CN114717207B - Yeast cell homologous recombination enzyme system, DNA in-vitro assembly reagent and application thereof - Google Patents

Yeast cell homologous recombination enzyme system, DNA in-vitro assembly reagent and application thereof Download PDF

Info

Publication number
CN114717207B
CN114717207B CN202210436102.2A CN202210436102A CN114717207B CN 114717207 B CN114717207 B CN 114717207B CN 202210436102 A CN202210436102 A CN 202210436102A CN 114717207 B CN114717207 B CN 114717207B
Authority
CN
China
Prior art keywords
leu
dna
ser
yeast
ala
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202210436102.2A
Other languages
Chinese (zh)
Other versions
CN114717207A (en
Inventor
柳伟强
杨平
付煜烨
许映冲
张斌
徐杰
齐金才
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Synbio Technologies
Original Assignee
Synbio Technologies
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Synbio Technologies filed Critical Synbio Technologies
Priority to CN202210436102.2A priority Critical patent/CN114717207B/en
Publication of CN114717207A publication Critical patent/CN114717207A/en
Application granted granted Critical
Publication of CN114717207B publication Critical patent/CN114717207B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • C07K14/395—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Saccharomyces
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1217—Phosphotransferases with a carboxyl group as acceptor (2.7.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1223—Phosphotransferases with a nitrogenous group as acceptor (2.7.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1229—Phosphotransferases with a phosphate group as acceptor (2.7.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
    • C12Y207/0104—Pyruvate kinase (2.7.1.40)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/02—Phosphotransferases with a carboxy group as acceptor (2.7.2)
    • C12Y207/02001—Acetate kinase (2.7.2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/03—Phosphotransferases with a nitrogenous group as acceptor (2.7.3)
    • C12Y207/03002—Creatine kinase (2.7.3.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/04—Phosphotransferases with a phosphate group as acceptor (2.7.4)
    • C12Y207/04001—Polyphosphate kinase (2.7.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/04—Phosphotransferases with a phosphate group as acceptor (2.7.4)
    • C12Y207/04006—Nucleoside-diphosphate kinase (2.7.4.6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07007—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/11—Exodeoxyribonucleases producing 5'-phosphomonoesters (3.1.11)
    • C12Y301/11001—Exodeoxyribonuclease I (3.1.11.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y306/00—Hydrolases acting on acid anhydrides (3.6)
    • C12Y306/04—Hydrolases acting on acid anhydrides (3.6) acting on acid anhydrides; involved in cellular and subcellular movement (3.6.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y605/00—Ligases forming phosphoric ester bonds (6.5)
    • C12Y605/01—Ligases forming phosphoric ester bonds (6.5) forming phosphoric ester bonds (6.5.1)
    • C12Y605/01001—DNA ligase (ATP) (6.5.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Mycology (AREA)
  • Immunology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Toxicology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

The invention provides a yeast cell homologous recombination enzyme system, a DNA in-vitro assembly reagent and application thereof, belonging to the technical field of molecular biology and synthetic biology. The invention provides a DNA in-vitro assembly reagent prepared by utilizing a yeast cell homologous recombination enzyme system and a part of prokaryotic cell expressed recombinase, and the DNA in-vitro assembly reagent is utilized to carry out in-vitro assembly on DNA segments with homologous arms at two ends, so as to realize rapid in-vitro assembly construction of single-stranded DNA and double-stranded DNA. Experiments prove that the method provided by the invention can realize seamless assembly of 4-40 single-stranded primers or 2-6 double-stranded fragments within 1h, has the characteristics of high efficiency and rapidness, and has obvious advantages compared with Gibsonassembly.

