CN113174353A - Genetic engineering strain, preparation thereof and directed evolution method based on antibody affinity maturation of strain - Google Patents

Abstract

The invention relates to the field of continuous directed evolution, and relates to a genetic engineering strain, preparation thereof and a continuous directed evolution method based on antibody affinity maturation of the strain. The strain is obtained by constructing a mutant plasmid and a screening target plasmid into a modified strain JS 200-delta thya delta fola; the mutant plasmid comprises pEP-mcs- (GGGGS)2-F1, 2-Flag; the screening target plasmid comprises pLA230-mcs- (GGGGS)2-F3-6 xHis. The antigen and antibody are fused to two functionally complementary fragments F1,2 and F3 of mDHFR (murine DHFR), respectively, to form Ag-F1,2 and Ab-F3. Through the graded and increasing Trimethoprim (TMP) resistance screening, the method can be realized in a solid/liquid space, and the antibody monoclonal strain with improved affinity is obtained after solid/liquid culture, so that the operation is simple and the flux is high.

Description

Genetic engineering strain, preparation thereof and directed evolution method based on antibody affinity maturation of strain

Technical Field

The invention relates to the technical field of directed evolution, in particular to a genetic engineering strain, preparation thereof and a directed evolution method based on antibody affinity maturation of the strain.

Background

Antibody affinity maturation (affinity maturation) is a process by which the affinity, activity, and antigen binding capacity of antibodies are improved. In the natural process, the antibody affinity maturation is realized by repeatedly stimulating and selecting B cell immunoglobulin genes for multiple times through antigens under the conditions of VDJ rearrangement and somatic cell mutation. The high affinity antibody has great application value in the first infection and the treatment process after infection. In 2005, Song et al first reported that antibody-mediated in vivo targeted gene therapy, in which antibody fragments were mixed with siRNA (c-myc, MDM2, VEGF) to form complexes that target in vivo cells containing the corresponding antigen, effectively inhibited tumor growth (Song E, et al Nature biotechnology,2005,23(6): 709-717). In addition, the PD1/PDL1 is used for treating tumors, namely, the antibodies act on PD1 or PDL1 to achieve the purposes of activating an immune system and removing tumors. The acquisition of high affinity antibodies has great application value in the treatment of tumors and other diseases.

According to the principle of in vivo antibody affinity maturation, people are always developing methods for accelerating maturation of antibody affinity, and at present, there are two main strategies, an antibody affinity maturation strategy for simulating high-frequency mutation of somatic cells and an antibody affinity maturation strategy based on an antibody library.

Antibody affinity maturation strategies that mimic somatic high-frequency mutations fall into two broad categories, B-cell based and other cell-based. Among them, Sarah reported that high affinity IgM for streptavidin was selected using Ramos as early as 2002 based on the strategy of B cells (Cumbers, et al. Nature biotechnology,2002,20(11): 1129-1134.). And fixing streptavidin on the magnetic beads to screen the IgM, and carrying out iterative evolution by reducing the density of the streptavidin on the magnetic beads to screen the IgM with high affinity. However, the use of B cells for antibody affinity maturation has disadvantages, such as difficult gene manipulation, inability of post-translational modification of proteins to meet the requirements of all foreign proteins, and the like. Attempts have therefore been made in other cells, such as the system designed by Gregory Coia et al (Coia G, et al. journal of immunological methods,2001,251(1-2):187-193.), to generate random libraries of mutations in E.coli followed by screening with the aid of phage display technology, and to generate high affinity mutants after several iterations. The system is carried out in vivo, reduces a part of labor capacity, and can realize continuous evolution to a certain extent. However, since the system involves two species, the system design is complicated, and human intervention is required in the experimental process, which cannot be performed continuously.

The antibody affinity maturation strategy based on the antibody library mainly focuses on the construction of the mutation library and the selection of a screening system. The choice of mutation sites and mutation patterns is a major issue when constructing a library of mutations. There are three commonly used mutation strategies: random mutagenesis, site-directed mutagenesis, DNA shuffling.

For random mutation, error-prone PCR is mainly adopted, and Mg can be adjusted during PCR²⁺The concentration, the proportion of dNTPs, the selection of error-prone Taq enzyme and the like introduce mutation on antibody genes, the mutation is accumulated in multiple PCR processes, the mutation diversity is enlarged, and the method has the advantages of easiness in operation, high mutation frequency, relatively uniform mutation spectrum and the like.

For site-directed mutagenesis, saturation mutagenesis can be performed on a specific site by designing a degenerated primer, so that the quality of a mutation library can be improved, and an amino acid saturation mutation library with the specific site can be obtained, but the site selection depends on analysis of an antibody structure and biochemical function analysis.

For DNA shuffling (DNA shuffling), it is a PCR-based technique for constructing mutant libraries. Fragmenting antibody genes by using deoxyribonuclease I, then in the renaturation process of PCR reaction, mismatching homologous sections, using the mismatched sections as templates to extend, and repeating denaturation, renaturation and extension until the full-length genes are amplified. These genes undergo recombination due to mismatches during the elongation process, resulting in a library of mutations. The subsequent StEP, SCRATCH, RACHITT and other methods are all similar principles. The process of affinity maturation of the natural antibody is simulated to a certain extent, and the in vitro evolution speed is accelerated.

The screening system mainly comprises systems such as phage display, yeast display, ribosome display and the like.

In the phage display technology, an exogenous DNA fragment encoding a polypeptide is fused with a gene encoding a phage surface protein and then is displayed on the surface of a phage in the form of fusion protein, and the displayed polypeptide or protein can maintain relative spatial structure and biological activity and is displayed on the surface of the phage.

For the yeast display technology, the target protein and the yeast surface protein are mainly subjected to fusion expression to realize the yeast surface display of the protein.

In the ribosome display technology, a correctly folded protein and mRNA thereof are simultaneously bound to a ribosome to form an mRNA-ribosome-protein trimer, so that the genotype and the phenotype of the target protein are directly related.

The antibody affinity maturation strategy based on the antibody library needs continuous iteration to construct a mutation library, transform plasmids and screen, and the antibody with the desired characteristics can be obtained through several rounds of experiments, so that the method is a work with huge labor.

The existing in vivo continuous directed evolution methods, like the OrthoRep system (Ravikumar a, et al cell,2018,175(7):1946-1957.e13.) proposed by Chang c.liu et al and the phage assisted continuous evolution system (PACE) (envelt K M, et al nature,2011,472(7344):499 and 503.) proposed by David Liu et al, can simultaneously achieve the construction and screening of antibody mutation libraries, while the former has been used for antibody affinity improvement (Wellner a, et al biorxiv,2020.) by combining yeast display and flow sorting techniques, but the workload is large, the time and labor are wasted, and the later system construction operation is complicated and has certain difficulty in repetition.

In contrast, the in vivo directed evolution system constructed by Manel Camps et al (Camps M, et al, proceedings of the National Academy of Sciences,2003,100(17): 9727-. Have been previously used to create libraries in directed evolution (Camps M, et al proceedings of the National Academy of Sciences,2003,100(17): 9727-. mDHFR is a murine dihydrofolate reductase that can be divided into two inactive portions (F1,2 and F3) that complement each other when in spatial proximity to exert mDHFR activity, conferring resistance to bacteria to Trimethoprim (TMP), a property that has been applied to study protein interactions (Pelletier J N, et al. proceedings of the National Academy of Sciences,1998,95(21):12141-12146.), detection of biological or drug interactions (patent: US19980017412), optimization of two interaction libraries (patent: US20050134253), and identification of antigen-antibody interactions (patent: US2003138850A1), among others. But have not been applied to antibody affinity maturation.

Therefore, although the continuous evolution system in vivo is rapidly developed and antibody affinity improvement methods also exist, none of them can be directed to a continuous evolution method of antibody affinity maturation. In view of the above, the present invention is particularly proposed.

Disclosure of Invention

Aiming at the technical problems of the existing antibody affinity maturation method and the great significance of the antibody affinity maturation, the invention provides a genetic engineering strain, the preparation thereof and an antibody affinity maturation directed evolution method based on the strain, and realizes the continuous directed evolution technology of the antibody affinity maturation by integrating mutation and screening with the assistance of prokaryotes.

The purpose of the invention is realized by the following technical scheme:

in a first aspect, the invention provides a genetically engineered strain obtained by constructing a mutant plasmid and a screening target plasmid into a modified strain;

the mutant plasmid comprises pEP-mcs- (GGGGS)2-F1,2-Flag, and the sequence is shown as SEQ ID NO. 1;

the screening target plasmid comprises pLA230-mcs- (GGGGS)2-F3-6xHis, and the sequence is shown as SEQ ID NO. 2.

Preferably, the modified strain is Escherichia coli JS 200-delta thyA delta folA after knocking out genes thyA and folA. To avoid possible leakage by endogenous dihydrofolate reductase, we knocked out endogenous dihydrofolate reductase (folA) and thymidylate synthase (thyA) of strain JS 200.

The mutant plasmid contains error-prone DNA polymerase I, mcs- (GGGGS)2-F1,2-Flag and thya genes, antigens can be uniformly constructed in a multi-cloning site mcs region, and error-prone DNA polymerase I can introduce random mutation to antibody genes when a screened target plasmid is copied to generate an in-vivo mutation library; the screened target plasmid is a ColE1 replicon, contains mcs- (GGGGS)2-F3-6xHis gene, and can uniformly construct the antibody in the mcc region of the multiple cloning site. F1,2 and F3 are mDHFR two fragments respectively. The promoter and RBS of two plasmids can be designed in various ways, namely, a plurality of groups of plasmids are established to form a toolbox.

Preferably, the construction method of the mutant plasmid comprises the following steps:

a1, PCR amplifying to obtain gene segment SEQ-mcs- (GGGGS)2-F1,2-Flag, the sequence is shown in SEQ ID NO. 3;

a2, inserting a gene sequence expressing thyA into an NdeI restriction site of a mutant plasmid pEP, and carrying out restriction enzyme reaction on the obtained pEP-thyA plasmid by adopting a Bsu36I single restriction enzyme site to obtain a restriction enzyme linear framework plasmid pEP-thyA-Bsu 36I;

a3, carrying out recombination reaction on the gene fragment obtained in the step A1 and the enzyme-digested linear skeleton plasmid obtained in the step A2 to obtain a mutant plasmid.

The invention constructs mcs- (GGGGS)2-F1,2-Flag segment on original mutation plasmid containing error-prone DNA polymerase I by using recombinase reaction; then, the thya gene is continuously constructed on the plasmid by using recombinase reaction to obtain a mutant plasmid.

Preferably, the construction method of the screening target plasmid comprises the following steps:

b1, PCR amplifying to obtain gene segment SEQ-mcs- (GGGGS)2-F3-6xHis, the sequence is shown in SEQ ID NO. 4;

b2, carrying out enzyme digestion reaction on the skeleton-containing plasmid pLA230 by adopting two enzyme digestion sites of AflIII and SacI to obtain an enzyme digestion linear skeleton plasmid pLA 230-AflIII/SacI;

and B3, carrying out recombination reaction on the gene fragment in the step B1 and the enzyme digestion linear skeleton plasmid in the step B2 to obtain the screening target plasmid.

The invention constructs the mcs- (GGGGS)2-F3-6xHis segment near the original target plasmid ColE1 replicon by using recombinase reaction to obtain the screening target plasmid.

In a second aspect, the invention provides a method for constructing a genetically engineered strain, comprising the following steps:

and (3) electrically transforming the mutant plasmid and the screening target plasmid into escherichia coli JS 200-delta thya delta fola, culturing by adopting a non-resistant LB culture medium, and then coating the escherichia coli on an LB-thymidine-corresponding resistant solid plate for culturing to obtain the genetic engineering strain.

In a third aspect, the invention provides an application of a genetic engineering strain in directed evolution of antibody affinity maturation.

Preferably, the method comprises screening the genetically engineered strain to obtain a mutant antibody with high affinity to the antigen.

In a fourth aspect, the present invention provides a directed evolution method for antibody affinity maturation, comprising the steps of:

s1, constructing antigen and antibody into the mcs region of the mutant plasmid and the screening target plasmid of the genetic engineering strain of claim 1 respectively;

s2, carrying out mutation screening on the strain obtained in the step S1 under the condition of certain TMP pressure;

s3, obtaining mutation site information, and cloning and expressing to obtain the mutation antibody with high affinity to the antigen.

Preferably, in step S2, thymine (thymine) is added to the culture medium used for the mutation screening; the mutation screening mode comprises mutation screening under liquid culture conditions, mutation screening on a solid plate and mutation screening alternately on a liquid-solid plate.

The invention uses recombinase reaction to construct antigen in the mutant plasmid mcs region, and constructs antibody in the screening target plasmid mcs region. An antigen antibody may also be replaced by two interacting proteins or peptides.

The invention provides two affinity maturation screening modes of directed evolution, which can be specifically divided into a liquid culture mode and a solid culture mode.

The solid culture screening mode is as follows:

preparing JS 200-delta thya delta fola electrotransformation competence, and co-electrotransfering the two mutant plasmids and the screening target plasmids which already contain the antigen and the antibody into a JS 200-delta thya delta fola strain and coating the strain on an LB-thymine-corresponding resistance plate;

selecting the monoclonal antibody to be cultured in an LB-thymidine-corresponding resistant liquid culture medium at 30 ℃ overnight;

washing the thallus with LB liquid culture medium without thymine, transferring to LB-resistant liquid culture medium, and culturing overnight at 37 deg.C to generate in vivo antibody mutation library;

coating the mixture into an LB-corresponding resistant solid culture medium containing low-concentration TMP according to a certain proportion, and carrying out overnight screening at the temperature of 30 ℃; selecting a monoclonal antibody to an LB-corresponding resistant liquid culture medium containing the TMP with the same low concentration as the previous stage, and culturing overnight at 30 ℃; taking a small amount of the bacterial liquid to carry out bacterial liquid PCR and Sanger sequencing, transferring a certain amount of the bacterial liquid into a liquid culture medium containing LB-corresponding resistance of the upper-stage TMP with the same low concentration, and culturing overnight at 37 ℃;

coating the mixture into LB-corresponding resistant solid culture medium containing correspondingly high-concentration TMP according to a certain proportion, and carrying out overnight screening at 30 ℃; selecting a monoclonal antibody to an LB-corresponding resistant liquid culture medium containing the TMP with the same high concentration as the previous stage, and culturing overnight at 30 ℃; taking a small amount of the bacterial liquid to carry out bacterial liquid PCR and Sanger sequencing, transferring a certain amount of the bacterial liquid into a liquid culture medium containing LB-corresponding resistance of the TMP with the same high concentration as the previous stage, and culturing overnight at 37 ℃;

repeating the process till the highest TMP screening pressure;

changing the solid culture medium into a liquid culture medium, namely a liquid culture screening mode;

the invention also obtains the corresponding table of the antibody affinity and the site mutation under different pressure conditions.

The antibody affinity and site mutation mapping table is obtained as follows:

the mutation site under each stress condition can be obtained by PCR and Sanger sequencing;

only copying the mutation site information with changed amino acids to an original antibody plasmid to obtain a mutant plasmid, and further constructing a mutant expression strain; respectively inducing, expressing and purifying wild type antibodies and mutant antibodies, and quantifying antibody proteins;

performing induced expression on the purified antigen, quantifying the antigen protein, and performing biotinylation marking;

by adopting a BLI method and using an SA probe to fix biotinylated antigen, different antibodies can be subjected to binding dissociation only at the same concentration point in a large batch, or each antibody has a plurality of antibodies with different concentrations, and finally the calculated K is obtained_D(M)；

Establishing a corresponding table of different affinity-mutation sites;

and searching different affinity-mutation site correspondence tables to obtain the corresponding mutation sites of a certain antibody under different affinities.

During the growth process of the engineering bacteria, when the affinity of the antibody to the antigen is remarkably improved due to mutation, the antigen and the antibody are combined with F1,2 and F3 to form more functional mDHFR to resist TMP, so that the engineering bacteria can survive better and realize positive screening.

Compared with the prior art, the invention has the following beneficial effects:

1. the directed evolution method for antibody affinity maturation provided by the invention integrates a mutation system and a screening system, only needs to construct an antibody antigen on a corresponding plasmid, can directly obtain an antibody with remarkably improved affinity only by a mode of solid culture or direct liquid culture screening for several times under the condition of certain TMP pressure, and realizes the maturation of the antibody affinity by directed evolution.

2. According to the directed evolution method for antibody affinity maturation provided by the invention, under different TMP pressure conditions, all strains can be stored in liquid wells or on a flat plate, and corresponding different affinity and site mutation corresponding tables under all pressure conditions can be obtained.

3. The directed evolution method for antibody affinity maturation provided by the invention has the advantages of simple evolution operation and high flux, can be used for carrying out a plurality of groups of evolution experiments at one time, and saves time and cost.

Drawings

Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:

FIG. 1 is a schematic diagram of the principle of directed evolution of antibody affinity maturation in an embodiment of the present invention;

FIG. 2 is a schematic diagram of the operation flow of antibody affinity maturation directed evolution in an embodiment of the present invention;

FIG. 3 shows the results of the construction of JS 200-. DELTA.thya.DELTA.fola strain in example 1 of the present invention;

FIG. 4 shows that mutations at three positions of GCN4 affect the formation of GCN4 dimer in example 1 of the present invention;

FIG. 5 is a diagram showing the results of the GCN4 screening evolution in example 1 of the present invention;

FIG. 6 shows the expression of split of mDHFR in example 2 of the present invention;

FIG. 7 shows 18A4Hu in example 3 of the present invention^scFvA multi-round screening result graph;

FIG. 8 shows 18A4Hu in example 3 of the present invention^scFvScreening a pressure and mutation site corresponding table;

FIG. 9 shows 18A4Hu in example 3 of the present invention^scFvThe result of the affinity determination of the mutant with AGR 2;

FIG. 10 shows 18A4Hu in example 3 of the present invention^scFvMutation site and affinity mapping table.

Detailed Description

The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention.

The principle of the invention is illustrated as follows:

1. the schematic diagram of directed evolution of affinity maturation is shown in FIG. 1. In e.coli JS200 Δ thya Δ fola, Ag-F1,2 and Ab-F3 were expressed from mutant and target plasmids, respectively, and mutant DNA polymerase i (error pro Pol i) on the mutant plasmid specifically targeted Ab-F3 to generate an Ab mutant library. When Ag-Ab interacts, F1,2 and F3 are close in space to form functional mDHFR, exert the activity of dihydrofolate reductase, enable the strain to grow normally under TMP screening conditions, and moreover, the concentration of TMP and the amount of mDHFR are in positive correlation.

2. The flow chart of the directed evolution of affinity maturation is shown in FIG. 2. On the first day, at the temperature of 30 ℃, the strain grows and breeds normally; the next day, starting to 37 ℃, the error prone Pol I targets Ab-F3 to generate a large amount of Ab mutants, the Ab mutants are coated on a TMP plate for screening, if the mutant Ab (mut) -F3 can be combined with Ag-F1,2, corresponding clones can grow on the plate, and the clones are activated at 30 ℃; the mutation is generated at 37 ℃ by sequential switching, TMP screening is carried out, and the process is repeated to finally screen the Ab mutant with higher affinity.

In the following examples, the JS200 strain and the backbone pEP/pWT of the mutation/control plasmid and the target plasmid pLA230 were obtained from Addgene, pACYC184 was obtained from the adeps saidsurage of wuhan virus, pACYC184-AMP was self-constructed based on pACYC184 (pACYC 184 plasmid was obtained by digesting pACYC184 plasmid with Bsu36I and inserting AMP gene by homologous recombination, the specific procedure was the same as in example 1.2), e.coli DH5 α/BL21(DE3) strain was obtained from holo-type gold biotechnology limited, pET28a vector and pET32a vector were common plasmids used in the laboratory, and nickel column was obtained from kakkiso technology limited.

Example 1 integration of mutation and screening systems and feasibility study

1. Plasmid construction integrated with mutation screening system (GCN 4/GCN4^3mutExploration of feasibility for example)

1.1 GCN4^3mutOf the site of mutationAnd (4) determining. The literature indicates that GCN4 is a yeast transcription factor, GCN4- "Leucine-Zipper" can form a dimer, and the region is selected for verification experiments and is also called GCN4 in the following. Previous researches found that the formation of GCN4 homomodimer can be greatly reduced after three-site mutation of GCN4, namely L12E, N16A and L19A, as shown in FIG. 4. The wild type of GCN4 (GCN 4 in FIG. 4 and FIG. 5) was determined after rational design^WT) And mutant form (GCN 4 in FIGS. 4 and 5)^3mut) The protein sequence is as follows.

GCN4 wild type: LEDKVEELLSKNYHLENEVARLKKLVGER (original SEQ ID NO: UniProtKB-P03069, SEQ ID NO.5)

GCN4^3mutMutant type: LEDKVEEELSKAYHAENEVARLKK(K)LVGER(SEQ ID NO.6)

To distinguish between GCN4 and GCN4^3mutIn GCN4^3mutThe sequence of (2) is added with an amino acid K, as shown in (K) above.

1.2 GCN4/GCN4^3mutMutation and screening of target plasmid construction

1.2.1 cloning of the fragment of interest. Designing upstream and downstream primers according to the principle of constructing plasmids by homologous recombinases: F1-pEP (pWT), R1-pEP (pWT), and F1-pLA230, R1-pLA230 were subjected to PCR reaction. Subjecting 2 μ LPCR products to agarose gel electrophoresis to identify products, and recovering the PCR products SEQ-GCN4- (GGGGS)2-F1,2 (shown in SEQ ID NO. 7), SEQ-GCN4- (GGGGGGS) 2-F3 (shown in SEQ ID NO. 8), and SEQ-GCN4 with Tiangen PCR product recovery kit^3mut- (GGGGS)2-F3 (the sequence is shown as SEQ ID NO. 9) is purified and recovered, and the recovered amount is directly used or frozen at-20 ℃ for standby after being measured by using Nanodrop.

The upstream primer F1-pEP (pWT): GCCTGAGCAAACTGGCCTCAGGTTTACACTTTATGCT (SEQ ID NO.10)

Downstream primer R1-pEP (pWT): TGTGCTTCTCAAATGCCTGAGGTTAACTACGTCGACA (SEQ ID NO.11)

Upstream primer F1-pLA 230: TGCTGGCCTTTTGCTCACATGTACAATTGTTCGAAT (SEQ ID NO.12)

The downstream primer R1-pLA 230: TCTGTGACTGGTACCGAGCTCGTTAACGGGGGATCC (SEQ ID NO.13)

1.2.2 backbone plasmid cleavage. Plasmids pEP and pWT contained error-prone DNA polymerase I and high fidelity DNA polymerase I, respectively, pEP as the mutant plasmid and pWT as the control plasmid. pLA230 contains the ColEI ori as a target plasmid specifically recognized by DNA polymerase I on pEP and pWT. Coli containing the backbone plasmids pEP, pWT and pLA230 were inoculated into 5mL of fresh medium, and cultured overnight at 37 ℃ respectively, and the backbone plasmids pEP, pWT and pLA230 were extracted using a Tiangen plasmid extraction kit, and quantified using Nanodrop. Adopting Bsu36I single enzyme cutting sites aiming at pEP and pWT plasmids and adopting AflIII and SacI two enzyme cutting sites aiming at pLA230 plasmids, respectively carrying out enzyme cutting reaction, carrying out reaction at 37 ℃ for 1h, then taking 5 mu L of agarose gel electrophoresis to identify the enzyme cutting effect, using a Tiangen enzyme cutting product recovery kit to recover the enzyme cutting product, and finally using Nanodrop to determine the recovery amount. The obtained restriction enzyme cutting linear skeleton plasmids are pEP-Bsu36I, pWT-Bsu36I and pLA 230-AflIII/SacI.

1.2.3 plasmid recombination ligation reaction. By using

II recombinant enzyme is used for plasmid construction, and the details are shown in the specification. The components are shown in table 1 below, and after the addition, the mixture is gently pipetted and uniformly mixed, and the reaction solution is collected to the bottom of the tube by short-time centrifugation. Reacting at 37 ℃ for 30min to obtain connection products pEP-GCN4- (GGGGGGS) 2-F1,2, pWT-GCN4- (GGGGGGS) 2-F1,2, pLA230-GCN4- (GGGGS)2-F3 and pLA230-GCN4^3mut- (GGGGS) 2-F3. Standing on ice for transformation or storing at-20 deg.C for one week.

TABLE 1

1.2.4 transformation. 10 μ L of each ligation product obtained in step 1.2.3 was taken and directly transformed into E.coli DH5 α competent, step universal plasmid transformation protocol. Finally coating on the surface of the substrate containing the corresponding resistance (KAN)⁺Or CAP⁺) The LB solid plate was gently spread. Optionally coating or coating with 1/3, and culturing at 37 deg.C for 12-16 h.

1.2.5 plate selection and validation. Plates cultured from step 1.2.4The single clones were selected against pEP-GCN4- (GGGGS)2-F1,2, pWT-GCN4- (GGGGS)2-F1,2, pLA230-GCN4- (GGGGS)2-F3 and pLA230-GCN4^3mut- (GGGGS)2-F3 was subjected to PCR/Sanger sequencing verification. Culturing 5mL of bacterial solution of each single clone with correct sequencing, and extracting plasmids pEP-GCN4- (GGGGS)2-F1,2 (shown in SEQ ID NO. 14), pLA230-GCN4- (GGGGGGS) 2-F3 (shown in SEQ ID NO. 15), pWT-GCN4- (GGGGGGS) 2-F1,2 (shown in SEQ ID NO. 16) and pLA230-GCN4^3mut- (GGGGS)2-F3 (the sequence is shown as SEQ ID NO. 17). And (4) detecting and quantifying Nanodrop, and freezing and storing at-20 ℃ for later use.

