacagaaa 10380

gtgatagcgt tgatgattct tcattggtca gaaaattatg aacggtttct tctattttgt 10440

ctctatatac tacgtatagg aaatgtttac attttcgtat tgttttcgat tcactctatg 10500

aatagttctt actacaattt ttttgtctaa agagtaatac tagagataaa cataaaaaat 10560

gtagaggtcg agtttagatg caagttcaag gagcgaaagg tggatgggta ggttatatag 10620

ggatatagca cagagatata tagcaaagag atacttttga gcaatgtttg tggaagcggt 10680

attcgcaata ttttagtagc tcgttacagt ccggtgcgtt tttggttttt tgaaagtgcg 10740

tcttcagagc gcttttggtt ttcaaaagcg ctctgaagtt cctatacttt ctagagaata 10800

ggaacttcgg aataggaact tcaaagcgtt tccgaaaacg agcgcttccg aaaatgcaac 10860

gcgagctgcg cacatacagc tcactgttca cgtcgcacct atatctgcgt gttgcctgta 10920

tatatatata catgagaaga acggcatagt gcgtgtttat gcttaaatgc gtacttatat 10980

gcgtctattt atgtaggatg aaaggtagtc tagtacctcc tgtgatatta tcccattcca 11040

tgcggggtat cgtatgcttc cttcagcact accctttagc tgttctatat gctgccactc 11100

ctcaattgga ttagtctcat ccttcaatgc tatcatttcc tttgatattg gatcatacta 11160

agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg 11220

tc 11222

<210> 9

<211> 11462

<212> DNA

<213> Artificial Sequence

<400> 9

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accataccac agcttttcaa ttcaattcat catttttttt ttattctttt ttttgatttc 240

ggtttctttg aaattttttt gattcggtaa tctccgaaca gaaggaagaa cgaaggaagg 300

agcacagact tagattggta tatatacgca tatgtagtgt tgaagaaaca tgaaattgcc 360

cagtattctt aacccaactg cacagaacaa aaacctgcag gaaacgaaga taaatctaaa 420

aaactgtatt ataagtaaat gcatgtatac taaactcaca aattagagct tcaatttaat 480

tatatcagtt attaccctat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac 540

cgcatcagga aattgtaaac gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa 600

tcagctcattttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat 660

agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg 720

tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac 780

catcacccta atcaagttttttggggtcga ggtgccgtaa agcactaaat cggaacccta 840

aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag 900

ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg 960

taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtccatt cgccattcag 1020

gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctgga 1080

taaaggcgcg ccgtcgacaa tatagaaatt tcttagtttt aatgattgaa tgaatccatt 1140

tcgaacacgt caatgtactt ggacaagctt tgtagaacat gaagtacttg agcaatggac 1200

gcccaactgg aatttacttt agcgacacta ttccaagacc agcaactcaa agaagtaaat 1260

tctttttttc acaatagttg tttttttgat gatgtacgtc ccagcagcac ctaatggtat 1320

ttttatgagt attggcagtt tgccttatta attaagcaaa ccggtcatat attttaaaaa 1380

ataggaactt tgtagtcctt attaatgctg aataaaaaaa taggaagaga aatcagcaag 1440

tataaacagt taaatgtttt ttaactataa tggttgcatt caagaggaca tgtaaattgc 1500

gtactcggta caagttggta aaaacgttaa agcactccta cgagatttag tcagtcctgg 1560

cattaataaa caaaaagacc tgtatctata tttcatgaac tgggtaaaat caccacacaa 1620

aaaagaataa acaaatataa tgaactactt ttggtcttgg actcgcagtt tatagtttgt 1680

taaaattgac aaacacacat gttctgatta acttacttcc gcttcaaagt actgaaaaca 1740

tcatacgaac tggtataaga aggaagatag caaataaaaa taaatcaaat attgcttgta 1800

aaaaattagg atataaaaaa atctctattc attaaagaca aaaagctaaa ggaataaaaa 1860

agtcattcgc tttacgccaa ataaaacgtc tatgcacgtt tttaaaggct gtgaacatta 1920

acaatgttgt ggaattcagg cttgagagaa gcaccgccaa ccaagaaacc gtcaatatcg 1980

tggaacttga ggaactcctt gcagttacca ccgttaacgg aaccaccgta gatgacacgg 2040

agaccctcgg caacagatgc accaagcttg ttggtagccc acttgcggat ctcagcgtga 2100

acctcttgag cttgctcagg ggtggcagtc ttaccagtac caatggccca gacaggctca 2160

taagcaatga caatcttgga ccagttctgg accttgtcag cgatggcgtt caattgacga 2220

acaacaacgt tgatggtctc gttagcctca cgctcggcca aagtctcacc aatgcaggca 2280

acgacagtaa gaccttgttc aagggcaaac ttggtcttgt cggcaacgaa ctcgtcagac 2340

tccttgaaga tggtacgacg ctcggagtga ccagtcaaag tgtaggtaat accagcatca 2400

atcaaagatt gagcactgtt ctcaccagtg taggcaccgt tcttcttgtc gaagacgttt 2460

tgggcaccaa cgccaatatc cttcttgact tgttggcggg tggtgatgag gtacatgttt 2520

tgagggaaga tgacagtttc gacatcacca acgttaagct tggtggtgtt caaaccctca 2580

ataatagtct tcatggactc caaagagcca ttcatcttaa agttaccacc gacaaagaat 2640

ttacgtgcca ttttgatcct agttctacta ttgaaatgta gttggggaga gacgagagtt 2700

ggcccgtccg ctcattatat ataacgtagc ggacagtcac acagttaagg ggaattaccg 2760

agcttcggca atttaccccg tcgatagcaa ccgttggcat ggatccggcc ggccagatct 2820

acgtatggtc atttcttctt cagattccct catggagaaa gtgcggcaga tgtatatgac 2880

agagtcgcca gtttccaaga gactttattc aggcacttcc atgataggca agagagaaga 2940

cccagagatg ttgttgtcct agttacacat ggtatttatt ccagagtatt cctgatgaaa 3000

tggtttagat ggacatacga agagtttgaa tcgtttacca atgttcctaa cgggagcgta 3060

atggtgatgg aactggacga atccatcaat agatacgtcc tgaggaccgt gctacccaaa 3120

tggactgatt gtgagggaga cctaactaca tagtgtttaa agattacgga tatttaactt 3180

acttagaata atgccatttttttgagttat aataatccta cgttagtgtg agcgggattt 3240

aaactgtgag gaccttaata cattcagaca cttctgcggt atcaccctac ttattccctt 3300

cgagattata tctaggaacc catcaggttg gtggaagatt acccgttcta agacttttca 3360

gcttcctcta ttgatgttac acctggacac cccttttctg gcatccagtt tttaatcttc 3420

agtggcatgt gagattctcc gaaattaatt aaagcaatca cacaattctc tcggatacca 3480

cctcggttga aactgacagg tggtttgtta cgcatgctaa tgcaaaggag cctatatacc 3540

tttggctcgg ctgctgtaac agggaatata aagggcagca taatttagga gtttagtgaa 3600

cttgcaacat ttactatttt cccttcttac gtaaatattt ttctttttaa ttctaaatca 3660

atctttttca attttttgtt tgtattcttt tcttgcttaa atctataact acaaaaaaca 3720