Description

Yeast cell homologous recombination enzyme system, DNA in-vitro assembly reagent and application thereof
Technical Field
The invention belongs to the technical field of molecular biology and synthetic biology, and particularly relates to a yeast cell homologous recombination enzyme system, a DNA in-vitro assembly reagent and application thereof.
Background
Yeast, a eukaryotic microorganism, has higher activity of homologous recombination enzyme system than that of prokaryotic homologous recombination enzyme such as bacteriophage and bacteria. The saccharomyces cerevisiae cell can assemble artificially synthesized short gene segments of thousands of base pairs into prokaryotic genomes of mycoplasma, bacteria and the like and eukaryotic genomes of yeast, mammals and the like by virtue of a strong homologous recombination enzyme system. However, the transformation efficiency is low because the DNA fragment is difficult to transform into yeast cells, and complicated operations such as preparation of protoplasts of yeast cells and electrotransformation are involved. In addition, the growth cycle of yeast is also relatively long and very time consuming. It can be seen that the assembly of DNA fragments using the yeast intracellular homologous recombinase system is time-consuming and laborious.
The existing in vitro DNA Assembly reagent is Gibson Assembly. The Gibson Assembly is specifically composed of T5 exonuclease, thermostable DNA polymerase and thermostable DNA ligase, and all enzyme components use enzymes of phage and bacteria, and annealing between fragments relies on random brownian motion to perform DNA fragment Assembly, which has a problem of low Assembly efficiency.
Disclosure of Invention
In view of this, the present invention provides a yeast cell homologous recombination enzyme system, a DNA in vitro assembly reagent, and applications thereof, which can achieve efficient and rapid assembly of multiple single-stranded or double-stranded DNA fragments.
The yeast cell homologous recombination enzyme system provided by the invention comprises yeast exonuclease ExoI, yeast recombinase RAD51, yeast recombinase RAD52 and yeast single-stranded DNA binding protein RPA.
Preferably, the yeast exonuclease ExoI, the yeast recombinase RAD51, the yeast recombinase RAD52 and the yeast single-stranded DNA binding protein RPA are obtained by codon optimization of a prokaryotic expression system;
the optimized nucleotide sequence of the yeast exonuclease ExoI is shown as SEQ ID NO. 1;
the nucleotide sequence of the optimized yeast recombinase RAD51 is shown as SEQ ID NO. 2;
the nucleotide sequence of the optimized yeast recombinase RAD52 is shown as SEQ ID NO. 3;
the nucleotide sequence of the optimized yeast single-stranded DNA binding protein RPA is shown in SEQ ID NO. 4.
Preferably, the in vitro homologous recombination enzyme system further comprises escherichia coli DNA polymerase I, escherichia coli DNA ligase and ATP regenerative enzyme;
the ATP regenerating enzyme comprises one or more of the following kinases: creatine kinase, pyruvate kinase, acetate kinase, polyphosphate kinase, and nucleoside diphosphate kinase.
Preferably, the activity ratio of the yeast exonuclease ExoI, the yeast recombinase RAD51, the yeast recombinase RAD52, the yeast single-stranded DNA binding protein RPA, the escherichia coli DNA polymerase I, the escherichia coli DNA ligase, and the ATP reproducing enzyme is (0.001 to 0.1): (10-100): (10-100): (10-100): (0.1-1): (0.1-1): (1-100).
The invention provides a DNA in vitro assembly reagent, which comprises an in vitro homologous recombination enzyme system and 2.6 multiplied by buffer solution;
the 2.6 multiplied buffer solution is a 10-200 mM Tris base buffer solution containing 1-50 mM magnesium ion source, 5-50 mM alkali metal ion source, 2-10% polyethylene glycol, 1-10 mM NAD, 2-20mM dithiothreitol, 1-10 mM MATP regeneration enzyme substrate and 1-10 mM deoxyribonucleoside triphosphate, and the pH value is 7.0-8.0.
Preferably, the concentration of the yeast exonuclease ExoI in the reagent for assembling DNA in vitro is 1 to 10U/ml, the concentration of yeast recombinase RAD51 is 10 to 100nM, the concentration of yeast recombinase RAD52 is 10 to 100nM, the concentration of yeast single-stranded DNA binding protein RPA is 100 to 500nM, the concentration of escherichia coli DNA polymerase I is 10 to 100U/ml, the concentration of escherichia coli DNA ligase is 1000 to 5000U/ml, and the concentration of ATP regenerating enzyme is 1 to 10mM.
The invention provides an application of the in vitro homologous recombination enzyme system or the DNA in vitro assembly reagent in DNA fragment in vitro assembly.
Preferably, the application comprises assembly of two or more DNA fragments;
the DNA fragment comprises a single-stranded or double-stranded DNA fragment;
the DNA fragment comprises one or more of the following components: animal-derived target genes, virus-derived target genes, and antibody library DNA fragments.
The invention provides a method for in vitro assembly of DNA fragments, which comprises the following steps:
mixing and reacting a DNA fragment to be assembled, a linear carrier and the DNA in-vitro assembly reagent to obtain a linear chain or annular DNA long fragment;
the DNA fragment to be assembled contains a base homologous or complementary region with the length of 15-200 bp.
Preferably, after the mixing reaction, the reaction product is introduced into prokaryotic cells to complete the amplification of DNA fragments, and double-stranded DNA molecules are obtained.
The yeast cell homologous recombination enzyme system provided by the invention comprises yeast exonuclease ExoI, yeast recombinase RAD51, yeast recombinase RAD52 and yeast single-stranded DNA binding protein RPA.
The yeast cell homologous recombination enzyme system provided by the invention comprises yeast exonuclease ExoI, yeast recombinase RAD51, yeast recombinase RAD52 and yeast single-stranded DNA binding protein RPA. The yeast exonuclease yExoI provided by the invention is used for carrying out enzymolysis on the tail ends of two sections of double-stranded DNA into single-stranded DNA; and (3) mutually cooperating yeast recombinase yRAD51, yeast recombinase yRAD52 and yeast single-stranded DNA binding protein yRPA to anneal the single-stranded parts of two homologous DNA fragments together so as to complete the assembly of the DNAs at two ends. Because the multienzyme synergistic reaction of the yeast homologous recombination enzyme system and the high activity of various enzymes and the elaborate reconstruction and research work of the in-vitro recombination system, the DNA in-vitro assembly of the application has higher assembly efficiency.
Furthermore, the application specifically limits the codon optimization of each enzyme in the yeast cell homologous recombination enzyme system through prokaryotic expression cells. The invention optimizes the codons of the core homologous recombinase gene of the homologous recombinase system of the yeast, obtains the recombinant protein of the homologous recombinase of the yeast through recombinant expression, and further realizes the efficient and rapid seamless assembly of a plurality of single-stranded or double-stranded DNA fragments under the in vitro condition.
The method for in vitro assembly of the DNA fragments provided by the invention mixes and reacts the DNA fragments to be assembled with the in vitro DNA assembly reagent to obtain straight chain or annular long DNA fragments. The in vitro assembly method provided by the invention can efficiently connect a plurality of single-stranded or double-stranded DNA fragments to obtain straight-chain or circular DNA, and has high connection accuracy. The method is adopted to carry out in-vitro assembly on the EGFP gene, the novel coronavirus mutant Omicron S protein gene and the yeast cell surface display nano antibody library, and the result shows that compared with the Gibson assembly efficiency, the method provided by the invention has obvious advantage in positive rate which is 50-95%.
Drawings
FIG. 1 is a schematic diagram showing the principle of in vitro recombination of a yeast cell homologous recombination enzyme system;
FIG. 2 is an electrophoresis diagram of the recombinant proteins of yeast exonuclease ExoI and yeast recombinase RAD51 in example 1 of the present invention;
FIG. 3 is an electrophoretogram of recombinant proteins of yeast recombinase RAD52 and yeast single-stranded DNA binding protein RPA in example 1 of the present invention;
FIG. 4 is a PCR verification diagram of EGFP gene assembly colonies;
FIGS. 5 to 10 show the alignment of the sequencing peak maps with a portion of the EGFP sequence;
FIG. 11 is a colony PCR identification chart of the Omicron S gene assembly efficiency (amplified partial sequence);
FIG. 12 shows the PCR amplification result of the assembled nano antibody library.
Detailed Description
The yeast cell homologous recombination enzyme system provided by the invention comprises yeast exonuclease ExoI, yeast recombinase RAD51, yeast recombinase RAD52 and yeast single-stranded DNA binding protein RPA.
In the invention, the yeast exonuclease ExoI, the yeast recombinase RAD51, the yeast recombinase RAD52 and the yeast single-stranded DNA binding protein RPA are preferably obtained by performing codon optimization by taking a prokaryotic expression system as a host and performing recombinant expression. The yeast cell homologous recombination enzyme system is preferably shown as SEQ ID NO. 1 according to the optimized nucleotide sequence of the yeast exonuclease ExoI of the prokaryotic expression system. The optimized nucleotide sequence of yeast recombinase RAD51 is preferably shown as SEQ ID NO. 2. The optimized nucleotide sequence of yeast recombinase RAD52 is preferably shown as SEQ ID NO. 3. The optimized nucleotide sequence of the yeast single-stranded DNA binding protein RPA is preferably shown as SEQ ID NO. 4. The method of recombinant expression is not particularly limited in the present invention, and a method of recombinant expression using a prokaryotic expression system known in the art may be used.
In the present invention, the working principle of the yeast exonuclease ExoI, the yeast recombinase RAD51, the yeast recombinase RAD52 and the yeast single-stranded DNA binding protein RPA is schematically shown in fig. 1, and the specific principle is as follows: h is a homologous sequence area existing in two DNA segments, namely two DNA segments needing to be assembled together, and the assembly joint needs to have a homologous sequence of 15-200 bp. And (2) carrying out enzymolysis on the tail ends of the two sections of double-stranded DNA into single-stranded DNA under the action of yeast exonuclease yExoI, carrying out mutual synergistic action on yeast recombinase yRAD51, yeast recombinase yRAD52 and yeast single-stranded DNA binding protein yRPA, annealing two single strands in the two single-stranded DNAs, connecting to form a DNA fragment, and finishing the assembly of the DNA fragment.
In the invention, after the in vitro homologous recombination enzyme system acts, gaps or pores in the recombined DNA fragment also need to be supplemented, and the process can be completed in an in vitro reaction or a prokaryotic cell expression system. When done under in vitro conditions, the kit also preferably includes E.coli DNA polymerase I, E.coli DNA ligase, and ATP regenerating enzyme. The ATP regenerating enzyme comprises one or more of the following kinases: creatine kinase, pyruvate kinase, acetate kinase, polyphosphate kinase, and nucleoside diphosphate kinase. The activity ratio of the yeast exonuclease ExoI, the yeast recombinase RAD51, the yeast recombinase RAD52, the yeast single-stranded DNA binding protein RPA, the Escherichia coli DNA polymerase I, the Escherichia coli DNA ligase and the ATP regenerating enzyme is preferably (0.001-0.1): (10-100): (10-100): (10-100): (0.1-1): (0.1-1): (1-100).
The invention provides a DNA in vitro assembly reagent, which comprises an in vitro homologous recombination enzyme system and 2.6 multiplied by buffer solution; the 2.6 multiplied buffer solution is a 10-200 mM Tris base buffer solution containing 1-50 mM magnesium ion source, 5-50 mM alkali metal ion source, 2-10% polyethylene glycol, 1-10 mM NAD, 2-20mM dithiothreitol, 1-10 mM MATP regenerative enzyme substrate and 1-10 mM deoxynucleoside triphosphate in percentage by mass, and the pH value is 7.0-8.0.
In the present embodiment, the source of magnesium ions is preferably MgAc2. The alkali metal ion source is preferably creatine phosphate sodium. The polyethylene glycol is preferably PEG-8000. When the ATP regenerative enzyme is creatine kinase, the ATP regenerative enzyme substrate is phosphocreatine; when the ATP regenerating enzyme is pyruvate kinase, the ATP regenerating enzyme substrate is phosphoenolpyruvate; when the ATP regenerating enzyme is acetate kinase, the ATP regenerating enzyme substrate is acetyl phosphate; when the ATP regenerative enzyme is polyphosphate kinase, the ATP regenerative enzyme substrate is polyphosphate; the ATP regenerating enzyme is nucleoside diphosphate kinase, and the ATP regenerating enzyme substrate is nucleoside triphosphate.
In the present invention, the concentration of the yeast exonuclease ExoI in the DNA in vitro assembly reagent is preferably 1 to 10U/ml, more preferably 2 to 8U/ml, even more preferably 4 to 7U/ml, and most preferably 5U/ml. The concentration of yeast recombinase RAD51 is preferably 10 to 100nM, more preferably 20 to 90nM, even more preferably 40 to 70nM, and most preferably 50nM. The concentration of yeast recombinase RAD52 is preferably 10 to 100nM, more preferably 20 to 90nM, even more preferably 40 to 70nM, and most preferably 50nM. The concentration of the yeast single-stranded DNA binding protein RPA is preferably 100 to 500nM, more preferably 200 to 400nM, still more preferably 250 to 350nM, and most preferably 300nM. The concentration of the E.coli DNA polymerase I is preferably 10 to 100U/ml, more preferably 20 to 90U/ml, still more preferably 40 to 70U/ml, and most preferably 50U/ml. The concentration of E.coli DNA ligase is preferably 1000 to 5000U/ml, more preferably 1500 to 4500U/ml, more preferably 2000 to 4000U/ml, even more preferably 2500 to 3500U/ml, and most preferably 3000U/ml. The concentration of the ATP regenerating enzyme is preferably 1 to 10mM, more preferably 2 to 8mM, still more preferably 4 to 7mM, and most preferably 5mM.
The invention provides an application of the in vitro homologous recombination enzyme system or the DNA in vitro assembly reagent in DNA fragment in vitro assembly.
In the present invention, the application preferably comprises assembly of two or more DNA fragments. In the present example, 4 to 14 DNA fragments were simultaneously assembled. The DNA fragment includes a single-stranded or double-stranded DNA fragment. The in vitro assembly of the DNA fragments comprises the assembly of target genes of animal origin, the assembly of target genes or genomes of virus origin and the assembly of DNA fragments of antibody libraries.
In the present invention, when assembling a short segment of a target gene, it is preferable to design a primer to be assembled based on the DNA sequence of the target gene, and complete the assembly by mixing the primer to a linker and the in vitro DNA assembly reagent. The design of the primer is preferably finished by adopting DNAworks software, the parameter during design is that the Tm value is 62 ℃, and the design principle is that the length of the primer is 65-70 nt. The sequence of the adaptor is selected from sequences complementary or homologous to the 3 'end sequence of the upstream primer and sequences complementary or homologous to the 5' end sequence of the downstream primer, respectively, according to the sequence of the primer to the primer. The upstream linker of the 5' segment primer of the target gene may be homologous or complementary to the vector sequence to be cloned. The downstream joint of the 3' segment primer of the target gene is homologous or complementary with the sequence of the vector to be cloned. The length of the joint is 15-200 bp.
The invention provides a method for in vitro assembly of DNA fragments, which comprises the following steps:
mixing and reacting a DNA fragment to be assembled, a linear carrier and the DNA in-vitro assembly reagent to obtain a linear chain or annular DNA long fragment;
the DNA fragment to be assembled contains a base homologous or complementary region with the length of 15-200 bp.
In the present invention, when different DNA fragments to be assembled are mixed, they are preferably mixed in equimolar amounts. The final concentration of the DNA fragment to be assembled is preferably 10 to 500 ng/. Mu.l, more preferably 100 to 200 ng/. Mu.l. The final concentration of the linear vector is preferably 10 to 500 ng/. Mu.l, more preferably 50 to 200 ng/. Mu.l. The temperature of the mixing reaction is preferably 20 to 27 ℃, more preferably 25 ℃. The time of the mixing reaction is preferably 1 to 1.5 hours. The system of the mixing reaction is preferably: mu.l of each 10 mu.M primer to be assembled, 6 mu.l of 50-200 ng/mu.l of linear vector, 20 mu.l of the DNA in-vitro assembly reagent, and 50 mu.l of water.
In the present invention, after the mixing reaction, the reaction product is preferably introduced into a prokaryotic expression system to complete the repair and amplification of the gap or nick of the DNA fragment, resulting in a double-stranded DNA molecule.
After obtaining the double-stranded DNA molecule, it is preferable to detect the positive assembly rate. The detection method is preferably performed by agarose gel electrophoresis and sequencing. And (3) cutting, recovering and purifying the band with the target length in the electrophoretogram, feeding the band into a sample for sequencing, comparing the sequencing result with the target gene sequence, and indicating that the sequences are completely consistent to successfully obtain the assembled sequence.