1.3 JS 200-delta thya delta fola Strain construction and competent preparation

1.3.1 Strain construction. Coli JS200- Δ thyA Δ folA was constructed based on JS200 strain by knocking out the conditionally essential genes thyA and folA using Lambda-Red homologous recombination technique, using PCR and sequencing verification, as shown in FIG. 3. Wherein JS200- Δ thya indicates a knockout of the thya gene, JS200- Δ thya fola: kana shows that fola, JS 200-delta thya delta fola-1, JS 200-delta thya delta fola-2, JS 200-delta thya delta fola-3, JS 200-delta thya delta fola-4 are knocked out by kana gene recombination on the basis of the knock-out of thya gene, and four parallel monoclonal strains after the thya and fola are knocked out are shown. The complete genotype generated was SC-18recA718 polA12 uvrA155 trpE65 lon-11 sulfoA 1 thyA folA. Furthermore, according to the screening principle: JS200- Δ thyA Δ folA Strain in the absence of thymosine or/and TMP, the strain requires the normal expression of folA and thyA for growth. The function of the dihydrofolate reductase folA depends on the interaction of GCN-F1,2 and GCN4-F3 to form functional F1,2-F3 (mDHFR) and play the role of dihydrofolate reductase. For thymidylate synthase thyA function, we obtained by complementing the thyA gene. According to the principle of plasmid construction in 1.2, we constructed the thyA gene on a pACYC184-AMP plasmid named pACYC184-AMP-thyA (post-optimization construction onto pEP/pWT plasmid).

1.3.2 electroporation competent cell preparation and electroporation. The basic method is shown in the molecular cloning experimental guidance. After the Escherichia coli JS 200-delta thya delta fola electrotransformation competence is prepared, the Escherichia coli JS 200-delta thya delta fola can be stored for half a year at the temperature of minus 80 ℃. The plasmids constructed in the above 1.2 and 1.3.1 were electroporated into JS200- Δ thya Δ fola cells by three-plasmid combination, respectively:

pWT-GCN4- (GGGGS)2-F1,2, pLA230-GCN4- (GGGGS)2-F3 and pACYC 184-AMP-thya;

pEP-GCN4- (GGGGS)2-F1,2, pLA230-GCN4- (GGGGS)2-F3 and pACYC 184-AMP-thya;

③pWT-GCN4-(GGGGS)2-F1,2、pLA230-GCN4^3mut- (GGGGS)2-F3 and pACYC 184-AMP-thya;

④pEP-GCN4-(GGGGS)2-F1,2、pLA230-GCN4^3mut- (GGGGS)2-F3 and pACYC 184-AMP-thya; adding 1mL of nonresistant LB-thymine culture medium, culturing for 1h at 30 ℃ in a shaking table, centrifuging to remove supernatant, and respectively coating 1/5 or all the supernatant on LB-thymine-KAN⁺/CAP⁺/AMP⁺Culturing on solid plate at 30 deg.C for 16-20 hr. Four JS200- Δ thya Δ fola strains were obtained containing the above three plasmids, respectively, as shown in the left panel of FIG. 5a, wherein each strain contained pACYC184-AMP-thya plasmid, not shown. Bacterium 1 contains pWT-GCN4- (GGGGGGS) 2-F1,2 and pLA230-GCN4- (GGGGS)2-F3 plasmids, bacterium 2 contains pEP-GCN4- (GGGGGGS) 2-F1,2 and pLA230-GCN4- (GGGGGGS) 2-F3 plasmids, and bacterium 3 contains pWT-GCN4- (GGGGGGS) 2-F1,2 and pLA230-GCN4 plasmids^3mut- (GGGGS)2-F3 plasmid, bacterium 4 containing pEP-GCN4- (GGGGS)2-F1,2 and pLA230-GCN4^3mut- (GGGGS)2-F3 plasmid.

2. Strain activation and mutagenesis

2.1 Strain activation. Selecting 5-10 monoclonals of the normally expressed three-plasmid JS 200-delta thya delta fola strain obtained in the step 1 to respectively contain LB-thymidine-KAN⁺/CAP⁺/AMP⁺The culture was carried out in a 96-deep well plate in liquid medium at 30 ℃ and 200rpm for 12 hours.

2.2 mutations were generated. Transferring the bacteria grown after the activation culture in the step 2.1 to a medium containing fresh LB-thymine-KAN at a ratio of 1:100⁺/CAP⁺/AMP⁺The liquid medium was cultured in a 96-deep well plate at 37 ℃ and 200rpm for 12 hours to generate mutations.

3. Primary screening and TMP pressure screening of mutant strains

3.1 cleaning of the strain. Centrifuging the bacterium solution with mutation generated in the step 2.2 at 5000rpm for 5min, removing supernatant, washing with LB medium without thymine for three times, and respectively switching to No. 1:100LB-KAN of thymine⁺/CAP⁺/AMP⁺Culturing in liquid culture medium at 30 deg.C for 16-20 hr.

3.2TMP pressure screening. And (3) reserving a sample of the bacterial liquid grown after the culture in the step 3.1 for carrying out PCR and sequencing on the bacterial liquid. The remaining bacterial solution was applied to LB-KAN containing 10. mu.g/mL TMP at a ratio of 1:100⁺/CAP⁺/AMP⁺After culturing the solid plate at 37 ℃ for 12 hours, the plate was observed for growth as shown in the right panel of FIG. 5a, and the single clone was picked up in sterile water.

4.GCN4^3mutCloning reversion sites determination.

And (4) determining mutation sites. And (3) respectively carrying out colony/bacteria liquid PCR and Sanger sequencing on the bacteria liquid containing the monoclone and the bacteria liquid containing the reserved sample obtained in the step 3.2. The mutation sites were determined according to the sequencing results, and it can be seen that the three sites of the mutant strain (strain 1-strain 4) all had reversion mutations, as shown in FIG. 5 b.

The results of this example illustrate that: at GCN4/GCN4^3mutFor example, the mutation screening integrated system contains control plasmid GCN4-F1,2 and screening target plasmid GCN4 under the condition of not adding thymine or/and adding TMP resistance screening^3mutStrain JS200- Δ thya Δ fola of-F3 was not viable. Containing only the mutant plasmid, GCN4^3mut-F3 reversion to GCN4^WTWhen F3 interacts with GCN4-F1,2 to ensure that F1,2 and F3 are close to form functional mDHFR (F1,2-F3) in space, the strain JS 200-delta thya delta fola can survive, namely the surviving strain contains GCN4^3mutBack-mutation of (3). By successfully implementing GCN4^3mutThe back mutation shows that the integrated directed evolution platform for mutation and screening (the engineering strain JS 200-delta thya delta fola, the mutant plasmid and the screening target plasmid) built by the platform can realize the improvement of the affinity of the protein pair or the antigen-antibody of the interaction.

In addition, in order to use the platform more universally and conveniently, under the condition that engineering strains are not changed, plasmids are optimized to a certain extent, three plasmids are fused into two plasmids, a gene promoter is replaced by a high-expression promoter to improve the expression quantity of proteins so as to avoid that proteins with low affinity generate less active mDHFR at the starting point and are filtered out and cannot be screened, and different protein labels are added to detect the protein expression condition.

Example 2 integrative backbone plasmid optimization of mutation and screening System

1. Construction of backbone plasmids pEP-mcs- (GGGGS)2-F1,2-Flag, pWT-mcs- (GGGGS)2-F1,2-Flag and pLA230-mcs- (GGGGS)2-F3-6XHis

1.1 thya was constructed into a mutant plasmid. pEP-thyA and pWT-thyA plasmids were obtained by designing upstream and downstream primers F1-thyA and R1-thyA according to the plasmid construction principles of example 1 and inserting the gene sequence for expression of thyA into the NdeI cleavage sites of the mutant plasmid pEP and the control plasmid pWT described in example 1.

The upstream primer F1-thya: AAAGGGAAAACTGTCCATATGCGCGGATCATATACA (SEQ ID NO.18)

The downstream primer R1-thya: CCGTTTTCATCTGTGCATATGTTAGTGGTGGTGGTG (SEQ ID NO.19)

1.2 cloning of the target fragment. The upstream and downstream primers F1-pEP (pWT) -2 and R1-pEP (pWT) -2, and F1-pLA230-2 and R1-pLA230-2 were designed according to the principle of constructing plasmids from homologous recombinases. PCR product identification, purification recovery and quantification were performed according to the same method as in step 1.2.1 of example 1. PCR products of SEQ-mcs- (GGGGS)2-F1,2-Flag (the sequence is shown as SEQ ID NO. 3) and SEQ-mcs- (GGGGS)2-F3-6xHis (the sequence is shown as SEQ ID NO. 4) are obtained and are directly used or frozen at-20 ℃ for standby.

The upstream primer F1-pEP (pWT) -2: GCCTGAGCAAACTGGCCTCAGGTAATGTGAGTTAGCT (SEQ ID NO.44)

The downstream primer R1-pEP (pWT) -2: TGTGCTTCTCAAATGCCTGAGGTTAACTACGTCGACA (SEQ ID NO.45)

The upstream primer F1-pLA 230-2: GCTGGCCTTTTGCTCACATGTTAATGTGAGTTAGCT (SEQ ID NO.46)

The downstream primer R1-pLA 230-2: TCTGTGACTGGTACCGAGCTCGTTAACACTAGTTCT (SEQ ID NO.47)

1.3 backbone plasmid digestion. The plasmid digestion gel was recovered and quantified according to the method 1.2.2 in example 1. Adopting Bsu36I single enzyme cutting site aiming at pWT-thya and pEP-thya plasmids in the step 1.1, adopting AflIII and SacI two enzyme cutting sites aiming at pLA230 plasmids, respectively carrying out enzyme cutting reaction, reacting for 1h at 37 ℃, and then recovering and quantifying nucleic acid gel. The obtained restriction enzyme cutting linear skeleton plasmids are pEP-thya-Bsu36I, pWT-thya-Bsu36I and pLA 230-AflIII/SacI.

1.4 recombinant construction of plasmids. The procedure was carried out in the same manner as in step 1.2.3 of example 1

II recombinase homologous recombination reaction, reacting for 30min at 37 ℃ to obtain connecting products pEP-mcs- (GGGGS)2-F1,2-Flag, pWT-mcs- (GGGGS)2-F1,2-Flag and pLA230-mcs- (GGGGS)2-F3-6 xHis.

And 1.5 transformation. Coli DH 5. alpha. competent by direct transformation of 10. mu.L of the recombinant ligation product described above according to step 1.2.3 of example 1, plated on plates containing the corresponding resistance (KAN)⁺Or CAP⁺) The LB solid plate was gently spread. Optionally coating or coating with 1/3, and culturing at 37 deg.C for 12-16 h.

1.6 plate selection and validation. Single clones were picked from plates of pEP-mcs- (GGGGS)2-F1,2-Flag, pWT-mcs- (GGGGGGS) 2-F1,2-Flag and pLA230-mcs- (GGGGGGS) 2-F3-6XHis after culture in step 1.4, PCR, sequencing verification and plasmid extraction, respectively, according to the method in step 1.2.3 of example 1. The concentration of the Nanodrop is measured to obtain recombinant plasmids pEP-mcs- (GGGGGGS) 2-F1,2-Flag (the sequence is shown as SEQ ID NO. 1), pWT-mcs- (GGGGGGS) 2-F1,2-Flag (the sequence is shown as SEQ ID NO. 20) and pLA230-mcs- (GGGGS)2-F3-6xHis (the sequence is shown as SEQ ID NO. 2), and the recombinant plasmids are frozen at the temperature of minus 20 ℃ for temporary use.

2. Expression of optimized mutant, control and selection plasmids in JS200- Δ thya Δ fola Strain

2.1 electroporation competent cell preparation and electroporation. Electrotransfer competent cells and transformation were prepared according to 1.3.2 of example 1 by electrotransforming the previously constructed 1.6 plasmids with the two plasmids pEP-mcs- (GGGGS)2-F1,2-Flag and pLA230-mcs- (GGGGGGS) 2-F3-6XHis and pWT-mcs- (GGGGGGS) 2-F1,2-Flag and pLA230-mcs- (GGGGGGGGS) 2-F3-6XHis, respectively, into JS200- Δ thya Δ fola cells, adding 1mL of nonresistant LB medium, shake culturing at 30 ℃ for 1h, centrifuging to remove supernatant, and taking 1/5 or spreading all LB-thymidine-KAN cells⁺/CAP⁺Culturing on/IPTG solid plate in 30 deg.C incubator 16-20 h. Obtaining JS 200-delta thya delta fola strain containing double plasmids pEP-mcs- (GGGGGGS) 2-F1,2-Flag and pLA230-mcs- (GGGGS)2-F3-6xHis, JS 200-delta thya delta fola strain containing double plasmids pWT-mcs- (GGGGGGS) 2-F1,2-Flag and pLA230-mcs- (GGGGGGS) 2-F3-6 xHis.

2.2Western blot to identify the expression condition. From the above-grown plate, single clones were picked and activated in 1mL of LB-thymine-KAN⁺/CAP⁺Carrying out shake culture at 30 ℃ for 12h in an IPTG liquid culture medium, and then collecting the strain. The strain is lysed, and 1/10 samples are taken to carry out Western blot by using His and Flag antibodies respectively to identify the expression condition, as shown in figure 6. The results of fig. 6 show that: the constructed plasmids pEP-mcs- (GGGGS)2-F1,2-Flag and pLA230-mcs- (GGGGS)2-F3-6XHis, pWT-mcs- (GGGGS)2-F1,2-Flag and pLA230-mcs- (GGGGGGS) 2-F3-6XHis can express F1,2 and F3 fragments containing multiple cloning sites, namely mcs- (GGGGGGGGS) 2-F1,2-Flag and mcs- (GGGGGGS) 2-F3-6XHis in JS200- Δ thya strain.

Thus, antigen and antibody or interacting protein pairs can be constructed separately to the mcs region of mutant plasmids pEP-mcs- (GGGGS)2-F1,2-Flag and target plasmid pLA230-mcs- (GGGGS)2-F3-6XHis and together with strain JS200- Δ thya Δ fola constitute the mutant-screened engineered strain.

Example 318A 4Hu antibody affinity maturation

1.18A4Hu mutant plasmids, target plasmids and Strain construction

Designing upstream and downstream primers according to the principle of constructing plasmid by homologous recombinase, and obtaining target fragments SEQ-AGR2 (the sequence is shown as SEQ ID NO. 21) and SEQ-18A4Hu by PCR^scFv(the sequence is shown as SEQ ID NO. 22), respectively inserting the target fragment into the backbone plasmids pEP-mcs- (GGGGGGS) 2-F1,2-Flag and pLA230-mcs- (GGGGS)2-F3-6XHis constructed in example 2, the restriction sites are BamHI/NotI and MfeI/NcoI, respectively, obtaining plasmids pEP-AGR2- (GGGGGGS) 2-F1,2-Flag (the sequence is shown as SEQ ID NO. 23) and pLA230-18A4Hu^scFv- (GGGGS)2-F3-6XHis (sequence shown in SEQ ID NO. 24). Then the plasmids pEP-AGR2- (GGGGS)2-F1,2-Flag and pLA230-18A4Hu^scFv- (GGGGS)2-F3-6XHis was constructed in the strain JS200- Δ thya Δ fola obtained in example 1, to obtain an engineered strain Escherichia coli containing these two plasmids.

2. Strain activation and mutagenesis

The procedure was the same as in 2 of example 2. The Escherichia coli engineering strain containing the two plasmids obtained in the step 1 is subjected to LB-thymine-CAP at the temperature of 30 DEG C⁺/KAN⁺Activation in culture Medium, and transfer to LB-thymine-CAP⁺/KAN⁺The mutation was generated in the medium by culturing at 37 ℃.

3. Primary screening and TMP pressure screening of strong interaction strains

3.1 preliminary screening of interacting strains

The procedure was the same as in step 3 of example 2. Cleaning of strains: centrifuging the mutant-producing bacterial liquid obtained in the step 2 at 5000rpm for 5min, removing supernatant, washing with a thynine-free LB culture medium for three times, and respectively inoculating the mutant-producing bacterial liquid to thynine-free LB-CAP at a ratio of 1:100⁺/KAN⁺And (3) performing shake culture at 30 ℃ for 16-20h in a liquid culture medium, reserving a sample, and performing bacterial liquid PCR and sequencing by using a universal primer on a pLA230 carrier.

3.2 mutagenesis and TMP pressure screening.

1) TMP pressure screen 1. The strain grown in the above step 3.1 was coated with LB-CAP containing 10. mu.g/mL TMP at a ratio of 1:100 and 1:1000, respectively⁺/KAN⁺Culturing on plate in 30 deg.C incubator for 16-20 hr. 5-10 monoclonals are picked from the grown plate and put into 10 mu L of sterile water to be mixed evenly, 1 mu L of bacterial liquid is taken to carry out bacterial liquid PCR by using the universal primer on the pLA230 carrier, and sequencing is carried out. The remaining 9. mu.L was added to LB-CAP containing 10. mu.g/mL TMP⁺/KAN⁺Culturing in liquid culture medium at 37 deg.C and 200rpm for 16-20 h.

2) TMP pressure screen 2. The strain grown in step 1) was coated with LB-CAP containing 100. mu.g/mL TMP at 1:100, 1:1000, respectively⁺/KAN⁺On a flat plate. Culturing at 30 deg.C for 16-20 h. 5-10 monoclonals are picked from the grown plate and put into 10 mu L of sterile water to be mixed evenly, 1 mu L of bacterial liquid is taken to carry out bacterial liquid PCR by using the universal primer on the pLA230 carrier, and sequencing is carried out. The remaining 9. mu.L was added to LB-CAP containing 10. mu.g/mL TMP⁺/KAN⁺Culturing in liquid culture medium at 37 deg.C and 200rpm for 16-20 h.

3) And (3) adopting the methods of the steps 1) and 2), and continuously carrying out the same treatment on the grown strains by sequentially adopting the TMP concentrations of the pressure screening 3, the pressure screening 4 and the pressure screening 5 in the following table to obtain screened strains.

The screening for TMP gradient boost pressure was performed sequentially as follows:

screening stage	Screening condition TMP ng/. mu.L	Plate coating dilution factor after liquid culture
			Pressure screening
1	10	1:1000
			Pressure screening 2	100	1:1000
Pressure screening 3	200	1:100
			Pressure screening 4	500	1:100
Pressure screening 5	1000	1:50

Note: for each screening stage, the plates were grown and transferred to liquid medium with the same TMP concentration for activation and further mutation, after which the plate spreading factor was adjusted according to the actual situation.

4. Affinity assay

4.1 18A4Hu^scFvCloning mutation site determination. And respectively reserving the bacteria liquid obtained after pressure screening for PCR identification and Sanger sequencing. From the sequencing site mutation profile, 18A4Hu is plotted as the TMP pressure increases^scFvThe divergent evolutionary trajectory of (2), as shown in fig. 7. The results of fig. 7 show that: the mutation sites of the same starting strain accumulate with increasing TMP concentration (0, 100, 500, 1000. mu.g/mL TMP), wherein the numbers are the strain clone numbers of the corresponding mutants and the information on the site mutations generated in the process is shown on the arrow. Starting strain 18A4Hu^scFvIs named as 1; the strains selected at a TMP concentration of 100. mu.g/mL were cloned as: 2, 4, 6; on this basis, the mutation sites accumulated at a TMP concentration of 500. mu.g/mL to form a strain clone: 3, 5, 7, 8; and strains with additional newly generated mutation sites were cloned as: 9, 11, strain clones were selected on the basis of strains 9 and 11 at a TMP concentration of 1000. mu.g/mL as: 12, 10. Of these, 5 mutants ( strains 6, 7, 8,9, 12) and the original strain (strain 1) were selected for further validation, and the mutation site information of the specific mutant strains is shown in FIG. 8 (18A4 Hu)^scFv-1、18A4Hu^scFv-6、18A4Hu^scFv-7、18A4Hu^scFv-8、18A4Hu^scFv-9、18A4Hu^scFv12-1 corresponds in turn to strains 1, 6, 7, 8,9, 12 in FIG. 7).

The construction of 18A4Hu was determined with reference to the plasmid construction procedure of example 1^scFv-1、18A4Hu^scFv-6、18A4Hu^scFv-7、18A4Hu^scFv-8、18A4Hu^scFv-9、18A4Hu^scFv12-1 plasmid, pET28a-18A4Hu was first constructed^scFv-1, the steps are as follows:

4.1.1 cloning of the fragment of interest. Designing upstream and downstream primers F1-pET28a and R1-pET28a according to the principle of constructing plasmids by using homologous recombinase, namely, respectively designing homologous sequences of about 15bp around the restriction enzyme cutting site, and carrying out PCR reaction. 2. mu.L of each PCR product was subjected to agarose gelAfter the gel electrophoresis identification of the product, the PCR product 18A4Hu is recovered by a Tiangen PCR product recovery kit^scFvAnd (4) purifying and recovering the-WT (the sequence is shown as SEQ ID NO. 25), and directly using the recovered quantity after the quantity is determined by using the Nanodrop or freezing and storing the recovered quantity at-20 ℃ for later use.

The upstream primer F1-pET28 a: CAGCAAATGGGTCGCGGATCCATGGACATGAGGGTT (SEQ ID NO.26)

The downstream primer R1-pET28 a: CTCGAGTGCGGCCGCAAGCTTTACGAAGTTATCATG (SEQ ID NO.27)

4.1.2 backbone plasmid cleavage. Escherichia coli containing plasmid pET28a was inoculated into 5mL of fresh medium and cultured overnight at 37 ℃ and plasmid pET28a was extracted using a Tiangen plasmid extraction kit and quantified using Nanodrop. Aiming at the pET28a plasmid, adopting BamHI and HindIII double enzyme cutting sites to carry out enzyme cutting reaction, carrying out reaction for 1h at 37 ℃, taking 5 mu L of agarose gel to identify the enzyme cutting effect, using a Tiangen enzyme cutting product recovery kit to recover the enzyme cutting product, and finally using Nanodrop to determine the recovery amount. The resulting digested linear backbone plasmid was pET28 a-BamHI/HindIII.

4.1.3 recombinant construction of plasmids. By using

II recombinant enzyme is used for plasmid construction, and the details are shown in the specification. The components are shown in table 2 below, and after the addition, the mixture is gently pipetted and uniformly mixed, and the reaction solution is collected to the bottom of the tube by short-time centrifugation. Reacting at 37 ℃ for 30min to obtain a ligation product pET28a-18A4Hu^scFv-a WT. Standing on ice for transformation or storing at-20 deg.C for one week.

TABLE 2

Components	Recombination reactions
		pET28a-BamHI/HindIII	100ng
18A4HuscFv-WT	80ng
		5×CE II buffer	4μL
Exnase II	2μL
		ddH2O	to 20μL

4.1.4 transformation. 10 μ L of the ligation product obtained in step 4.1.3 was used to transform DH5 α directly, a general plasmid transformation protocol. Finally coating on the surface of the substrate containing the corresponding resistance (KAN)⁺) The LB solid plate was gently spread. Optionally coating or coating with 1/3, and culturing at 37 deg.C for 12-16 h.

4.1.5 plate selection and validation. Single clones were picked from the plates after the culture in step 4.1.4 and directed against pET28a-18A4Hu^scFvWT for PCR/sequencing validation. 5mL of bacterial solution is respectively cultured on the monoclonals with correct sequencing, and plasmids pET28a-18A4Hu are extracted^scFvWT, Nanodrop assay concentration, obtained recombinant plasmid pET28a-18A4Hu^scFvWT (SEQ ID NO.28) frozen at-20 ℃ for use.