catacataaa ctaaaaggta ccaacaaaat gagatttcca tctattttta ctgctgtttt 3780

gtttgctgct tcttctgctt tggctgctcc agttaatact actactgaag atgaaactgc 3840

tcaaattcca gctgaagctg ttattggtta ttctgatttg gagggtgact ttgatgttgc 3900

tgttttgcca ttttctaact ctactaacaa cggtttgcta ttcatcaaca ctactatcgc 3960

ttctatcgct gctaaagaag aaggtgtttc tttggataaa agagaggctg aagctaagcc 4020

aatcgaaaac actaacgaca ccagcatcaa gaacgttgaa aagttgagaa acgccccaaa 4080

cgaagaaaac tctaagaaag ttgaagactc taagaatgat aaggttgaac acgttaagaa 4140

cattgaagaa gctaaggtcg aacaagttgc tccagaagtc aagtctaaaa gtaccttgag 4200

atctgcttct atagctaata caaattccga aaagtacgac ttcgaatatc ttaatggttt 4260

gagttacacc gaattgacta acttgatcaa gaacatcaag tggaaccaaa tcaacggttt 4320

attcaactac tccaccggtt cgcaaaaatt tttcggtgac aagaacagag tccaagccat 4380

catcaacgct ctacaagaat ctggtagaac ttacactgcc aatgacatga agggtattga 4440

aactttcacc gaagttttga gagccggttt ttacttgggc tattacaacg atggtctctc 4500

ctacctgaac gaccgtaatt tccaagacaa gtgtatccca gccatgattg ctattcaaaa 4560

gaacccaaac ttcaagttag gtactgctgt tcaagacgaa gttatcacat ctttgggtaa 4620

gttgattggt aacgcctctg ctaacgctga agttgttaat aactgtgtcc cagttttgaa 4680

gcaattcaga gaaaacttaa accaatacgc tccagactac gtcaaaggta ctgccgtaaa 4740

tgaattgatc aagggtattg aatttgactt ttccggtgct gcttacgaaa aggacgtcaa 4800

gaccatgcca tggtacggta agattgaccc attcatcaat gaattaaagg ccttgggtct 4860

ctacggtaac atcacttctg ccactgaatg ggcttcagat gttggtatct actacttgtc 4920

taagttcggt ttgtactcaa ccaatcgtaa cgacattgtt caatccttag aaaaggctgt 4980

tgatatgtac aaatatggta agatcgcttt cgttgctatg gaaagaatca cctgggatta 5040

cgatggtatc ggttccaacg ggaagaaggt cgaccacgat aagttcttgg acgatgcaga 5100

aaagcattatttgccaaaga cctatacttt cgacaacgga accttcatca tcagagctgg 5160

agataaggtt tctgaagaaa agattaagag attgtactgg gcttcccgtg aagttaagtc 5220

tcaattccac agagttgttg gtaacgataa ggctttggaa gtgggtaacg ctgacgatgt 5280

cttgaccatg aaaatcttca actctccaga agaatacaag ttcaacacca acattaacgg 5340

tgtctccact gataacggtg gtttgtacat cgaaccaaga ggtactttct acacttacga 5400

aagaacccca caacaatcta ttttctcctt ggaagaactg ttcagacacg aatacaccca 5460

ctacttgcaa gctagatact tggttgacgg tttgtggggt caaggtcctt tctacgaaaa 5520

aaacagattg acttggttcg atgaaggtac tgctgaattt ttcgctggtt ccaccagaac 5580

ttctggtgtg ttgccaagaa agtccatctt gggttacctt gccaaggata aggtcgatca 5640

tagatactcc ttgaagaaaa ctttgaactc tggttacgat gactctgact ggatgttcta 5700

caactacggt tttgctgttg ctcactactt atacgaaaag gacatgccaa cttttattaa 5760

gatgaacaag gccatcttga acactgatgt taagtcttac gacgaaatca ttaaaaaact 5820

gtctgacgac gctaacaaga acactgaata ccaaaaccac atccaagaac tcgctgataa 5880

gtaccaaggt gccggtatac cattggtgtc tgatgactac ctgaaggacc atggttacaa 5940

gaaggcttcc gaagtttact cagaaatctc caaggctgcg tctttaacta acacttctgt 6000

taccgctgaa aagtctcaat acttcaacac attcactttg aggggtactt acactggtga 6060

aacctctaag ggtgaattta aggactggga cgaaatgtct aagaagttgg acggtacttt 6120

ggagtcttta gctaagaact cttggtctgg ttacaagact ttgaccgctt acttcaccaa 6180

ctacagagtc acttctgata acaaagtcca atatgatgtt gtcttccacg gtgttttgac 6240

tgacaacgcc gatatttcta acaacaaggc tcctattgct aaggtcaccg gtccatctac 6300

tggtgctgtt ggtagaaaca ttgaattttc tggtaaggat tccaaggacg aagatggcaa 6360

gattgtctct tatgactggg atttcggtga tggtgctacc tccagaggta agaactctgt 6420

ccacgcttac aagaaggctg gtacttacaa cgtcactttg aaggtcaccg atgacaaggg 6480

tgctacggct actgaaagtt tcactattga aattaaaaac gaagacacta ccaccccaat 6540

caccaaggaa atggaaccaa acgatgacat taaggaagcc aacggcccga ttgtcgaagg 6600

cgtcacggtc aaaggtgact tgaacggttc tgatgacgct gacactttct actttgatgt 6660

caaggaagac ggtgacgtta ccatcgaatt gccatactct ggttcctcta actttacctg 6720

gttagtctac aaggaaggtg acgaccaaaa ccacatcgcc tccggtatcg ataaaaacaa 6780

ctccaaggtt ggtactttca agtccactaa gggtcgtcat tacgttttca tctacaagca 6840