The yeast cell homologous recombination enzyme system, the DNA in vitro assembly reagent and the application thereof provided by the present invention will be described in detail with reference to the following examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Expression and purification method of yeast cell homologous recombinase core enzyme component
1. The yeast cell homologous recombination enzyme system core enzyme component: the yeast exonuclease ExoI, the yeast recombinase RAD51, the yeast single-stranded DNA annealing protein RAD52 and the yeast single-stranded DNA binding protein RPA are subjected to codon optimization to obtain an optimized DNA sequence, wherein the specific sequence is shown as SEQ ID NO 1-4 in a sequence table, and the amino acid sequence is shown as SEQ ID NO 5-8 in a sequence table.
2. DNA fragments containing restriction enzyme sites were artificially synthesized based on the DNA sequences of 4 enzyme genes, loaded into pET28a through NdeI and XhoI sites, sequenced correctly, and then subjected to inducible expression and recombinant protein purification according to the following procedures.
3. Transformation and shake flask culture of plasmids
1) Mu.l of the plasmid was added to 100. Mu.l of an Arctic Expression (DE 3) competent bacteria and placed on ice for 20min;
2) Thermally shocking at 42 deg.C for 90sec, and rapidly placing in ice for 3min; adding 600 mul LB culture liquid;
3) Shaking at 220rpm at 37 deg.C for 1h, spreading 200 μ l bacterial solution on LB plate containing 50 μ g/ml Kan, and culturing at 37 deg.C in inverted mode overnight;
4) The next morning, the single colonies on the plate were picked and inoculated into 3 tubes containing 4ml LB medium with 50. Mu.g/ml Kan, and cultured at 37 ℃ and 220rpm with shaking to about 1 p.m. with an OD of about 0.6;
5) According to the following steps: 250 proportion, inoculated into 3 bottles of 1L LB culture solution containing 100. Mu.g/ml Kan, shaken at 37 ℃ and 220rpm until the cell OD 600 0.5 to 0.6 (about 4 hours);
6) Adding inducer IPTG into 1L fermentation medium to a final concentration of 0.1mM, and culturing at 220rpm at 16 deg.C overnight;
7) Centrifuging at 5000rpm for 5min to remove supernatant, collecting fermented thallus, and storing at-20 deg.C for crushing and purification.
4. Disruption of bacterial cells and purification of proteins
1) Ultrasonic crushing thallus
Crushing conditions: 350W power, crushing for 4s, interval of 7s, and 120 cycles in total, and a bacteria-breaking buffer solution: 20mM Tris,500mM NaCl, pH8.0.
2) Ni-IMAC purified recombinant proteins
The crushed thallus is centrifuged at 12000rpm for 20min at 4 ℃, and the supernatant is collected for nickel column purification, wherein the filler type is Ni-IMAC. Balance liquid: 20mM Tris,500mM NaCl, pH8.0, eluent: 20mM Tris,500mM NaCl,500mM imidazole, pH8.0. Elution was carried out using 4 kinds of eluents containing 20mM, 50mM, 250mM, and 500mM imidazole, respectively, and the eluents were collected according to the absorption peaks.
The results are shown in FIGS. 2 to 3. As can be seen from the figure, the yeast exonuclease ExoI, the yeast recombinase RAD51, the yeast single-stranded DNA annealing protein RAD52 and the yeast single-stranded DNA binding protein RPA are successfully obtained by recombinant expression in the invention.
Example 2
The yeast cell homologous recombination enzyme system prepared in example 1 was used to prepare DNA in vitro assembly reagents, as shown in tables 1 and 2:
TABLE 1 buffer for in vitro assembly of DNA
Composition (I) Concentration of
Trisbase 200mM
MgAc2 20mM
DTT 20mM
NAD 2mM
Creatine phosphate sodium salt 10mM
ATP sodium salt 2mM
PEG-8000 10％
dNTP，10mM 0.2mM
ddH 2 O Make up to 500ml
Glacial acetic acid Adjusting pH to 7.5
TABLE 2 DNA in vitro Assembly reagents
2.6 Xbuffer 300ml
Creatine kinase, 1mg/ml 10ml
Yeast exonuclease ExoI,1mg/ml 0.5ml
Yeast recombinase RAD51,1mg/ml 20ml
Yeast Single Strand annealing protein RAD52,1mg/ml 20ml
Yeast Single-stranded DNA binding protein RPA,1mg/ml 30ml
E.coli polymerase I,0.25mg/ml 10ml
E.coli DNA ligase, 0.25mg/ml 10ml
Example 3
The EGFP gene assembly was carried out using the DNA in vitro assembly reagent prepared in example 2, the specific method was as follows:
the EGFP gene sequence and the pUC57 plasmid vector sequence are as follows and cloned into the pUC57 plasmid vector by EcoRI and HindIII sites.
EGFP sequence is shown inSEQ ID NO:9。
The pUC57 plasmid vector sequence is shown in SEQ ID NO 10.
Among these, the pUC57 plasmid vector EcoRI containing underlining marks and the homologous sequence near the HindIII site):
based on the EGFP gene sequence, DNAworks software was used to design primers for assembly, as shown in Table 3.
TABLE 3 primer sequences to be assembled
Figure BDA0003612929390000061
Figure BDA0003612929390000071
Add ddH to 200. Mu.l PCR tube 2 And (3) diluting the 14 primers for assembling the EGFP to 10 mu M, adding 1 mu l of each primer, adding 6 mu l (50-200 ng) of the pUC57 vector digested by EcoRI/HindIII, adding 20 mu l of the mixed solution for assembling the 2 XDNA fragment, uniformly mixing, centrifuging at low speed, ensuring that all the liquid is deposited to the bottom of a PCR tube, and reacting at constant temperature of 25 ℃ for 1h. And then transforming the assembly product after reaction into an escherichia coli competent cell, coating an LB solid culture medium, culturing overnight at 37 ℃, and identifying the assembly efficiency by colony PCR the next day.
The method for converting escherichia coli competence by the assembly reaction solution comprises the following steps:
1. taking out the competent cells of the escherichia coli from the temperature of minus 80 ℃, quickly inserting the competent cells into ice, melting the bacterial block after 5 minutes, adding the assembled reaction liquid, stirring the EP tube bottom by hands, gently mixing the mixture, and standing the mixture in the ice for 30 minutes.
The water bath was heat-shocked at 2.42 ℃ for 90 seconds, quickly returned to ice and allowed to stand for 2 minutes.
3. 600. Mu.l of antibiotic-free sterilized LB medium was added to the centrifuge tube, mixed well and then thawed at 37 ℃ and 200rpm for 60 minutes.
The cells were collected by centrifugation at 4.5000 rpm for 1 minute, and about 100. Mu.l of the supernatant was left to gently blow and beat the resuspended pellet and spread on LB medium containing Amp antibiotics.
5. The plates were placed in an incubator at 37 ℃ upside down for overnight incubation.
The colony PCR identification method comprises the following steps:
1. single colony picking
The LB medium was poured into a gun-discharging tank, 400. Mu.L of LB medium was then added to a 96-well deep-well plate using a gun, a single colony on the plate was picked with a sterilized toothpick using tweezers and placed in the 96-well deep-well plate, and cultured on a shaker at 37 ℃ for one hour.
2. Colony PCR reaction
A reaction system of the PCR reaction is prepared, the prepared reaction liquid is added into a 96-hole PCR reaction plate, and 2.5 mu L of the bacterial liquid is added into the reaction liquid by a liquid transfer device. The 96-well reaction plate was placed on a PCR instrument with a rubber pad covered, and the reaction procedure was set according to the PCR reaction conditions.
3. Agarose gel electrophoresis
Preparing 1.5% agarose gel, adding 5 μ L Loading buffer in each tube of a 96-well reaction plate, shaking uniformly, then carrying out sample application electrophoresis, and taking pictures of the electrophoresed agarose gel.
4. Determination of Positive clones
The successful DNA assembly clones were judged by agarose gel electrophoresis pattern.
The agarose gel electrophoresis pattern is shown in FIG. 4. As can be seen from fig. 4, 42 of 48 clones contained the EGFP gene of the expected length by duplication of the fragment size, and the positive rate was 87.5%.
And preparing plasmids of positive clones, and carrying out sequencing verification. The sequencing results were aligned to the expected requirements and are shown in FIGS. 5-10.
Sequencing results showed that the assembled EGFP DNA conformed to the expected designed sequence.
Example 4
The new coronavirus mutant Omicron S protein gene is assembled and loaded into a mammalian cell expression vector pMT7-CMV through EcoRI/ApaI.
The amino acid sequence of the Omicron S protein is shown as SEQ ID NO. 25.
The DNA sequence of the Omicron S protein is shown in SEQ ID NO. 26.
The pMT7-CMV sequence is shown as SEQ ID NO: 27.
Four sequences A, B, C and D are respectively designed on the DNA sequence of the Omicron S protein, four DNA fragments are obtained by the assembly method similar to the embodiment 1, and the full-length gene sequence of the Omicron S protein is assembled again by using 2X DNA assembly mixed liquor.
Fragment A is shown in SEQ ID NO 28.
Fragment B is shown in SEQ ID NO. 29.
Fragment C is shown in SEQ ID NO:30.
Fragment D is shown in SEQ ID NO. 31.
The four fragments were obtained by PCR amplification, purified and recovered by agarose gel electrophoresis, and the concentration was 50 ng/. Mu.l after recovery. The vector pMT7-CMV was digested with EcoRI/ApaI, purified and recovered by agarose gel electrophoresis in the same manner, and the recovered concentration was 100 ng/. Mu.l. The assembly was performed simultaneously with the Gibson assembly reagent.
The assembly reaction is shown in table 4.
TABLE 4 Assembly reaction System
pMT7-CMV(EcoRI/ApaI) 10μl
Fragment A 2.5μl
Fragment B 2.5μl
C fragment 2.5μl
D fragment 2.5μl
2 XDNA assembling mixed liquor 20μl
Is totaled 40μl
The reaction was carried out at a constant temperature of 25 ℃ for 1 hour. The assembly product after the reaction was then transformed into E.coli competent cells in the same manner as in example 1, cultured overnight at 37 ℃ the following day, and the assembly efficiency was confirmed by colony PCR.
The results are shown in FIG. 11. From the colony PCR identification chart, the upper row is the identification result assembled by Gibson assembly (purchased from NEB), and only 2 positive clones out of 24 clones were found, with a positive rate of 8.3%. The lower row is the identification result assembled by the DNA assembly mixed liquor of the invention, 13 positive clones in 24 clones have positive rate more than 50%.
Example 5
Assembly of yeast cell surface display nano antibody library
The sequence of the nano antibody library is shown in SEQ ID NO. 32.
The codon sequences corresponding to the amino acids are shown in Table 5.
TABLE 5 codon sequence corresponding to amino acids
Figure BDA0003612929390000081
Figure BDA0003612929390000091
The vector pSBT-YD2 is shown in SEQ ID NO:33 and assembled into the yeast cell surface through an EcoRI site.
In a 1.5nl centrifuge tube, the reagents shown in Table 6 were added:
TABLE 6 reaction System
pMT7-CMV(EcoRI)，100ng/μl 125μl
Library Assembly Individual fragment mixtures 125μl
2 XDNA assembling mixed liquor 250μl
Is totaled 500μl
The reaction was carried out at a constant temperature of 25 ℃ for 3 hours. Then extracting with phenol and chloroform, purifying the assembly product by isopropanol precipitation, electrically shocking the purified assembly product to transform into escherichia coli competent cells, and culturing overnight at 37 ℃. The next day, assembly efficiency was identified by colony PCR.
And (3) purifying an assembly product:
1. mu.l of the assembly product (less than 500. Mu.l of the recombinant product was replenished with water) was transferred to a 1.5ml centrifuge tube, and 500. Mu.l of phenol chloroform isoamyl alcohol (25.
2. Centrifuging at 12000rpm for 5min at room temperature;
3. the upper aqueous phase was taken, 500. Mu.l of chloroform isoamyl alcohol was added thereto, and mixed with shaking.
4. Centrifuging at 12000rpm for 5min at room temperature;
5. collecting upper water phase, adding 10% of 3M NaAC (pH 5.2), mixing by inversion, adding equal volume of isopropanol, mixing by inversion, standing at room temperature for 15min, and standing at-20 deg.C for 2 hr.
Centrifuging at 12000rpm for 30 minutes at 6.4 ℃;
and 7 carefully remove the supernatant to avoid contact with the sediment at the bottom of the tube.
8. 1ml of 70% ethanol at room temperature was added, and the precipitate was washed by gently inverting several times.
Centrifuge at 12000rpm for 10min at 9.4 ℃.
10. The supernatant was carefully removed and steps 8, 9 were repeated, rinsing the pellet again.
11. Carefully remove the supernatant, centrifuge instantaneously, remove the residual liquid, place Eppendorf tube in a clean bench to blow dry for 5-10min until there is no smell of ethanol.
12. Add 10-15. Mu.l ddH 2 The precipitate was redissolved by O and stored at-20 ℃ until use.
Electric conversion
1. Electric rotating device
Taking 1 μ g of the purified product, adding into electric rotating competence (ice water bath for 10min, transferring to a precooled electric rotating cup, performing electric shock transformation, and recovering and culturing at 30 ℃ for 1.5h.
2. Coating flat plate
The reservoir capacity measurement was calculated according to formula I. The specific method comprises the following steps: 100 mul of 6.4.1 product is taken and added into 900 mul of LB culture medium, 100 mul of spread plate is evenly mixed, the bacterial liquid is diluted by 100 times, and the bacterial liquid is sequentially diluted according to 10 times of gradient. The remaining original bacterial solution was spread on the plate. Incubated at 30 ℃ for about 20h or at 37 ℃ overnight.
Storage capacity = number of long spots × dilution factor formula I
According to the method of example 1, 24 clones were picked for colony PCR and the assembly efficiency was verified.
The colony PCR electrophorogram is shown in FIG. 12. As can be seen from the figure, it was found that the efficiency of the group length of the library was high, and 22 clones out of 24 clones contained the library sequence, with a positive rate of 91.6%.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and amendments can be made without departing from the principle of the present invention, and these modifications and amendments should also be considered as the protection scope of the present invention.
Sequence listing
<110> Suzhou Hongxn Biotechnology Ltd
<120> yeast cell homologous recombination enzyme system, DNA in-vitro assembly reagent and application thereof
<160> 33
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2109
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
atgggtattc aaggtctgct gccgcaactg aaaccgattc agaatccggt ctctctgcgt 60
cgttatgaag gcgaagttct ggccattgac ggttatgctt ggctgcatcg cgcagcttgt 120
tcttgcgctt acgaactggc gatgggtaaa ccgaccgaca aatacctgca gttcttcatc 180
aaacgcttca gcctgctgaa aaccttcaaa gtcgaaccgt acctggtttt tgacggcgat 240
gcgattccgg tcaaaaaatc caccgaaagc aaacgccgcg acaaacgcaa agagaacaaa 300
gcaattgctg aacgtctgtg ggcttgcggc gagaaaaaga acgcgatgga ctacttccag 360
aaatgcgtcg atatcacccc ggaaatggcg aaatgcatca tctgctactg caaactgaac 420
ggcatccgtt acattgttgc gccgtttgaa gccgattcac agatggttta cctggaacag 480
aaaaacatcg tccagggcat catcagcgag gatagcgatc tgctggtttt tggttgccgt 540
cgtctgatca ccaaactgaa cgattacggc gagtgtctgg aaatctgccg cgataacttc 600
atcaaactgc cgaaaaaatt cccgctgggt agcctgacca acgaagaaat tatcaccatg 660
gtctgtctga gcggttgcga ttataccaac ggtattccga aagtcggtct gattaccgca 720
atgaaactgg tccgtcgctt caacaccatc gaacgtatca tcctgagcat tcagcgcgaa 780
ggtaaactga tgatcccgga cacctacatc aacgaatacg aagcagcggt tctggcgttt 840
caatttcagc gcgtattttg cccgattcgc aaaaagattg tcagcctgaa cgagattccg 900
ctgtacctga aagacaccga aagcaaacgc aaacgcctgt acgcttgcat tggctttgtc 960
atccaccgcg aaacccagaa aaaacagatc gtgcacttcg acgacgatat cgatcaccat 1020
ctgcacctga aaattgcaca gggcgatctg aacccgtacg attttcatca gccgctggca 1080
aatcgcgaac ataaactgca gctggcaagc aaaagcaaca tcgagttcgg caaaaccaac 1140
accaccaact ctgaagcgaa agtcaaaccg atcgagagct tcttccagaa aatgaccaaa 1200
ctggaccaca acccgaaagt tgcgaacaat atccacagcc tgcgtcaggc agaagataaa 1260
ctgaccatgg cgatcaaacg tcgcaaactg tctaacgcga acgttgttca ggaaaccctg 1320
aaagacaccc gcagcaaatt cttcaacaaa ccgagcatga ccgtcgtcga aaacttcaaa 1380
gagaaaggcg acagcatcca ggacttcaaa gaggatacca atagccagag cctggaagaa 1440
ccggtttctg aaagtcagct gagtacccaa attccgagca gctttatcac caccaacctg 1500
gaagacgacg ataacctgag cgaagaagtc agcgaagttg tcagcgatat cgaagaagat 1560
cgcaaaaaca gcgaaggtaa aaccatcggc aacgagatct ataataccga cgacgacggc 1620
gacggcgata ccagcgaaga ttatagcgaa accgcagaaa gtcgcgttcc gaccagtagt 1680
accaccagtt ttccgggtag tagtcaacgt agcattagcg gttgcaccaa agttctgcag 1740
aaattccgct atagcagcag ctttagcggc gttaacgcaa atcgtcaacc gctgtttccg 1800
cgtcacgtta atcagaaaag ccgcggtatg gtctacgtta accagaatcg cgacgacgat 1860
tgcgacgata acgacggcaa aaaccagatc acccaacgtc cgagtctgcg taaaagtctg 1920
attggcgcac gtagccagcg tattgtcatt gacatgaaaa gcgtcgacga acgcaaaagc 1980
ttcaatagca gcccgattct gcacgaagag agcaaaaaac gcgacatcga gaccaccaaa 2040
agcagtcaag cacgtccggc agttcgtagt attagcctgc tgagccagtt cgtctacaaa 2100
ggcaaataa 2109
<210> 2
<211> 1203
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
atgagccagg tgcaggagca gcatattagc gaaagccagc tgcagtatgg caacggcagc 60
ctgatgagca ccgtgccagc ggatctgagc cagtctgtgg ttgatggcaa cggcaatggc 120
agcagcgaag atattgaagc gaccaacggc agcggcgatg gcggtggctt acaggaacaa 180
gcagaagcgc aaggtgaaat ggaagatgaa gcgtatgatg aagcggcgct gggcagcttt 240
gtgccgattg aaaagctgca ggtgaacggc attaccatgg cggatgtgaa aaagctgcgc 300
gaaagcggcc tgcataccgc ggaagcagtt gcatatgcac cgcgcaaaga tctgctggaa 360
atcaaaggca ttagcgaagc gaaagcggat aaactgctga acgaagcggc gcgcctggtg 420
ccgatgggct ttgttactgc agcagatttt catatgcgcc gcagcgaact gatttgcctg 480
accaccggca gcaaaaacct ggataccctg ctgggcggcg gtgttgaaac cggcagcatt 540
accgagctgt ttggcgaatt tcgcaccggc aaaagccagc tgtgccatac cctggcggtg 600
acctgccaga ttccgctgga tattggcggc ggtgaaggta aatgcctgta tattgatacc 660
gaaggcacct ttcgcccggt gcgcctggtg agcattgcgc aacgctttgg tctggatcca 720
gatgatgcgc tgaacaacgt ggcgtatgcg cgcgcgtata acgcggatca tcagctgcgc 780
ttattagatg cggcggcgca gatgatgagc gaaagccgct ttagcctgat tgtggtggat 840
agcgtgatgg cgctgtatcg caccgatttt agcggccgcg gcgaactgtc tgcacgtcag 900
atgcatctgg cgaaatttat gcgcgcgctg cagcgcctgg cggatcagtt tggcgtggca 960
gttgttgtta ccaaccaggt ggtggcgcag gtggatggcg gcatggcgtt taatccggat 1020
ccgaaaaagc cgattggcgg caacattatg gcgcatagca gcaccacccg cctgggcttt 1080