4.1.6 plasmid point mutations. Based on pET28a-18A4Hu obtained in the 4.1.5 step^scFvWT plasmid, point mutated using PCR. Reserving homologous sequences of about 15bp at the upstream and downstream of a mutation site to design a primer pair, wherein a mutation primer group comprises the following components:

(1)L12R

18A4HU-mut-1-up:TGCTCAGCTCCTGGGACGCCTGCTGCTC(SEQ ID NO.29)

18A4HU-mut-1-down:GGAGCCAGAGCAGCAGGCGTCCCAGGAGCT(SEQ ID NO.30)

(2)L14R

18A4HU-mut-2-2-up:TCCTGGGACTCCTGCGGCTCTGGCTCCCAGGTGCCA(SEQ ID NO.31)

18A4HU-mut-2-2-down:CCTGGGAGCCAGAGCCGCAGGAGTCCCAGGAGCT(SEQ ID NO.32)

(3)D226N

18A4HU-mut-3-up:CAGAGTGACAATGACTGTGAACAAGTCCACGAGCA(SEQ ID NO.33)

18A4HU-mut-3-down:CTGTGCTCGTGGACTTGTTCACAGTCATTGTCACT(SEQ ID NO.34)

(4)E131K

18A4HU-mut-4-up:TGGAGGCACCAAGCTGAAAATCAAAGG(SEQ ID NO.35)

18A4HU-mut-4-down:GCCGCCGCCTTTGATTTTCAGCTTGGTGCC(SEQ ID NO.36)

(5)S102G

18A4HU-mut-5-up:ACTTCACACTCACCATCGGCAGGCTGGAA(SEQ ID NO.37)

18A4HU-mut-5-down:CTCAGGTTCCAGCCTGCCGATGGTGAGTGT(SEQ ID NO.38)

plasmid PCR was performed using this primer pair in a 50. mu.L system as shown in Table 3 below:

TABLE 3

If the plasmid contains two point mutation sites, two rounds of plasmid PCR are performed. After 12cycles, 2. mu.L of PCR product is taken for agarose gel electrophoresis verification, and 0.5. mu.L of DpnI enzyme digestion template plasmid is added into the residual PCR product; then, the digested product was directly transformed into DH 5. alpha. and KAN was applied by reference to the conventional plasmid transformation procedure⁺Plates were incubated overnight at 37 ℃; transformants were picked again for sequencing validation. Finally, the plasmid pET28a-18A4Hu with point mutation is obtained^scFv-L12R(SEQ ID NO.39)、pET28a-18A4Hu^scFv-L12R,D226N(SEQ ID NO.40)、pET28a-18A4Hu^scFv-L12R,E131K(SEQ ID NO.41)、pET28a-18A4Hu^scFv-L14R(SEQ ID NO.42)、pET28a-18A4Hu^scFv-L12R,S102G(SEQ ID NO.43)。

4.1.7 transformation of the expression strains. The correct recombinant plasmids obtained in steps 4.1.5 and 4.1.6BL21 was directly transformed, and the expression strain BL21-18A4Hu was obtained by reference to the conventional plasmid transformation procedure^scFv-WT、BL21-18A4Hu^scFv-D226N、BL21-18A4Hu^scFv-E131K、BL21-18A4Hu^scFv-S102G。

4.2 18A4Hu^scFvSingle chain antibody and AGR2 antigen induced expression purification. 18A4Hu purified by induction expression in Escherichia coli^scFvSingle chain antibody mutants and the antigen AGR 2. The method comprises the following specific steps:

expression strain BL21-18A4Hu constructed by 1:100 transfer 4.1^scFv-WT、BL21-18A4Hu^scFv-D226N、BL21-18A4Hu^scFv-E131K、BL21-18A4Hu^scFv-S102G was separately cultured in 500ml of fresh LB medium at 37 ℃ for about 2h to OD600 ═ 0.4-0.6, IPTG was added to the final concentration of 1mM, and induction was carried out overnight at 16 ℃. 7000rpm, centrifugate for 3min to collect the bacteria. Resuspend to 30ml Buffer A, use high pressure breaker to break up bacteria for 2-3min, then at 4 degrees C, 8000rpm, centrifuge for 10min, supernatant adding 500 u L Ni-NTA beads, 4 degrees C coupling 1 h. Then, the mixture was centrifuged at 2000rpm for 2min at 4 ℃ to remove the supernatant, and the beads were washed with Buffer B and Buffer C, 5ml each, 5min each, 2000rpm, 4 ℃ and 2min to remove the supernatant. And finally eluting with Buffer C for three times, incubating for 15min at the temperature of 4 ℃ and 300 ul each time, centrifuging for 2-3min at the temperature of 4 ℃ and 2000rpm, and collecting the supernatant. After the concentration is determined by SDS-PAGE electrophoresis, the mixture is temporarily stored at-80 ℃. Wherein Buffer A: 10mM imidazole, 0.5M NaCl, 20mM Tris-HCl, 4mM MgCl ₂5% glycerol, pH 7.9; buffer B: 40mM imidazole, 0.5M NaCl, 20mM Tris-HCl, 4mM MgCl ₂5% glycerol, pH 7.9; buffer C: 300mM imidazole, 0.5M NaCl, 20mM Tris-HCl, 10% glycerol, 0.01% Triton-100, pH 7.9.

4.3 18A4Hu^scFvAffinity assay of mutants and AGR 2.

4.3.1 antigen Biotin labelling. The antigen AGR2 was biotinylated and labeled with a thermobiotinylation kit at a molar ratio of antigen to biotin of 1:3, incubated at 4 ℃ or on ice for 2 h; gly termination reaction is carried out according to the molar ratio of 10:1(Glycine: Biotin), and incubation is carried out for 1h at room temperature; and (4) desalting. Obtaining biotinylated antigen AGR 2.

4.3.2 BLI affinity assay. First SA sensBiotinylated antigen AGR 260 s was immobilized or, then baseline 60s in SD Buffer (1 XPBS, 0.02% tween20, 0.1% BSA), association 120s was performed with 4-5 concentration points set according to the concentration of the antibody, and finally association 120s in SD Buffer. The affinity constants were calculated by curve fitting using Data Analysis 10.0 software, as shown in fig. 9. The final screening yielded 18A4Hu with the highest binding affinity to AGR2^scFvMutant 18A4Hu of (1)^scFv-L12R,S102G。

5. And establishing an affinity-mutation site corresponding table.

18A4Hu determined as described above, except for the mutation that gave the highest affinity^scFvAffinity of the different mutants of (A) to AGR2 and 18A4Hu^scFvThe mutation sites under different stress conditions of (2) can be mapped to affinity, as shown in FIG. 10.

The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.

Sequence listing

<110> Shanghai university of transportation

<120> a genetic engineering strain, preparation thereof and directed evolution method based on antibody affinity maturation of the strain

<130> KAG45915

<160> 47

<170> SIPOSequenceListing 1.0

<210> 1

<211> 7840

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atgaccatga ttacgccaag cttatggttc agatccccca aaatccactt atccttgtag 60

atggttcatc ttatctttat cgcgcatatc acgcgtttcc cccgctgact aacagcgcag 120

gcgagccgac cggtgcgatg tatggtgtcc tcaacatgct gcgcagtctg atcatgcaat 180

ataaaccgac gcatgcagcg gtggtctttg acgccaaggg aaaaaccttt cgtgatgaac 240

tgtttgaaca ttacaaatca catcgcccgc caatgccgga cgatctgcgt gcacaaatcg 300

aacccttgca cgcgatggtt aaagcgatgg gactgccgct gctggcggtt tctggcgtag 360

aagcggacga cgttatcggt actctggcgc gcgaagccga aaaagccggg cgtccggtgc 420

tgatcagcac tggcgataaa gatatggcgc agctggtgac gccaaatatt acgcttatca 480

ataccatgac gaataccatc ctcggaccgg aagaggtggt gaataagtac ggcgtgccgc 540

cagaactgat catcgatttc ctggcgctga tgggtgactc ctctgataac attcctggcg 600

taccgggcgt cggtgaaaaa accgcgcagg cattgctgca aggtcttggc ggactggata 660

cgctgtatgc cgagccagaa aaaattgctg ggttgagctt ccgtggcgcg aaaacaatgg 720

cagcgaagct cgagcaaaac aaagaagttg cttatctctc ataccagctg gcgacgatta 780

aaaccgacgt tgaactggag ctgacctgtg aacaactgga agtgcagcaa ccggcagcgg 840

aagagttgtt ggggctgttc aaaaagtatg agttcaaacg ctggactgct gatgtcgaag 900

cgggcaaatg gttacaggcc aaaggggcaa aaccagccgc gaagccacag gaaaccagtg 960

ttgcagacga agcaccagaa gtgacggcaa cggtgatttc ttatgacaac tacgtcacca 1020

tccttgatga agaaacactg aaagcgtgga ttgcgaagct ggaaaaagcg ccggtatttg 1080

catttgatac cgaaaccgac agccttgata acatctctgc taacctggtc gggctttctt 1140

ttgctatcga gccaggcgta gcggcatata ttccggttgc tcatgattat cttgatgcgc 1200

ccgatcaaat ctctcgcgag cgtgcactcg agttgctaaa accgctgctg gaagatgaaa 1260

aggcgctgaa ggtcgggcaa aacctgaaat acgctcgcgg tattctggcg aactacggca 1320

ttgaactgcg tgggattgcg tttgatacca tgctggagtc ctacattctc aatagcgttg 1380

ccgggcgtca cgatatggac agcctcgcgg aacgttggtt gaagcacaaa accatcactt 1440

ttgaagagat tgctggtaaa ggcaaaaatc aactgacctt taaccagatt gccctcgaag 1500

aagccggacg ttacgccgcc gaagatgcag atgtcacctt gcagttgcat ctgaaaatgt 1560

ggccggatct gcaaaaacac aaagggccgt tgaacgtctt cgagaatatc gaaatgccgc 1620

tggtgccggt gctttcacgc attgaacgta acggtgtgaa gatcgatccg aaagtgctgc 1680

acaatcattc tgaagagctc acccttcgtc tggctgagct ggaaaagaaa gcgcatgaaa 1740

ttgcaggtga ggaatttaac ctttcttcca ccaaggagtt acaaaccatt ctctttgaaa 1800

aacagggcat taaaccgctg aagaaaacgc cgggtggcgc gccgtcaacg tcggaagagg 1860

tactggaaga actggcgctg gactatccgt tgccaaaagt gattctggag tatcgtggtc 1920

tggcgaagct gaaatcgacc tacaccgaca agctgccgct gatgatcaac ccgaaaaccg 1980

ggcgtgtgca tacctcttat caccaggcag taactgcaac gggacgttta tcgtcaaccg 2040

atcctaacct gcaaaacatt ccggtgcgta acgaagaagg tcgtcgtata cgccaggcgt 2100

ttattgcgcc agaggattat gtgattgtct cagccgacta ctcgcagaat gaactgcgca 2160

ttatggcgta tctttcgcgt gacaaaggct tgctgaccgc attcgcggaa ggaaaagata 2220

tccaccgggc aacggccgca gaagtgtttg gtttgccact ggaaaccgtc accagcgagc 2280

aacgccgtag cgcgaaacgg atcaactttg gtctgattta tggcatgagt gctttcggtc 2340

tggcgcggca attgaacatt ccacgtaaag aagcgcagaa gtacatggac ctttacttcg 2400

aacgctaccc tggcgtgctg gagtatatgg aacgcacccg tgctcaggcg aaagagcagg 2460

gctacgttga aacgctggac ggacgccgtc tgtatctgcc ggatatcaaa tccagcaatg 2520

gtgctcgtcg tgcagcggct gaacgtgcag ccattaacgc gccaatgcag ggaaccgccg 2580

ccgacattat caaacgggcg atgattgccg ttgatgcgtg gttacaggct gagcaaccgc 2640

gtgtacgtat gatcatgcag gtacacgatg aactggtatt tgaagttcat aaagatgatg 2700

ttgatgccgt cgcgaagcag attcatcaac tgatggaaaa ctgtacccgt ctggatgtgc 2760

cgttgctggt ggaagtgggg agtggcgaaa actgggatca ggcgcactaa gaattcactg 2820

gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt 2880

gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct 2940

tcccaacagt tgcgcagcct gaatggcgaa tggcgctaac cgtttttatc aggctctggg 3000

aggcagaata aatgatcata tcgtcaatta ttacctccac ggggagagcc tgagcaaact 3060

ggcctcaggt aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct 3120

tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag aattcattaa 3180

agaggagaaa ttaactatga gaggatctgg atcccatcat caccatgcgg ccgcattcga 3240

acatggcgaa cgcatcgatg gtggcggtgg ctctggaggt ggtgggtcct ccggaatggt 3300

tcgaccattg aactgcatcg tcgccgtgtc ccaaaatatg gggattggca agaacggaga 3360

cctaccctgg cctccgctca ggaacgagtt caagtacttc caaagaatga ccacaacctc 3420

ttcagtggaa ggtaaacaga atctggtgat tatgggtagg aaaacctggt tctccattcc 3480

tgagaagaat cgacctttaa aggacagaat taatatagtt ctcagtagag aactcaaaga 3540

accaccacga ggagctcatt ttcttgccaa aagtttggat gatgccttaa gacttattga 3600

acaaccggaa ttgggtaccc tggttgacta caaggacgac gatgacaagt aatctagaac 3660

tagtggagtc gatgtcgacg tagttaacct caggcatttg agaagcacac ggtcacactg 3720

cttccggtag tcaataaacc ggtaaaccag caatagacat aagcggctat ttaacgaccc 3780

tgccctgaac cgacgaccgg gtcgaatttg ctttcgaatt tctgccattc atccgcttat 3840

tatcacttat tcaggcgtag caccaggcgt ttaagggcac caataactgc cttaaaaaaa 3900

ttacgccccg ccctgccact catcgcagta ctgttgtaat tcattaagca ttctgccgac 3960

atggaagcca tcacagacgg catgatgaac ctgaatcgcc agcggcatca gcaccttgtc 4020

gccttgcgta taatatttgc ccatggtgaa aacgggggcg aagaagttgt ccatattggc 4080

cacgtttaaa tcaaaactgg tgaaactcac ccagggattg gctgagacga aaaacatatt 4140

ctcaataaac cctttaggga aataggccag gttttcaccg taacacgcca catcttgcga 4200

atatatgtgt agaaactgcc ggaaatcgtc gtggtattca ctccagagcg atgaaaacgt 4260

ttcagtttgc tcatggaaaa cggtgtaaca agggtgaaca ctatcccata tcaccagctc 4320

accgtctttc attgccatac gaaattccgg atgagcattc atcaggcggg caagaatgtg 4380

aataaaggcc ggataaaact tgtgcttatt tttctttacg gtctttaaaa aggccgtaat 4440

atccagctga acggtctggt tataggtaca ttgagcaact gactgaaatg cctcaaaatg 4500

ttctttacga tgccattggg atatatcaac ggtggtatat ccagtgattt ttttctccat 4560

tttagcttcc ttagctcctg aaaatctcga taactcaaaa aatacgcccg gtagtgatct 4620

tatttcatta tggtgaaagt tggaacctct tacgtgccga tcaacgtctc attttcgcca 4680

aaagttggcc cagggcttcc cggtatcaac agggacacca ggatttattt attctgcgaa 4740

gtgatcttcc gtcacaggta tttattcggc gcctgtagtg ccatttaccc ccattcactg 4800

ccagagccgt gagcgcagcg aactgaatgt cacgaaaaag acagcgactc aggtgcctga 4860

tggtcggaga caaaaggaat attcagcgat ttgcccgagc ttgcgagggt gctacttaag 4920

cctttagggt tttaaggtct gttttgtaga ggagcaaaca gcgtttgcga catccttttg 4980

taatactgcg gaactgacta aagtagtgag ttatacacag ggctgggatc tattcttttt 5040

atcttttttt attctttctt tattctataa attataacca cttgaatata aacaaaaaaa 5100

acacacaaag gtctagcgga atttacagag ggtctagcag aatttacaag ttttccagca 5160

aaggtctagc agaatttaca gatacccaca actcaaagga aaaggactag taattatcat 5220

tgactagccc atctcaattg gtatagtgat taaaatcacc tagaccaatt gagatgtatg 5280

tctgaattag ttgttttcaa agcaaatgaa ctagcgatta gtcgctatga cttaacggag 5340

catgaaacca agctaatttt atgctgtgtg gcactactca accccacgat tgaaaaccct 5400

acaaggaaag aacggacggt atcgttcact tataaccaat acgctcagat gatgaacatc 5460

agtagggaaa atgcttatgg tgtattagct aaagcaacca gagagctgat gacgagaact 5520

gtggaaatca ggaatccttt ggttaaaggc tttgagattt tccagtggac aaactatgcc 5580

aagttctcaa gcgaaaaatt agaattagtt tttagtgaag agatattgcc ttatcttttc 5640

cagttaaaaa aattcataaa atataatctg gaacatgtta agtcttttga aaacaaatac 5700

tctatgagga tttatgagtg gttattaaaa gaactaacac aaaagaaaac tcacaaggca 5760

aatatagaga ttagccttga tgaatttaag ttcatgttaa tgcttgaaaa taactaccat 5820

gagtttaaaa ggcttaacca atgggttttg aaaccaataa gtaaagattt aaacacttac 5880

agcaatatga aattggtggt tgataagcga ggccgcccga ctgatacgtt gattttccaa 5940

gttgaactag atagacaaat ggatctcgta accgaacttg agaacaacca gataaaaatg 6000

aatggtgaca aaataccaac aaccattaca tcagattcct acctacataa cggactaaga 6060

aaaacactac acgatgcttt aactgcaaaa attcagctca ccagttttga ggcaaaattt 6120

ttgagtgaca tgcaaagtaa gtatgatctc aatggttcgt tctcatggct cacgcaaaaa 6180

caacgaacca cactagagaa catactggct aaatacggaa ggatctgagg ttcttatggc 6240

tcttgtatct atcagtgaag catcaagact aacaaacaaa agtagaacaa ctgttcaccg 6300

ttacatatca aagggaaaac tgtccatatg cgcggatcat atacatatct tttaacggta 6360

tccggcaacc agccaggtcc ccttgtgcta ttattcgcac ctttggagcg cctgaaacct 6420

gcggcgcgca tttcaatcgc tgttctcttt cagcgaaata acaagaactt gtggtgacag 6480

gaggaaccat gaaacagtat ttagaactga tgcaaaaagt gctcgacgaa ggcacacaga 6540

aaaacgaccg taccggaacc ggaacgcttt ccatttttgg tcatcagatg cgttttaacc 6600

tgcaagatgg attcccgctg gtgacaacta aacgttgcca cctgcgttcc atcatccatg 6660

aactgctgtg gtttctgcag ggcgacacta acattgctta tctacacgaa aacaatgtca 6720

ccatctggga cgaatgggcc gatgaaaacg gcgacctcgg gccagtgtat ggtaaacagt 6780

ggcgcgcctg gccaacgcca gatggtcgtc atattgacca gatcactacg gtactgaacc 6840

agctgaaaaa cgacccggat tcgcgccgca ttattgtttc agcgtggaac gtaggcgaac 6900

tggataaaat ggcgctggca ccgtgccatg cattcttcca gttctatgtg gcagacggca 6960

aactctcttg ccagctttat cagcgctcct gtgacgtctt cctcggcctg ccgttcaaca 7020

ttgccagcta cgcgttattg gtgcatatga tggcgcagca gtgcgatctg gaagtgggtg 7080

attttgtctg gaccggtggc gacacgcatc tgtacagcaa ccatatggat caaactcatc 7140

tgcaattaag ccgcgaaccg cgtccgctgc cgaagttgat tatcaaacgt aaacccgaat 7200

ccatcttcga ctaccgtttc gaagactttg agattgaagg ctacgatccg catccgggca 7260

ttaaagcgcc ggtggctatc caccaccacc accaccacta acatatgcac agatgaaaac 7320

ggtgtaaaaa agatagatac atcagagctt ttacgagttt ttggtgcatt caaagctgtt 7380

caccatgaac agatcgacaa tgtaacagat gaacagcatg taacacctaa tagaacaggt 7440

gaaaccagta aaacaaagca actagaacat gaaattgaac acctgagaca acttgttaca 7500

gctcaacagt cacacataga cagcctgaaa caggcgatgc tgcttatcga atcaaagctg 7560

ccgacaacac gggagccagt gacgcctccc gtggggaaaa aatcatggca attctggaag 7620

aaatagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc 7680

tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt 7740

tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt 7800

ggaattgtga gcggataaca atttcacaca ggaaacagct 7840

<210> 2

<211> 3222

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

agtcacagaa aagcatctta cggtgtgggc ggacaaaata gttgggaact gggaggggtg 60

gaaatggagt ttttaaggat tatttaggga agagtgacaa aatagatggg aactgggtgt 120

agcgtcgtaa gctaatacga aaattaaaaa tgacaaaata gtttggaact agatttcact 180

tatctggttg gtcgacacta gtattaccct gttatcccta gatttaaatg atatcggatc 240

ctagtaagcc acgttgtgtc tcaaaatctc tgatgttaca ttgcacaaga taaaaatata 300

tcatcatgaa caataaaact gtctgcttac ataaacagta atacaagggg tgttatgagc 360

catattcaac gggaaacgtc ttgctcgagg ccgcgattaa attccaacat ggatgctgat 420

ttatatgggt ataaatgggc tcgcgataat gtcgggcaat caggtgcgac aatctatcga 480

ttgtatggga agcccgatgc gccagagttg tttctgaaac atggcaaagg tagcgttgcc 540

aatgatgtta cagatgagat ggtcagacta aactggctga cggaatttat gcctcttccg 600

accatcaagc attttatccg tactcctgat gatgcatggt tactcaccac tgcgatcccc 660

gggaaaacag cattccaggt attagaagaa tatcctgatt caggtgaaaa tattgttgat 720

gcgctggcag tgttcctgcg ccggttgcat tcgattcctg tttgtaattg tccttttaac 780

agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa tgaataacgg tttggttgat 840

gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg 900

cataagcttt tgccattctc accggattca gtcgtcactc atggtgattt ctcacttgat 960

aaccttattt ttgacgaggg gaaattaata ggttgtattg atgttggacg agtcggaatc 1020

gcagaccgat accaggatct tgccatccta tggaactgcc tcggtgagtt ttctccttca 1080

ttacagaaac ggctttttca aaaatatggt attgataatc ctgatatgaa taaattgcag 1140

tttcatttga tgctcgatga gtttttctaa tcagaattgg ttaattggtt gtaacactgg 1200

cagagcatta cgctgacttg acgggacggc ggctttgttg aataaatcga acttttgctg 1260

agttgaagga tcagatcacg catcttcccg acaacgcaga ccgttccgtg gcaaagcaaa 1320

agttcaaaat caccaactgg tccacctaca acaaagctct catcaaccgt ggctccctca 1380

ctttctggct ggatgatggg gcgattcagg cctggtatga gtcagcaaca ccttcttcac 1440

gaggcagacc tcagcggcgg ccggatccga tatcgtttaa actcgctacc ttaggaccgt 1500

tatagttagc ggccgcgtcg accccacgcc cctctttaat acgacgggca atttgcactt 1560

cagaaaatga agagtttgct ttagccataa caaaagtcca gtatgctttt tcacagcata 1620

actggactga tttcagttta caactattct gtctagttta agactttatt gtcatagttt 1680

agatctgatc gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg 1740

tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt 1800

tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat 1860

aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta 1920

gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa 1980

tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga 2040

aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac 2100

aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt 2160

tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc 2220

gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat 2280

cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag 2340

acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc 2400

cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag 2460

cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac 2520

aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg 2580

gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct 2640

atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc 2700

tcacatgtta atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt 2760

ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacaga attcattaaa 2820

gaggagaaat taactatgag aggatctcaa ttgcatcatc accatccatg gttcttagtt 2880

ggcgaacgca tcgatggtgg cggtggctct ggaggtggtg ggtcctccgg aagtaaagta 2940

gacatggttt ggatagtcgg aggcagttct gtttaccagg aagccatgaa tcaaccaggc 3000

caccttagac tctttgtgac aaggatcatg caggaatttg aaagtgacac gtttttccca 3060

gaaattgatt tggggaaata taaacttctc ccagaatacc caggcgtcct ctctgaggtc 3120

caggaggaaa aaggcatcaa gtataagttt gaagtctacg agaagaaaga ccatcatcac 3180

catcaccatt aatctagaac tagtgttaac gagctcggta cc 3222

<210> 3

<211> 662

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gcctgagcaa actggcctca ggtaatgtga gttagctcac tcattaggca ccccaggctt 60

tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca 120

cagaattcat taaagaggag aaattaacta tgagaggatc tggatcccat catcaccatg 180

cggccgcatt cgaacatggc gaacgcatcg atggtggcgg tggctctgga ggtggtgggt 240

cctccggaat ggttcgacca ttgaactgca tcgtcgccgt gtcccaaaat atggggattg 300

gcaagaacgg agacctaccc tggcctccgc tcaggaacga gttcaagtac ttccaaagaa 360

tgaccacaac ctcttcagtg gaaggtaaac agaatctggt gattatgggt aggaaaacct 420

ggttctccat tcctgagaag aatcgacctt taaaggacag aattaatata gttctcagta 480

gagaactcaa agaaccacca cgaggagctc attttcttgc caaaagtttg gatgatgcct 540

taagacttat tgaacaaccg gaattgggta ccctggttga ctacaaggac gacgatgaca 600

agtaatctag aactagtgga gtcgatgtcg acgtagttaa cctcaggcat ttgagaagca 660

ca 662

<210> 4

<211> 544

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gctggccttt tgctcacatg ttaatgtgag ttagctcact cattaggcac cccaggcttt 60

acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac 120

agaattcatt aaagaggaga aattaactat gagaggatct caattgcatc atcaccatcc 180

atggttctta gttggcgaac gcatcgatgg tggcggtggc tctggaggtg gtgggtcctc 240

cggaagtaaa gtagacatgg tttggatagt cggaggcagt tctgtttacc aggaagccat 300

gaatcaacca ggccacctta gactctttgt gacaaggatc atgcaggaat ttgaaagtga 360

cacgtttttc ccagaaattg atttggggaa atataaactt ctcccagaat acccaggcgt 420

cctctctgag gtccaggagg aaaaaggcat caagtataag tttgaagtct acgagaagaa 480

agaccatcat caccatcacc attaatctag aactagtgtt aacgagctcg gtaccagtca 540

caga 544

<210> 5

<211> 29

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Leu Glu Asp Lys Val Glu Glu Leu Leu Ser Lys Asn Tyr His Leu Glu

1 5 10 15

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

20 25

<210> 6

<211> 30

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Leu Glu Asp Lys Val Glu Glu Leu Leu Ser Lys Asn Tyr His Leu Glu