cgactctgct tccaacattt catactcctt gaacatcaag ggtttgggta acgaaaagtt 6900

gaaagagaag gaaaacaatg actcctctga caaggcaacc gttattccaa atttcaatac 6960

caccatgcaa ggttctctat taggggacga ttctagagac tactactcct tcgaagtcaa 7020

ggaagaaggt gaagttaaca ttgaattgga taaaaaggat gaatttggtg ttacttggac 7080

cttgcaccca gaaagcaaca tcaacgacag aattacctac ggtcaagtcg atggtaacaa 7140

ggtatccaac aaggtcaaat tgagaccagg taagtactac ttgttggttt acaagtacag 7200

tggttccggt aactacgaat tgcgggttaa caagcaccac caccaccacc actaagctag 7260

cctcgagtct agaaactaag attaatataa ttatataaaa atattatctt cttttcttta 7320

tatctagtgt tatgtaaaat aaattgatga ctacggaaag ctttttttata ttgtttcttt 7380

ttcattctga gccacttaaa tttcgtgaat gttcttataa gggacggtag atttacaagt 7440

gatacaacaa aaagcaaggc gctttttcta ataaaaagaa gaaaagcatt taacaattga 7500

acacctctat atcaacgaag aatattactt tgtctctaaa tccttgtaaa atgtgtacga 7560

tctctatatg ggttactcag aagtgtaccg aagactgcat tgaaagttta tgttttttca 7620

ctgcaagcgt cattttcgct ttgagaagat gttcttattc aaatttcaac tgttatatag 7680

aagagcaaaa aattgccaaa aaaaacaaca tttattcatt taaaatataa aatttgggct 7740

tctatatttt aatattgctt ttcaattact gttattaaat gtaagtactg cgtctatgaa 7800

aatatatgca aatgctaaga aaaatcctaa aaatttgaat atgagatatt cctcagtatt 7860

tctttttcat cctttcttct gcggctctag ccctttgttc tctcatcaat ctgcgtctct 7920

gttcatcggt caaagaattc aaattttgtt gctgaattga aggaataacg cgtgtacgca 7980

tgtaacatta tactgaaaac cttgcttgag aaggttttgg gacgctcgaa gatcctccgg 8040

atcgtttcgc cggcgtttat ccagctgcat taatgaatcg gccaacgcgc ggggagaggc 8100

ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt 8160

cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca 8220

ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa 8280

aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat 8340

cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc 8400

cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc 8460

gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt 8520

tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac 8580

cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg 8640

ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca 8700

gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc 8760

gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa 8820

accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa 8880

ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac 8940

tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta 9000

aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt 9060

taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata 9120

gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc 9180

agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac 9240

cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag 9300

tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac 9360

gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc 9420

agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg 9480

gttagctccttcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc 9540

atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct 9600

gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc 9660

tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc 9720

atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc 9780

agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc 9840

gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca 9900

cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt 9960

tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt 10020

ccgcgcacat ttccccgaaa agtgccacct gaacgaagca tctgtgcttc attttgtaga 10080

acaaaaatgc aacgcgagag cgctaatttt tcaaacaaag aatctgagct gcatttttac 10140

agaacagaaa tgcaacgcga aagcgctatt ttaccaacga agaatctgtg cttcattttt 10200