aagaagggca aaggctgcca gcgcttatgc aaagtggtgg atagcccgtg cctgccggaa 1140
gcggaatgcg tgtttgcgat ttatgaagat ggcgtgggcg atccgcgcga agaagatgaa 1200
taa 1203
<210> 3
<211> 1416
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
atgaacgaaa ttatggacat ggatgaaaag aaaccggtgt ttggcaacca tagcgaagat 60
attcagacca aactggataa aaagctgggc ccggaatata ttagcaaacg cgtgggcttt 120
ggcaccagcc gcattgcgta tattgaaggc tggcgcgtga ttaacctggc gaaccagatt 180
tttggctata acggctggag caccgaagtg aaaagcgtgg tgattgattt tctggatgaa 240
cgccagggca aatttagcat tggctgcacc gcgattgtgc gcgtgaccct gaccagcggc 300
acttatcgtg aagacattgg ctatggcacc gtggaaaacg aacgccgcaa accggcggcg 360
tttgaacgcg cgaaaaagag cgcggtgact gatgcgctga aacgcagcct gcgtggcttt 420
ggtaacgcgc tgggcaactg cctgtatgat aaagactttc tggcgaaaat cgacaaagtg 480
aagtttgacc cgccggattt tgatgaaaac aacctgtttc gcccgaccga tgaaattagc 540
gaaagcagcc gcaccaacac cctgcatgaa aaccaggaac agcagcagta tccgaacaaa 600
cgccgccagc tgaccaaagt gaccaacacc aatccggata gcaccaagaa cctggtgaaa 660
attgaaaaca ccgtgagccg cggcactcca atgatggcag caccagcaga ggcaaacagc 720
aagaacagca gcaacaaaga taccgatctg aaaagcctgg atgcgagcaa acaggatcag 780
gacgatctgc tggatgatag cctgatgttt agcgatgatt ttcaggacga tgacctgatt 840
aacatgggca acaccaacag caacgtgctg accaccgaaa aagatccggt ggtggcgaaa 900
cagagcccga ccgcgagctc taatccggaa gcagaacaga ttacctttgt gaccgcgaaa 960
gcggcgacca gcgtgcagaa cgaacgctat atcggcgaag aaagcatttt tgatccgaaa 1020
tatcaggcgc agagcattcg ccataccgtg gatcagacca ccagcaaaca tattccggcg 1080
agcgtgctga aagataaaac catgaccacc gcgcgcgata gcgtgtatga aaaattcgcg 1140
ccgaaaggca aacagctgag catgaaaaac aacgataaag aactgggtcc gcatatgctg 1200
gaaggcgcgg gcaaccaggt tccacgcgaa accactccga ttaaaaccaa cgcgaccgcg 1260
tttccgccag cagcagcacc acgttttgca ccaccaagca aagtggtgca tccgaatggc 1320
aacggcgcgg tgccagcggt tccacaacaa cgctctactc gtcgtgaagt gggccgcccg 1380
aaaattaacc cgctgcatgc gcgcaaaccg acctaa 1416
<210> 4
<211> 1866
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
atgagttcag tacaactatc taggggagat ttccatagca tttttactaa caaacaacgt 60
tacgataacc caacgggtgg cgtgtaccag gtgtacaaca cccgtaaatc cgacggcgca 120
aacagcaacc gcaagaacct tataatgatt agcgatggta tttatcatat gaaagcgtta 180
ttgcgtaatc aagctgcatc gaagtttcag agtatggaac tgcagcgtgg tgatattatt 240
cgcgtgatta tcgcggaacc ggcgatcgtg cgtgaacgta agaaatacgt tctcttagtt 300
gacgacttcg agctggtgca gtctcgtgcc gacatggtga atcaaaccag cacctttctg 360
gataattact tcagcgagca cccgaatgaa accctgaagg acgaagacat caccgattcc 420
ggtaatgttg cgaaccagac caacgcttcc aacgcgggtg ttccggatat gctgcactcg 480
aactccaact tgaatgctaa cgagcgcaag ttcgcgaacg agaacccgaa ctcgcaaaaa 540
actcgtccca tctttgccat cgaacagctg tccccgtatc agaacgtgtg gaccatcaaa 600
gcgagagtct cctataaagg tgaaatcaaa acctggcata atcaacgcgg cgatggcaaa 660
ctgtttaacg tcaacttctt ggacaccagc ggtgaaatcc gtgcgaccgc atttaacgac 720
ttcgcaacta agttcaacga gatcctgcaa gaaggtaagg tttactacgt tagcaaggcg 780
aaactgcaac cggcgaaacc gcagtttacc aatctgacgc acccgtacga attgaatttg 840
gaccgcgata ccgtcatcga agagtgcttc gacgagagca atgtgccgaa aacgcacttt 900
aacttcatca agctggacgc aattcaaaac caagaagtga atagcaacgt ggacgttctg 960
ggcatcatcc agaccattaa cccgcatttt gaattgacgt cccgtgccgg caagaaattt 1020
gatcgtcgtg atattacgat cgttgatgat tctggcttca gcatttccgt tggtctgtgg 1080
aatcaacagg ctctggactt caacctgccg gagggcagcg tggccgcgat taagggtgtt 1140
cgtgtcaccg acttcggtgg caagagcctg tctatgggtt tttcaagcac cctgatcccg 1200
aacccagaga tcccggaggc atatgcatta aagggctggt atgactctaa ggggcgtaac 1260
gctaatttca tcaccttgaa gcaggagccg ggtatgggcg gtcagagcgc tgcatctctg 1320
acgaagttca tcgcgcaacg tattacgatc gcgcgtgcgc aggcggaaaa tttgggtcgt 1380
agcgagaaag gcgacttttt ctctgttaaa gctgccattt cctttttgaa ggttgacaac 1440
ttcgcgtacc cggcctgcag caacgagaac tgtaataaaa aagtacttga gcagccggat 1500
ggtacctggc gctgcgagaa atgtgatacc aataacgccc gccctaactg gcgttacatt 1560
ctgaccattt ctatcattga tgaaaccaac caactctggc tgaccctgtt cgacgaccag 1620
gctaaacagc tgttgggcgt ggacgctaat accttaatga gcctgaaaga agaagacccg 1680
aacgagttta ctaagatcac ccagagcatc caaatgaatg agtacgactt ccgcatccgc 1740
gcgagagagg acacatataa tgatcagagc cgtattcgct ataccgttgc gaatcttcac 1800
tccctgaact atcgtgctga ggcggactat ctggcagatg aactgagcaa ggcgctgctg 1860
gcgtaa 1866
<210> 5
<211> 702
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Met Gly Ile Gln Gly Leu Leu Pro Gln Leu Lys Pro Ile Gln Asn Pro
1 5 10 15
Val Ser Leu Arg Arg Tyr Glu Gly Glu Val Leu Ala Ile Asp Gly Tyr
20 25 30
Ala Trp Leu His Arg Ala Ala Cys Ser Cys Ala Tyr Glu Leu Ala Met
35 40 45
Gly Lys Pro Thr Asp Lys Tyr Leu Gln Phe Phe Ile Lys Arg Phe Ser
50 55 60
Leu Leu Lys Thr Phe Lys Val Glu Pro Tyr Leu Val Phe Asp Gly Asp
65 70 75 80
Ala Ile Pro Val Lys Lys Ser Thr Glu Ser Lys Arg Arg Asp Lys Arg
85 90 95
Lys Glu Asn Lys Ala Ile Ala Glu Arg Leu Trp Ala Cys Gly Glu Lys
100 105 110
Lys Asn Ala Met Asp Tyr Phe Gln Lys Cys Val Asp Ile Thr Pro Glu
115 120 125
Met Ala Lys Cys Ile Ile Cys Tyr Cys Lys Leu Asn Gly Ile Arg Tyr
130 135 140
Ile Val Ala Pro Phe Glu Ala Asp Ser Gln Met Val Tyr Leu Glu Gln
145 150 155 160
Lys Asn Ile Val Gln Gly Ile Ile Ser Glu Asp Ser Asp Leu Leu Val
165 170 175
Phe Gly Cys Arg Arg Leu Ile Thr Lys Leu Asn Asp Tyr Gly Glu Cys
180 185 190
Leu Glu Ile Cys Arg Asp Asn Phe Ile Lys Leu Pro Lys Lys Phe Pro
195 200 205
Leu Gly Ser Leu Thr Asn Glu Glu Ile Ile Thr Met Val Cys Leu Ser
210 215 220
Gly Cys Asp Tyr Thr Asn Gly Ile Pro Lys Val Gly Leu Ile Thr Ala
225 230 235 240
Met Lys Leu Val Arg Arg Phe Asn Thr Ile Glu Arg Ile Ile Leu Ser
245 250 255
Ile Gln Arg Glu Gly Lys Leu Met Ile Pro Asp Thr Tyr Ile Asn Glu
260 265 270
Tyr Glu Ala Ala Val Leu Ala Phe Gln Phe Gln Arg Val Phe Cys Pro
275 280 285
Ile Arg Lys Lys Ile Val Ser Leu Asn Glu Ile Pro Leu Tyr Leu Lys
290 295 300
Asp Thr Glu Ser Lys Arg Lys Arg Leu Tyr Ala Cys Ile Gly Phe Val
305 310 315 320
Ile His Arg Glu Thr Gln Lys Lys Gln Ile Val His Phe Asp Asp Asp
325 330 335
Ile Asp His His Leu His Leu Lys Ile Ala Gln Gly Asp Leu Asn Pro
340 345 350
Tyr Asp Phe His Gln Pro Leu Ala Asn Arg Glu His Lys Leu Gln Leu
355 360 365
Ala Ser Lys Ser Asn Ile Glu Phe Gly Lys Thr Asn Thr Thr Asn Ser
370 375 380
Glu Ala Lys Val Lys Pro Ile Glu Ser Phe Phe Gln Lys Met Thr Lys
385 390 395 400
Leu Asp His Asn Pro Lys Val Ala Asn Asn Ile His Ser Leu Arg Gln
405 410 415
Ala Glu Asp Lys Leu Thr Met Ala Ile Lys Arg Arg Lys Leu Ser Asn
420 425 430
Ala Asn Val Val Gln Glu Thr Leu Lys Asp Thr Arg Ser Lys Phe Phe
435 440 445
Asn Lys Pro Ser Met Thr Val Val Glu Asn Phe Lys Glu Lys Gly Asp
450 455 460
Ser Ile Gln Asp Phe Lys Glu Asp Thr Asn Ser Gln Ser Leu Glu Glu
465 470 475 480
Pro Val Ser Glu Ser Gln Leu Ser Thr Gln Ile Pro Ser Ser Phe Ile
485 490 495
Thr Thr Asn Leu Glu Asp Asp Asp Asn Leu Ser Glu Glu Val Ser Glu
500 505 510
Val Val Ser Asp Ile Glu Glu Asp Arg Lys Asn Ser Glu Gly Lys Thr
515 520 525
Ile Gly Asn Glu Ile Tyr Asn Thr Asp Asp Asp Gly Asp Gly Asp Thr
530 535 540
Ser Glu Asp Tyr Ser Glu Thr Ala Glu Ser Arg Val Pro Thr Ser Ser
545 550 555 560
Thr Thr Ser Phe Pro Gly Ser Ser Gln Arg Ser Ile Ser Gly Cys Thr
565 570 575
Lys Val Leu Gln Lys Phe Arg Tyr Ser Ser Ser Phe Ser Gly Val Asn
580 585 590
Ala Asn Arg Gln Pro Leu Phe Pro Arg His Val Asn Gln Lys Ser Arg
595 600 605
Gly Met Val Tyr Val Asn Gln Asn Arg Asp Asp Asp Cys Asp Asp Asn
610 615 620
Asp Gly Lys Asn Gln Ile Thr Gln Arg Pro Ser Leu Arg Lys Ser Leu
625 630 635 640
Ile Gly Ala Arg Ser Gln Arg Ile Val Ile Asp Met Lys Ser Val Asp
645 650 655
Glu Arg Lys Ser Phe Asn Ser Ser Pro Ile Leu His Glu Glu Ser Lys
660 665 670
Lys Arg Asp Ile Glu Thr Thr Lys Ser Ser Gln Ala Arg Pro Ala Val
675 680 685
Arg Ser Ile Ser Leu Leu Ser Gln Phe Val Tyr Lys Gly Lys
690 695 700
<210> 6
<211> 400
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Met Ser Gln Val Gln Glu Gln His Ile Ser Glu Ser Gln Leu Gln Tyr
1 5 10 15
Gly Asn Gly Ser Leu Met Ser Thr Val Pro Ala Asp Leu Ser Gln Ser
20 25 30
Val Val Asp Gly Asn Gly Asn Gly Ser Ser Glu Asp Ile Glu Ala Thr
35 40 45
Asn Gly Ser Gly Asp Gly Gly Gly Leu Gln Glu Gln Ala Glu Ala Gln
50 55 60
Gly Glu Met Glu Asp Glu Ala Tyr Asp Glu Ala Ala Leu Gly Ser Phe
65 70 75 80
Val Pro Ile Glu Lys Leu Gln Val Asn Gly Ile Thr Met Ala Asp Val
85 90 95
Lys Lys Leu Arg Glu Ser Gly Leu His Thr Ala Glu Ala Val Ala Tyr
100 105 110
Ala Pro Arg Lys Asp Leu Leu Glu Ile Lys Gly Ile Ser Glu Ala Lys
115 120 125
Ala Asp Lys Leu Leu Asn Glu Ala Ala Arg Leu Val Pro Met Gly Phe
130 135 140
Val Thr Ala Ala Asp Phe His Met Arg Arg Ser Glu Leu Ile Cys Leu
145 150 155 160
Thr Thr Gly Ser Lys Asn Leu Asp Thr Leu Leu Gly Gly Gly Val Glu
165 170 175
Thr Gly Ser Ile Thr Glu Leu Phe Gly Glu Phe Arg Thr Gly Lys Ser
180 185 190
Gln Leu Cys His Thr Leu Ala Val Thr Cys Gln Ile Pro Leu Asp Ile
195 200 205
Gly Gly Gly Glu Gly Lys Cys Leu Tyr Ile Asp Thr Glu Gly Thr Phe
210 215 220
Arg Pro Val Arg Leu Val Ser Ile Ala Gln Arg Phe Gly Leu Asp Pro
225 230 235 240
Asp Asp Ala Leu Asn Asn Val Ala Tyr Ala Arg Ala Tyr Asn Ala Asp
245 250 255
His Gln Leu Arg Leu Leu Asp Ala Ala Ala Gln Met Met Ser Glu Ser
260 265 270
Arg Phe Ser Leu Ile Val Val Asp Ser Val Met Ala Leu Tyr Arg Thr
275 280 285
Asp Phe Ser Gly Arg Gly Glu Leu Ser Ala Arg Gln Met His Leu Ala
290 295 300
Lys Phe Met Arg Ala Leu Gln Arg Leu Ala Asp Gln Phe Gly Val Ala
305 310 315 320
Val Val Val Thr Asn Gln Val Val Ala Gln Val Asp Gly Gly Met Ala
325 330 335
Phe Asn Pro Asp Pro Lys Lys Pro Ile Gly Gly Asn Ile Met Ala His
340 345 350
Ser Ser Thr Thr Arg Leu Gly Phe Lys Lys Gly Lys Gly Cys Gln Arg
355 360 365
Leu Cys Lys Val Val Asp Ser Pro Cys Leu Pro Glu Ala Glu Cys Val
370 375 380
Phe Ala Ile Tyr Glu Asp Gly Val Gly Asp Pro Arg Glu Glu Asp Glu
385 390 395 400
<210> 7
<211> 471
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Met Asn Glu Ile Met Asp Met Asp Glu Lys Lys Pro Val Phe Gly Asn
1 5 10 15
His Ser Glu Asp Ile Gln Thr Lys Leu Asp Lys Lys Leu Gly Pro Glu
20 25 30
Tyr Ile Ser Lys Arg Val Gly Phe Gly Thr Ser Arg Ile Ala Tyr Ile
35 40 45
Glu Gly Trp Arg Val Ile Asn Leu Ala Asn Gln Ile Phe Gly Tyr Asn
50 55 60
Gly Trp Ser Thr Glu Val Lys Ser Val Val Ile Asp Phe Leu Asp Glu
65 70 75 80
Arg Gln Gly Lys Phe Ser Ile Gly Cys Thr Ala Ile Val Arg Val Thr
85 90 95
Leu Thr Ser Gly Thr Tyr Arg Glu Asp Ile Gly Tyr Gly Thr Val Glu
100 105 110
Asn Glu Arg Arg Lys Pro Ala Ala Phe Glu Arg Ala Lys Lys Ser Ala
115 120 125
Val Thr Asp Ala Leu Lys Arg Ser Leu Arg Gly Phe Gly Asn Ala Leu
130 135 140
Gly Asn Cys Leu Tyr Asp Lys Asp Phe Leu Ala Lys Ile Asp Lys Val
145 150 155 160
Lys Phe Asp Pro Pro Asp Phe Asp Glu Asn Asn Leu Phe Arg Pro Thr
165 170 175
Asp Glu Ile Ser Glu Ser Ser Arg Thr Asn Thr Leu His Glu Asn Gln
180 185 190
Glu Gln Gln Gln Tyr Pro Asn Lys Arg Arg Gln Leu Thr Lys Val Thr
195 200 205
Asn Thr Asn Pro Asp Ser Thr Lys Asn Leu Val Lys Ile Glu Asn Thr
210 215 220
Val Ser Arg Gly Thr Pro Met Met Ala Ala Pro Ala Glu Ala Asn Ser
225 230 235 240
Lys Asn Ser Ser Asn Lys Asp Thr Asp Leu Lys Ser Leu Asp Ala Ser
245 250 255
Lys Gln Asp Gln Asp Asp Leu Leu Asp Asp Ser Leu Met Phe Ser Asp
260 265 270
Asp Phe Gln Asp Asp Asp Leu Ile Asn Met Gly Asn Thr Asn Ser Asn
275 280 285
Val Leu Thr Thr Glu Lys Asp Pro Val Val Ala Lys Gln Ser Pro Thr
290 295 300
Ala Ser Ser Asn Pro Glu Ala Glu Gln Ile Thr Phe Val Thr Ala Lys
305 310 315 320
Ala Ala Thr Ser Val Gln Asn Glu Arg Tyr Ile Gly Glu Glu Ser Ile
325 330 335
Phe Asp Pro Lys Tyr Gln Ala Gln Ser Ile Arg His Thr Val Asp Gln
340 345 350
Thr Thr Ser Lys His Ile Pro Ala Ser Val Leu Lys Asp Lys Thr Met
355 360 365
Thr Thr Ala Arg Asp Ser Val Tyr Glu Lys Phe Ala Pro Lys Gly Lys
370 375 380
Gln Leu Ser Met Lys Asn Asn Asp Lys Glu Leu Gly Pro His Met Leu
385 390 395 400
Glu Gly Ala Gly Asn Gln Val Pro Arg Glu Thr Thr Pro Ile Lys Thr
405 410 415
Asn Ala Thr Ala Phe Pro Pro Ala Ala Ala Pro Arg Phe Ala Pro Pro
420 425 430
Ser Lys Val Val His Pro Asn Gly Asn Gly Ala Val Pro Ala Val Pro
435 440 445
Gln Gln Arg Ser Thr Arg Arg Glu Val Gly Arg Pro Lys Ile Asn Pro
450 455 460
Leu His Ala Arg Lys Pro Thr
465 470
<210> 8
<211> 621
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
Met Ser Ser Val Gln Leu Ser Arg Gly Asp Phe His Ser Ile Phe Thr
1 5 10 15
Asn Lys Gln Arg Tyr Asp Asn Pro Thr Gly Gly Val Tyr Gln Val Tyr
20 25 30
Asn Thr Arg Lys Ser Asp Gly Ala Asn Ser Asn Arg Lys Asn Leu Ile
35 40 45
Met Ile Ser Asp Gly Ile Tyr His Met Lys Ala Leu Leu Arg Asn Gln
50 55 60
Ala Ala Ser Lys Phe Gln Ser Met Glu Leu Gln Arg Gly Asp Ile Ile
65 70 75 80
Arg Val Ile Ile Ala Glu Pro Ala Ile Val Arg Glu Arg Lys Lys Tyr
85 90 95
Val Leu Leu Val Asp Asp Phe Glu Leu Val Gln Ser Arg Ala Asp Met
100 105 110
Val Asn Gln Thr Ser Thr Phe Leu Asp Asn Tyr Phe Ser Glu His Pro
115 120 125
Asn Glu Thr Leu Lys Asp Glu Asp Ile Thr Asp Ser Gly Asn Val Ala
130 135 140
Asn Gln Thr Asn Ala Ser Asn Ala Gly Val Pro Asp Met Leu His Ser
145 150 155 160
Asn Ser Asn Leu Asn Ala Asn Glu Arg Lys Phe Ala Asn Glu Asn Pro
165 170 175
Asn Ser Gln Lys Thr Arg Pro Ile Phe Ala Ile Glu Gln Leu Ser Pro
180 185 190
Tyr Gln Asn Val Trp Thr Ile Lys Ala Arg Val Ser Tyr Lys Gly Glu
195 200 205
Ile Lys Thr Trp His Asn Gln Arg Gly Asp Gly Lys Leu Phe Asn Val
210 215 220
Asn Phe Leu Asp Thr Ser Gly Glu Ile Arg Ala Thr Ala Phe Asn Asp
225 230 235 240
Phe Ala Thr Lys Phe Asn Glu Ile Leu Gln Glu Gly Lys Val Tyr Tyr
245 250 255
Val Ser Lys Ala Lys Leu Gln Pro Ala Lys Pro Gln Phe Thr Asn Leu
260 265 270
Thr His Pro Tyr Glu Leu Asn Leu Asp Arg Asp Thr Val Ile Glu Glu
275 280 285
Cys Phe Asp Glu Ser Asn Val Pro Lys Thr His Phe Asn Phe Ile Lys
290 295 300
Leu Asp Ala Ile Gln Asn Gln Glu Val Asn Ser Asn Val Asp Val Leu
305 310 315 320
Gly Ile Ile Gln Thr Ile Asn Pro His Phe Glu Leu Thr Ser Arg Ala
325 330 335
Gly Lys Lys Phe Asp Arg Arg Asp Ile Thr Ile Val Asp Asp Ser Gly
340 345 350
Phe Ser Ile Ser Val Gly Leu Trp Asn Gln Gln Ala Leu Asp Phe Asn
355 360 365
Leu Pro Glu Gly Ser Val Ala Ala Ile Lys Gly Val Arg Val Thr Asp
370 375 380
Phe Gly Gly Lys Ser Leu Ser Met Gly Phe Ser Ser Thr Leu Ile Pro
385 390 395 400
Asn Pro Glu Ile Pro Glu Ala Tyr Ala Leu Lys Gly Trp Tyr Asp Ser
405 410 415
Lys Gly Arg Asn Ala Asn Phe Ile Thr Leu Lys Gln Glu Pro Gly Met
420 425 430
Gly Gly Gln Ser Ala Ala Ser Leu Thr Lys Phe Ile Ala Gln Arg Ile
435 440 445
Thr Ile Ala Arg Ala Gln Ala Glu Asn Leu Gly Arg Ser Glu Lys Gly
450 455 460
Asp Phe Phe Ser Val Lys Ala Ala Ile Ser Phe Leu Lys Val Asp Asn
465 470 475 480
Phe Ala Tyr Pro Ala Cys Ser Asn Glu Asn Cys Asn Lys Lys Val Leu
485 490 495
Glu Gln Pro Asp Gly Thr Trp Arg Cys Glu Lys Cys Asp Thr Asn Asn
500 505 510
Ala Arg Pro Asn Trp Arg Tyr Ile Leu Thr Ile Ser Ile Ile Asp Glu
515 520 525
Thr Asn Gln Leu Trp Leu Thr Leu Phe Asp Asp Gln Ala Lys Gln Leu
530 535 540
Leu Gly Val Asp Ala Asn Thr Leu Met Ser Leu Lys Glu Glu Asp Pro
545 550 555 560
Asn Glu Phe Thr Lys Ile Thr Gln Ser Ile Gln Met Asn Glu Tyr Asp
565 570 575
Phe Arg Ile Arg Ala Arg Glu Asp Thr Tyr Asn Asp Gln Ser Arg Ile
580 585 590
Arg Tyr Thr Val Ala Asn Leu His Ser Leu Asn Tyr Arg Ala Glu Ala
595 600 605
Asp Tyr Leu Ala Asp Glu Leu Ser Lys Ala Leu Leu Ala
610 615 620
<210> 9
<211> 772
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
gttgtaaaac gacggccagt gaattcatgg tgagcaaggg cgaggagctg ttcaccgggg 60
tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg 120
gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg 180
gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct 240
tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag 300
gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg 360
aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca 420
aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct 480
atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca 540
tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg 600
gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc 660
ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc 720
tcggcatgga cgagctgtac aagtaaaagc ttggcgtaat catggtcata gc 772
<210> 10
<211> 2710
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accagatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300
tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acctcgcgaa 420
tgcatctaga tatcggatcc cgggcccgtc gactgcagag gcctgcatgc aagcttggcg 480
taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac 540
atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca 600
ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat 660
taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc 720
tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca 780
aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca 840
aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 900
ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 960
acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 1020
ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 1080
tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 1140
tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 1200
gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 1260
agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 1320
tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 1380
agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 1440
tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 1500
acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 1560
tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 1620
agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc 1680
tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact 1740
acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg actcccacgc 1800
tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt 1860
ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta 1920
agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg 1980
tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt 2040
acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc 2100
agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt 2160
actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc 2220
tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc 2280
gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 2340
ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac 2400
tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa 2460
aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt 2520
tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa 2580
tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct 2640
gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg 2700
ccctttcgtc 2710
<210> 11