1 5 10 15

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

20 25 30

<210> 7

<211> 667

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gcctgagcaa actggcctca ggtttacact ttatgcttcc ggctcgtatg ttgtgtggaa 60

ttgtgagcgg ataacaattc cagcggatcc ttcgaacatg aacactgaag ccgccaggcg 120

ttctcgtgcg agaaagttgc aaagaatgaa acaacttgaa gacaaggttg aagaattgct 180

ttcgaaaaat tatcacttgg aaaatgaggt tgccagatta aagaaattag ttggcgaacg 240

catcgatggt ggcggtggct ctggaggtgg tgggtcctcc ggaatggttc gaccattgaa 300

ctgcatcgtc gccgtgtccc aaaatatggg gattggcaag aacggagacc taccctggcc 360

tccgctcagg aacgagttca agtacttcca aagaatgacc acaacctctt cagtggaagg 420

taaacagaat ctggtgatta tgggtaggaa aacctggttc tccattcctg agaagaatcg 480

acctttaaag gacagaatta atatagttct cagtagagaa ctcaaagaac caccacgagg 540

agctcatttt cttgccaaaa gtttggatga tgccttaaga cttattgaac aaccggaatt 600

gggtacctaa tctagaacta gtggagtcga tgtcgacgta gttaacctca ggcatttgag 660

aagcaca 667

<210> 8

<211> 510

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gctggccttt tgctcacatg tacaattgtt cgaatgaaca ctgaagccgc caggcgttct 60

cgtgcgagaa agttgcaaag aatgaaacaa cttgaagaca aggttgaaga attgctttcg 120

aaaaattatc acttggaaaa tgaggttgcc agattaaaga aattagttgg cgaacgcatc 180

gatggtggcg gtggctctgg aggtggtggg tcctccggaa gtaaagtaga catggtttgg 240

atagtcggag gcagttctgt ttaccaggaa gccatgaatc aaccaggcca ccttagactc 300

tttgtgacaa ggatcatgca ggaatttgaa agtgacacgt ttttcccaga aattgatttg 360

gggaaatata aacttctccc agaataccca ggcgtcctct ctgaggtcca ggaggaaaaa 420

ggcatcaagt ataagtttga agtctacgag aagaaagact aatctagaac tagtggatcc 480

cccgttaacg agctcggtac cagtcacaga 510

<210> 9

<211> 513

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gctggccttt tgctcacatg tacaattgtt cgaatgaaca ctgaagccgc caggcgttct 60

cgtgcgagaa agttgcaaag aatgaaacaa cttgaagaca aggttgaaga agagctttcg 120

aaagcttatc acgcggaaaa tgaggttgcc agattaaaga aaaaattagt tggcgaacgc 180

atcgatggtg gcggtggctc tggaggtggt gggtcctccg gaagtaaagt agacatggtt 240

tggatagtcg gaggcagttc tgtttaccag gaagccatga atcaaccagg ccaccttaga 300

ctctttgtga caaggatcat gcaggaattt gaaagtgaca cgtttttccc agaaattgat 360

ttggggaaat ataaacttct cccagaatac ccaggcgtcc tctctgaggt ccaggaggaa 420

aaaggcatca agtataagtt tgaagtctac gagaagaaag actaatctag aactagtgga 480

tcccccgtta acgagctcgg taccagtcac aga 513

<210> 10

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gcctgagcaa actggcctca ggtttacact ttatgct 37

<210> 11

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tgtgcttctc aaatgcctga ggttaactac gtcgaca 37

<210> 12

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

tgctggcctt ttgctcacat gtacaattgt tcgaat 36

<210> 13

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

tctgtgactg gtaccgagct cgttaacggg ggatcc 36

<210> 14

<211> 6868

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

atgaccatga ttacgccaag cttatggttc agatccccca aaatccactt atccttgtag 60

atggttcatc ttatctttat cgcgcatatc acgcgtttcc cccgctgact aacagcgcag 120

gcgagccgac cggtgcgatg tatggtgtcc tcaacatgct gcgcagtctg atcatgcaat 180

ataaaccgac gcatgcagcg gtggtctttg acgccaaggg aaaaaccttt cgtgatgaac 240

tgtttgaaca ttacaaatca catcgcccgc caatgccgga cgatctgcgt gcacaaatcg 300

aacccttgca cgcgatggtt aaagcgatgg gactgccgct gctggcggtt tctggcgtag 360

aagcggacga cgttatcggt actctggcgc gcgaagccga aaaagccggg cgtccggtgc 420

tgatcagcac tggcgataaa gatatggcgc agctggtgac gccaaatatt acgcttatca 480

ataccatgac gaataccatc ctcggaccgg aagaggtggt gaataagtac ggcgtgccgc 540

cagaactgat catcgatttc ctggcgctga tgggtgactc ctctgataac attcctggcg 600

taccgggcgt cggtgaaaaa accgcgcagg cattgctgca aggtcttggc ggactggata 660

cgctgtatgc cgagccagaa aaaattgctg ggttgagctt ccgtggcgcg aaaacaatgg 720

cagcgaagct cgagcaaaac aaagaagttg cttatctctc ataccagctg gcgacgatta 780

aaaccgacgt tgaactggag ctgacctgtg aacaactgga agtgcagcaa ccggcagcgg 840

aagagttgtt ggggctgttc aaaaagtatg agttcaaacg ctggactgct gatgtcgaag 900

cgggcaaatg gttacaggcc aaaggggcaa aaccagccgc gaagccacag gaaaccagtg 960

ttgcagacga agcaccagaa gtgacggcaa cggtgatttc ttatgacaac tacgtcacca 1020

tccttgatga agaaacactg aaagcgtgga ttgcgaagct ggaaaaagcg ccggtatttg 1080

catttgatac cgaaaccgac agccttgata acatctctgc taacctggtc gggctttctt 1140

ttgctatcga gccaggcgta gcggcatata ttccggttgc tcatgattat cttgatgcgc 1200

ccgatcaaat ctctcgcgag cgtgcactcg agttgctaaa accgctgctg gaagatgaaa 1260

aggcgctgaa ggtcgggcaa aacctgaaat acgctcgcgg tattctggcg aactacggca 1320

ttgaactgcg tgggattgcg tttgatacca tgctggagtc ctacattctc aatagcgttg 1380

ccgggcgtca cgatatggac agcctcgcgg aacgttggtt gaagcacaaa accatcactt 1440

ttgaagagat tgctggtaaa ggcaaaaatc aactgacctt taaccagatt gccctcgaag 1500

aagccggacg ttacgccgcc gaagatgcag atgtcacctt gcagttgcat ctgaaaatgt 1560

ggccggatct gcaaaaacac aaagggccgt tgaacgtctt cgagaatatc gaaatgccgc 1620

tggtgccggt gctttcacgc attgaacgta acggtgtgaa gatcgatccg aaagtgctgc 1680

acaatcattc tgaagagctc acccttcgtc tggctgagct ggaaaagaaa gcgcatgaaa 1740

ttgcaggtga ggaatttaac ctttcttcca ccaaggagtt acaaaccatt ctctttgaaa 1800

aacagggcat taaaccgctg aagaaaacgc cgggtggcgc gccgtcaacg tcggaagagg 1860

tactggaaga actggcgctg gactatccgt tgccaaaagt gattctggag tatcgtggtc 1920

tggcgaagct gaaatcgacc tacaccgaca agctgccgct gatgatcaac ccgaaaaccg 1980

ggcgtgtgca tacctcttat caccaggcag taactgcaac gggacgttta tcgtcaaccg 2040

atcctaacct gcaaaacatt ccggtgcgta acgaagaagg tcgtcgtata cgccaggcgt 2100

ttattgcgcc agaggattat gtgattgtct cagccgacta ctcgcagaat gaactgcgca 2160

ttatggcgta tctttcgcgt gacaaaggct tgctgaccgc attcgcggaa ggaaaagata 2220

tccaccgggc aacggccgca gaagtgtttg gtttgccact ggaaaccgtc accagcgagc 2280

aacgccgtag cgcgaaacgg atcaactttg gtctgattta tggcatgagt gctttcggtc 2340

tggcgcggca attgaacatt ccacgtaaag aagcgcagaa gtacatggac ctttacttcg 2400

aacgctaccc tggcgtgctg gagtatatgg aacgcacccg tgctcaggcg aaagagcagg 2460

gctacgttga aacgctggac ggacgccgtc tgtatctgcc ggatatcaaa tccagcaatg 2520

gtgctcgtcg tgcagcggct gaacgtgcag ccattaacgc gccaatgcag ggaaccgccg 2580

ccgacattat caaacgggcg atgattgccg ttgatgcgtg gttacaggct gagcaaccgc 2640

gtgtacgtat gatcatgcag gtacacgatg aactggtatt tgaagttcat aaagatgatg 2700

ttgatgccgt cgcgaagcag attcatcaac tgatggaaaa ctgtacccgt ctggatgtgc 2760

cgttgctggt ggaagtgggg agtggcgaaa actgggatca ggcgcactaa gaattcactg 2820

gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt 2880

gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct 2940

tcccaacagt tgcgcagcct gaatggcgaa tggcgctaac cgtttttatc aggctctggg 3000

aggcagaata aatgatcata tcgtcaatta ttacctccac ggggagagcc tgagcaaact 3060

ggcctcaggt ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata 3120

acaattccag cggatccttc gaacatgaac actgaagccg ccaggcgttc tcgtgcgaga 3180

aagttgcaaa gaatgaaaca acttgaagac aaggttgaag aattgctttc gaaaaattat 3240

cacttggaaa atgaggttgc cagattaaag aaattagttg gcgaacgcat cgatggtggc 3300

ggtggctctg gaggtggtgg gtcctccgga atggttcgac cattgaactg catcgtcgcc 3360

gtgtcccaaa atatggggat tggcaagaac ggagacctac cctggcctcc gctcaggaac 3420

gagttcaagt acttccaaag aatgaccaca acctcttcag tggaaggtaa acagaatctg 3480

gtgattatgg gtaggaaaac ctggttctcc attcctgaga agaatcgacc tttaaaggac 3540

agaattaata tagttctcag tagagaactc aaagaaccac cacgaggagc tcattttctt 3600

gccaaaagtt tggatgatgc cttaagactt attgaacaac cggaattggg tacctaatct 3660

agaactagtg gagtcgatgt cgacgtagtt aacctcaggc atttgagaag cacacggtca 3720

cactgcttcc ggtagtcaat aaaccggtaa accagcaata gacataagcg gctatttaac 3780

gaccctgccc tgaaccgacg accgggtcga atttgctttc gaatttctgc cattcatccg 3840

cttattatca cttattcagg cgtagcacca ggcgtttaag ggcaccaata actgccttaa 3900

aaaaattacg ccccgccctg ccactcatcg cagtactgtt gtaattcatt aagcattctg 3960

ccgacatgga agccatcaca gacggcatga tgaacctgaa tcgccagcgg catcagcacc 4020

ttgtcgcctt gcgtataata tttgcccatg gtgaaaacgg gggcgaagaa gttgtccata 4080

ttggccacgt ttaaatcaaa actggtgaaa ctcacccagg gattggctga gacgaaaaac 4140

atattctcaa taaacccttt agggaaatag gccaggtttt caccgtaaca cgccacatct 4200

tgcgaatata tgtgtagaaa ctgccggaaa tcgtcgtggt attcactcca gagcgatgaa 4260

aacgtttcag tttgctcatg gaaaacggtg taacaagggt gaacactatc ccatatcacc 4320

agctcaccgt ctttcattgc catacgaaat tccggatgag cattcatcag gcgggcaaga 4380

atgtgaataa aggccggata aaacttgtgc ttatttttct ttacggtctt taaaaaggcc 4440

gtaatatcca gctgaacggt ctggttatag gtacattgag caactgactg aaatgcctca 4500

aaatgttctt tacgatgcca ttgggatata tcaacggtgg tatatccagt gatttttttc 4560

tccattttag cttccttagc tcctgaaaat ctcgataact caaaaaatac gcccggtagt 4620

gatcttattt cattatggtg aaagttggaa cctcttacgt gccgatcaac gtctcatttt 4680

cgccaaaagt tggcccaggg cttcccggta tcaacaggga caccaggatt tatttattct 4740

gcgaagtgat cttccgtcac aggtatttat tcggcgcctg tagtgccatt tacccccatt 4800

cactgccaga gccgtgagcg cagcgaactg aatgtcacga aaaagacagc gactcaggtg 4860

cctgatggtc ggagacaaaa ggaatattca gcgatttgcc cgagcttgcg agggtgctac 4920

ttaagccttt agggttttaa ggtctgtttt gtagaggagc aaacagcgtt tgcgacatcc 4980

ttttgtaata ctgcggaact gactaaagta gtgagttata cacagggctg ggatctattc 5040

tttttatctt tttttattct ttctttattc tataaattat aaccacttga atataaacaa 5100

aaaaaacaca caaaggtcta gcggaattta cagagggtct agcagaattt acaagttttc 5160

cagcaaaggt ctagcagaat ttacagatac ccacaactca aaggaaaagg actagtaatt 5220

atcattgact agcccatctc aattggtata gtgattaaaa tcacctagac caattgagat 5280

gtatgtctga attagttgtt ttcaaagcaa atgaactagc gattagtcgc tatgacttaa 5340

cggagcatga aaccaagcta attttatgct gtgtggcact actcaacccc acgattgaaa 5400

accctacaag gaaagaacgg acggtatcgt tcacttataa ccaatacgct cagatgatga 5460

acatcagtag ggaaaatgct tatggtgtat tagctaaagc aaccagagag ctgatgacga 5520

gaactgtgga aatcaggaat cctttggtta aaggctttga gattttccag tggacaaact 5580

atgccaagtt ctcaagcgaa aaattagaat tagtttttag tgaagagata ttgccttatc 5640

ttttccagtt aaaaaaattc ataaaatata atctggaaca tgttaagtct tttgaaaaca 5700

aatactctat gaggatttat gagtggttat taaaagaact aacacaaaag aaaactcaca 5760

aggcaaatat agagattagc cttgatgaat ttaagttcat gttaatgctt gaaaataact 5820

accatgagtt taaaaggctt aaccaatggg ttttgaaacc aataagtaaa gatttaaaca 5880

cttacagcaa tatgaaattg gtggttgata agcgaggccg cccgactgat acgttgattt 5940

tccaagttga actagataga caaatggatc tcgtaaccga acttgagaac aaccagataa 6000

aaatgaatgg tgacaaaata ccaacaacca ttacatcaga ttcctaccta cataacggac 6060

taagaaaaac actacacgat gctttaactg caaaaattca gctcaccagt tttgaggcaa 6120

aatttttgag tgacatgcaa agtaagtatg atctcaatgg ttcgttctca tggctcacgc 6180

aaaaacaacg aaccacacta gagaacatac tggctaaata cggaaggatc tgaggttctt 6240

atggctcttg tatctatcag tgaagcatca agactaacaa acaaaagtag aacaactgtt 6300

caccgttaca tatcaaaggg aaaactgtcc atatgcacag atgaaaacgg tgtaaaaaag 6360

atagatacat cagagctttt acgagttttt ggtgcattca aagctgttca ccatgaacag 6420

atcgacaatg taacagatga acagcatgta acacctaata gaacaggtga aaccagtaaa 6480

acaaagcaac tagaacatga aattgaacac ctgagacaac ttgttacagc tcaacagtca 6540

cacatagaca gcctgaaaca ggcgatgctg cttatcgaat caaagctgcc gacaacacgg 6600

gagccagtga cgcctcccgt ggggaaaaaa tcatggcaat tctggaagaa atagcgccca 6660

atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg 6720

tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat 6780

taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc 6840

ggataacaat ttcacacagg aaacagct 6868

<210> 15

<211> 3188

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

agtcacagaa aagcatctta cggtgtgggc ggacaaaata gttgggaact gggaggggtg 60

gaaatggagt ttttaaggat tatttaggga agagtgacaa aatagatggg aactgggtgt 120

agcgtcgtaa gctaatacga aaattaaaaa tgacaaaata gtttggaact agatttcact 180

tatctggttg gtcgacacta gtattaccct gttatcccta gatttaaatg atatcggatc 240

ctagtaagcc acgttgtgtc tcaaaatctc tgatgttaca ttgcacaaga taaaaatata 300

tcatcatgaa caataaaact gtctgcttac ataaacagta atacaagggg tgttatgagc 360

catattcaac gggaaacgtc ttgctcgagg ccgcgattaa attccaacat ggatgctgat 420

ttatatgggt ataaatgggc tcgcgataat gtcgggcaat caggtgcgac aatctatcga 480

ttgtatggga agcccgatgc gccagagttg tttctgaaac atggcaaagg tagcgttgcc 540

aatgatgtta cagatgagat ggtcagacta aactggctga cggaatttat gcctcttccg 600

accatcaagc attttatccg tactcctgat gatgcatggt tactcaccac tgcgatcccc 660

gggaaaacag cattccaggt attagaagaa tatcctgatt caggtgaaaa tattgttgat 720

gcgctggcag tgttcctgcg ccggttgcat tcgattcctg tttgtaattg tccttttaac 780

agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa tgaataacgg tttggttgat 840

gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg 900

cataagcttt tgccattctc accggattca gtcgtcactc atggtgattt ctcacttgat 960

aaccttattt ttgacgaggg gaaattaata ggttgtattg atgttggacg agtcggaatc 1020

gcagaccgat accaggatct tgccatccta tggaactgcc tcggtgagtt ttctccttca 1080

ttacagaaac ggctttttca aaaatatggt attgataatc ctgatatgaa taaattgcag 1140

tttcatttga tgctcgatga gtttttctaa tcagaattgg ttaattggtt gtaacactgg 1200

cagagcatta cgctgacttg acgggacggc ggctttgttg aataaatcga acttttgctg 1260

agttgaagga tcagatcacg catcttcccg acaacgcaga ccgttccgtg gcaaagcaaa 1320

agttcaaaat caccaactgg tccacctaca acaaagctct catcaaccgt ggctccctca 1380

ctttctggct ggatgatggg gcgattcagg cctggtatga gtcagcaaca ccttcttcac 1440

gaggcagacc tcagcggcgg ccggatccga tatcgtttaa actcgctacc ttaggaccgt 1500

tatagttagc ggccgcgtcg accccacgcc cctctttaat acgacgggca atttgcactt 1560

cagaaaatga agagtttgct ttagccataa caaaagtcca gtatgctttt tcacagcata 1620

actggactga tttcagttta caactattct gtctagttta agactttatt gtcatagttt 1680

agatctgatc gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg 1740

tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt 1800

tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat 1860

aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta 1920

gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa 1980

tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga 2040

aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac 2100

aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt 2160

tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc 2220

gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat 2280

cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag 2340

acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc 2400

cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag 2460

cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac 2520

aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg 2580

gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct 2640

atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc 2700

tcacatgtac aattgttcga atgaacactg aagccgccag gcgttctcgt gcgagaaagt 2760

tgcaaagaat gaaacaactt gaagacaagg ttgaagaatt gctttcgaaa aattatcact 2820

tggaaaatga ggttgccaga ttaaagaaat tagttggcga acgcatcgat ggtggcggtg 2880

gctctggagg tggtgggtcc tccggaagta aagtagacat ggtttggata gtcggaggca 2940

gttctgttta ccaggaagcc atgaatcaac caggccacct tagactcttt gtgacaagga 3000

tcatgcagga atttgaaagt gacacgtttt tcccagaaat tgatttgggg aaatataaac 3060

ttctcccaga atacccaggc gtcctctctg aggtccagga ggaaaaaggc atcaagtata 3120

agtttgaagt ctacgagaag aaagactaat ctagaactag tggatccccc gttaacgagc 3180

tcggtacc 3188

<210> 16

<211> 6874

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

atgaccatga ttacgccaag cttatggttc agatccccca aaatccactt atccttgtag 60

atggttcatc ttatctttat cgcgcatatc acgcgtttcc cccgctgact aacagcgcag 120

gcgagccgac cggtgcgatg tatggtgtcc tcaacatgct gcgcagtctg atcatgcaat 180

ataaaccgac gcatgcagcg gtggtctttg acgccaaggg aaaaaccttt cgtgatgaac 240

tgtttgaaca ttacaaatca catcgcccgc caatgccgga cgatctgcgt gcacaaatcg 300

aacccttgca cgcgatggtt aaagcgatgg gactgccgct gctggcggtt tctggcgtag 360

aagcggacga cgttatcggt actctggcgc gcgaagccga aaaagccggg cgtccggtgc 420

tgatcagcac tggcgataaa gatatggcgc agctggtgac gccaaatatt acgcttatca 480

ataccatgac gaataccatc ctcggaccgg aagaggtggt gaataagtac ggcgtgccgc 540

cagaactgat catcgatttc ctggcgctga tgggtgactc ctctgataac attcctggcg 600

taccgggcgt cggtgaaaaa accgcgcagg cattgctgca aggtcttggc ggactggata 660

cgctgtatgc cgagccagaa aaaattgctg ggttgagctt ccgtggcgcg aaaacaatgg 720

cagcgaagct cgagcaaaac aaagaagttg cttatctctc ataccagctg gcgacgatta 780

aaaccgacgt tgaactggag ctgacctgtg aacaactgga agtgcagcaa ccggcagcgg 840

aagagttgtt ggggctgttc aaaaagtatg agttcaaacg ctggactgct gatgtcgaag 900

cgggcaaatg gttacaggcc aaaggggcaa aaccagccgc gaagccacag gaaaccagtg 960

ttgcagacga agcaccagaa gtgacggcaa cggtgatttc ttatgacaac tacgtcacca 1020

tccttgatga agaaacactg aaagcgtgga ttgcgaagct ggaaaaagcg ccggtatttg 1080

catttgatac cgaaaccgac agccttgata acatctctgc taacctggtc gggctttctt 1140

ttgctatcga gccaggcgta gcggcatata ttccggttgc tcatgattat cttgatgcgc 1200

ccgatcaaat ctctcgcgag cgtgcactcg agttgctaaa accgctgctg gaagatgaaa 1260

aggcgctgaa ggtcgggcaa aacctgaaat acgatcgcgg tattctggcg aactacggca 1320

ttgaactgcg tgggattgcg tttgatacca tgctggagtc ctacattctc aatagcgttg 1380

ccgggcgtca cgatatggac agcctcgcgg aacgttggtt gaagcacaaa accatcactt 1440

ttgaagagat tgctggtaaa ggcaaaaatc aactgacctt taaccagatt gccctcgaag 1500

aagccggacg ttacgccgcc gaagatgcag atgtcacctt gcagttgcat ctgaaaatgt 1560

ggccggatct gcaaaaacac aaagggccgt tgaacgtctt cgagaatatc gaaatgccgc 1620

tggtgccggt gctttcacgc attgaacgta acggtgtgaa gatcgatccg aaagtgctgc 1680

acaatcattc tgaagagctc acccttcgtc tggctgagct ggaaaagaaa gcgcatgaaa 1740

ttgcaggtga ggaatttaac ctttcttcca ccaagcagtt acaaaccatt ctctttgaaa 1800

aacagggcat taaaccgctg aagaaaacgc cgggtggcgc gccgtcaacg tcggaagagg 1860

tactggaaga actggcgctg gactatccgt tgccaaaagt gattctggag tatcgtggtc 1920

tggcgaagct gaaatcgacc tacaccgaca agctgccgct gatgatcaac ccgaaaaccg 1980

ggcgtgtgca tacctcttat caccaggcag taactgcaac gggacgttta tcgtcaaccg 2040

atcctaacct gcaaaacatt ccggtgcgta acgaagaagg tcgtcgtata cgccaggcgt 2100

ttattgcgcc agaggattat gtgattgtct cagcggacta ctcgcagatt gaactgcgca 2160

ttatggcgca tctttcgcgt gacaaaggct tgctgaccgc attcgcggaa ggaaaagata 2220

tccaccgggc aacggcggca gaagtgtttg gtttgccact ggaaaccgtc accagcgagc 2280

aacgccgtag cgcgaaagcg atcaactttg gtctgattta tggcatgagt gctttcggtc 2340

tggcgcggca attgaacatt ccacgtaaag aagcgcagaa gtacatggac ctttacttcg 2400

aacgctaccc tggcgtgctg gagtatatgg aacgcacccg tgctcaggcg aaagagcagg 2460

gctacgttga aacgctggac ggacgccgtc tgtatctgcc ggatatcaaa tccagcaatg 2520

gtgctcgtcg tgcagcggct gaacgtgcag ccattaacgc gccaatgcag ggaaccgccg 2580

ccgacattat caaacgggcg atgattgccg ttgatgcgtg gttacaggct gagcaaccgc 2640

gtgtacgtat gatcatgcag gtacacgatg aactggtatt tgaagttcat aaagatgatg 2700

ttgatgccgt cgcgaagcag attcatcaac tgatggaaaa ctgtacccgt ctggatgtgc 2760

cgttgctggt ggaagtgggg agtggcgaaa actgggatca ggcgcactaa gaattcactg 2820

gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt 2880

gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct 2940

tcccaacagt tgcgcagcct gaatggcgaa tggcgctaac cgtttttatc aggctctggg 3000

aggcagaata aatgatcata tcgtcaatta ttacctccac ggggagagcc tgagcaaact 3060

ggcctcaggt ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata 3120

acaattccag cggatccttc gaacatgaac actgaagccg ccaggcgttc tcgtgcgaga 3180

aagttgcaaa gaatgaaaca acttgaagac aaggttgaag aattgctttc gaaaaattat 3240

cacttggaaa atgaggttgc cagattaaag aaattagttg gcgaacgcat cgatggtggc 3300

ggtggctctg gaggtggtgg gtcctccgga atggttcgac cattgaactg catcgtcgcc 3360

gtgtcccaaa atatggggat tggcaagaac ggagacctac cctggcctcc gctcaggaac 3420

gagttcaagt acttccaaag aatgaccaca acctcttcag tggaaggtaa acagaatctg 3480

gtgattatgg gtaggaaaac ctggttctcc attcctgaga agaatcgacc tttaaaggac 3540

agaattaata tagttctcag tagagaactc aaagaaccac cacgaggagc tcattttctt 3600

gccaaaagtt tggatgatgc cttaagactt attgaacaac cggaattggg tacctaatct 3660

agaactagtg gagtcgatgt cgacgtagtt aacctcaggc atttgagaag cacacggtca 3720

cactgcttcc ggtagtcaat aaaccggtaa accagcaata gacataagcg gctatttaac 3780

gaccctgccc tgaaccgacg accgggtcga atttgctttc gaatttctgc cattcatccg 3840

cttattatca cttattcagg cgtagcacca ggcgtttaag ggcaccaata actgccttaa 3900

aaaaattacg ccccgccctg ccactcatcg cagtactgtt gtaattcatt aagcattctg 3960

ccgacatgga agccatcaca gacggcatga tgaacctgaa tcgccagcgg catcagcacc 4020

ttgtcgcctt gcgtataata tttgcccatg gtgaaaacgg gggcgaagaa gttgtccata 4080

ttggccacgt ttaaatcaaa actggtgaaa ctcacccagg gattggctga gacgaaaaac 4140

atattctcaa taaacccttt agggaaatag gccaggtttt caccgtaaca cgccacatct 4200

tgcgaatata tgtgtagaaa ctgccggaaa tcgtcgtggt attcactcca gagcgatgaa 4260

aacgtttcag tttgctcatg gaaaacggtg taacaagggt gaacactatc ccatatcacc 4320

agctcaccgt ctttcattgc catacgaaat tccggatgag cattcatcag gcgggcaaga 4380

atgtgaataa aggccggata aaacttgtgc ttatttttct ttacggtctt taaaaaggcc 4440

gtaatatcca gctgaacggt ctggttatag gtacattgag caactgactg aaatgcctca 4500

aaatgttctt tacgatgcca ttgggatata tcaacggtgg tatatccagt gatttttttc 4560

tccattttag cttccttagc tcctgaaaat ctcgataact caaaaaatac gcccggtagt 4620

gatcttattt cattatggtg aaagttggaa cctcttacgt gccgatcaac gtctcatttt 4680

cgccaaaagt tggcccaggg cttcccggta tcaacaggga caccaggatt tatttattct 4740

gcgaagtgat cttccgtcac aggtatttat tcggcgcctg tagtgccatt tacccccatt 4800

cactgccaga gccgtgagcg cagcgaactg aatgtcacga aaaagacagc gactcaggtg 4860

cctgatggtc ggagacaaaa ggaatattca gcgatttgcc cgagcttgcg agggtgctac 4920

ttaagccttt agggttttaa ggtctgtttt gtagaggagc aaacagcgtt tgcgacatcc 4980

ttttgtaata ctgcggaact gactaaagta gtgagttata cacagggctg ggatctattc 5040

tttttatctt tttttattct ttctttattc tataaattat aaccacttga atataaacaa 5100

aaaaaacaca caaaggtcta gcggaattta cagagggtct agcagaattt acaagttttc 5160

cagcaaaggt ctagcagaat ttacagatac ccacaactca aaggaaaagg actagtaatt 5220

atcattgact agcccatctc aattggtata gtgattaaaa tcacctagac caattgagat 5280

gtatgtctga attagttgtt ttcaaagcaa atgaactagc gattagtcgc tatgacttaa 5340

cggagcatga aaccaagcta attttatgct gtgtggcact actcaacccc acgattgaaa 5400

accctacaag gaaagaacgg acggtatcgt tcacttataa ccaatacgct cagatgatga 5460

acatcagtag ggaaaatgct tatggtgtat tagctaaagc aaccagagag ctgatgacga 5520

gaactgtgga aatcaggaat cctttggtta aaggctttga gattttccag tggacaaact 5580

atgccaagtt ctcaagcgaa aaattagaat tagtttttag tgaagagata ttgccttatc 5640

ttttccagtt aaaaaaattc ataaaatata atctggaaca tgttaagtct tttgaaaaca 5700

aatactctat gaggatttat gagtggttat taaaagaact aacacaaaag aaaactcaca 5760

aggcaaatat agagattagc cttgatgaat ttaagttcat gttaatgctt gaaaataact 5820

accatgagtt taaaaggctt aaccaatggg ttttgaaacc aataagtaaa gatttaaaca 5880

cttacagcaa tatgaaattg gtggttgata agcgaggccg cccgactgat acgttgattt 5940

tccaagttga actagataga caaatggatc tcgtaaccga acttgagaac aaccagataa 6000

aaatgaatgg tgacaaaata ccaacaacca ttacatcaga ttcctaccta cataacggac 6060

taagaaaaac actacacgat gctttaactg caaaaattca gctcaccagt tttgaggcaa 6120

aatttttgag tgacatgcaa agtaagtatg atctcaatgg ttcgttctca tggctcacgc 6180

aaaaacaacg aaccacacta gagaacatac tggctaaata cggaaggatc tgaggttctt 6240

atggctcttg tatctatcag tgaagcatca agactaacaa acaaaagtag aacaactgtt 6300

caccgttaca tatcaaaggg aaaactgtcc atatgcatat gcacagatga aaacggtgta 6360

aaaaagatag atacatcaga gcttttacga gtttttggtg cattcaaagc tgttcaccat 6420

gaacagatcg acaatgtaac agatgaacag catgtaacac ctaatagaac aggtgaaacc 6480

agtaaaacaa agcaactaga acatgaaatt gaacacctga gacaacttgt tacagctcaa 6540

cagtcacaca tagacagcct gaaacaggcg atgctgctta tcgaatcaaa gctgccgaca 6600

acacgggagc cagtgacgcc tcccgtgggg aaaaaatcat ggcaattctg gaagaaatag 6660

cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac 6720

gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc 6780

actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt 6840

gtgagcggat aacaatttca cacaggaaac agct 6874

<210> 17

<211> 3191

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

agtcacagaa aagcatctta cggtgtgggc ggacaaaata gttgggaact gggaggggtg 60

gaaatggagt ttttaaggat tatttaggga agagtgacaa aatagatggg aactgggtgt 120

agcgtcgtaa gctaatacga aaattaaaaa tgacaaaata gtttggaact agatttcact 180

tatctggttg gtcgacacta gtattaccct gttatcccta gatttaaatg atatcggatc 240

ctagtaagcc acgttgtgtc tcaaaatctc tgatgttaca ttgcacaaga taaaaatata 300

tcatcatgaa caataaaact gtctgcttac ataaacagta atacaagggg tgttatgagc 360

catattcaac gggaaacgtc ttgctcgagg ccgcgattaa attccaacat ggatgctgat 420

ttatatgggt ataaatgggc tcgcgataat gtcgggcaat caggtgcgac aatctatcga 480

ttgtatggga agcccgatgc gccagagttg tttctgaaac atggcaaagg tagcgttgcc 540

aatgatgtta cagatgagat ggtcagacta aactggctga cggaatttat gcctcttccg 600

accatcaagc attttatccg tactcctgat gatgcatggt tactcaccac tgcgatcccc 660

gggaaaacag cattccaggt attagaagaa tatcctgatt caggtgaaaa tattgttgat 720

gcgctggcag tgttcctgcg ccggttgcat tcgattcctg tttgtaattg tccttttaac 780

agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa tgaataacgg tttggttgat 840

gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg 900

cataagcttt tgccattctc accggattca gtcgtcactc atggtgattt ctcacttgat 960

aaccttattt ttgacgaggg gaaattaata ggttgtattg atgttggacg agtcggaatc 1020

gcagaccgat accaggatct tgccatccta tggaactgcc tcggtgagtt ttctccttca 1080

ttacagaaac ggctttttca aaaatatggt attgataatc ctgatatgaa taaattgcag 1140

tttcatttga tgctcgatga gtttttctaa tcagaattgg ttaattggtt gtaacactgg 1200

cagagcatta cgctgacttg acgggacggc ggctttgttg aataaatcga acttttgctg 1260

agttgaagga tcagatcacg catcttcccg acaacgcaga ccgttccgtg gcaaagcaaa 1320

agttcaaaat caccaactgg tccacctaca acaaagctct catcaaccgt ggctccctca 1380

ctttctggct ggatgatggg gcgattcagg cctggtatga gtcagcaaca ccttcttcac 1440

gaggcagacc tcagcggcgg ccggatccga tatcgtttaa actcgctacc ttaggaccgt 1500

tatagttagc ggccgcgtcg accccacgcc cctctttaat acgacgggca atttgcactt 1560

cagaaaatga agagtttgct ttagccataa caaaagtcca gtatgctttt tcacagcata 1620

actggactga tttcagttta caactattct gtctagttta agactttatt gtcatagttt 1680

agatctgatc gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg 1740

tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt 1800

tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat 1860

aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta 1920

gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa 1980

tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga 2040

aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac 2100

aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt 2160

tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc 2220

gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat 2280

cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag 2340

acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc 2400

cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag 2460

cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac 2520

aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg 2580

gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct 2640

atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc 2700

tcacatgtac aattgttcga atgaacactg aagccgccag gcgttctcgt gcgagaaagt 2760

tgcaaagaat gaaacaactt gaagacaagg ttgaagaaga gctttcgaaa gcttatcacg 2820

cggaaaatga ggttgccaga ttaaagaaaa aattagttgg cgaacgcatc gatggtggcg 2880

gtggctctgg aggtggtggg tcctccggaa gtaaagtaga catggtttgg atagtcggag 2940

gcagttctgt ttaccaggaa gccatgaatc aaccaggcca ccttagactc tttgtgacaa 3000

ggatcatgca ggaatttgaa agtgacacgt ttttcccaga aattgatttg gggaaatata 3060

aacttctccc agaataccca ggcgtcctct ctgaggtcca ggaggaaaaa ggcatcaagt 3120

ataagtttga agtctacgag aagaaagact aatctagaac tagtggatcc cccgttaacg 3180

agctcggtac c 3191

<210> 18

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

aaagggaaaa ctgtccatat gcgcggatca tataca 36

<210> 19

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

ccgttttcat ctgtgcatat gttagtggtg gtggtg 36

<210> 20

<211> 7840

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

atgaccatga ttacgccaag cttatggttc agatccccca aaatccactt atccttgtag 60

atggttcatc ttatctttat cgcgcatatc acgcgtttcc cccgctgact aacagcgcag 120

gcgagccgac cggtgcgatg tatggtgtcc tcaacatgct gcgcagtctg atcatgcaat 180

ataaaccgac gcatgcagcg gtggtctttg acgccaaggg aaaaaccttt cgtgatgaac 240

tgtttgaaca ttacaaatca catcgcccgc caatgccgga cgatctgcgt gcacaaatcg 300

aacccttgca cgcgatggtt aaagcgatgg gactgccgct gctggcggtt tctggcgtag 360

aagcggacga cgttatcggt actctggcgc gcgaagccga aaaagccggg cgtccggtgc 420

tgatcagcac tggcgataaa gatatggcgc agctggtgac gccaaatatt acgcttatca 480

ataccatgac gaataccatc ctcggaccgg aagaggtggt gaataagtac ggcgtgccgc 540

cagaactgat catcgatttc ctggcgctga tgggtgactc ctctgataac attcctggcg 600

taccgggcgt cggtgaaaaa accgcgcagg cattgctgca aggtcttggc ggactggata 660

cgctgtatgc cgagccagaa aaaattgctg ggttgagctt ccgtggcgcg aaaacaatgg 720

cagcgaagct cgagcaaaac aaagaagttg cttatctctc ataccagctg gcgacgatta 780

aaaccgacgt tgaactggag ctgacctgtg aacaactgga agtgcagcaa ccggcagcgg 840

aagagttgtt ggggctgttc aaaaagtatg agttcaaacg ctggactgct gatgtcgaag 900

cgggcaaatg gttacaggcc aaaggggcaa aaccagccgc gaagccacag gaaaccagtg 960

ttgcagacga agcaccagaa gtgacggcaa cggtgatttc ttatgacaac tacgtcacca 1020

tccttgatga agaaacactg aaagcgtgga ttgcgaagct ggaaaaagcg ccggtatttg 1080

catttgatac cgaaaccgac agccttgata acatctctgc taacctggtc gggctttctt 1140

ttgctatcga gccaggcgta gcggcatata ttccggttgc tcatgattat cttgatgcgc 1200

ccgatcaaat ctctcgcgag cgtgcactcg agttgctaaa accgctgctg gaagatgaaa 1260

aggcgctgaa ggtcgggcaa aacctgaaat acgatcgcgg tattctggcg aactacggca 1320

ttgaactgcg tgggattgcg tttgatacca tgctggagtc ctacattctc aatagcgttg 1380

ccgggcgtca cgatatggac agcctcgcgg aacgttggtt gaagcacaaa accatcactt 1440

ttgaagagat tgctggtaaa ggcaaaaatc aactgacctt taaccagatt gccctcgaag 1500

aagccggacg ttacgccgcc gaagatgcag atgtcacctt gcagttgcat ctgaaaatgt 1560

ggccggatct gcaaaaacac aaagggccgt tgaacgtctt cgagaatatc gaaatgccgc 1620

tggtgccggt gctttcacgc attgaacgta acggtgtgaa gatcgatccg aaagtgctgc 1680

acaatcattc tgaagagctc acccttcgtc tggctgagct ggaaaagaaa gcgcatgaaa 1740

ttgcaggtga ggaatttaac ctttcttcca ccaagcagtt acaaaccatt ctctttgaaa 1800

aacagggcat taaaccgctg aagaaaacgc cgggtggcgc gccgtcaacg tcggaagagg 1860

tactggaaga actggcgctg gactatccgt tgccaaaagt gattctggag tatcgtggtc 1920

tggcgaagct gaaatcgacc tacaccgaca agctgccgct gatgatcaac ccgaaaaccg 1980

ggcgtgtgca tacctcttat caccaggcag taactgcaac gggacgttta tcgtcaaccg 2040

atcctaacct gcaaaacatt ccggtgcgta acgaagaagg tcgtcgtata cgccaggcgt 2100

ttattgcgcc agaggattat gtgattgtct cagcggacta ctcgcagatt gaactgcgca 2160

ttatggcgca tctttcgcgt gacaaaggct tgctgaccgc attcgcggaa ggaaaagata 2220

tccaccgggc aacggcggca gaagtgtttg gtttgccact ggaaaccgtc accagcgagc 2280

aacgccgtag cgcgaaagcg atcaactttg gtctgattta tggcatgagt gctttcggtc 2340

tggcgcggca attgaacatt ccacgtaaag aagcgcagaa gtacatggac ctttacttcg 2400

aacgctaccc tggcgtgctg gagtatatgg aacgcacccg tgctcaggcg aaagagcagg 2460

gctacgttga aacgctggac ggacgccgtc tgtatctgcc ggatatcaaa tccagcaatg 2520

gtgctcgtcg tgcagcggct gaacgtgcag ccattaacgc gccaatgcag ggaaccgccg 2580

ccgacattat caaacgggcg atgattgccg ttgatgcgtg gttacaggct gagcaaccgc 2640

gtgtacgtat gatcatgcag gtacacgatg aactggtatt tgaagttcat aaagatgatg 2700

ttgatgccgt cgcgaagcag attcatcaac tgatggaaaa ctgtacccgt ctggatgtgc 2760

cgttgctggt ggaagtgggg agtggcgaaa actgggatca ggcgcactaa gaattcactg 2820

gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt 2880

gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct 2940

tcccaacagt tgcgcagcct gaatggcgaa tggcgctaac cgtttttatc aggctctggg 3000

aggcagaata aatgatcata tcgtcaatta ttacctccac ggggagagcc tgagcaaact 3060

ggcctcaggt aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct 3120

tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag aattcattaa 3180

agaggagaaa ttaactatga gaggatctgg atcccatcat caccatgcgg ccgcattcga 3240

acatggcgaa cgcatcgatg gtggcggtgg ctctggaggt ggtgggtcct ccggaatggt 3300

tcgaccattg aactgcatcg tcgccgtgtc ccaaaatatg gggattggca agaacggaga 3360

cctaccctgg cctccgctca ggaacgagtt caagtacttc caaagaatga ccacaacctc 3420

ttcagtggaa ggtaaacaga atctggtgat tatgggtagg aaaacctggt tctccattcc 3480

tgagaagaat cgacctttaa aggacagaat taatatagtt ctcagtagag aactcaaaga 3540

accaccacga ggagctcatt ttcttgccaa aagtttggat gatgccttaa gacttattga 3600

acaaccggaa ttgggtaccc tggttgacta caaggacgac gatgacaagt aatctagaac 3660

tagtggagtc gatgtcgacg tagttaacct caggcatttg agaagcacac ggtcacactg 3720

cttccggtag tcaataaacc ggtaaaccag caatagacat aagcggctat ttaacgaccc 3780

tgccctgaac cgacgaccgg gtcgaatttg ctttcgaatt tctgccattc atccgcttat 3840

tatcacttat tcaggcgtag caccaggcgt ttaagggcac caataactgc cttaaaaaaa 3900

ttacgccccg ccctgccact catcgcagta ctgttgtaat tcattaagca ttctgccgac 3960

atggaagcca tcacagacgg catgatgaac ctgaatcgcc agcggcatca gcaccttgtc 4020

gccttgcgta taatatttgc ccatggtgaa aacgggggcg aagaagttgt ccatattggc 4080

cacgtttaaa tcaaaactgg tgaaactcac ccagggattg gctgagacga aaaacatatt 4140

ctcaataaac cctttaggga aataggccag gttttcaccg taacacgcca catcttgcga 4200

atatatgtgt agaaactgcc ggaaatcgtc gtggtattca ctccagagcg atgaaaacgt 4260

ttcagtttgc tcatggaaaa cggtgtaaca agggtgaaca ctatcccata tcaccagctc 4320

accgtctttc attgccatac gaaattccgg atgagcattc atcaggcggg caagaatgtg 4380

aataaaggcc ggataaaact tgtgcttatt tttctttacg gtctttaaaa aggccgtaat 4440

atccagctga acggtctggt tataggtaca ttgagcaact gactgaaatg cctcaaaatg 4500

ttctttacga tgccattggg atatatcaac ggtggtatat ccagtgattt ttttctccat 4560

tttagcttcc ttagctcctg aaaatctcga taactcaaaa aatacgcccg gtagtgatct 4620

tatttcatta tggtgaaagt tggaacctct tacgtgccga tcaacgtctc attttcgcca 4680

aaagttggcc cagggcttcc cggtatcaac agggacacca ggatttattt attctgcgaa 4740

gtgatcttcc gtcacaggta tttattcggc gcctgtagtg ccatttaccc ccattcactg 4800

ccagagccgt gagcgcagcg aactgaatgt cacgaaaaag acagcgactc aggtgcctga 4860

tggtcggaga caaaaggaat attcagcgat ttgcccgagc ttgcgagggt gctacttaag 4920

cctttagggt tttaaggtct gttttgtaga ggagcaaaca gcgtttgcga catccttttg 4980

taatactgcg gaactgacta aagtagtgag ttatacacag ggctgggatc tattcttttt 5040

atcttttttt attctttctt tattctataa attataacca cttgaatata aacaaaaaaa 5100

acacacaaag gtctagcgga atttacagag ggtctagcag aatttacaag ttttccagca 5160

aaggtctagc agaatttaca gatacccaca actcaaagga aaaggactag taattatcat 5220

tgactagccc atctcaattg gtatagtgat taaaatcacc tagaccaatt gagatgtatg 5280

tctgaattag ttgttttcaa agcaaatgaa ctagcgatta gtcgctatga cttaacggag 5340

catgaaacca agctaatttt atgctgtgtg gcactactca accccacgat tgaaaaccct 5400

acaaggaaag aacggacggt atcgttcact tataaccaat acgctcagat gatgaacatc 5460

agtagggaaa atgcttatgg tgtattagct aaagcaacca gagagctgat gacgagaact 5520

gtggaaatca ggaatccttt ggttaaaggc tttgagattt tccagtggac aaactatgcc 5580

aagttctcaa gcgaaaaatt agaattagtt tttagtgaag agatattgcc ttatcttttc 5640

cagttaaaaa aattcataaa atataatctg gaacatgtta agtcttttga aaacaaatac 5700

tctatgagga tttatgagtg gttattaaaa gaactaacac aaaagaaaac tcacaaggca 5760

aatatagaga ttagccttga tgaatttaag ttcatgttaa tgcttgaaaa taactaccat 5820

gagtttaaaa ggcttaacca atgggttttg aaaccaataa gtaaagattt aaacacttac 5880

agcaatatga aattggtggt tgataagcga ggccgcccga ctgatacgtt gattttccaa 5940

gttgaactag atagacaaat ggatctcgta accgaacttg agaacaacca gataaaaatg 6000

aatggtgaca aaataccaac aaccattaca tcagattcct acctacataa cggactaaga 6060

aaaacactac acgatgcttt aactgcaaaa attcagctca ccagttttga ggcaaaattt 6120

ttgagtgaca tgcaaagtaa gtatgatctc aatggttcgt tctcatggct cacgcaaaaa 6180

caacgaacca cactagagaa catactggct aaatacggaa ggatctgagg ttcttatggc 6240

tcttgtatct atcagtgaag catcaagact aacaaacaaa agtagaacaa ctgttcaccg 6300

ttacatatca aagggaaaac tgtccatatg cgcggatcat atacatatct tttaacggta 6360

tccggcaacc agccaggtcc ccttgtgcta ttattcgcac ctttggagcg cctgaaacct 6420

gcggcgcgca tttcaatcgc tgttctcttt cagcgaaata acaagaactt gtggtgacag 6480

gaggaaccat gaaacagtat ttagaactga tgcaaaaagt gctcgacgaa ggcacacaga 6540

aaaacgaccg taccggaacc ggaacgcttt ccatttttgg tcatcagatg cgttttaacc 6600

tgcaagatgg attcccgctg gtgacaacta aacgttgcca cctgcgttcc atcatccatg 6660

aactgctgtg gtttctgcag ggcgacacta acattgctta tctacacgaa aacaatgtca 6720

ccatctggga cgaatgggcc gatgaaaacg gcgacctcgg gccagtgtat ggtaaacagt 6780

ggcgcgcctg gccaacgcca gatggtcgtc atattgacca gatcactacg gtactgaacc 6840

agctgaaaaa cgacccggat tcgcgccgca ttattgtttc agcgtggaac gtaggcgaac 6900

tggataaaat ggcgctggca ccgtgccatg cattcttcca gttctatgtg gcagacggca 6960

aactctcttg ccagctttat cagcgctcct gtgacgtctt cctcggcctg ccgttcaaca 7020

ttgccagcta cgcgttattg gtgcatatga tggcgcagca gtgcgatctg gaagtgggtg 7080

attttgtctg gaccggtggc gacacgcatc tgtacagcaa ccatatggat caaactcatc 7140

tgcaattaag ccgcgaaccg cgtccgctgc cgaagttgat tatcaaacgt aaacccgaat 7200

ccatcttcga ctaccgtttc gaagactttg agattgaagg ctacgatccg catccgggca 7260

ttaaagcgcc ggtggctatc caccaccacc accaccacta acatatgcac agatgaaaac 7320

ggtgtaaaaa agatagatac atcagagctt ttacgagttt ttggtgcatt caaagctgtt 7380

caccatgaac agatcgacaa tgtaacagat gaacagcatg taacacctaa tagaacaggt 7440

gaaaccagta aaacaaagca actagaacat gaaattgaac acctgagaca acttgttaca 7500

gctcaacagt cacacataga cagcctgaaa caggcgatgc tgcttatcga atcaaagctg 7560

ccgacaacac gggagccagt gacgcctccc gtggggaaaa aatcatggca attctggaag 7620

aaatagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc 7680

tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt 7740

tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt 7800

ggaattgtga gcggataaca atttcacaca ggaaacagct 7840

<210> 21

<211> 468

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

atgagagata ccacagtcaa acctggagcc aaaaaggaca caaaggactc tcgacccaaa 60

ctgccccaga ccctctccag aggttggggt gaccaactca tctggactca gacatatgaa 120

gaagctctat ataaatccaa gacaagcaac aaacccttga tgattattca tcacttggat 180

gagtgcccac acagtcaagc tttaaagaaa gtgtttgctg aaaataaaga aatccagaaa 240

ttggcagagc agtttgtcct cctcaatctg gtttatgaaa caactgacaa acacctttct 300

cctgatggcc agtatgtccc caggattatg tttgttgacc catctctgac agttagagcc 360

gatatcactg gaagatattc aaatcgtctc tatgcttacg aacctgcaga tacagctctg 420

ttgcttgaca acatgaagaa agctctcaag ttgctgaaga ctgaattg 468

<210> 22

<211> 1533

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

atggacatga gggttcctgc tcagctcctg ggactcctgc tgctctggct cccaggtgcc 60

agatgtgaga ttgtgctgac acagtctcct gccaccttat ctctctctcc aggggagagg 120

gccaccctga gctgcagggc cagcaagagt gtcagtacat ctggctatag ttatatgcac 180

tggtaccaac agaaaccagg ccaggccccc agactcctca tctatcttgc atctaaccta 240

gaatctggga tccctgctag attcagtggc agtgggtctg ggacagactt cacactcacc 300

atcagcaggc tggaacctga ggacttcgct gtgtattact gtcagcacat tagagagctt 360

cctcggacgt tcggtggagg caccaagctg gaaatcaaag gcggcggcgg ctccggcggc 420

ggcggctccg gcggcggcgg ctccggcggc ggcggctccc aggtccagct ggtgcagtct 480

ggagctgagg tgaagaagcc tggagcttca gtgaaggttt cctgcaaggc ttctggatac 540

acattcactg actacaacat ggactgggtt cgacaggccc ctggacaggg ccttgagtgg 600

attggagata ttaatcctaa ctatgacact actagctaca accagaagtt ccagggcaga 660

gtgacaatga ctgtggacaa gtccacgagc acagcctaca tggagctcag cagcctgaga 720

tctgaggaca ctgcagtcta ttactgtgca agatcgatga tgggatatgg ttcccctatg 780

gactactggg gtcaaggcac actggtcacc gtctcctcag cctccaccaa gggcccatcg 840

gtcttccccc tggcaccctc ctccaagagc acctctgggg gcacagcagc cctgggctgc 900

ctggtcaagg actacttccc cgaaccggtg acggtgtcgt ggaactcagg cgccctgacc 960

agcggcgtgc acaccttccc ggctgtccta cagtcctcag gactctactc cctcagcagc 1020

gtggtgaccg tgccctccag cagcttggac acccagacct acatctgcaa cgtgaatcac 1080

aagcccagca acaccaaggt ggacaagaaa gttgagccca aatcttgtga caaaactcac 1140

acatgcccac cgtgcccagc acctgaactc ctggggggac cgtcagtctt cctcttcccc 1200

ccaaaaccca aggacaccct catgatctcc cggacccctg aggtcacatg cgtggtggtg 1260

gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt acgtggacgg cgtggaggtg 1320