gtaaaacaaa aatgcaacgc gagagcgcta atttttcaaa caaagaatct gagctgcatt 10260

tttacagaac agaaatgcaa cgcgagagcg ctattttacc aacaaagaat ctatacttct 10320

tttttgttct acaaaaatgc atcccgagag cgctattttt ctaacaaagc atcttagatt 10380

actttttttc tcctttgtgc gctctataat gcagtctctt gataactttt tgcactgtag 10440

gtccgttaag gttagaagaa ggctactttg gtgtctattt tctcttccat aaaaaaagcc 10500

tgactccact tcccgcgttt actgattact agcgaagctg cgggtgcatt ttttcaagat 10560

aaaggcatcc ccgattatat tctataccga tgtggattgc gcatactttg tgaacagaaa 10620

gtgatagcgt tgatgattct tcattggtca gaaaattatg aacggtttct tctattttgt 10680

ctctatatac tacgtatagg aaatgtttac attttcgtat tgttttcgat tcactctatg 10740

aatagttctt actacaattt ttttgtctaa agagtaatac tagagataaa cataaaaaat 10800

gtagaggtcg agtttagatg caagttcaag gagcgaaagg tggatgggta ggttatatag 10860

ggatatagca cagagatata tagcaaagag atacttttga gcaatgtttg tggaagcggt 10920

attcgcaata ttttagtagc tcgttacagt ccggtgcgtt tttggttttt tgaaagtgcg 10980

tcttcagagc gcttttggtt ttcaaaagcg ctctgaagtt cctatacttt ctagagaata 11040

ggaacttcgg aataggaact tcaaagcgtt tccgaaaacg agcgcttccg aaaatgcaac 11100

gcgagctgcg cacatacagc tcactgttca cgtcgcacct atatctgcgt gttgcctgta 11160

tatatatata catgagaaga acggcatagt gcgtgtttat gcttaaatgc gtacttatat 11220

gcgtctattt atgtaggatg aaaggtagtc tagtacctcc tgtgatatta tcccattcca 11280

tgcggggtat cgtatgcttc cttcagcact accctttagc tgttctatat gctgccactc 11340

ctcaattgga ttagtctcat ccttcaatgc tatcatttcc tttgatattg gatcatacta 11400

agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg 11460

tc 11462

Claims (6)

1. A recombinant plasmid for expressing collagenase, characterized in that: the leader sequence of α factor and the leader peptide sequence and enzyme functional region sequence propeptide-Chain of Clostridium histolytica collagenase ColG are inserted into the expression plasmid; or It is to insert the leader sequence and spacer sequence leader-spacer of α factor and the leader peptide sequence and enzyme functional region sequence pro peptide-Chain of Clostridium histolytica collagenase ColH into the expression plasmid; The leader sequence of the α factor is shown in SEQ ID No. 1; The leader peptide sequence and enzyme functional region sequence pro peptide-Chain of ColG are shown in SEQ ID No. 2; The leader sequence and spacer sequence leader-spacer of the α factor are shown in SEQ ID No. 3; The leader peptide sequence and enzyme functional region sequence pro peptide-Chain of ColH are shown in SEQ ID No. 4; The expression plasmid is CPOTud. 2. A Saccharomyces cerevisiae that secretes collagenase, characterized in that: it is obtained by transforming the recombinant plasmid of claim 1 into a yeast strain; The starting yeast strain is Saccharomyces cerevisiae B184M. 3. Use of a fermentation medium for culturing the brewer's yeast according to claim 2, characterized in that Ca ²⁺ and Zn ²⁺ are added to the YPD medium; The concentration of Ca ²⁺ is 10 mM, and the concentration of Zn ²⁺ is 0.6 mM. 4. A fermentation culture method of Saccharomyces cerevisiae according to claim 2, characterized in that it includes the following steps: Saccharomyces cerevisiae according to claim 2 is inoculated into YPD medium supplemented with Ca ²⁺ and Zn ^{2+ and} cultured; The concentration of Ca ²⁺ is 10 mM, and the concentration of Zn ²⁺ is 0.6 mM; The described culture is to culture at 30-37°C for at least 96 hours, and then the recombinant collagenase can be collected. 5. Use of the recombinant plasmid according to claim 1 and the Saccharomyces cerevisiae according to claim 2 in producing collagenase. 6. Application of the recombinant plasmid of claim 1 and the Saccharomyces cerevisiae of claim 2 in degrading gelatin and collagen.