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
gttgtaaaac gacggccag 19
<210> 12
<211> 75
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
ccaggatggg caccaccccg gtgaacagct cctcgccctt gctcaccatg aattcactgg 60
ccgtcgtttt acaac 75
<210> 13
<211> 75
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc 60
ggcgagggcg agggc 75
<210> 14
<211> 75
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
gcacgggcag cttgccggtg gtgcagatga acttcagggt cagcttgccg taggtggcat 60
cgccctcgcc ctcgc 75
<210> 15
<211> 75
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct 60
tcagccgcta ccccg 75
<210> 16
<211> 75
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
gctcctggac gtagccttcg ggcatggcgg acttgaagaa gtcgtgctgc ttcatgtggt 60
cggggtagcg gctga 75
<210> 17
<211> 75
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc 60
cgaggtgaag ttcga 75
<210> 18
<211> 75
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
ccgtcctcct tgaagtcgat gcccttcagc tcgatgcggt tcaccagggt gtcgccctcg 60
aacttcacct cggcg 75
<210> 19
<211> 75
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca 60
caacgtctat atcat 75
<210> 20
<211> 75
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
tgttgtggcg gatcttgaag ttcaccttga tgccgttctt ctgcttgtcg gccatgatat 60
agacgttgtg gctgt 75
<210> 21
<211> 75
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag 60
aacaccccca tcggc 75
<210> 22
<211> 75
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
ttgctcaggg cggactgggt gctcaggtag tggttgtcgg gcagcagcac ggggccgtcg 60
ccgatggggg tgttc 75
<210> 23
<211> 75
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg 60
tgaccgccgc cggga 75
<210> 24
<211> 70
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
gctatgacca tgattacgcc aagcttttac ttgtacagct cgtccatgcc gagagtgatc 60
ccggcggcgg 70
<210> 25
<211> 1270
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 25
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val
1 5 10 15
Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe
20 25 30
Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu
35 40 45
His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp
50 55 60
Phe His Val Ile Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp Asn Pro
65 70 75 80
Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Ile Glu Lys Ser
85 90 95
Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser Lys Thr
100 105 110
Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile Lys Val
115 120 125
Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Asp His Lys Asn Asn
130 135 140
Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr Ser Ser Ala Asn Asn
145 150 155 160
Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu Met Asp Leu Glu Gly
165 170 175
Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe Val Phe Lys Asn Ile
180 185 190
Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr Pro Ile Ile Val Arg
195 200 205
Glu Pro Glu Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu Pro Leu Val
210 215 220
Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr Leu Leu Ala
225 230 235 240
Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser Gly Trp Thr
245 250 255
Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro Arg Thr Phe
260 265 270
Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala Val Asp Cys
275 280 285
Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys Ser Phe Thr
290 295 300
Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val Gln Pro Thr
305 310 315 320
Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Asp
325 330 335
Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg
340 345 350
Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Leu
355 360 365
Ala Pro Phe Phe Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu
370 375 380
Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg
385 390 395 400
Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Asn Ile Ala
405 410 415
Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala
420 425 430
Trp Asn Ser Asn Lys Leu Asp Ser Lys Val Ser Gly Asn Tyr Asn Tyr
435 440 445
Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp
450 455 460
Ile Ser Thr Glu Ile Tyr Gln Ala Gly Asn Lys Pro Cys Asn Gly Val
465 470 475 480
Ala Gly Phe Asn Cys Tyr Phe Pro Leu Arg Ser Tyr Ser Phe Arg Pro
485 490 495
Thr Tyr Gly Val Gly His Gln Pro Tyr Arg Val Val Val Leu Ser Phe
500 505 510
Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr
515 520 525
Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn Gly Leu Lys
530 535 540
Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu Pro Phe Gln
545 550 555 560
Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val Arg Asp Pro
565 570 575
Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe Gly Gly Val
580 585 590
Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val Ala Val Leu
595 600 605
Tyr Gln Gly Val Asn Cys Thr Glu Val Pro Val Ala Ile His Ala Asp
610 615 620
Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser Asn Val Phe
625 630 635 640
Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu Tyr Val Asn Asn Ser
645 650 655
Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala Ser Tyr Gln
660 665 670
Thr Gln Thr Lys Ser His Arg Arg Ala Arg Ser Val Ala Ser Gln Ser
675 680 685
Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser Val Ala Tyr
690 695 700
Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile Ser Val Thr
705 710 715 720
Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val Asp Cys Thr
725 730 735
Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu Leu Leu Gln
740 745 750
Tyr Gly Ser Phe Cys Thr Gln Leu Lys Arg Ala Leu Thr Gly Ile Ala
755 760 765
Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln Val Lys Gln
770 775 780
Ile Tyr Lys Thr Pro Pro Ile Lys Tyr Phe Gly Gly Phe Asn Phe Ser
785 790 795 800
Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser Phe Ile Glu
805 810 815
Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly Phe Ile Lys
820 825 830
Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp Leu Ile Cys
835 840 845
Ala Gln Lys Phe Lys Gly Leu Thr Val Leu Pro Pro Leu Leu Thr Asp
850 855 860
Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly Thr Ile Thr
865 870 875 880
Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe Ala
885 890 895
Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn Val
900 905 910
Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn Ser Ala Ile
915 920 925
Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala Leu Gly Lys
930 935 940
Leu Gln Asp Val Val Asn His Asn Ala Gln Ala Leu Asn Thr Leu Val
945 950 955 960
Lys Gln Leu Ser Ser Lys Phe Gly Ala Ile Ser Ser Val Leu Asn Asp
965 970 975
Ile Phe Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln Ile Asp Arg
980 985 990
Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln Gln
995 1000 1005
Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala Thr
1010 1015 1020
Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val Asp Phe Cys
1025 1030 1035 1040
Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ser Ala Pro His Gly
1045 1050 1055
Val Val Phe Leu His Val Thr Tyr Val Pro Ala Gln Glu Lys Asn Phe
1060 1065 1070
Thr Thr Ala Pro Ala Ile Cys His Asp Gly Lys Ala His Phe Pro Arg
1075 1080 1085
Glu Gly Val Phe Val Ser Asn Gly Thr His Trp Phe Val Thr Gln Arg
1090 1095 1100
Asn Phe Tyr Glu Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val Ser
1105 1110 1115 1120
Gly Asn Cys Asp Val Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp
1125 1130 1135
Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr
1140 1145 1150
Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly
1155 1160 1165
Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn
1170 1175 1180
Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu
1185 1190 1195 1200
Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu Gly
1205 1210 1215
Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met Leu Cys
1220 1225 1230
Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys Ser Cys Gly
1235 1240 1245
Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro Val Leu Lys Gly
1250 1255 1260
Val Lys Leu His Tyr Thr
1265 1270
<210> 26
<211> 3862
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
tccagcctcc ggactctaga gaattcatgt ttgtttttct tgttttattg ccactagtct 60
ctagtcagtg tgttaatctt acaaccagaa ctcaattacc ccctgcatac actaattctt 120
tcacacgtgg tgtttattac cctgacaaag ttttcagatc ctcagtttta cattcaactc 180
aggacttgtt cttacctttc ttttccaatg ttacttggtt ccatgttatc tctgggacca 240
atggtactaa gaggtttgat aaccctgtcc taccatttaa tgatggtgtt tattttgctt 300
ccattgagaa gtctaacata ataagaggct ggatttttgg tactacttta gattcgaaga 360
cccagtccct acttattgtt aataacgcta ctaatgttgt tattaaagtc tgtgaatttc 420
aattttgtaa tgatccattt ttggaccaca aaaacaacaa aagttggatg gaaagtgagt 480
tcagagttta ttctagtgcg aataattgca cttttgaata tgtctctcag ccttttctta 540
tggaccttga aggaaaacag ggtaatttca aaaatcttag ggaatttgtg tttaagaata 600
ttgatggtta ttttaaaata tattctaagc acacgcctat tatagtgcgt gagccagaag 660
atctccctca gggtttttcg gctttagaac cattggtaga tttgccaata ggtattaaca 720
tcactaggtt tcaaacttta cttgctttac atagaagtta tttgactcct ggtgattctt 780
cttcaggttg gacagctggt gctgcagctt attatgtggg ttatcttcaa cctaggactt 840
ttctattaaa atataatgaa aatggaacca ttacagatgc tgtagactgt gcacttgacc 900
ctctctcaga aacaaagtgt acgttgaaat ccttcactgt agaaaaagga atctatcaaa 960
cttctaactt tagagtccaa ccaacagaat ctattgttag atttcctaat attacaaact 1020
tgtgcccttt tgatgaagtt tttaacgcca ccagatttgc atctgtttat gcttggaaca 1080
ggaagagaat cagcaactgt gttgctgatt attctgtcct atataatctc gcaccatttt 1140
tcacttttaa gtgttatgga gtgtctccta ctaaattaaa tgatctctgc tttactaatg 1200
tctatgcaga ttcatttgta attagaggtg atgaagtcag acaaatcgct ccagggcaaa 1260
ctggaaatat tgctgattat aattataaat taccagatga ttttacaggc tgcgttatag 1320
cttggaattc taacaagctt gattctaagg ttagtggtaa ttataattac ctgtatagat 1380
tgtttaggaa gtctaatctc aaaccttttg agagagatat ttcaactgaa atctatcagg 1440
ccggtaacaa accttgtaat ggtgttgcag gttttaattg ttactttcct ttacgatcat 1500
atagtttccg acccacttat ggtgttggtc accaaccata cagagtagta gtactttctt 1560
ttgaacttct acatgcacca gcaactgttt gtggacctaa aaagtctact aatttggtta 1620
aaaacaaatg tgtcaatttc aacttcaatg gtttaaaagg cacaggtgtt cttactgagt 1680
ctaacaaaaa gtttctgcct ttccaacaat ttggcagaga cattgctgac actactgatg 1740
ctgtccgtga tccacagaca cttgagattc ttgacattac accatgttct tttggtggtg 1800
tcagtgttat aacaccagga acaaatactt ctaaccaggt tgctgttctt tatcagggtg 1860
ttaactgcac agaagtccct gttgctattc atgcagatca acttactcct acttggcgtg 1920
tttattctac aggttctaat gtttttcaaa cacgtgcagg ctgtttaata ggggctgaat 1980
atgtcaacaa ctcatatgag tgtgacatac ccattggtgc aggtatatgc gctagttatc 2040
agactcagac taagtctcat cggcgggcac gtagtgtagc tagtcaatcc atcattgcct 2100
acactatgtc acttggtgca gaaaattcag ttgcttactc taataactct attgccatac 2160
ccacaaattt tactattagt gttaccacag aaattctacc agtgtctatg accaagacat 2220
cagtagattg tacaatgtac atttgtggtg attcaactga atgcagcaat cttttgttgc 2280
aatatggcag tttttgtaca caattaaaac gtgctttaac tggaatagct gttgaacaag 2340
acaaaaacac ccaagaagtt tttgcacaag tcaaacaaat ttacaaaaca ccaccaatta 2400
aatattttgg tggttttaat ttttcacaaa tattaccaga tccatcaaaa ccaagcaaga 2460
ggtcatttat tgaagatcta cttttcaaca aagtgacact tgcagatgct ggcttcatca 2520
aacaatatgg tgattgcctt ggtgatattg ctgctagaga cctcatttgt gcacaaaagt 2580
ttaaaggcct tactgttttg ccacctttgc tcacagatga aatgattgct caatacactt 2640
ctgcactgtt agcgggtaca atcacttctg gttggacctt tggtgcaggt gctgcattac 2700
aaataccatt tgctatgcaa atggcttata ggtttaatgg tattggagtt acacagaatg 2760
ttctctatga gaaccaaaaa ttgattgcca accaatttaa tagtgctatt ggcaaaattc 2820
aagactcact ttcttccaca gcaagtgcac ttggaaaact tcaagatgtg gtcaaccata 2880
atgcacaagc tttaaacacg cttgttaaac aacttagctc caaatttggt gcaatttcaa 2940
gtgttttaaa tgatatcttt tcacgtcttg acaaagttga ggctgaagtg caaattgata 3000
ggttgatcac aggcagactt caaagtttgc agacatatgt gactcaacaa ttaattagag 3060
ctgcagaaat cagagcttct gctaatcttg ctgctactaa aatgtcagag tgtgtacttg 3120
gacaatcaaa aagagttgat ttttgtggaa agggctatca tcttatgtcc ttccctcagt 3180
cagcacctca tggtgtagtc ttcttgcatg tgacttatgt ccctgcacaa gaaaagaact 3240
tcacaactgc tcctgccatt tgtcatgatg gaaaagcaca ctttcctcgt gaaggtgtct 3300
ttgtttcaaa tggcacacac tggtttgtaa cacaaaggaa tttttatgaa ccacaaatca 3360
ttactacaga caacacattt gtgtctggta actgtgatgt tgtaatagga attgtcaaca 3420
acacagttta tgatcctttg caacctgaat tagattcatt caaggaggag ttagataaat 3480
attttaagaa tcatacatca ccagatgttg atttaggtga catctctggc attaatgctt 3540
cagttgtaaa cattcaaaaa gaaattgacc gcctcaatga ggttgccaag aatttaaatg 3600
aatctctcat cgatctccaa gaacttggaa agtatgagca gtatataaaa tggccatggt 3660
acatttggct aggttttata gctggcttga ttgccatagt aatggtgaca attatgcttt 3720
gctgtatgac cagttgctgt agttgtctca agggctgttg ttcttgtgga tcctgctgca 3780
aatttgatga agacgactct gagccagtgc tcaaaggagt caaattacat tacacagggc 3840
ccggttcttc tgaaaacctg ta 3862
<210> 27
<211> 7451
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atgggcagag cgcacatcgc 60
ccacagtccc cgagaagttg gggggagggg tcggcaattg aacgggtgcc tagagaaggt 120
ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg 180
ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg 240
ccgccagaac acagctgaag cttcgagggg ctcgcatctc tccttcacgc gcccgccgcc 300
ctacctgagg ccgccatcca cgccggttga gtcgcgttct gccgcctccc gcctgtggtg 360
cctcctgaac tgcgtccgcc gtctaggtaa gtttaaagct caggtcgaga ccgggccttt 420
gtccggcgct cccttggagc ctacctagac tcagccggct ctccacgctt tgcctgaccc 480
tgcttgctca actctacgtc tttgtttcgt tttctgttct gcgccgttac agatccaagc 540
tgtgaccggc gcctacacgc gtgccaccat ggtgagcaag ggcgaggagc tgttcaccgg 600
ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc 660
cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac 720
cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg 780
cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga 840
aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc 900
cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt 960
caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt 1020
ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa 1080
catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga 1140
cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga 1200
ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac 1260
tctcggcatg gacgagctgt acaagtaacc gcgctagcgc ctcgactgtg ccttctagtt 1320
gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc 1380
ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt 1440
ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gagaatagca 1500
ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct 1560
ctagggggta tcccctgaca ttgattattg actagttatt aatagtaatc aattacgggg 1620
tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg 1680
cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata 1740
gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc 1800
cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac 1860
ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg 1920
cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc 1980
aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc 2040
aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc 2100
gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct 2160
cgtttagtga accgtcagat cgcctggaga cgccatccac gctgttttga cctccataga 2220
agacaccggg accgatccag cctccggact ctagagaatt cgagctcggt acctcgcgaa 2280
tgcatctaga tatcggatcc cgggcccggt tcttctgaaa acctgtactt ccaatccaaa 2340
tcttctcacc atcaccatca ccatcaccat caccattagt gataaaccgg ttagtaatga 2400
gtttgatatc tcgacaatca acctctggat tacaaaattt gtgaaagatt gactggtatt 2460
cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat 2520
gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct 2580
ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct 2640
gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc 2700
gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg 2760
acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa gctgacgtcc 2820
tttccatggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac 2880
gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg 2940
cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc 3000
ccgcctggaa acgggggagg ctaactgaaa cacggaagga gacaataccg gaaggaaccc 3060
gcgctatgac ggcaataaaa agacagaata aaacgcacgg gtgttgggtc gtttgttcat 3120
aaacgcgggg ttcggtccca gggctggcac tctgtcgata ccccaccgag accccattgg 3180
ggccaatacg cccgcgtttc ttccttttcc ccaccccacc ccccaagttc gggtgaaggc 3240
ccagggctcg cagccaacgt cggggcggca ggccctgcca tagcagatct gcgcagctgg 3300
ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg 3360
gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc 3420
ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcggggcatc 3480
cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt 3540
gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag 3600
tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg 3660
gtctattctt ttgatttata agggattttg gggatttcgg cctattggtt aaaaaatgag 3720
ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg 3780
gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag 3840
caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc 3900
tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc 3960
ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg 4020
aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag 4080
gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat caagagacag 4140
gatgaggatc gtttcgcatg atgaccgagt acaagcccac ggtgcgcctc gccacccgcg 4200
acgacgtccc ccgggccgta cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc 4260
gccacaccgt cgacccggac cgccacatcg agcgggtcac cgagctgcaa gaactcttcc 4320
tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg 4380
cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag atcggcccgc 4440
gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa ggcctcctgg 4500
cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc 4560
accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg 4620
ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc tacgagcggc 4680
tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga 4740
cccgcaagcc cggtgcctag tgagcgggac tctggggttc gcgaaatgac cgaccaagcg 4800
acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc 4860
ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg 4920
gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat 4980
agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc 5040
aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg 5100
taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac 5160
atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca 5220
ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat 5280
taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc 5340
tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca 5400
aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca 5460
aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 5520
ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 5580
acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 5640
ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 5700
tctcaatgct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 5760
tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 5820
gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 5880
agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 5940
tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 6000
agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 6060
tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 6120
acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 6180
tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 6240
agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc 6300
tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact 6360
acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc 6420
tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt 6480
ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta 6540
agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg 6600
tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt 6660
acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc 6720
agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt 6780
actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc 6840
tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc 6900
gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 6960
ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac 7020
tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa 7080
aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt 7140
tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa 7200
tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct 7260
gacgtcgacg gatcgggaga tctcccgatc ccctatggtc gactctcagt acaatctgct 7320
ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt 7380
agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga 7440
atctgcttag g 7451
<210> 28
<211> 1040
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
tccagcctcc ggactctaga gaattcatgt ttgtttttct tgttttattg ccactagtct 60
ctagtcagtg tgttaatctt acaaccagaa ctcaattacc ccctgcatac actaattctt 120
tcacacgtgg tgtttattac cctgacaaag ttttcagatc ctcagtttta cattcaactc 180
aggacttgtt cttacctttc ttttccaatg ttacttggtt ccatgttatc tctgggacca 240
atggtactaa gaggtttgat aaccctgtcc taccatttaa tgatggtgtt tattttgctt 300
ccattgagaa gtctaacata ataagaggct ggatttttgg tactacttta gattcgaaga 360
cccagtccct acttattgtt aataacgcta ctaatgttgt tattaaagtc tgtgaatttc 420
aattttgtaa tgatccattt ttggaccaca aaaacaacaa aagttggatg gaaagtgagt 480
tcagagttta ttctagtgcg aataattgca cttttgaata tgtctctcag ccttttctta 540
tggaccttga aggaaaacag ggtaatttca aaaatcttag ggaatttgtg tttaagaata 600
ttgatggtta ttttaaaata tattctaagc acacgcctat tatagtgcgt gagccagaag 660
atctccctca gggtttttcg gctttagaac cattggtaga tttgccaata ggtattaaca 720
tcactaggtt tcaaacttta cttgctttac atagaagtta tttgactcct ggtgattctt 780
cttcaggttg gacagctggt gctgcagctt attatgtggg ttatcttcaa cctaggactt 840
ttctattaaa atataatgaa aatggaacca ttacagatgc tgtagactgt gcacttgacc 900
ctctctcaga aacaaagtgt acgttgaaat ccttcactgt agaaaaagga atctatcaaa 960
cttctaactt tagagtccaa ccaacagaat ctattgttag atttcctaat attacaaact 1020
tgtgcccttt tgatgaagtt 1040
<210> 29
<211> 1040
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
tgtgcccttt tgatgaagtt tttaacgcca ccagatttgc atctgtttat gcttggaaca 60
ggaagagaat cagcaactgt gttgctgatt attctgtcct atataatctc gcaccatttt 120
tcacttttaa gtgttatgga gtgtctccta ctaaattaaa tgatctctgc tttactaatg 180
tctatgcaga ttcatttgta attagaggtg atgaagtcag acaaatcgct ccagggcaaa 240
ctggaaatat tgctgattat aattataaat taccagatga ttttacaggc tgcgttatag 300
cttggaattc taacaagctt gattctaagg ttagtggtaa ttataattac ctgtatagat 360
tgtttaggaa gtctaatctc aaaccttttg agagagatat ttcaactgaa atctatcagg 420
ccggtaacaa accttgtaat ggtgttgcag gttttaattg ttactttcct ttacgatcat 480
atagtttccg acccacttat ggtgttggtc accaaccata cagagtagta gtactttctt 540
ttgaacttct acatgcacca gcaactgttt gtggacctaa aaagtctact aatttggtta 600
aaaacaaatg tgtcaatttc aacttcaatg gtttaaaagg cacaggtgtt cttactgagt 660
ctaacaaaaa gtttctgcct ttccaacaat ttggcagaga cattgctgac actactgatg 720
ctgtccgtga tccacagaca cttgagattc ttgacattac accatgttct tttggtggtg 780
tcagtgttat aacaccagga acaaatactt ctaaccaggt tgctgttctt tatcagggtg 840
ttaactgcac agaagtccct gttgctattc atgcagatca acttactcct acttggcgtg 900
tttattctac aggttctaat gtttttcaaa cacgtgcagg ctgtttaata ggggctgaat 960
atgtcaacaa ctcatatgag tgtgacatac ccattggtgc aggtatatgc gctagttatc 1020
agactcagac taagtctcat 1040
<210> 30
<211> 1040
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
agactcagac taagtctcat cggcgggcac gtagtgtagc tagtcaatcc atcattgcct 60
acactatgtc acttggtgca gaaaattcag ttgcttactc taataactct attgccatac 120
ccacaaattt tactattagt gttaccacag aaattctacc agtgtctatg accaagacat 180
cagtagattg tacaatgtac atttgtggtg attcaactga atgcagcaat cttttgttgc 240
aatatggcag tttttgtaca caattaaaac gtgctttaac tggaatagct gttgaacaag 300
acaaaaacac ccaagaagtt tttgcacaag tcaaacaaat ttacaaaaca ccaccaatta 360
aatattttgg tggttttaat ttttcacaaa tattaccaga tccatcaaaa ccaagcaaga 420
ggtcatttat tgaagatcta cttttcaaca aagtgacact tgcagatgct ggcttcatca 480
aacaatatgg tgattgcctt ggtgatattg ctgctagaga cctcatttgt gcacaaaagt 540
ttaaaggcct tactgttttg ccacctttgc tcacagatga aatgattgct caatacactt 600
ctgcactgtt agcgggtaca atcacttctg gttggacctt tggtgcaggt gctgcattac 660
aaataccatt tgctatgcaa atggcttata ggtttaatgg tattggagtt acacagaatg 720
ttctctatga gaaccaaaaa ttgattgcca accaatttaa tagtgctatt ggcaaaattc 780
aagactcact ttcttccaca gcaagtgcac ttggaaaact tcaagatgtg gtcaaccata 840
atgcacaagc tttaaacacg cttgttaaac aacttagctc caaatttggt gcaatttcaa 900
gtgttttaaa tgatatcttt tcacgtcttg acaaagttga ggctgaagtg caaattgata 960
ggttgatcac aggcagactt caaagtttgc agacatatgt gactcaacaa ttaattagag 1020
ctgcagaaat cagagcttct 1040
<210> 31
<211> 802
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
ctgcagaaat cagagcttct gctaatcttg ctgctactaa aatgtcagag tgtgtacttg 60
gacaatcaaa aagagttgat ttttgtggaa agggctatca tcttatgtcc ttccctcagt 120
cagcacctca tggtgtagtc ttcttgcatg tgacttatgt ccctgcacaa gaaaagaact 180
tcacaactgc tcctgccatt tgtcatgatg gaaaagcaca ctttcctcgt gaaggtgtct 240
ttgtttcaaa tggcacacac tggtttgtaa cacaaaggaa tttttatgaa ccacaaatca 300
ttactacaga caacacattt gtgtctggta actgtgatgt tgtaatagga attgtcaaca 360
acacagttta tgatcctttg caacctgaat tagattcatt caaggaggag ttagataaat 420
attttaagaa tcatacatca ccagatgttg atttaggtga catctctggc attaatgctt 480
cagttgtaaa cattcaaaaa gaaattgacc gcctcaatga ggttgccaag aatttaaatg 540
aatctctcat cgatctccaa gaacttggaa agtatgagca gtatataaaa tggccatggt 600
acatttggct aggttttata gctggcttga ttgccatagt aatggtgaca attatgcttt 660
gctgtatgac cagttgctgt agttgtctca agggctgttg ttcttgtgga tcctgctgca 720
aatttgatga agacgactct gagccagtgc tcaaaggagt caaattacat tacacagggc 780
ccggttcttc tgaaaacctg ta 802
<210> 32
<211> 355
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (99)..(99)
<223> N=Y，R，S，T，F，G，AorD；
<220>
<221> misc_feature
<222> (100)..(100)
<223> n = Y, S, T, F, G, toRT (the proportion of T is three times that of the others)
<220>
<221> misc_feature
<222> (101)..(101)
<223> n = Y, S, S, S, F or W (the proportion of S is three times that of the others)
<220>
<221> misc_feature
<222> (102)..(102)
<223> n=Y，R，S，T，F，G，A，W，D，E，K or N
<220>
<221> misc_feature
<222> (103)..(103)
<223> n=S，T，F，G，A，W，D，E，N，I，H，R，Q or L
<220>
<221> misc_feature
<222> (104)..(104)
<223> n=S，T，Y，D or E
<220>
<221> misc_feature
<222> (105)..(105)
<223> n=S，T，G，A，D，E，N，I or V
<220>
<221> misc_feature
<222> (154)..(154)
<223> n=R，S，F，G，A，W，D，E or Y
<220>
<221> misc_feature
<222> (155)..(155)
<223> n=S，T，F，G，A，W，D，E，N，H，R，Q，L or Y
<220>
<221> misc_feature
<222> (156)..(156)
<223> n=S，T，F，G，A，W，D，E，N，H，Q or P
<220>
<221> misc_feature
<222> (157)..(157)
<223> n=G，S，T，N or D
<220>
<221> misc_feature
<222> (158)..(158)
<223> n=S，T，F，G，A，Y，D，E，N，I，H，R，Q，L，P，V，W，K or M
<220>
<221> misc_feature
<222> (159)..(159)
<223> n=S，T，F，G，A，Y，D，E，N，I，H，R，Q，L，P，V，W or K
<220>
<221> misc_feature
<222> (160)..(160)
<223> n=S，T，F，G，A，Y，D，E，N，I，H，R，Q，L，P or V
<220>
<221> misc_feature
<222> (278)..(293)
<223> n=S，T，F，G，A，Y，D，E，N，I，H，R，Q，L，P，V，W，K or M
<400> 32
acaagagaga agctgaagct caagttcaac tggttgaatc tggcggcggt ctggttcaac 60
cgggcggtag tctgcgtctg agttgcgcag catctggtnn nnnnnatggg ttggtttcgt 120
caagcaccgg gtcaaggtct ggaagcagtt gcannnnnnn tactacgcgg atagcgtcaa 180
aggtcgcttt accatcagcc gcgataacag caaaaacacc ctgtacctgc agatgaattc 240
tctgcgcgca gaagataccg cggtttatta ttgcgcgnnn nnnnnnnnnn nnntattggg 300
gtcaaggtac gctggttacc gttagtagtg aattcggtaa gcctatccct aaccc 355
<210> 33
<211> 8165
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 60
catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 120
caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 180
ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt 240
aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 300
gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 360
cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 420
ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta 480
tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 540
tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg 600
cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag 660
tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc 720
tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact 780
tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt 840
cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta 900
ccatctggcc ccagtgctgc aatgataccg cgactcccac gctcaccggc tccagattta 960
tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc 1020
gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat 1080
agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt 1140
atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg 1200
tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca 1260
gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta 1320
agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg 1380
cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact 1440
ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg 1500
ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt 1560
actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga 1620
ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc 1680
atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa 1740
caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgaacg aagcatctgt 1800
gcttcatttt gtagaacaaa aatgcaacgc gagagcgcta atttttcaaa caaagaatct 1860
gagctgcatt tttacagaac agaaatgcaa cgcgaaagcg ctattttacc aacgaagaat 1920
ctgtgcttca tttttgtaaa acaaaaatgc aacgcgacga gagcgctaat ttttcaaaca 1980
aagaatctga gctgcatttt tacagaacag aaatgcaacg cgagagcgct attttaccaa 2040
caaagaatct atacttcttt tttgttctac aaaaatgcat cccgagagcg ctatttttct 2100
aacaaagcat cttagattac tttttttctc ctttgtgcgc tctataatgc agtctcttga 2160
taactttttg cactgtaggt ccgttaaggt tagaagaagg ctactttggt gtctattttc 2220
tcttccataa aaaaagcctg actccacttc ccgcgtttac tgattactag cgaagctgcg 2280
ggtgcatttt ttcaagataa aggcatcccc gattatattc tataccgatg tggattgcgc 2340
atactttgtg aacagaaagt gatagcgttg atgattcttc attggtcaga aaattatgaa 2400
cggtttcttc tattttgtct ctatatacta cgtataggaa atgtttacat tttcgtattg 2460
ttttcgattc actctatgaa tagttcttac tacaattttt ttgtctaaag agtaatacta 2520
gagataaaca taaaaaatgt agaggtcgag tttagatgca agttcaagga gcgaaaggtg 2580
gatgggtagg ttatataggg atatagcaca gagatatata gcaaagagat acttttgagc 2640
aatgtttgtg gaagcggtat tcgcaatgat gtgctgcaag gcgattaagt tgggtaacgc 2700
cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgccgcaaat taaagccttc 2760
gagcgtccca aaaccttctc aagcaaggtt ttcagtataa tgttacatgc gtacacgcgt 2820
ctgtacagaa aaaaaagaaa aatttgaaat ataaataacg ttcttaatac taacataact 2880
ataaaaaaat aaatagggac ctagacttca ggttgtctaa ctccttcctt ttcggttaga 2940
gcggatgtgg ggggagggcg tgaatgtaag cgtgacataa ctaattacat gactttaact 3000
gaaaattaca ttgcaagcaa ctgccatgat ggcgaagacc aactttccta atggcaaacg 3060
cgtttgcgat ccactgccca tatattggct aattgttacc attgatgaag tttggtattg 3120
tatggagatt gaagtagtgg aaacttctgt agtggtaaca gatgtttgta gagaagaggg 3180
agcagatgat gatgagagga aaatagaaga taccgtcgga gtcaatgatg aagattcaat 3240
agtcgattcc acagaggtag atggtgtaga gatagatgtt gcggaaagtg atgatgtaga 3300
ggcagatatt gtagcaatag ctgtggaaga agtgggttgt aaaatctctg atgttgaaga 3360
aatttgaagt gtggtaactg gtgactgttc gacacttagt gatgatacag aactgaatag 3420
caaaatagta ctgtcagagg ataatgtagc agacaaggag gaggatgaaa ggaccgatgt 3480
cgataataag tgtgatgatt gttcttgaga ttctgagtca atgcttgatg tactcgtggc 3540
ttcacttaca ctagtcgaaa tggttgaggt acctgaagat gttacatgat acttaccggt 3600
agcgtgctta ttgaaatctt catccgaagt tgtcattgaa cttacccaac tctttgtgga 3660
tgtggtctgc tgtgttacag aacttgatga agctgtttgc acggaagaat acgagtgtga 3720
cgtttcagca catgtggagc atgcatatga ggattcaacc gttgaggtgc ttacagaagt 3780
tgcgacgttt ttagtggaca tttttataga agatgtaatc aaacttgttg tctgcgaggg 3840
catgcaagac tcgtgagtgc agactgtagc gtctgttgta accgtttcat gcatactggt 3900
taatgtgcta gtctcagatt cagagctata aggacaccat gtcgtataca ttgtagtgac 3960
tccactaaca gtggtgaaat atgtagacat ggaaaatgat gggatagtag tagaaattga 4020
ggatttggtg atatattcag acgaactttg tgaactaaca gttgtttcaa cagttgaaga 4080
agtcatagaa ggagttgcaa catttgacga agtcgaaggg acggagtaaa caggtgtgga 4140
tgggctgtat aatgacacag acgttgatga agatgctata gaggagccaa gggatgaagt 4200
tgaatcagta aaagtagagc taatagatgt tgaacttgga gaagtggaag ccaaagttgg 4260
agatgaagaa gtcaaacttg gggatgtaga tgttgaatat gaagaagtgg aagtggagct 4320
tgcagaagta gatttagaac ttggagatgt agatgttgaa cttgatgagg tagatgtaga 4380
gcttggagat gtagatgtcg aacttgatga ggtagatgta gaactttggg atgtagatgt 4440
agaacttgaa gatgtggatg tcaaacttga agatgtggat gtcgaacttg gagatgtaga 4500
tgtcgaactt gatgaggtag atgtcgaact tgatgaggta gatgtcgaac ttgatgaggt 4560
agatgtagag cttggagatg tagatgtaga gcttggagat gtagatgtcg aacttaggga 4620
ggtagtagtt ggattggacg atgttgtact cgaaagtgta gaggtgacag gtgagattat 4680
agaggcactt gatggttcaa tggcgctgga tgataccacg gttgtagttc ctacttcgga 4740
taatgagctg atagcggttg tgtgacaagt agggcaaata tatgaagtga acttggaagt 4800
cacagataag gtggtggttg gcaacgtcga tgacgaagga catgcctcat gggagcagac 4860
ttctgtagat aatgtcaaag tactaaatct ggatagggta gtagcgtacg atattgaagg 4920
acttatttcg gcagcagatg aagtggatac cgtcaatgga caccaagtcg tatacaatgt 4980
cgtagtgcca gcttgaacga tagtggatgt ggagactagc gcgggtgaaa ctgtagttgt 5040
aacaacccca tttgcatcgt ttgtcttggt tatcgtcact agaatcgttt ctggatcaga 5100
tgccaaggca atattagtta atcccaacaa tattgtgaac aggtaggtaa aatgagcgaa 5160
agataatgtc atggtggcgg cgtcgactta tattgaattt tcaaaaattc ttactttttt 5220
tttggatgga cgcaaagaag tttaataatc atattacatg gcattaccac catatacata 5280
tccatataca tatccatatc taatcttact tatatgttgt ggaaatgtaa agagccccat 5340
tatcttagcc taaaaaaacc ttctctttgg aactttcagt aatacgctta actgctcatt 5400
gctatattga agtacggatt agaagccgcc gagcgggtga cagccctccg aaggaagact 5460
ctcctccgtg cgtcctcgtc ttcaccggtc gcgttcctga aacgcagatg tgcctcgcgc 5520
cgcactgctc cgaacaataa agattctaca atactagctt ttatggttat gaagaggaaa 5580
aattggcagt aacctggccc cacaaacctt caaatgaacg aatcaaatta acaaccatag 5640
gatgataatg cgattagttt tttagcctta tttctggggt aattaatcag cgaagcgatg 5700
atttttgatc tattaacaga tatataaatg caaaaactgc ataaccactt taactaatac 5760
tttcaacatt ttcggtttgt attacttctt attcaaatgt aataaaagta tcaacaaaaa 5820
attgttaata tacctctata ctttaacgtc aaggagaaaa aaccccggat cggactacta 5880
gcagctgtaa tacgactcac tatagggaat attaagctaa ttccctactt catacatttt 5940
caattaagat gagattccca tctatcttca ccgctgtttt atttgctgct tcttctgcct 6000
tggccgctcc agctaacaca actactgaag atgaaaccgc tcaaatccca gctgaagccg 6060
tcattgacta ctcagatttg gaaggtgact tcgatgctgc tgctttgcca ctctctaact 6120
ccaccaacaa cggtttatcc tctactaata ccaccattgc ttctattgcc gctaaggaag 6180
aaggtgttca attggacaag agagaagctg aagctgaatt ctattggggt caaggtacgc 6240
tggttaccgt tagtagtgaa ttcggtaagc ctatccctaa ccctctcctc ggtctcgatt 6300
ctacgggtgg tggtggttct ggtggtggtg gttctggtgg tggtggttct caggaactga 6360
caactatatg cgagcaaatc ccctcaccaa ctttagaatc gacgccgtac tctttgtcaa 6420
cgactactat tttggccaac gggaaggcaa tgcaaggagt ttttgaatat tacaaatcag 6480
taacgtttgt cagtaattgc ggttctcacc cctcaacgac tagcaaaggc agccccataa 6540
acacacagta tgtttttaag cttctgcagg ctagttgata ataggtttaa acccgctgat 6600
ctgataacaa cagtgtagat gtaacaaaat cgactttgtt cccactgtac ttttagctcg 6660
tacaaaatac aatatacttt tcatttctcc gtaaacaaca tgttttccca tgtaatatcc 6720
ttttctattt ttcgttccgt taccaacttt acacatactt tatatagcta ttcacttcta 6780
tacactaaaa aactaagaca attttaattt tgctgcctgc catatttcaa tttgttataa 6840
attcctataa tttatcctat tagtagctaa aaaaagatga atgtgaatcg aatcctaaga 6900
gaattggcaa gtgcacaaac aatacttaaa taaatactac tcagtaataa ctgcagaggc 6960
ctgcatgcaa gcttggctcg agcatggtca tagctgtttc ctgtgtgaaa ttgttatccg 7020
ctcacaattc cacacaacat acgagccgga agcataacta gtacactcta tattttttta 7080
tgcctcggta atgattttca tttttttttt tccacctagc ggatgactct ttttttttct 7140
tagcgattgg cattatcaca taatgaatta tacattatat aaagtaatgt gatttcttcg 7200
aagaatatac taaaaaatga gcaggcaaga taaacgaagg caaagatgac agagcagaaa 7260
gccctagtaa agcgtattac aaatgaaacc aagattcaga ttgcgatctc tttaaagggt 7320
ggtcccctag cgatagagca ctcgatcttc ccagaaaaag aggcagaagc agtagcagaa 7380
caggccacac aatcgcaagt gattaacgtc cacacaggta tagggtttct ggaccatatg 7440
atacatgctc tggccaagca ttccggctgg tcgctaatcg ttgagtgcat tggtgactta 7500
cacatagacg accatcacac cactgaagac tgcgggattg ctctcggtca agcttttaaa 7560
gaggccctac tggcgcgtgg agtaaaaagg tttggatcag gatttgcgcc tttggatgag 7620
gcactttcca gagcggtggt agatctttcg aacaggccgt acgcagttgt cgaacttggt 7680
ttgcaaaggg agaaagtagg agatctctct tgcgagatga tcccgcattt tcttgaaagc 7740
tttgcagagg ctagcagaat taccctccac gttgattgtc tgcgaggcaa gaatgatcat 7800
caccgtagtg agagtgcgtt caaggctctt gcggttgcca taagagaagc cacctcgccc 7860
aatggtacca acgatgttcc ctccaccaaa ggtgttctta tgtagtctct tctcgagtca 7920
tgtaattagt tatgtcacgc ttacattcac gccctccccc cacatccgct ctaaccgaaa 7980
aggaaggagt tagacaacct gaagtctagg tccctattta tttttttata gttatgttag 8040
tattaagaac gttatttata tttcaaattt ttcttttttt tctgtacaga cgcgtgtacg 8100
catgtaacat tatactgaaa accttgcttg agaaggtttt gggacgctcg aaggctttaa 8160
tttgc 8165