cataatgcca agacaaagcc gcgggaggag cagtacaaca gcacgtaccg tgtggtcagc 1380

gtcctcaccg tcctgcacca ggactggctg aatggcaagg agtacaagtg caaggtctcc 1440

aacaaagccc tcccagcccc catcgagaaa accatctcca aagccaaagg tgagtggacc 1500

cgtggggtgc gagggccaca tgataacttc gta 1533

<210> 23

<211> 8296

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

atgaccatga ttacgccaag cttatggttc agatccccca aaatccactt atccttgtag 60

atggttcatc ttatctttat cgcgcatatc acgcgtttcc cccgctgact aacagcgcag 120

gcgagccgac cggtgcgatg tatggtgtcc tcaacatgct gcgcagtctg atcatgcaat 180

ataaaccgac gcatgcagcg gtggtctttg acgccaaggg aaaaaccttt cgtgatgaac 240

tgtttgaaca ttacaaatca catcgcccgc caatgccgga cgatctgcgt gcacaaatcg 300

aacccttgca cgcgatggtt aaagcgatgg gactgccgct gctggcggtt tctggcgtag 360

aagcggacga cgttatcggt actctggcgc gcgaagccga aaaagccggg cgtccggtgc 420

tgatcagcac tggcgataaa gatatggcgc agctggtgac gccaaatatt acgcttatca 480

ataccatgac gaataccatc ctcggaccgg aagaggtggt gaataagtac ggcgtgccgc 540

cagaactgat catcgatttc ctggcgctga tgggtgactc ctctgataac attcctggcg 600

taccgggcgt cggtgaaaaa accgcgcagg cattgctgca aggtcttggc ggactggata 660

cgctgtatgc cgagccagaa aaaattgctg ggttgagctt ccgtggcgcg aaaacaatgg 720

cagcgaagct cgagcaaaac aaagaagttg cttatctctc ataccagctg gcgacgatta 780

aaaccgacgt tgaactggag ctgacctgtg aacaactgga agtgcagcaa ccggcagcgg 840

aagagttgtt ggggctgttc aaaaagtatg agttcaaacg ctggactgct gatgtcgaag 900

cgggcaaatg gttacaggcc aaaggggcaa aaccagccgc gaagccacag gaaaccagtg 960

ttgcagacga agcaccagaa gtgacggcaa cggtgatttc ttatgacaac tacgtcacca 1020

tccttgatga agaaacactg aaagcgtgga ttgcgaagct ggaaaaagcg ccggtatttg 1080

catttgatac cgaaaccgac agccttgata acatctctgc taacctggtc gggctttctt 1140

ttgctatcga gccaggcgta gcggcatata ttccggttgc tcatgattat cttgatgcgc 1200

ccgatcaaat ctctcgcgag cgtgcactcg agttgctaaa accgctgctg gaagatgaaa 1260

aggcgctgaa ggtcgggcaa aacctgaaat acgctcgcgg tattctggcg aactacggca 1320

ttgaactgcg tgggattgcg tttgatacca tgctggagtc ctacattctc aatagcgttg 1380

ccgggcgtca cgatatggac agcctcgcgg aacgttggtt gaagcacaaa accatcactt 1440

ttgaagagat tgctggtaaa ggcaaaaatc aactgacctt taaccagatt gccctcgaag 1500

aagccggacg ttacgccgcc gaagatgcag atgtcacctt gcagttgcat ctgaaaatgt 1560

ggccggatct gcaaaaacac aaagggccgt tgaacgtctt cgagaatatc gaaatgccgc 1620

tggtgccggt gctttcacgc attgaacgta acggtgtgaa gatcgatccg aaagtgctgc 1680

acaatcattc tgaagagctc acccttcgtc tggctgagct ggaaaagaaa gcgcatgaaa 1740

ttgcaggtga ggaatttaac ctttcttcca ccaaggagtt acaaaccatt ctctttgaaa 1800

aacagggcat taaaccgctg aagaaaacgc cgggtggcgc gccgtcaacg tcggaagagg 1860

tactggaaga actggcgctg gactatccgt tgccaaaagt gattctggag tatcgtggtc 1920

tggcgaagct gaaatcgacc tacaccgaca agctgccgct gatgatcaac ccgaaaaccg 1980

ggcgtgtgca tacctcttat caccaggcag taactgcaac gggacgttta tcgtcaaccg 2040

atcctaacct gcaaaacatt ccggtgcgta acgaagaagg tcgtcgtata cgccaggcgt 2100

ttattgcgcc agaggattat gtgattgtct cagccgacta ctcgcagaat gaactgcgca 2160

ttatggcgta tctttcgcgt gacaaaggct tgctgaccgc attcgcggaa ggaaaagata 2220

tccaccgggc aacggccgca gaagtgtttg gtttgccact ggaaaccgtc accagcgagc 2280

aacgccgtag cgcgaaacgg atcaactttg gtctgattta tggcatgagt gctttcggtc 2340

tggcgcggca attgaacatt ccacgtaaag aagcgcagaa gtacatggac ctttacttcg 2400

aacgctaccc tggcgtgctg gagtatatgg aacgcacccg tgctcaggcg aaagagcagg 2460

gctacgttga aacgctggac ggacgccgtc tgtatctgcc ggatatcaaa tccagcaatg 2520

gtgctcgtcg tgcagcggct gaacgtgcag ccattaacgc gccaatgcag ggaaccgccg 2580

ccgacattat caaacgggcg atgattgccg ttgatgcgtg gttacaggct gagcaaccgc 2640

gtgtacgtat gatcatgcag gtacacgatg aactggtatt tgaagttcat aaagatgatg 2700

ttgatgccgt cgcgaagcag attcatcaac tgatggaaaa ctgtacccgt ctggatgtgc 2760

cgttgctggt ggaagtgggg agtggcgaaa actgggatca ggcgcactaa gaattcactg 2820

gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt 2880

gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct 2940

tcccaacagt tgcgcagcct gaatggcgaa tggcgctaac cgtttttatc aggctctggg 3000

aggcagaata aatgatcata tcgtcaatta ttacctccac ggggagagcc tgagcaaact 3060

ggcctcaggt aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct 3120

tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag aattcattaa 3180

agaggagaaa ttaactatga gaggatctgg atccatgaga gataccacag tcaaacctgg 3240

agccaaaaag gacacaaagg actctcgacc caaactgccc cagaccctct ccagaggttg 3300

gggtgaccaa ctcatctgga ctcagacata tgaagaagct ctatataaat ccaagacaag 3360

caacaaaccc ttgatgatta ttcatcactt ggatgagtgc ccacacagtc aagctttaaa 3420

gaaagtgttt gctgaaaata aagaaatcca gaaattggca gagcagtttg tcctcctcaa 3480

tctggtttat gaaacaactg acaaacacct ttctcctgat ggccagtatg tccccaggat 3540

tatgtttgtt gacccatctc tgacagttag agccgatatc actggaagat attcaaatcg 3600

tctctatgct tacgaacctg cagatacagc tctgttgctt gacaacatga agaaagctct 3660

caagttgctg aagactgaat tggcggccgc attcgaacat ggcgaacgca tcgatggtgg 3720

cggtggctct ggaggtggtg ggtcctccgg aatggttcga ccattgaact gcatcgtcgc 3780

cgtgtcccaa aatatgggga ttggcaagaa cggagaccta ccctggcctc cgctcaggaa 3840

cgagttcaag tacttccaaa gaatgaccac aacctcttca gtggaaggta aacagaatct 3900

ggtgattatg ggtaggaaaa cctggttctc cattcctgag aagaatcgac ctttaaagga 3960

cagaattaat atagttctca gtagagaact caaagaacca ccacgaggag ctcattttct 4020

tgccaaaagt ttggatgatg ccttaagact tattgaacaa ccggaattgg gtaccctggt 4080

tgactacaag gacgacgatg acaagtaatc tagaactagt ggagtcgatg tcgacgtagt 4140

taacctcagg catttgagaa gcacacggtc acactgcttc cggtagtcaa taaaccggta 4200

aaccagcaat agacataagc ggctatttaa cgaccctgcc ctgaaccgac gaccgggtcg 4260

aatttgcttt cgaatttctg ccattcatcc gcttattatc acttattcag gcgtagcacc 4320

aggcgtttaa gggcaccaat aactgcctta aaaaaattac gccccgccct gccactcatc 4380

gcagtactgt tgtaattcat taagcattct gccgacatgg aagccatcac agacggcatg 4440

atgaacctga atcgccagcg gcatcagcac cttgtcgcct tgcgtataat atttgcccat 4500

ggtgaaaacg ggggcgaaga agttgtccat attggccacg tttaaatcaa aactggtgaa 4560

actcacccag ggattggctg agacgaaaaa catattctca ataaaccctt tagggaaata 4620

ggccaggttt tcaccgtaac acgccacatc ttgcgaatat atgtgtagaa actgccggaa 4680

atcgtcgtgg tattcactcc agagcgatga aaacgtttca gtttgctcat ggaaaacggt 4740

gtaacaaggg tgaacactat cccatatcac cagctcaccg tctttcattg ccatacgaaa 4800

ttccggatga gcattcatca ggcgggcaag aatgtgaata aaggccggat aaaacttgtg 4860

cttatttttc tttacggtct ttaaaaaggc cgtaatatcc agctgaacgg tctggttata 4920

ggtacattga gcaactgact gaaatgcctc aaaatgttct ttacgatgcc attgggatat 4980

atcaacggtg gtatatccag tgattttttt ctccatttta gcttccttag ctcctgaaaa 5040

tctcgataac tcaaaaaata cgcccggtag tgatcttatt tcattatggt gaaagttgga 5100

acctcttacg tgccgatcaa cgtctcattt tcgccaaaag ttggcccagg gcttcccggt 5160

atcaacaggg acaccaggat ttatttattc tgcgaagtga tcttccgtca caggtattta 5220

ttcggcgcct gtagtgccat ttacccccat tcactgccag agccgtgagc gcagcgaact 5280

gaatgtcacg aaaaagacag cgactcaggt gcctgatggt cggagacaaa aggaatattc 5340

agcgatttgc ccgagcttgc gagggtgcta cttaagcctt tagggtttta aggtctgttt 5400

tgtagaggag caaacagcgt ttgcgacatc cttttgtaat actgcggaac tgactaaagt 5460

agtgagttat acacagggct gggatctatt ctttttatct ttttttattc tttctttatt 5520

ctataaatta taaccacttg aatataaaca aaaaaaacac acaaaggtct agcggaattt 5580

acagagggtc tagcagaatt tacaagtttt ccagcaaagg tctagcagaa tttacagata 5640

cccacaactc aaaggaaaag gactagtaat tatcattgac tagcccatct caattggtat 5700

agtgattaaa atcacctaga ccaattgaga tgtatgtctg aattagttgt tttcaaagca 5760

aatgaactag cgattagtcg ctatgactta acggagcatg aaaccaagct aattttatgc 5820

tgtgtggcac tactcaaccc cacgattgaa aaccctacaa ggaaagaacg gacggtatcg 5880

ttcacttata accaatacgc tcagatgatg aacatcagta gggaaaatgc ttatggtgta 5940

ttagctaaag caaccagaga gctgatgacg agaactgtgg aaatcaggaa tcctttggtt 6000

aaaggctttg agattttcca gtggacaaac tatgccaagt tctcaagcga aaaattagaa 6060

ttagttttta gtgaagagat attgccttat cttttccagt taaaaaaatt cataaaatat 6120

aatctggaac atgttaagtc ttttgaaaac aaatactcta tgaggattta tgagtggtta 6180

ttaaaagaac taacacaaaa gaaaactcac aaggcaaata tagagattag ccttgatgaa 6240

tttaagttca tgttaatgct tgaaaataac taccatgagt ttaaaaggct taaccaatgg 6300

gttttgaaac caataagtaa agatttaaac acttacagca atatgaaatt ggtggttgat 6360

aagcgaggcc gcccgactga tacgttgatt ttccaagttg aactagatag acaaatggat 6420

ctcgtaaccg aacttgagaa caaccagata aaaatgaatg gtgacaaaat accaacaacc 6480

attacatcag attcctacct acataacgga ctaagaaaaa cactacacga tgctttaact 6540

gcaaaaattc agctcaccag ttttgaggca aaatttttga gtgacatgca aagtaagtat 6600

gatctcaatg gttcgttctc atggctcacg caaaaacaac gaaccacact agagaacata 6660

ctggctaaat acggaaggat ctgaggttct tatggctctt gtatctatca gtgaagcatc 6720

aagactaaca aacaaaagta gaacaactgt tcaccgttac atatcaaagg gaaaactgtc 6780

catatgcgcg gatcatatac atatctttta acggtatccg gcaaccagcc aggtcccctt 6840

gtgctattat tcgcaccttt ggagcgcctg aaacctgcgg cgcgcatttc aatcgctgtt 6900

ctctttcagc gaaataacaa gaacttgtgg tgacaggagg aaccatgaaa cagtatttag 6960

aactgatgca aaaagtgctc gacgaaggca cacagaaaaa cgaccgtacc ggaaccggaa 7020

cgctttccat ttttggtcat cagatgcgtt ttaacctgca agatggattc ccgctggtga 7080

caactaaacg ttgccacctg cgttccatca tccatgaact gctgtggttt ctgcagggcg 7140

acactaacat tgcttatcta cacgaaaaca atgtcaccat ctgggacgaa tgggccgatg 7200

aaaacggcga cctcgggcca gtgtatggta aacagtggcg cgcctggcca acgccagatg 7260

gtcgtcatat tgaccagatc actacggtac tgaaccagct gaaaaacgac ccggattcgc 7320

gccgcattat tgtttcagcg tggaacgtag gcgaactgga taaaatggcg ctggcaccgt 7380

gccatgcatt cttccagttc tatgtggcag acggcaaact ctcttgccag ctttatcagc 7440

gctcctgtga cgtcttcctc ggcctgccgt tcaacattgc cagctacgcg ttattggtgc 7500

atatgatggc gcagcagtgc gatctggaag tgggtgattt tgtctggacc ggtggcgaca 7560

cgcatctgta cagcaaccat atggatcaaa ctcatctgca attaagccgc gaaccgcgtc 7620

cgctgccgaa gttgattatc aaacgtaaac ccgaatccat cttcgactac cgtttcgaag 7680

actttgagat tgaaggctac gatccgcatc cgggcattaa agcgccggtg gctatccacc 7740

accaccacca ccactaacat atgcacagat gaaaacggtg taaaaaagat agatacatca 7800

gagcttttac gagtttttgg tgcattcaaa gctgttcacc atgaacagat cgacaatgta 7860

acagatgaac agcatgtaac acctaataga acaggtgaaa ccagtaaaac aaagcaacta 7920

gaacatgaaa ttgaacacct gagacaactt gttacagctc aacagtcaca catagacagc 7980

ctgaaacagg cgatgctgct tatcgaatca aagctgccga caacacggga gccagtgacg 8040

cctcccgtgg ggaaaaaatc atggcaattc tggaagaaat agcgcccaat acgcaaaccg 8100

cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg 8160

aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag 8220

gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt 8280

cacacaggaa acagct 8296

<210> 24

<211> 4743

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

agtcacagaa aagcatctta cggtgtgggc ggacaaaata gttgggaact gggaggggtg 60

gaaatggagt ttttaaggat tatttaggga agagtgacaa aatagatggg aactgggtgt 120

agcgtcgtaa gctaatacga aaattaaaaa tgacaaaata gtttggaact agatttcact 180

tatctggttg gtcgacacta gtattaccct gttatcccta gatttaaatg atatcggatc 240

ctagtaagcc acgttgtgtc tcaaaatctc tgatgttaca ttgcacaaga taaaaatata 300

tcatcatgaa caataaaact gtctgcttac ataaacagta atacaagggg tgttatgagc 360

catattcaac gggaaacgtc ttgctcgagg ccgcgattaa attccaacat ggatgctgat 420

ttatatgggt ataaatgggc tcgcgataat gtcgggcaat caggtgcgac aatctatcga 480

ttgtatggga agcccgatgc gccagagttg tttctgaaac atggcaaagg tagcgttgcc 540

aatgatgtta cagatgagat ggtcagacta aactggctga cggaatttat gcctcttccg 600

accatcaagc attttatccg tactcctgat gatgcatggt tactcaccac tgcgatcccc 660

gggaaaacag cattccaggt attagaagaa tatcctgatt caggtgaaaa tattgttgat 720

gcgctggcag tgttcctgcg ccggttgcat tcgattcctg tttgtaattg tccttttaac 780

agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa tgaataacgg tttggttgat 840

gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg 900

cataagcttt tgccattctc accggattca gtcgtcactc atggtgattt ctcacttgat 960

aaccttattt ttgacgaggg gaaattaata ggttgtattg atgttggacg agtcggaatc 1020

gcagaccgat accaggatct tgccatccta tggaactgcc tcggtgagtt ttctccttca 1080

ttacagaaac ggctttttca aaaatatggt attgataatc ctgatatgaa taaattgcag 1140

tttcatttga tgctcgatga gtttttctaa tcagaattgg ttaattggtt gtaacactgg 1200

cagagcatta cgctgacttg acgggacggc ggctttgttg aataaatcga acttttgctg 1260

agttgaagga tcagatcacg catcttcccg acaacgcaga ccgttccgtg gcaaagcaaa 1320

agttcaaaat caccaactgg tccacctaca acaaagctct catcaaccgt ggctccctca 1380

ctttctggct ggatgatggg gcgattcagg cctggtatga gtcagcaaca ccttcttcac 1440

gaggcagacc tcagcggcgg ccggatccga tatcgtttaa actcgctacc ttaggaccgt 1500

tatagttagc ggccgcgtcg accccacgcc cctctttaat acgacgggca atttgcactt 1560

cagaaaatga agagtttgct ttagccataa caaaagtcca gtatgctttt tcacagcata 1620

actggactga tttcagttta caactattct gtctagttta agactttatt gtcatagttt 1680

agatctgatc gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg 1740

tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt 1800

tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat 1860

aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta 1920

gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa 1980

tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga 2040

aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac 2100

aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt 2160

tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc 2220

gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat 2280

cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag 2340

acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc 2400

cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag 2460

cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac 2520

aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg 2580

gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct 2640

atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc 2700

tcacatgtta atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt 2760

ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacaga attcattaaa 2820

gaggagaaat taactatgag aggatctcaa ttgatggaca tgagggttcc tgctcagctc 2880

ctgggactcc tgctgctctg gctcccaggt gccagatgtg agattgtgct gacacagtct 2940

cctgccacct tatctctctc tccaggggag agggccaccc tgagctgcag ggccagcaag 3000

agtgtcagta catctggcta tagttatatg cactggtacc aacagaaacc aggccaggcc 3060

cccagactcc tcatctatct tgcatctaac ctagaatctg ggatccctgc tagattcagt 3120

ggcagtgggt ctgggacaga cttcacactc accatcagca ggctggaacc tgaggacttc 3180

gctgtgtatt actgtcagca cattagagag cttcctcgga cgttcggtgg aggcaccaag 3240

ctggaaatca aaggcggcgg cggctccggc ggcggcggct ccggcggcgg cggctccggc 3300

ggcggcggct cccaggtcca gctggtgcag tctggagctg aggtgaagaa gcctggagct 3360

tcagtgaagg tttcctgcaa ggcttctgga tacacattca ctgactacaa catggactgg 3420

gttcgacagg cccctggaca gggccttgag tggattggag atattaatcc taactatgac 3480

actactagct acaaccagaa gttccagggc agagtgacaa tgactgtgga caagtccacg 3540

agcacagcct acatggagct cagcagcctg agatctgagg acactgcagt ctattactgt 3600

gcaagatcga tgatgggata tggttcccct atggactact ggggtcaagg cacactggtc 3660

accgtctcct cagcctccac caagggccca tcggtcttcc ccctggcacc ctcctccaag 3720

agcacctctg ggggcacagc agccctgggc tgcctggtca aggactactt ccccgaaccg 3780

gtgacggtgt cgtggaactc aggcgccctg accagcggcg tgcacacctt cccggctgtc 3840

ctacagtcct caggactcta ctccctcagc agcgtggtga ccgtgccctc cagcagcttg 3900

gacacccaga cctacatctg caacgtgaat cacaagccca gcaacaccaa ggtggacaag 3960

aaagttgagc ccaaatcttg tgacaaaact cacacatgcc caccgtgccc agcacctgaa 4020

ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 4080

tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 4140

aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag 4200

gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 4260

ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag 4320

aaaaccatct ccaaagccaa aggtgagtgg acccgtgggg tgcgagggcc acatgataac 4380

ttcgtaccat ggttcttagt tggcgaacgc atcgatggtg gcggtggctc tggaggtggt 4440

gggtcctccg gaagtaaagt agacatggtt tggatagtcg gaggcagttc tgtttaccag 4500

gaagccatga atcaaccagg ccaccttaga ctctttgtga caaggatcat gcaggaattt 4560

gaaagtgaca cgtttttccc agaaattgat ttggggaaat ataaacttct cccagaatac 4620

ccaggcgtcc tctctgaggt ccaggaggaa aaaggcatca agtataagtt tgaagtctac 4680

gagaagaaag accatcatca ccatcaccat taatctagaa ctagtgttaa cgagctcggt 4740

acc 4743

<210> 25

<211> 1575

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

cagcaaatgg gtcgcggatc catggacatg agggttcctg ctcagctcct gggactcctg 60

ctgctctggc tcccaggtgc cagatgtgag attgtgctga cacagtctcc tgccacctta 120

tctctctctc caggggagag ggccaccctg agctgcaggg ccagcaagag tgtcagtaca 180

tctggctata gttatatgca ctggtaccaa cagaaaccag gccaggcccc cagactcctc 240

atctatcttg catctaacct agaatctggg atccctgcta gattcagtgg cagtgggtct 300

gggacagact tcacactcac catcagcagg ctggaacctg aggacttcgc tgtgtattac 360

tgtcagcaca ttagagagct tcctcggacg ttcggtggag gcaccaagct ggaaatcaaa 420

ggcggcggcg gctccggcgg cggcggctcc ggcggcggcg gctccggcgg cggcggctcc 480

caggtccagc tggtgcagtc tggagctgag gtgaagaagc ctggagcttc agtgaaggtt 540

tcctgcaagg cttctggata cacattcact gactacaaca tggactgggt tcgacaggcc 600

cctggacagg gccttgagtg gattggagat attaatccta actatgacac tactagctac 660

aaccagaagt tccagggcag agtgacaatg actgtggaca agtccacgag cacagcctac 720

atggagctca gcagcctgag atctgaggac actgcagtct attactgtgc aagatcgatg 780

atgggatatg gttcccctat ggactactgg ggtcaaggca cactggtcac cgtctcctca 840

gcctccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 900

ggcacagcag ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 960

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 1020

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttgga cacccagacc 1080

tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 1140

aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 1200

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 1260

gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 1320

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 1380

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 1440

gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1500

aaagccaaag gtgagtggac ccgtggggtg cgagggccac atgataactt cgtaaagctt 1560

gcggccgcac tcgag 1575

<210> 26

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

cagcaaatgg gtcgcggatc catggacatg agggtt 36

<210> 27

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

ctcgagtgcg gccgcaagct ttacgaagtt atcatg 36

<210> 28

<211> 6883

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa 60

ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt 120

tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttta 180

cgaagttatc atgtggccct cgcaccccac gggtccactc acctttggct ttggagatgg 240

ttttctcgat gggggctggg agggctttgt tggagacctt gcacttgtac tccttgccat 300

tcagccagtc ctggtgcagg acggtgagga cgctgaccac acggtacgtg ctgttgtact 360

gctcctcccg cggctttgtc ttggcattat gcacctccac gccgtccacg taccagttga 420

acttgacctc agggtcttcg tggctcacgt ccaccaccac gcatgtgacc tcaggggtcc 480

gggagatcat gagggtgtcc ttgggttttg gggggaagag gaagactgac ggtcccccca 540

ggagttcagg tgctgggcac ggtgggcatg tgtgagtttt gtcacaagat ttgggctcaa 600

ctttcttgtc caccttggtg ttgctgggct tgtgattcac gttgcagatg taggtctggg 660

tgtccaagct gctggagggc acggtcacca cgctgctgag ggagtagagt cctgaggact 720

gtaggacagc cgggaaggtg tgcacgccgc tggtcagggc gcctgagttc cacgacaccg 780

tcaccggttc ggggaagtag tccttgacca ggcagcccag ggctgctgtg cccccagagg 840

tgctcttgga ggagggtgcc agggggaaga ccgatgggcc cttggtggag gctgaggaga 900

cggtgaccag tgtgccttga ccccagtagt ccatagggga accatatccc atcatcgatc 960

ttgcacagta atagactgca gtgtcctcag atctcaggct gctgagctcc atgtaggctg 1020

tgctcgtgga cttgtccaca gtcattgtca ctctgccctg gaacttctgg ttgtagctag 1080

tagtgtcata gttaggatta atatctccaa tccactcaag gccctgtcca ggggcctgtc 1140

gaacccagtc catgttgtag tcagtgaatg tgtatccaga agccttgcag gaaaccttca 1200

ctgaagctcc aggcttcttc acctcagctc cagactgcac cagctggacc tgggagccgc 1260

cgccgccgga gccgccgccg ccggagccgc cgccgccgga gccgccgccg cctttgattt 1320

ccagcttggt gcctccaccg aacgtccgag gaagctctct aatgtgctga cagtaataca 1380

cagcgaagtc ctcaggttcc agcctgctga tggtgagtgt gaagtctgtc ccagacccac 1440

tgccactgaa tctagcaggg atcccagatt ctaggttaga tgcaagatag atgaggagtc 1500

tgggggcctg gcctggtttc tgttggtacc agtgcatata actatagcca gatgtactga 1560

cactcttgct ggccctgcag ctcagggtgg ccctctcccc tggagagaga gataaggtgg 1620

caggagactg tgtcagcaca atctcacatc tggcacctgg gagccagagc agcaggagtc 1680

ccaggagctg agcaggaacc ctcatgtcca tggatccgcg acccatttgc tgtccaccag 1740

tcatgctagc catatggctg ccgcgcggca ccaggccgct gctgtgatga tgatgatgat 1800

ggctgctgcc catggtatat ctccttctta aagttaaaca aaattatttc tagaggggaa 1860

ttgttatccg ctcacaattc ccctatagtg agtcgtatta atttcgcggg atcgagatct 1920

cgatcctcta cgccggacgc atcgtggccg gcatcaccgg cgccacaggt gcggttgctg 1980

gcgcctatat cgccgacatc accgatgggg aagatcgggc tcgccacttc gggctcatga 2040

gcgcttgttt cggcgtgggt atggtggcag gccccgtggc cgggggactg ttgggcgcca 2100

tctccttgca tgcaccattc cttgcggcgg cggtgctcaa cggcctcaac ctactactgg 2160

gctgcttcct aatgcaggag tcgcataagg gagagcgtcg agatcccgga caccatcgaa 2220

tggcgcaaaa cctttcgcgg tatggcatga tagcgcccgg aagagagtca attcagggtg 2280

gtgaatgtga aaccagtaac gttatacgat gtcgcagagt atgccggtgt ctcttatcag 2340

accgtttccc gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg 2400

gaagcggcga tggcggagct gaattacatt cccaaccgcg tggcacaaca actggcgggc 2460

aaacagtcgt tgctgattgg cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa 2520

attgtcgcgg cgattaaatc tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg 2580

gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc 2640

gtcagtgggc tgatcattaa ctatccgctg gatgaccagg atgccattgc tgtggaagct 2700

gcctgcacta atgttccggc gttatttctt gatgtctctg accagacacc catcaacagt 2760

attattttct cccatgaaga cggtacgcga ctgggcgtgg agcatctggt cgcattgggt 2820

caccagcaaa tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg tctgcgtctg 2880

gctggctggc ataaatatct cactcgcaat caaattcagc cgatagcgga acgggaaggc 2940

gactggagtg ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt 3000

cccactgcga tgctggttgc caacgatcag atggcgctgg gcgcaatgcg cgccattacc 3060

gagtccgggc tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga taccgaagac 3120

agctcatgtt atatcccgcc gttaaccacc atcaaacagg attttcgcct gctggggcaa 3180

accagcgtgg accgcttgct gcaactctct cagggccagg cggtgaaggg caatcagctg 3240

ttgcccgtct cactggtgaa aagaaaaacc accctggcgc ccaatacgca aaccgcctct 3300

ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc 3360

gggcagtgag cgcaacgcaa ttaatgtaag ttagctcact cattaggcac cgggatctcg 3420

accgatgccc ttgagagcct tcaacccagt cagctccttc cggtgggcgc ggggcatgac 3480

tatcgtcgcc gcacttatga ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc 3540

agcgctctgg gtcattttcg gcgaggaccg ctttcgctgg agcgcgacga tgatcggcct 3600

gtcgcttgcg gtattcggaa tcttgcacgc cctcgctcaa gccttcgtca ctggtcccgc 3660

caccaaacgt ttcggcgaga agcaggccat tatcgccggc atggcggccc cacgggtgcg 3720

catgatcgtg ctcctgtcgt tgaggacccg gctaggctgg cggggttgcc ttactggtta 3780

gcagaatgaa tcaccgatac gcgagcgaac gtgaagcgac tgctgctgca aaacgtctgc 3840

gacctgagca acaacatgaa tggtcttcgg tttccgtgtt tcgtaaagtc tggaaacgcg 3900

gaagtcagcg ccctgcacca ttatgttccg gatctgcatc gcaggatgct gctggctacc 3960

ctgtggaaca cctacatctg tattaacgaa gcgctggcat tgaccctgag tgatttttct 4020

ctggtcccgc cgcatccata ccgccagttg tttaccctca caacgttcca gtaaccgggc 4080

atgttcatca tcagtaaccc gtatcgtgag catcctctct cgtttcatcg gtatcattac 4140

ccccatgaac agaaatcccc cttacacgga ggcatcagtg accaaacagg aaaaaaccgc 4200

ccttaacatg gcccgcttta tcagaagcca gacattaacg cttctggaga aactcaacga 4260

gctggacgcg gatgaacagg cagacatctg tgaatcgctt cacgaccacg ctgatgagct 4320

ttaccgcagc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct 4380

cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg 4440

cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag 4500

cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat 4560

atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgctctt 4620

ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag 4680

ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca 4740

tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt 4800

tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc 4860

gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct 4920

ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg 4980

tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca 5040

agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact 5100

atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta 5160

acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta 5220

actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct 5280

tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt 5340

tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga 5400

tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca 5460

tgaacaataa aactgtctgc ttacataaac agtaatacaa ggggtgttat gagccatatt 5520

caacgggaaa cgtcttgctc taggccgcga ttaaattcca acatggatgc tgatttatat 5580

gggtataaat gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgattgtat 5640

gggaagcccg atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat 5700

gttacagatg agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc 5760

aagcatttta tccgtactcc tgatgatgca tggttactca ccactgcgat ccccgggaaa 5820

acagcattcc aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg 5880

gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat 5940

cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt 6000

gattttgatg acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataaa 6060

cttttgccat tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt 6120

atttttgacg aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac 6180

cgataccagg atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag 6240

aaacggcttt ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat 6300

ttgatgctcg atgagttttt ctaagaatta attcatgagc ggatacatat ttgaatgtat 6360

ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgaaat 6420

tgtaaacgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt 6480

taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg 6540

gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt 6600

caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc 6660

aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg 6720

atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa 6780

aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc 6840

cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg cca 6883

<210> 29

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

tgctcagctc ctgggacgcc tgctgctc 28

<210> 30

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

ggagccagag cagcaggcgt cccaggagct 30

<210> 31

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

tcctgggact cctgcggctc tggctcccag gtgcca 36

<210> 32

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

cctgggagcc agagccgcag gagtcccagg agct 34

<210> 33

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

cagagtgaca atgactgtga acaagtccac gagca 35

<210> 34

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

ctgtgctcgt ggacttgttc acagtcattg tcact 35

<210> 35

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 35

tggaggcacc aagctgaaaa tcaaagg 27

<210> 36

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

gccgccgcct ttgattttca gcttggtgcc 30

<210> 37

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 37

acttcacact caccatcggc aggctggaa 29

<210> 38

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 38

ctcaggttcc agcctgccga tggtgagtgt 30

<210> 39

<211> 6883

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 39

atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa 60

ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt 120

tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttta 180

cgaagttatc atgtggccct cgcaccccac gggtccactc acctttggct ttggagatgg 240

ttttctcgat gggggctggg agggctttgt tggagacctt gcacttgtac tccttgccat 300

tcagccagtc ctggtgcagg acggtgagga cgctgaccac acggtacgtg ctgttgtact 360

gctcctcccg cggctttgtc ttggcattat gcacctccac gccgtccacg taccagttga 420

acttgacctc agggtcttcg tggctcacgt ccaccaccac gcatgtgacc tcaggggtcc 480

gggagatcat gagggtgtcc ttgggttttg gggggaagag gaagactgac ggtcccccca 540

ggagttcagg tgctgggcac ggtgggcatg tgtgagtttt gtcacaagat ttgggctcaa 600

ctttcttgtc caccttggtg ttgctgggct tgtgattcac gttgcagatg taggtctggg 660

tgtccaagct gctggagggc acggtcacca cgctgctgag ggagtagagt cctgaggact 720

gtaggacagc cgggaaggtg tgcacgccgc tggtcagggc gcctgagttc cacgacaccg 780

tcaccggttc ggggaagtag tccttgacca ggcagcccag ggctgctgtg cccccagagg 840

tgctcttgga ggagggtgcc agggggaaga ccgatgggcc cttggtggag gctgaggaga 900

cggtgaccag tgtgccttga ccccagtagt ccatagggga accatatccc atcatcgatc 960

ttgcacagta atagactgca gtgtcctcag atctcaggct gctgagctcc atgtaggctg 1020

tgctcgtgga cttgtccaca gtcattgtca ctctgccctg gaacttctgg ttgtagctag 1080

tagtgtcata gttaggatta atatctccaa tccactcaag gccctgtcca ggggcctgtc 1140

gaacccagtc catgttgtag tcagtgaatg tgtatccaga agccttgcag gaaaccttca 1200

ctgaagctcc aggcttcttc acctcagctc cagactgcac cagctggacc tgggagccgc 1260

cgccgccgga gccgccgccg ccggagccgc cgccgccgga gccgccgccg cctttgattt 1320

ccagcttggt gcctccaccg aacgtccgag gaagctctct aatgtgctga cagtaataca 1380

cagcgaagtc ctcaggttcc agcctgctga tggtgagtgt gaagtctgtc ccagacccac 1440

tgccactgaa tctagcaggg atcccagatt ctaggttaga tgcaagatag atgaggagtc 1500

tgggggcctg gcctggtttc tgttggtacc agtgcatata actatagcca gatgtactga 1560

cactcttgct ggccctgcag ctcagggtgg ccctctcccc tggagagaga gataaggtgg 1620

caggagactg tgtcagcaca atctcacatc tggcacctgg gagccagagc agcaggcgtc 1680

ccaggagctg agcaggaacc ctcatgtcca tggatccgcg acccatttgc tgtccaccag 1740

tcatgctagc catatggctg ccgcgcggca ccaggccgct gctgtgatga tgatgatgat 1800

ggctgctgcc catggtatat ctccttctta aagttaaaca aaattatttc tagaggggaa 1860

ttgttatccg ctcacaattc ccctatagtg agtcgtatta atttcgcggg atcgagatct 1920

cgatcctcta cgccggacgc atcgtggccg gcatcaccgg cgccacaggt gcggttgctg 1980

gcgcctatat cgccgacatc accgatgggg aagatcgggc tcgccacttc gggctcatga 2040

gcgcttgttt cggcgtgggt atggtggcag gccccgtggc cgggggactg ttgggcgcca 2100

tctccttgca tgcaccattc cttgcggcgg cggtgctcaa cggcctcaac ctactactgg 2160

gctgcttcct aatgcaggag tcgcataagg gagagcgtcg agatcccgga caccatcgaa 2220

tggcgcaaaa cctttcgcgg tatggcatga tagcgcccgg aagagagtca attcagggtg 2280

gtgaatgtga aaccagtaac gttatacgat gtcgcagagt atgccggtgt ctcttatcag 2340

accgtttccc gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg 2400

gaagcggcga tggcggagct gaattacatt cccaaccgcg tggcacaaca actggcgggc 2460

aaacagtcgt tgctgattgg cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa 2520

attgtcgcgg cgattaaatc tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg 2580

gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc 2640

gtcagtgggc tgatcattaa ctatccgctg gatgaccagg atgccattgc tgtggaagct 2700

gcctgcacta atgttccggc gttatttctt gatgtctctg accagacacc catcaacagt 2760

attattttct cccatgaaga cggtacgcga ctgggcgtgg agcatctggt cgcattgggt 2820

caccagcaaa tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg tctgcgtctg 2880

gctggctggc ataaatatct cactcgcaat caaattcagc cgatagcgga acgggaaggc 2940

gactggagtg ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt 3000

cccactgcga tgctggttgc caacgatcag atggcgctgg gcgcaatgcg cgccattacc 3060

gagtccgggc tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga taccgaagac 3120

agctcatgtt atatcccgcc gttaaccacc atcaaacagg attttcgcct gctggggcaa 3180

accagcgtgg accgcttgct gcaactctct cagggccagg cggtgaaggg caatcagctg 3240

ttgcccgtct cactggtgaa aagaaaaacc accctggcgc ccaatacgca aaccgcctct 3300

ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc 3360

gggcagtgag cgcaacgcaa ttaatgtaag ttagctcact cattaggcac cgggatctcg 3420

accgatgccc ttgagagcct tcaacccagt cagctccttc cggtgggcgc ggggcatgac 3480

tatcgtcgcc gcacttatga ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc 3540

agcgctctgg gtcattttcg gcgaggaccg ctttcgctgg agcgcgacga tgatcggcct 3600

gtcgcttgcg gtattcggaa tcttgcacgc cctcgctcaa gccttcgtca ctggtcccgc 3660

caccaaacgt ttcggcgaga agcaggccat tatcgccggc atggcggccc cacgggtgcg 3720

catgatcgtg ctcctgtcgt tgaggacccg gctaggctgg cggggttgcc ttactggtta 3780

gcagaatgaa tcaccgatac gcgagcgaac gtgaagcgac tgctgctgca aaacgtctgc 3840

gacctgagca acaacatgaa tggtcttcgg tttccgtgtt tcgtaaagtc tggaaacgcg 3900

gaagtcagcg ccctgcacca ttatgttccg gatctgcatc gcaggatgct gctggctacc 3960

ctgtggaaca cctacatctg tattaacgaa gcgctggcat tgaccctgag tgatttttct 4020

ctggtcccgc cgcatccata ccgccagttg tttaccctca caacgttcca gtaaccgggc 4080

atgttcatca tcagtaaccc gtatcgtgag catcctctct cgtttcatcg gtatcattac 4140

ccccatgaac agaaatcccc cttacacgga ggcatcagtg accaaacagg aaaaaaccgc 4200

ccttaacatg gcccgcttta tcagaagcca gacattaacg cttctggaga aactcaacga 4260

gctggacgcg gatgaacagg cagacatctg tgaatcgctt cacgaccacg ctgatgagct 4320

ttaccgcagc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct 4380

cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg 4440

cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag 4500

cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat 4560

atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgctctt 4620

ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag 4680

ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca 4740

tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt 4800

tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc 4860

gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct 4920

ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg 4980

tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca 5040

agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact 5100

atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta 5160

acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta 5220

actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct 5280

tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt 5340

tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga 5400

tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca 5460

tgaacaataa aactgtctgc ttacataaac agtaatacaa ggggtgttat gagccatatt 5520

caacgggaaa cgtcttgctc taggccgcga ttaaattcca acatggatgc tgatttatat 5580

gggtataaat gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgattgtat 5640

gggaagcccg atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat 5700

gttacagatg agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc 5760

aagcatttta tccgtactcc tgatgatgca tggttactca ccactgcgat ccccgggaaa 5820

acagcattcc aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg 5880

gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat 5940

cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt 6000

gattttgatg acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataaa 6060

cttttgccat tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt 6120

atttttgacg aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac 6180

cgataccagg atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag 6240

aaacggcttt ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat 6300

ttgatgctcg atgagttttt ctaagaatta attcatgagc ggatacatat ttgaatgtat 6360

ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgaaat 6420

tgtaaacgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt 6480

taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg 6540

gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt 6600

caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc 6660

aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg 6720

atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa 6780

aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc 6840

cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg cca 6883

<210> 40

<211> 6883

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 40

atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa 60

ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt 120

tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttta 180

cgaagttatc atgtggccct cgcaccccac gggtccactc acctttggct ttggagatgg 240

ttttctcgat gggggctggg agggctttgt tggagacctt gcacttgtac tccttgccat 300

tcagccagtc ctggtgcagg acggtgagga cgctgaccac acggtacgtg ctgttgtact 360

gctcctcccg cggctttgtc ttggcattat gcacctccac gccgtccacg taccagttga 420

acttgacctc agggtcttcg tggctcacgt ccaccaccac gcatgtgacc tcaggggtcc 480

gggagatcat gagggtgtcc ttgggttttg gggggaagag gaagactgac ggtcccccca 540

ggagttcagg tgctgggcac ggtgggcatg tgtgagtttt gtcacaagat ttgggctcaa 600

ctttcttgtc caccttggtg ttgctgggct tgtgattcac gttgcagatg taggtctggg 660

tgtccaagct gctggagggc acggtcacca cgctgctgag ggagtagagt cctgaggact 720

gtaggacagc cgggaaggtg tgcacgccgc tggtcagggc gcctgagttc cacgacaccg 780

tcaccggttc ggggaagtag tccttgacca ggcagcccag ggctgctgtg cccccagagg 840

tgctcttgga ggagggtgcc agggggaaga ccgatgggcc cttggtggag gctgaggaga 900

cggtgaccag tgtgccttga ccccagtagt ccatagggga accatatccc atcatcgatc 960

ttgcacagta atagactgca gtgtcctcag atctcaggct gctgagctcc atgtaggctg 1020

tgctcgtgga cttgttcaca gtcattgtca ctctgccctg gaacttctgg ttgtagctag 1080

tagtgtcata gttaggatta atatctccaa tccactcaag gccctgtcca ggggcctgtc 1140

gaacccagtc catgttgtag tcagtgaatg tgtatccaga agccttgcag gaaaccttca 1200

ctgaagctcc aggcttcttc acctcagctc cagactgcac cagctggacc tgggagccgc 1260

cgccgccgga gccgccgccg ccggagccgc cgccgccgga gccgccgccg cctttgattt 1320

ccagcttggt gcctccaccg aacgtccgag gaagctctct aatgtgctga cagtaataca 1380

cagcgaagtc ctcaggttcc agcctgctga tggtgagtgt gaagtctgtc ccagacccac 1440

tgccactgaa tctagcaggg atcccagatt ctaggttaga tgcaagatag atgaggagtc 1500

tgggggcctg gcctggtttc tgttggtacc agtgcatata actatagcca gatgtactga 1560

cactcttgct ggccctgcag ctcagggtgg ccctctcccc tggagagaga gataaggtgg 1620

caggagactg tgtcagcaca atctcacatc tggcacctgg gagccagagc agcaggcgtc 1680

ccaggagctg agcaggaacc ctcatgtcca tggatccgcg acccatttgc tgtccaccag 1740

tcatgctagc catatggctg ccgcgcggca ccaggccgct gctgtgatga tgatgatgat 1800

ggctgctgcc catggtatat ctccttctta aagttaaaca aaattatttc tagaggggaa 1860

ttgttatccg ctcacaattc ccctatagtg agtcgtatta atttcgcggg atcgagatct 1920

cgatcctcta cgccggacgc atcgtggccg gcatcaccgg cgccacaggt gcggttgctg 1980

gcgcctatat cgccgacatc accgatgggg aagatcgggc tcgccacttc gggctcatga 2040

gcgcttgttt cggcgtgggt atggtggcag gccccgtggc cgggggactg ttgggcgcca 2100

tctccttgca tgcaccattc cttgcggcgg cggtgctcaa cggcctcaac ctactactgg 2160

gctgcttcct aatgcaggag tcgcataagg gagagcgtcg agatcccgga caccatcgaa 2220

tggcgcaaaa cctttcgcgg tatggcatga tagcgcccgg aagagagtca attcagggtg 2280

gtgaatgtga aaccagtaac gttatacgat gtcgcagagt atgccggtgt ctcttatcag 2340

accgtttccc gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg 2400

gaagcggcga tggcggagct gaattacatt cccaaccgcg tggcacaaca actggcgggc 2460

aaacagtcgt tgctgattgg cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa 2520

attgtcgcgg cgattaaatc tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg 2580

gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc 2640

gtcagtgggc tgatcattaa ctatccgctg gatgaccagg atgccattgc tgtggaagct 2700

gcctgcacta atgttccggc gttatttctt gatgtctctg accagacacc catcaacagt 2760

attattttct cccatgaaga cggtacgcga ctgggcgtgg agcatctggt cgcattgggt 2820

caccagcaaa tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg tctgcgtctg 2880

gctggctggc ataaatatct cactcgcaat caaattcagc cgatagcgga acgggaaggc 2940

gactggagtg ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt 3000

cccactgcga tgctggttgc caacgatcag atggcgctgg gcgcaatgcg cgccattacc 3060

gagtccgggc tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga taccgaagac 3120

agctcatgtt atatcccgcc gttaaccacc atcaaacagg attttcgcct gctggggcaa 3180

accagcgtgg accgcttgct gcaactctct cagggccagg cggtgaaggg caatcagctg 3240

ttgcccgtct cactggtgaa aagaaaaacc accctggcgc ccaatacgca aaccgcctct 3300

ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc 3360

gggcagtgag cgcaacgcaa ttaatgtaag ttagctcact cattaggcac cgggatctcg 3420

accgatgccc ttgagagcct tcaacccagt cagctccttc cggtgggcgc ggggcatgac 3480

tatcgtcgcc gcacttatga ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc 3540

agcgctctgg gtcattttcg gcgaggaccg ctttcgctgg agcgcgacga tgatcggcct 3600

gtcgcttgcg gtattcggaa tcttgcacgc cctcgctcaa gccttcgtca ctggtcccgc 3660

caccaaacgt ttcggcgaga agcaggccat tatcgccggc atggcggccc cacgggtgcg 3720

catgatcgtg ctcctgtcgt tgaggacccg gctaggctgg cggggttgcc ttactggtta 3780

gcagaatgaa tcaccgatac gcgagcgaac gtgaagcgac tgctgctgca aaacgtctgc 3840

gacctgagca acaacatgaa tggtcttcgg tttccgtgtt tcgtaaagtc tggaaacgcg 3900

gaagtcagcg ccctgcacca ttatgttccg gatctgcatc gcaggatgct gctggctacc 3960

ctgtggaaca cctacatctg tattaacgaa gcgctggcat tgaccctgag tgatttttct 4020

ctggtcccgc cgcatccata ccgccagttg tttaccctca caacgttcca gtaaccgggc 4080

atgttcatca tcagtaaccc gtatcgtgag catcctctct cgtttcatcg gtatcattac 4140

ccccatgaac agaaatcccc cttacacgga ggcatcagtg accaaacagg aaaaaaccgc 4200

ccttaacatg gcccgcttta tcagaagcca gacattaacg cttctggaga aactcaacga 4260

gctggacgcg gatgaacagg cagacatctg tgaatcgctt cacgaccacg ctgatgagct 4320

ttaccgcagc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct 4380

cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg 4440

cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag 4500

cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat 4560

atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgctctt 4620

ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag 4680

ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca 4740

tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt 4800

tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc 4860

gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct 4920

ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg 4980

tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca 5040

agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact 5100

atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta 5160

acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta 5220

actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct 5280

tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt 5340

tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga 5400

tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca 5460

tgaacaataa aactgtctgc ttacataaac agtaatacaa ggggtgttat gagccatatt 5520

caacgggaaa cgtcttgctc taggccgcga ttaaattcca acatggatgc tgatttatat 5580

gggtataaat gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgattgtat 5640

gggaagcccg atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat 5700

gttacagatg agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc 5760

aagcatttta tccgtactcc tgatgatgca tggttactca ccactgcgat ccccgggaaa 5820

acagcattcc aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg 5880

gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat 5940

cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt 6000

gattttgatg acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataaa 6060

cttttgccat tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt 6120

atttttgacg aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac 6180

cgataccagg atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag 6240

aaacggcttt ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat 6300

ttgatgctcg atgagttttt ctaagaatta attcatgagc ggatacatat ttgaatgtat 6360

ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgaaat 6420

tgtaaacgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt 6480

taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg 6540

gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt 6600

caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc 6660

aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg 6720

atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa 6780

aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc 6840

cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg cca 6883

<210> 41

<211> 6883

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 41

atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa 60

ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt 120

tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttta 180

cgaagttatc atgtggccct cgcaccccac gggtccactc acctttggct ttggagatgg 240

ttttctcgat gggggctggg agggctttgt tggagacctt gcacttgtac tccttgccat 300

tcagccagtc ctggtgcagg acggtgagga cgctgaccac acggtacgtg ctgttgtact 360

gctcctcccg cggctttgtc ttggcattat gcacctccac gccgtccacg taccagttga 420

acttgacctc agggtcttcg tggctcacgt ccaccaccac gcatgtgacc tcaggggtcc 480

gggagatcat gagggtgtcc ttgggttttg gggggaagag gaagactgac ggtcccccca 540

ggagttcagg tgctgggcac ggtgggcatg tgtgagtttt gtcacaagat ttgggctcaa 600

ctttcttgtc caccttggtg ttgctgggct tgtgattcac gttgcagatg taggtctggg 660

tgtccaagct gctggagggc acggtcacca cgctgctgag ggagtagagt cctgaggact 720

gtaggacagc cgggaaggtg tgcacgccgc tggtcagggc gcctgagttc cacgacaccg 780

tcaccggttc ggggaagtag tccttgacca ggcagcccag ggctgctgtg cccccagagg 840

tgctcttgga ggagggtgcc agggggaaga ccgatgggcc cttggtggag gctgaggaga 900

cggtgaccag tgtgccttga ccccagtagt ccatagggga accatatccc atcatcgatc 960

ttgcacagta atagactgca gtgtcctcag atctcaggct gctgagctcc atgtaggctg 1020

tgctcgtgga cttgtccaca gtcattgtca ctctgccctg gaacttctgg ttgtagctag 1080

tagtgtcata gttaggatta atatctccaa tccactcaag gccctgtcca ggggcctgtc 1140

gaacccagtc catgttgtag tcagtgaatg tgtatccaga agccttgcag gaaaccttca 1200

ctgaagctcc aggcttcttc acctcagctc cagactgcac cagctggacc tgggagccgc 1260

cgccgccgga gccgccgccg ccggagccgc cgccgccgga gccgccgccg cctttgattt 1320

tcagcttggt gcctccaccg aacgtccgag gaagctctct aatgtgctga cagtaataca 1380

cagcgaagtc ctcaggttcc agcctgctga tggtgagtgt gaagtctgtc ccagacccac 1440

tgccactgaa tctagcaggg atcccagatt ctaggttaga tgcaagatag atgaggagtc 1500

tgggggcctg gcctggtttc tgttggtacc agtgcatata actatagcca gatgtactga 1560

cactcttgct ggccctgcag ctcagggtgg ccctctcccc tggagagaga gataaggtgg 1620

caggagactg tgtcagcaca atctcacatc tggcacctgg gagccagagc agcaggcgtc 1680

ccaggagctg agcaggaacc ctcatgtcca tggatccgcg acccatttgc tgtccaccag 1740

tcatgctagc catatggctg ccgcgcggca ccaggccgct gctgtgatga tgatgatgat 1800

ggctgctgcc catggtatat ctccttctta aagttaaaca aaattatttc tagaggggaa 1860

ttgttatccg ctcacaattc ccctatagtg agtcgtatta atttcgcggg atcgagatct 1920

cgatcctcta cgccggacgc atcgtggccg gcatcaccgg cgccacaggt gcggttgctg 1980

gcgcctatat cgccgacatc accgatgggg aagatcgggc tcgccacttc gggctcatga 2040

gcgcttgttt cggcgtgggt atggtggcag gccccgtggc cgggggactg ttgggcgcca 2100

tctccttgca tgcaccattc cttgcggcgg cggtgctcaa cggcctcaac ctactactgg 2160

gctgcttcct aatgcaggag tcgcataagg gagagcgtcg agatcccgga caccatcgaa 2220

tggcgcaaaa cctttcgcgg tatggcatga tagcgcccgg aagagagtca attcagggtg 2280

gtgaatgtga aaccagtaac gttatacgat gtcgcagagt atgccggtgt ctcttatcag 2340

accgtttccc gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg 2400

gaagcggcga tggcggagct gaattacatt cccaaccgcg tggcacaaca actggcgggc 2460

aaacagtcgt tgctgattgg cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa 2520

attgtcgcgg cgattaaatc tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg 2580

gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc 2640

gtcagtgggc tgatcattaa ctatccgctg gatgaccagg atgccattgc tgtggaagct 2700

gcctgcacta atgttccggc gttatttctt gatgtctctg accagacacc catcaacagt 2760

attattttct cccatgaaga cggtacgcga ctgggcgtgg agcatctggt cgcattgggt 2820

caccagcaaa tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg tctgcgtctg 2880

gctggctggc ataaatatct cactcgcaat caaattcagc cgatagcgga acgggaaggc 2940

gactggagtg ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt 3000

cccactgcga tgctggttgc caacgatcag atggcgctgg gcgcaatgcg cgccattacc 3060

gagtccgggc tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga taccgaagac 3120

agctcatgtt atatcccgcc gttaaccacc atcaaacagg attttcgcct gctggggcaa 3180

accagcgtgg accgcttgct gcaactctct cagggccagg cggtgaaggg caatcagctg 3240

ttgcccgtct cactggtgaa aagaaaaacc accctggcgc ccaatacgca aaccgcctct 3300

ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc 3360

gggcagtgag cgcaacgcaa ttaatgtaag ttagctcact cattaggcac cgggatctcg 3420

accgatgccc ttgagagcct tcaacccagt cagctccttc cggtgggcgc ggggcatgac 3480

tatcgtcgcc gcacttatga ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc 3540

agcgctctgg gtcattttcg gcgaggaccg ctttcgctgg agcgcgacga tgatcggcct 3600

gtcgcttgcg gtattcggaa tcttgcacgc cctcgctcaa gccttcgtca ctggtcccgc 3660

caccaaacgt ttcggcgaga agcaggccat tatcgccggc atggcggccc cacgggtgcg 3720

catgatcgtg ctcctgtcgt tgaggacccg gctaggctgg cggggttgcc ttactggtta 3780

gcagaatgaa tcaccgatac gcgagcgaac gtgaagcgac tgctgctgca aaacgtctgc 3840

gacctgagca acaacatgaa tggtcttcgg tttccgtgtt tcgtaaagtc tggaaacgcg 3900

gaagtcagcg ccctgcacca ttatgttccg gatctgcatc gcaggatgct gctggctacc 3960

ctgtggaaca cctacatctg tattaacgaa gcgctggcat tgaccctgag tgatttttct 4020

ctggtcccgc cgcatccata ccgccagttg tttaccctca caacgttcca gtaaccgggc 4080

atgttcatca tcagtaaccc gtatcgtgag catcctctct cgtttcatcg gtatcattac 4140

ccccatgaac agaaatcccc cttacacgga ggcatcagtg accaaacagg aaaaaaccgc 4200

ccttaacatg gcccgcttta tcagaagcca gacattaacg cttctggaga aactcaacga 4260

gctggacgcg gatgaacagg cagacatctg tgaatcgctt cacgaccacg ctgatgagct 4320

ttaccgcagc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct 4380

cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg 4440

cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag 4500

cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat 4560

atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgctctt 4620

ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag 4680

ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca 4740

tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt 4800

tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc 4860

gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct 4920

ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg 4980

tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca 5040

agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact 5100

atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta 5160

acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta 5220

actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct 5280

tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt 5340

tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga 5400

tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca 5460

tgaacaataa aactgtctgc ttacataaac agtaatacaa ggggtgttat gagccatatt 5520

caacgggaaa cgtcttgctc taggccgcga ttaaattcca acatggatgc tgatttatat 5580

gggtataaat gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgattgtat 5640

gggaagcccg atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat 5700

gttacagatg agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc 5760

aagcatttta tccgtactcc tgatgatgca tggttactca ccactgcgat ccccgggaaa 5820

acagcattcc aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg 5880

gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat 5940

cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt 6000

gattttgatg acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataaa 6060

cttttgccat tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt 6120

atttttgacg aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac 6180

cgataccagg atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag 6240

aaacggcttt ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat 6300

ttgatgctcg atgagttttt ctaagaatta attcatgagc ggatacatat ttgaatgtat 6360

ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgaaat 6420

tgtaaacgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt 6480

taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg 6540

gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt 6600

caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc 6660

aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg 6720

atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa 6780

aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc 6840

cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg cca 6883

<210> 42

<211> 6883

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 42

atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa 60

ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt 120

tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttta 180

cgaagttatc atgtggccct cgcaccccac gggtccactc acctttggct ttggagatgg 240

ttttctcgat gggggctggg agggctttgt tggagacctt gcacttgtac tccttgccat 300

tcagccagtc ctggtgcagg acggtgagga cgctgaccac acggtacgtg ctgttgtact 360

gctcctcccg cggctttgtc ttggcattat gcacctccac gccgtccacg taccagttga 420

acttgacctc agggtcttcg tggctcacgt ccaccaccac gcatgtgacc tcaggggtcc 480

gggagatcat gagggtgtcc ttgggttttg gggggaagag gaagactgac ggtcccccca 540

ggagttcagg tgctgggcac ggtgggcatg tgtgagtttt gtcacaagat ttgggctcaa 600

ctttcttgtc caccttggtg ttgctgggct tgtgattcac gttgcagatg taggtctggg 660

tgtccaagct gctggagggc acggtcacca cgctgctgag ggagtagagt cctgaggact 720

gtaggacagc cgggaaggtg tgcacgccgc tggtcagggc gcctgagttc cacgacaccg 780

tcaccggttc ggggaagtag tccttgacca ggcagcccag ggctgctgtg cccccagagg 840

tgctcttgga ggagggtgcc agggggaaga ccgatgggcc cttggtggag gctgaggaga 900

cggtgaccag tgtgccttga ccccagtagt ccatagggga accatatccc atcatcgatc 960

ttgcacagta atagactgca gtgtcctcag atctcaggct gctgagctcc atgtaggctg 1020

tgctcgtgga cttgtccaca gtcattgtca ctctgccctg gaacttctgg ttgtagctag 1080

tagtgtcata gttaggatta atatctccaa tccactcaag gccctgtcca ggggcctgtc 1140

gaacccagtc catgttgtag tcagtgaatg tgtatccaga agccttgcag gaaaccttca 1200

ctgaagctcc aggcttcttc acctcagctc cagactgcac cagctggacc tgggagccgc 1260

cgccgccgga gccgccgccg ccggagccgc cgccgccgga gccgccgccg cctttgattt 1320

ccagcttggt gcctccaccg aacgtccgag gaagctctct aatgtgctga cagtaataca 1380

cagcgaagtc ctcaggttcc agcctgctga tggtgagtgt gaagtctgtc ccagacccac 1440

tgccactgaa tctagcaggg atcccagatt ctaggttaga tgcaagatag atgaggagtc 1500

tgggggcctg gcctggtttc tgttggtacc agtgcatata actatagcca gatgtactga 1560

cactcttgct ggccctgcag ctcagggtgg ccctctcccc tggagagaga gataaggtgg 1620

caggagactg tgtcagcaca atctcacatc tggcacctgg gagccagagc cgcaggagtc 1680

ccaggagctg agcaggaacc ctcatgtcca tggatccgcg acccatttgc tgtccaccag 1740

tcatgctagc catatggctg ccgcgcggca ccaggccgct gctgtgatga tgatgatgat 1800

ggctgctgcc catggtatat ctccttctta aagttaaaca aaattatttc tagaggggaa 1860

ttgttatccg ctcacaattc ccctatagtg agtcgtatta atttcgcggg atcgagatct 1920

cgatcctcta cgccggacgc atcgtggccg gcatcaccgg cgccacaggt gcggttgctg 1980

gcgcctatat cgccgacatc accgatgggg aagatcgggc tcgccacttc gggctcatga 2040

gcgcttgttt cggcgtgggt atggtggcag gccccgtggc cgggggactg ttgggcgcca 2100

tctccttgca tgcaccattc cttgcggcgg cggtgctcaa cggcctcaac ctactactgg 2160

gctgcttcct aatgcaggag tcgcataagg gagagcgtcg agatcccgga caccatcgaa 2220

tggcgcaaaa cctttcgcgg tatggcatga tagcgcccgg aagagagtca attcagggtg 2280

gtgaatgtga aaccagtaac gttatacgat gtcgcagagt atgccggtgt ctcttatcag 2340

accgtttccc gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg 2400

gaagcggcga tggcggagct gaattacatt cccaaccgcg tggcacaaca actggcgggc 2460

aaacagtcgt tgctgattgg cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa 2520

attgtcgcgg cgattaaatc tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg 2580

gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc 2640

gtcagtgggc tgatcattaa ctatccgctg gatgaccagg atgccattgc tgtggaagct 2700

gcctgcacta atgttccggc gttatttctt gatgtctctg accagacacc catcaacagt 2760

attattttct cccatgaaga cggtacgcga ctgggcgtgg agcatctggt cgcattgggt 2820

caccagcaaa tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg tctgcgtctg 2880

gctggctggc ataaatatct cactcgcaat caaattcagc cgatagcgga acgggaaggc 2940

gactggagtg ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt 3000

cccactgcga tgctggttgc caacgatcag atggcgctgg gcgcaatgcg cgccattacc 3060

gagtccgggc tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga taccgaagac 3120

agctcatgtt atatcccgcc gttaaccacc atcaaacagg attttcgcct gctggggcaa 3180

accagcgtgg accgcttgct gcaactctct cagggccagg cggtgaaggg caatcagctg 3240

ttgcccgtct cactggtgaa aagaaaaacc accctggcgc ccaatacgca aaccgcctct 3300

ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc 3360

gggcagtgag cgcaacgcaa ttaatgtaag ttagctcact cattaggcac cgggatctcg 3420

accgatgccc ttgagagcct tcaacccagt cagctccttc cggtgggcgc ggggcatgac 3480

tatcgtcgcc gcacttatga ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc 3540

agcgctctgg gtcattttcg gcgaggaccg ctttcgctgg agcgcgacga tgatcggcct 3600

gtcgcttgcg gtattcggaa tcttgcacgc cctcgctcaa gccttcgtca ctggtcccgc 3660

caccaaacgt ttcggcgaga agcaggccat tatcgccggc atggcggccc cacgggtgcg 3720

catgatcgtg ctcctgtcgt tgaggacccg gctaggctgg cggggttgcc ttactggtta 3780

gcagaatgaa tcaccgatac gcgagcgaac gtgaagcgac tgctgctgca aaacgtctgc 3840

gacctgagca acaacatgaa tggtcttcgg tttccgtgtt tcgtaaagtc tggaaacgcg 3900

gaagtcagcg ccctgcacca ttatgttccg gatctgcatc gcaggatgct gctggctacc 3960

ctgtggaaca cctacatctg tattaacgaa gcgctggcat tgaccctgag tgatttttct 4020

ctggtcccgc cgcatccata ccgccagttg tttaccctca caacgttcca gtaaccgggc 4080

atgttcatca tcagtaaccc gtatcgtgag catcctctct cgtttcatcg gtatcattac 4140

ccccatgaac agaaatcccc cttacacgga ggcatcagtg accaaacagg aaaaaaccgc 4200

ccttaacatg gcccgcttta tcagaagcca gacattaacg cttctggaga aactcaacga 4260

gctggacgcg gatgaacagg cagacatctg tgaatcgctt cacgaccacg ctgatgagct 4320

ttaccgcagc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct 4380

cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg 4440

cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag 4500

cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat 4560

atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgctctt 4620

ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag 4680

ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca 4740

tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt 4800

tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc 4860

gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct 4920

ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg 4980

tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca 5040

agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact 5100

atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta 5160

acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta 5220

actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct 5280

tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt 5340

tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga 5400

tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca 5460

tgaacaataa aactgtctgc ttacataaac agtaatacaa ggggtgttat gagccatatt 5520

caacgggaaa cgtcttgctc taggccgcga ttaaattcca acatggatgc tgatttatat 5580

gggtataaat gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgattgtat 5640

gggaagcccg atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat 5700

gttacagatg agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc 5760

aagcatttta tccgtactcc tgatgatgca tggttactca ccactgcgat ccccgggaaa 5820

acagcattcc aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg 5880

gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat 5940

cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt 6000

gattttgatg acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataaa 6060

cttttgccat tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt 6120

atttttgacg aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac 6180

cgataccagg atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag 6240

aaacggcttt ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat 6300

ttgatgctcg atgagttttt ctaagaatta attcatgagc ggatacatat ttgaatgtat 6360

ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgaaat 6420

tgtaaacgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt 6480

taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg 6540

gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt 6600

caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc 6660

aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg 6720

atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa 6780

aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc 6840

cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg cca 6883

<210> 43

<211> 6882

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 43

atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa 60

ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt 120

tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttta 180

cgaagttatc atgtggccct cgcaccccac gggtccactc acctttggct ttggagatgg 240

ttttctcgat gggggctggg agggctttgt tggagacctt gcacttgtac tccttgccat 300

tcagccagtc ctggtgcagg acggtgagga cgctgaccac acggtacgtg ctgttgtact 360

gctcctcccg cggctttgtc ttggcattat gcacctccac gccgtccacg taccagttga 420

acttgacctc agggtcttcg tggctcacgt ccaccaccac gcatgtgacc tcaggggtcc 480

gggagatcat gagggtgtcc ttgggttttg gggggaagag gaagactgac ggtcccccca 540

ggagttcagg tgctgggcac ggtgggcatg tgtgagtttt gtcacaagat ttgggctcaa 600

ctttcttgtc caccttggtg ttgctgggct tgtgattcac gttgcagatg taggtctggg 660

tgtccaagct gctggagggc acggtcacca cgctgctgag ggagtagagt cctgaggact 720

gtaggacagc cgggaaggtg tgcacgccgc tggtcagggc gcctgagttc cacgacaccg 780

tcaccggttc ggggaagtag tccttgacca ggcagcccag ggctgctgtg cccccagagg 840

tgctcttgga ggagggtgcc agggggaaga ccgatgggcc cttggtggag gctgaggaga 900

cggtgaccag tgtgccttga ccccagtagt ccatagggga accatatccc atcatcgatc 960

ttgcacagta atagactgca gtgtcctcag atctcaggct gctgagctcc atgtaggctg 1020

tgctcgtgga cttgtccaca gtcattgtca ctctgccctg gaacttctgg ttgtagctag 1080

tagtgtcata gttaggatta atatctccaa tccactcaag gccctgtcca ggggcctgtc 1140

gaacccagtc catgttgtag tcagtgaatg tgtatccaga agccttgcag gaaaccttca 1200

ctgaagctcc aggcttcttc acctcagctc cagactgcac cagctggacc tgggagccgc 1260

cgccgccgga gccgccgccg ccggagccgc cgccgccgga gccgccgccg cctttgattt 1320

cagcttggtg cctccaccga acgtccgagg aagctctcta atgtgctgac agtaatacac 1380

agcgaagtcc tcaggttcca gcctgccgat ggtgagtgtg aagtctgtcc cagacccact 1440

gccactgaat ctagcaggga tcccagattc taggttagat gcaagataga tgaggagtct 1500

gggggcctgg cctggtttct gttggtacca gtgcatataa ctatagccag atgtactgac 1560

actcttgctg gccctgcagc tcagggtggc cctctcccct ggagagagag ataaggtggc 1620

aggagactgt gtcagcacaa tctcacatct ggcacctggg agccagagca gcaggcgtcc 1680

caggagctga gcaggaaccc tcatgtccat ggatccgcga cccatttgct gtccaccagt 1740

catgctagcc atatggctgc cgcgcggcac caggccgctg ctgtgatgat gatgatgatg 1800

gctgctgccc atggtatatc tccttcttaa agttaaacaa aattatttct agaggggaat 1860

tgttatccgc tcacaattcc cctatagtga gtcgtattaa tttcgcggga tcgagatctc 1920

gatcctctac gccggacgca tcgtggccgg catcaccggc gccacaggtg cggttgctgg 1980

cgcctatatc gccgacatca ccgatgggga agatcgggct cgccacttcg ggctcatgag 2040

cgcttgtttc ggcgtgggta tggtggcagg ccccgtggcc gggggactgt tgggcgccat 2100

ctccttgcat gcaccattcc ttgcggcggc ggtgctcaac ggcctcaacc tactactggg 2160

ctgcttccta atgcaggagt cgcataaggg agagcgtcga gatcccggac accatcgaat 2220

ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga agagagtcaa ttcagggtgg 2280

tgaatgtgaa accagtaacg ttatacgatg tcgcagagta tgccggtgtc tcttatcaga 2340

ccgtttcccg cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg 2400

aagcggcgat ggcggagctg aattacattc ccaaccgcgt ggcacaacaa ctggcgggca 2460

aacagtcgtt gctgattggc gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa 2520

ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg 2580

tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg 2640

tcagtgggct gatcattaac tatccgctgg atgaccagga tgccattgct gtggaagctg 2700

cctgcactaa tgttccggcg ttatttcttg atgtctctga ccagacaccc atcaacagta 2760

ttattttctc ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc 2820

accagcaaat cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg 2880

ctggctggca taaatatctc actcgcaatc aaattcagcc gatagcggaa cgggaaggcg 2940

actggagtgc catgtccggt tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc 3000

ccactgcgat gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc gccattaccg 3060

agtccgggct gcgcgttggt gcggatatct cggtagtggg atacgacgat accgaagaca 3120

gctcatgtta tatcccgccg ttaaccacca tcaaacagga ttttcgcctg ctggggcaaa 3180

ccagcgtgga ccgcttgctg caactctctc agggccaggc ggtgaagggc aatcagctgt 3240

tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc caatacgcaa accgcctctc 3300

cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg 3360

ggcagtgagc gcaacgcaat taatgtaagt tagctcactc attaggcacc gggatctcga 3420

ccgatgccct tgagagcctt caacccagtc agctccttcc ggtgggcgcg gggcatgact 3480

atcgtcgccg cacttatgac tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca 3540

gcgctctggg tcattttcgg cgaggaccgc tttcgctgga gcgcgacgat gatcggcctg 3600

tcgcttgcgg tattcggaat cttgcacgcc ctcgctcaag ccttcgtcac tggtcccgcc 3660

accaaacgtt tcggcgagaa gcaggccatt atcgccggca tggcggcccc acgggtgcgc 3720

atgatcgtgc tcctgtcgtt gaggacccgg ctaggctggc ggggttgcct tactggttag 3780

cagaatgaat caccgatacg cgagcgaacg tgaagcgact gctgctgcaa aacgtctgcg 3840

acctgagcaa caacatgaat ggtcttcggt ttccgtgttt cgtaaagtct ggaaacgcgg 3900

aagtcagcgc cctgcaccat tatgttccgg atctgcatcg caggatgctg ctggctaccc 3960

tgtggaacac ctacatctgt attaacgaag cgctggcatt gaccctgagt gatttttctc 4020

tggtcccgcc gcatccatac cgccagttgt ttaccctcac aacgttccag taaccgggca 4080

tgttcatcat cagtaacccg tatcgtgagc atcctctctc gtttcatcgg tatcattacc 4140

cccatgaaca gaaatccccc ttacacggag gcatcagtga ccaaacagga aaaaaccgcc 4200

cttaacatgg cccgctttat cagaagccag acattaacgc ttctggagaa actcaacgag 4260

ctggacgcgg atgaacaggc agacatctgt gaatcgcttc acgaccacgc tgatgagctt 4320

taccgcagct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc 4380

ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc 4440

gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc 4500

ggagtgtata ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata 4560

tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgctcttc 4620

cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc 4680

tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat 4740

gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt 4800

ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg 4860

aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc 4920

tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt 4980

ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa 5040

gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta 5100

tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa 5160

caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa 5220

ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt 5280

cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt 5340

ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat 5400

cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat 5460

gaacaataaa actgtctgct tacataaaca gtaatacaag gggtgttatg agccatattc 5520

aacgggaaac gtcttgctct aggccgcgat taaattccaa catggatgct gatttatatg 5580

ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgattgtatg 5640

ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg 5700

ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca 5760

agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc cccgggaaaa 5820

cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg 5880

cagtgttcct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc 5940

gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg 6000

attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataaac 6060

ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta 6120

tttttgacga ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc 6180

gataccagga tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga 6240

aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt 6300

tgatgctcga tgagtttttc taagaattaa ttcatgagcg gatacatatt tgaatgtatt 6360

tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgaaatt 6420

gtaaacgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt 6480

aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg 6540

ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc 6600

aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca 6660

agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga 6720

tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa 6780

ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc 6840

gccgcgctta atgcgccgct acagggcgcg tcccattcgc ca 6882

<210> 44

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 44

gcctgagcaa actggcctca ggtaatgtga gttagct 37

<210> 45

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 45

tgtgcttctc aaatgcctga ggttaactac gtcgaca 37

<210> 46

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 46

gctggccttt tgctcacatg ttaatgtgag ttagct 36

<210> 47

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 47

tctgtgactg gtaccgagct cgttaacact agttct 36

Claims (9)

1. A genetic engineering strain is characterized in that the strain is obtained by constructing mutant plasmids and screening target plasmids into a modified strain;

the mutant plasmid comprises pEP-mcs- (GGGGS)2-F1,2-Flag, and the sequence is shown as SEQ ID NO. 1;

the screening target plasmid comprises pLA230-mcs- (GGGGS)2-F3-6xHis, and the sequence is shown as SEQ ID NO. 2.

2. The genetically engineered strain of claim 1, wherein the engineered strain is escherichia coli JS200- Δ thyA Δ folA after knockout of genes thyA and folA.

3. The genetically engineered strain of claim 1, wherein the mutant plasmid is constructed by a method comprising the steps of:

a1, PCR amplifying to obtain gene segment SEQ-mcs- (GGGGS)2-F1,2-Flag, the sequence is shown in SEQ ID NO. 3;

a2, inserting a gene sequence expressing thyA into an NdeI restriction site of a mutant plasmid pEP, and carrying out restriction enzyme reaction on the obtained pEP-thyA plasmid by adopting a Bsu36I single restriction enzyme site to obtain a restriction enzyme linear framework plasmid pEP-thyA-Bsu 36I;

a3, carrying out recombination reaction on the gene fragment obtained in the step A1 and the enzyme-digested linear skeleton plasmid obtained in the step A2 to obtain a mutant plasmid.

4. The genetically engineered strain of claim 1, wherein the construction method of the screening target plasmid comprises the following steps:

b1, PCR amplifying to obtain gene segment SEQ-mcs- (GGGGS)2-F3-6xHis, the sequence is shown in SEQ ID NO. 4;

b2, performing enzyme digestion reaction on the screened target plasmid skeleton pLA230 by adopting two enzyme digestion sites of AflIII and SacI to obtain enzyme digestion linear skeleton plasmid pLA 230-AflIII/SacI;

and B3, carrying out recombination reaction on the gene fragment in the step B1 and the enzyme digestion linear skeleton plasmid in the step B2 to obtain the screening target plasmid.

5. A method for constructing the genetically engineered strain of claim 1, comprising the steps of:

and (3) electrically transforming the mutant plasmid and the screening target plasmid into escherichia coli JS 200-delta thya delta fola, culturing by adopting a non-resistant LB culture medium, and then coating the escherichia coli on an LB-thymidine-corresponding resistant solid plate for culturing to obtain the genetic engineering strain.

6. Use of the genetically engineered strain of claim 1 for directed evolution for antibody affinity maturation.

7. The use of claim 6, which comprises screening the genetically engineered strain of claim 1 to obtain a mutant antibody with high affinity for an antigen.

8. An directed evolution method of antibody affinity maturation, comprising the steps of:

s1, constructing antigen and antibody into the mcs region of the mutant plasmid and the screening target plasmid of the genetic engineering strain of claim 1 respectively;

s2, carrying out mutation screening on the strain obtained in the step S1 under the condition of certain TMP pressure;

and S3, obtaining mutation site information, and cloning and expressing to obtain the mutant antibody with higher affinity with the antigen.

9. The directed evolution method for antibody affinity maturation according to claim 8, wherein in step S2, thymine is added to the culture medium used for said mutation screening; the mutation screening mode comprises mutation screening under liquid culture conditions, mutation screening on a solid plate and mutation screening alternately on a liquid-solid plate.

Images