Claims (9)

1. A yeast cell homologous recombination enzyme system is characterized by comprising yeast exonucleaseExoI. Yeast recombinase RAD51, yeast recombinase RAD52, and yeast single-stranded DNA binding protein RPA;
the yeast exonucleaseExoI. The yeast recombinase RAD51, the yeast recombinase RAD52 and the yeast single-stranded DNA binding protein RPA are obtained by codon optimization of a prokaryotic expression system;
optimized yeast exonucleaseExoThe nucleotide sequence of the I is shown as SEQ ID NO. 1;
the nucleotide sequence of the optimized yeast recombinase RAD51 is shown as SEQ ID NO. 2;
the nucleotide sequence of the optimized yeast recombinase RAD52 is shown as SEQ ID NO. 3;
the nucleotide sequence of the optimized yeast single-stranded DNA binding protein RPA is shown in SEQ ID NO. 4.
2. The homologous recombination enzyme system according to claim 1, wherein said homologous recombination enzyme system further comprises E.coli DNA polymerase I, E.coli DNA ligase, and ATP regenerating enzyme;
the ATP regenerating enzyme comprises one or more of the following kinases: creatine kinase, pyruvate kinase, acetate kinase, polyphosphate kinase, and nucleoside diphosphate kinase.
3. The homologous recombinant enzyme system according to claim 2, wherein the yeast exonucleaseExoI. The activity ratio of yeast recombinase RAD51, yeast recombinase RAD52, yeast single-stranded DNA binding protein RPA, escherichia coli DNA polymerase I, escherichia coli DNA ligase and ATP regenerating enzyme is (0.001 to 0.1): (10 to 100): (10 to 100): (10 to 100): (0.1 to 1): (0.1 to 1): (1 to 100).
4. An in vitro DNA assembly reagent comprising the homologous recombination enzyme system of any one of claims 1 to 3 and 2.6 Xbuffer;
the 2.6 multiplied buffer solution is 10 to 200mM Tris base buffer solution containing 1 to 50mM magnesium ion source, 5 to 50mM alkali metal ion source, 2 to 10 mass percent of polyethylene glycol, 1 to 10mM NAD, 2 to 20mM dithiothreitol, 1 to 10mM ATP regeneration enzyme substrate and 1 to 10mM deoxyribonucleoside triphosphate, and the pH value is 7.0 to 8.0.
5. The in vitro DNA assembly reagent of claim 4, wherein the in vitro DNA assembly reagent comprises yeast exonucleaseExoThe concentration of I is 1-10U/ml, the concentration of yeast recombinase RAD51 is 10-100nM, the concentration of yeast recombinase RAD52 is 10-100nM, the concentration of yeast single-stranded DNA binding protein RPA is 100-500nM, the concentration of escherichia coli DNA polymerase I is 10-100U/ml, the concentration of escherichia coli DNA ligase is 1000-5000U/ml, and the concentration of ATP regenerative enzyme is 1-10 mM.
6. Use of the homologous recombinase system of any one of claims 1 to 3 or the reagent for in vitro assembly of DNA fragments of claim 4 or 5 for in vitro assembly of DNA fragments.
7. The use of claim 6, wherein the use comprises assembly of two or more DNA fragments;
the DNA fragment comprises a single-stranded or double-stranded DNA fragment;
the in vitro assembly of the DNA fragments comprises one or more of the following applications: the assembly of target genes of animal origin, the assembly of target genes or genomes of viral origin and the assembly of DNA fragments of antibody libraries.
8. A method for in vitro assembly of DNA fragments, comprising the steps of:
mixing and reacting the DNA fragments to be assembled, the linear vector and the DNA in-vitro assembly reagent according to claim 4 or 5 to obtain linear or circular DNA long fragments;
the DNA fragment to be assembled contains a base homologous or complementary region with the length of 10 to 200bp.
9. The method of claim 8, wherein after the mixing reaction, the reaction product is introduced into a prokaryotic cell to complete the amplification of the DNA fragment, resulting in a double-stranded DNA molecule.
CN202210436102.2A 2022-04-25 2022-04-25 Yeast cell homologous recombination enzyme system, DNA in-vitro assembly reagent and application thereof Active CN114717207B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210436102.2A CN114717207B (en) 2022-04-25 2022-04-25 Yeast cell homologous recombination enzyme system, DNA in-vitro assembly reagent and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210436102.2A CN114717207B (en) 2022-04-25 2022-04-25 Yeast cell homologous recombination enzyme system, DNA in-vitro assembly reagent and application thereof

Publications (2)

Publication Number Publication Date
CN114717207A CN114717207A (en) 2022-07-08
CN114717207B true CN114717207B (en) 2023-03-07

Family

ID=82246465

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210436102.2A Active CN114717207B (en) 2022-04-25 2022-04-25 Yeast cell homologous recombination enzyme system, DNA in-vitro assembly reagent and application thereof

Country Status (1)

Country Link
CN (1) CN114717207B (en)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107699550A (en) * 2017-08-11 2018-02-16 山东大学 One group of Burkholderia homologous recombination enzyme and its expression vector and application
CN110691848A (en) * 2017-04-10 2020-01-14 马克斯·普朗克科学促进学会 Compounds for improving efficiency of genome editing
CN111630175A (en) * 2018-01-22 2020-09-04 庆尚大学校产学协力团 Method for improving gene editing efficiency in plants based on homologous recombination
WO2021178898A1 (en) * 2020-03-05 2021-09-10 Flagship Pioneering Innovations Vi, Llc Host defense suppressing methods and compositions for modulating a genome

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110691848A (en) * 2017-04-10 2020-01-14 马克斯·普朗克科学促进学会 Compounds for improving efficiency of genome editing
CN107699550A (en) * 2017-08-11 2018-02-16 山东大学 One group of Burkholderia homologous recombination enzyme and its expression vector and application
CN111630175A (en) * 2018-01-22 2020-09-04 庆尚大学校产学协力团 Method for improving gene editing efficiency in plants based on homologous recombination
WO2021178898A1 (en) * 2020-03-05 2021-09-10 Flagship Pioneering Innovations Vi, Llc Host defense suppressing methods and compositions for modulating a genome

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Pathways and assays for DNA double-strand break repair by homologous recombination;Jinbao Li等;《Acta Biochim Biophys Sin (Shanghai)》;20190906;第51卷(第9期);第879-889页 *
酵母细胞中构建HL-60细胞的酵母双杂交cDNA文库;黄功华等;《广东医学院学报》;20041009(第1期);第1-3页 *

Also Published As

Publication number Publication date
CN114717207A (en) 2022-07-08

Similar Documents

Publication Publication Date Title
US20020025561A1 (en) Vectors for gene-self-assembly
CN113481327B (en) Novel coronavirus ORF1ab gene detection method based on RAA amplification and CRISPR-Cas12a
CN113549618B (en) SARS-CoV-2 nucleic acid detection method based on RAA amplification and CRISPR-Cas13a system
CN108395996B (en) Classical swine fever virus subunit vaccine and preparation method and application thereof
CN108531471B (en) Long gene synthesis method
CN109609579B (en) Genetically engineered bacterium for producing beta-carotene and construction method thereof
CN108285886A (en) The method that recombined bacillus subtilis resting cell produces N-acetyl-neuraminate
CN114901816A (en) Lipase-modified strains
CN108531495A (en) A kind of light-operated gene expression system and its application
CN114717207B (en) Yeast cell homologous recombination enzyme system, DNA in-vitro assembly reagent and application thereof
CN113604505A (en) pSFV-p32 virus-like particle and preparation method and application thereof
CN109652352B (en) Genetically engineered bacterium for efficiently immobilizing enterococcus faecium glutamate decarboxylase and immobilization method
CN114292864B (en) Bacillus bailii mutant strain with high surfactant yield, construction method and application thereof
CN111979134B (en) Construction and application of recombinant saccharomyces cerevisiae for synthesizing carminic acid
CN113584223B (en) Identification method of D614G mutation in SARS-CoV-2 based on CRISPR-Cas12a
CN112626116B (en) Method for site-specific integration of large-fragment exogenous DNA
CN111321163B (en) Construction and application of bacillus subtilis linear plasmid system
CN109456930A (en) One plant for rapidly purifying the engineering bacteria and purification process of the preparation pure enzyme of enterococcus faecium glutamate decarboxylase native enzyme
CN107661496A (en) A kind of pig parvoviral immune composition and preparation method and application
CN112322706A (en) Specific human gene fragment, primer probe and application thereof
CN111378718A (en) Construction method of gene sequencing library
CN114540345B (en) Label fluorescent probe with hairpin structure and fluorescent detection method
CN114214347B (en) Plasmid system for tracing liver precursor cells and application
CN110607380B (en) Mulberry phytoplasma ltrA gene and application thereof in molecular detection of mulberry phytoplasma
CN114214346B (en) Plasmid system for targeting liver precursor cells and application

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant