CN109943584B - Recombinant vector and recombinant yeast strain for producing sabinene, and construction method and application thereof - Google Patents

Abstract

The invention relates to the technical field of genetic engineering, and discloses a recombinant vector and a recombinant yeast strain for producing sabinene, as well as a construction method and application thereof. The recombinant plasmid of the invention comprises the sequence ERG20 of ERG20 encoding the double mutation of F96W and N127W^wwAnd a sabinene synthase coding sequence derived from Salvia pomifera and/or Citrus jamshiri. The invention selects the proper sequence ERG20 of the double mutated ERG20^wwAnd the exogenous sabinene synthase gene is matched and combined to construct a recombinant vector capable of producing sabinene and the recombinant vector is transformed into a yeast strain, so that the capability of the strain for producing sabinene can be obviously improved, and the yield of the sabinene produced by the strain can be further improved by continuously optimizing and transforming on the basis.

Description

Recombinant vector and recombinant yeast strain for producing sabinene, and construction method and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a recombinant vector and a recombinant yeast strain for producing sabinene, and a construction method and application thereof.

Background

Sabinene (Sabinene, 1-isopropyl-4-methylenebicyclo [3.1.0 ]]Hexane, C₁₀H₁₆Molecular weight 136.23) is a naturally occurring bicyclic unsaturated monoterpene. Sabinene has long been used as perfume additive and fine chemical, and has excellent anti-inflammatory and antifungal activity applied in pharmaceutical industry. Researchers are currently developing it as a biofuel: the sabinene has a chemical structure more compact than that of common hydrocarbon, so that it has very high density and combustion heat, and can be used as an important component of jet aircraft mixed fuel.

At present, the main source of sabinene is extracted from plants, but the content of sabinene extracted from the plants is very low, so that the industrial requirement is difficult to meet, and a large amount of natural resources are consumed. Therefore, microbial synthesis is considered to be the most promising production method with low cost, high yield and product safety. In the research of microbial synthesis of sabinene, the hosts used were mainly Escherichia coli and Saccharomyces cerevisiae. In 2014, Chinese scientists Ximo et al constructed a heterologous pathway for producing sabinene in Escherichia coli: firstly, escherichia coli has a natural MEP pathway and can provide a rich precursor substance GPP for the synthesis of sabinene. In addition, the natural gene IspA (encoding farnesyl diphosphate synthase) in E.coli was overexpressed to increase the content of GPP; the mevalonate pathway present in s.cerevisiae was introduced to further enrich the precursor material. Finally, screening and over-expressing GPP synthetase, introducing sabinene synthase to obtain high-yield sabinene Escherichia coli strain, wherein the shake flask fermentation can reach 82.18mg/L, and the fermentation tank yield can reach 2.65 g/L; in 2017, the strain obtains the Escherichia coli with the shake flask fermentation yield of 150mg/L by a substrate MVA feeding mode. In 2017, Bowie et al designed an in vitro synthesis system of monoterpene, which introduced 27 enzymes for converting glucose into monoterpene and synthesized terpene using two cofactors NAD (P) H and ATP, finally synthesized 15.9g/L of sabinene.

The saccharomyces cerevisiae, as a well-known safe mode microorganism, has clear genetic background and simple gene operation, can be used for large-scale fermentation production, has stronger protein expression and a post-translational modification system and a complete endomembrane system, and is more suitable for the expression of the P450 protein. Compared with Escherichia coli, it has high content of vitamins and proteins, and can be used as edible medicinal and feed yeast; compared with in vitro synthesis, the method has the advantages of simple system, low cost and high stability of enzyme in the system. Therefore, it is very important to realize the high yield of sabinene in saccharomyces cerevisiae. However, few reports have been published up to now on the synthesis of sabinene using Saccharomyces cerevisiae.

Disclosure of Invention

In view of the above, the present invention aims to provide a recombinant vector and a recombinant yeast strain for producing sabinene, and a construction method and application thereof, so that the ability of the strain to produce sabinene can be significantly improved by transforming the recombinant vector into the yeast strain.

In order to achieve the above purpose, the invention provides the following technical scheme:

a recombinant vector for the production of sabinene comprising the sequence ERG20 encoding the F96W and N127W double mutant ERG20^wwAnd a sabinene synthase coding sequence derived from Salvia pomifera and/or Citrus jamshiri.

Aiming at the existing sabinene-producing recombinant yeast strain, the invention selects a suitable sequence ERG20 of ERG20 with double mutation^wwAnd the exogenous sabinene synthase gene is matched and combined to construct a recombinant vector capable of producing sabinene, and the recombinant vector is transformed into a yeast strain, so that the capability of the strain for producing sabinene can be obviously improved.

Preferably, the ERG20^wwAnd a sabinene synthase encoding sequence is inserted into the recombinant vector according to any one or more of the following forms of gene modules:

(1) terminator-ERG 20^ww-bidirectional promoter-sabinene synthase coding sequence-terminator;

(2) terminator-sabinene synthase coding sequence-bidirectional promoter-ERG 20^ww-a terminator;

(3) promoter-ERG 20^ww-terminator-promoter-sabinene synthase coding sequence-terminator;

(4) promoter-sabinene synthase coding sequence-terminator-promoter-ERG 20^ww-a terminator;

(5) promoter-ERG 20^ww-sabinene synthase coding sequence-terminator;

(6) promoter-sabinene synthase coding sequence-ERG 20^ww-a terminator;

(7) terminator-ERG 20^ww-sabinene synthase coding sequence-reverse promoter;

(8) terminator-sabinene synthase coding sequence-ERG 20^ww-a reverse promoter;

(9) terminator-bidirectional promoter-ERG 20^wwAnd a terminator, a fusion protein coding sequence of sabinene synthase;

(10) terminator-ERG 20^wwAnd a fusion protein coding sequence of sabinene synthase-bidirectional promoter-terminator;

(11) promoter-ERG 20^wwAnd sabinene synthaseThe fusion protein coding sequence of (1) -terminator;

(12) terminator-ERG 20^wwAnd a fusion protein coding sequence of sabinene synthase, a reverse promoter;

wherein, the bidirectional promoter is preferably GAL1& GAL10 promoter; the terminator is preferably selected from the ADH1t terminator and the TDH2t terminator, and in general, the plurality of terminators and promoters in each gene module are different from each other.

In a particular embodiment of the invention, the invention employs ADH1t-ERG20^ww-GAL1&A gene module in the form of GAL 10-sabinene synthase coding sequence-TDH 2 t; the YGG415 plasmid is taken as a basic plasmid of the recombinant vector, the map of the YGG415 plasmid is shown in figure 1, and the whole gene sequence of the plasmid is shown in SEQ ID NO: 1;

the invention selects 7 sabinene synthases from different sources, prepares different recombinant vectors under the same condition, and the sources comprise Salvia pomifera, Salvia officinalis, Picea silchensis, Thuja plicata, Murraya koeniii, Citrus jambhiri and Litsea cubeba which are abbreviated as SpSabS1, SoSabS1, PsSabS1, TpSABS1, MkSabS1, CjSabS1 and LcSabS1 in sequence; the result of detecting sabinene by shaking flask fermentation of the recombinant yeast strains transformed with different recombinant vectors and the blank yeast strains shows that the strains transformed with the recombinant vectors containing SpSabS1 and CjSabS1 can produce sabinene, and the other strains do not produce sabinene, which shows that the recombinant vector can obviously improve the sabinene production capacity of the strains.

In addition, the invention compares the coding sequences of different truncated sabinene synthases, the truncation modes are a truncated sabinene synthase coding sequence with 1-43 bit amino acid residue at the N end of the sabinene synthase removed, a truncated sabinene synthase coding sequence with 1-34 bit amino acid residue at the N end of the sabinene synthase removed, a truncated sabinene synthase coding sequence with 1-52 bit amino acid residue at the N end of the sabinene synthase removed, and a truncated sabinene synthase coding sequence with 423-449 bit amino acid residue removed, different recombinant vectors are prepared by each truncated sabinene synthase coding sequence under the same other conditions, recombinant yeast strains for transforming different recombinant vectors and recombinant yeast strains containing complete sabinene synthase coding sequences are converted to detect sabinene by shake flask fermentation, and the result shows that compared with the recombinant yeast strains containing complete sabinene synthase coding sequences, the yield of sabinene in the transformed recombinant yeast strain containing the coding sequence of truncated sabinene synthase with the amino acid residue at position 1-34 of N-terminal of complete sabinene synthase removed is increased by 5 times, and the rest truncation modes have no sabinene output.

According to the technical effects, the sabinene synthase coding sequence of the present invention may be either a complete sabinene synthase coding sequence or a truncated sabinene synthase coding sequence in which 1-34 amino acid residues from the N-terminus of the sabinene synthase are removed.

Preferably, the ERG20^wwThe fusion protein of the sabinene synthase is subjected to forward fusion or reverse fusion by a rigid linker or a flexible linker; wherein the rigid linker amino acid sequence is GGGGS, and the flexible linker amino acid sequence is PAP; the forward fusion is ERG20^ww-rigid/flexible linker coding sequence-sabinene synthase coding sequence, said reverse fusion being sabinene synthase coding sequence-rigid/flexible linker coding sequence-ERG 20^ww；

Preferably, the recombinant vector of the invention can additionally express 1 part or more than 2 parts of ERG20^wwI.e., the recombinant vector further comprises 1 or more than 2 copies of additional ERG20^ww. For simplicity, the 1 or more than 2 copies of additional ERG20^wwThe insertion position is between the promoter and the terminator in the 12 gene modules, and the insertion mode is realized by designing the same enzyme cutting site. More specifically, the 1 or more than 2 copies of the additional ERG20^wwInserted between the promoter and the terminator in the aforementioned gene module of (9) or (10).

According to the sabinene production effect of the recombinant vector transformed into a yeast strain, the invention provides an application of the recombinant vector in preparation of a recombinant yeast strain for producing sabinene; preferably, the yeast strain is preferably a saccharomyces cerevisiae strain, more preferably a saccharomyces cerevisiae strain of the cen.pk series or a saccharomyces cerevisiae strain of the BY series.

According to the application of the recombinant vector, the invention also correspondingly provides a recombinant yeast strain for producing sabinene, which is transformed with the yeast strain of the recombinant vector. On this basis, the promoter of the endogenous ERG20 of the yeast strain can be replaced by a weak promoter, which means a weak promoter relative to the original promoter of the endogenous ERG20 of the yeast strain. In a specific embodiment of the invention, the weak promoter is the HXT1p promoter.

Further improvements to the recombinant yeast strain also include transformation with a vector expressing a heterologous efflux protein. Wherein the heterologous efflux protein is from the ascomycete Grosmania clavigera and/or an efflux protein from Yarrowia lipolytica; in a specific embodiment of the invention, the vector for expressing the heterologous efflux protein is based on PRS424, and is embedded with a promoter-efflux protein coding sequence-terminator gene module; more specifically, the promoter-efflux protein coding sequence-terminator gene module is GAL 7-efflux protein coding sequence-GPDt; in a specific embodiment of the invention, the coding sequence of the efflux protein of the pathogenic bacterium Grosmania clavigera from pine is shown as SEQ ID NO. 2, and is abbreviated as SC-GcABCG 1; the coding sequence of the efflux protein from Yarrowia lipolytica is shown in SEQ ID NO 3 or 4, and is abbreviated as SC-YL-ABC2 and SC-YL-ABC 3;

meanwhile, based on the result of a sabinene shake flask fermentation test of the recombinant vector, the invention also provides an application of the recombinant yeast strain in sabinene production. The specific method for producing sabinene comprises the following steps:

step 1, inoculating the recombinant yeast strain into a culture medium for activation to prepare a seed solution;

and 2, inoculating the seed solution into a culture medium, adding isopropyl myristate to perform two-phase culture, and collecting an upper organic phase of the culture medium after culture to separate sabinene.

Wherein the medium contains 40g/L glucose, 6.7g/LYNB, 2g/Ldrop-out leucine-depleted amino acid mixture (SC-leu medium).

In a specific embodiment of the present invention, the step 1 is:

inoculating a single colony of the recombinant yeast strain into 3mL of SC-leu culture medium, and culturing at 30 ℃ and 220rpm for 24h to obtain a primary seed solution; transferring the primary seeds to 5mL of SC-leu culture medium at an initial OD600 of 0.2, and culturing at 30 ℃ and 220rpm for 12-18 h to obtain secondary seed liquid;

in a specific embodiment of the present invention, the step 2 is:

the secondary seed liquid was transferred to 50mL of SC-leu medium at an initial OD600 of 0.1, and two-phase culture was carried out by adding 20% isopropyl myristate (220rpm, 30 ℃ c., 96 hours).

In addition, the invention also provides a construction method of the recombinant vector, firstly constructing an expression cassette containing a promoter and a terminator, designing enzyme cutting sites at two ends of the expression cassette and between the promoter and the terminator, and inoculating the expression cassette into the vector by enzyme cutting the enzyme cutting sites at two ends of the expression cassette and the vector with the same enzyme cutting site;

respectively at ERG20^wwDesigning the same enzyme cutting site as that between the promoter and terminator at both ends of the coding sequence of sabinene synthase, or constructing ERG20^wwAnd the coding sequence of a fusion protein of sabinene synthase, in ERG20^wwAnd designing enzyme cutting sites which are the same as the enzyme cutting sites between the promoter and the terminator at two ends of the sabinene synthase coding sequence, and inoculating the enzyme cutting sites into an expression box in an enzyme cutting mode so as to connect the enzyme cutting sites into a vector to obtain the recombinant vector.

Preferably, a first enzyme cutting site-terminator-second enzyme cutting site-bidirectional promoter-third enzyme cutting site-terminator-first enzyme cutting site, first enzyme cutting site-promoter-second enzyme cutting site-terminator-promoter-third enzyme cutting site-terminator-first enzyme cutting site, first enzyme cutting site-promoter-second enzyme cutting site-third enzyme cutting site-terminator-first enzyme cutting site or first enzyme cutting site-terminator-second enzyme cutting site-third enzyme cutting site-reverse promoter-first enzyme cutting site expression cassette is constructed;

the expression cassette is accessed into the vector through enzyme digestion of two ends of the expression cassette and a first enzyme digestion site in the vector;

at ERG20^wwDesigning second enzyme cutting site at both ends, designing third enzyme cutting site at both ends of sabinene synthase coding sequence, or designing the second enzyme cutting site at both ends of ERG20^wwDesigning third enzyme cutting sites at two ends, designing second enzyme cutting sites at two ends of the sabinene synthase coding sequence, and inoculating the second enzyme cutting sites into an expression box in an enzyme cutting mode so as to connect the second enzyme cutting sites into a vector to obtain the recombinant vector.

Any suitable enzyme cutting site can be selected from the first to the third enzyme cutting sites, and the first enzyme cutting site is determined according to a vector to be accessed; in a specific embodiment of the present invention, the second enzyme cutting site and the third enzyme cutting site are both selected from a BsmBI enzyme cutting site and a BsaI enzyme cutting site, and the second enzyme cutting site or the third enzyme cutting site is in a back-to-back mode, that is, two second enzyme cutting sites or two third enzyme cutting sites are directly connected in a back-to-back manner, and a schematic diagram can be shown in fig. 2 by taking the BsmBI enzyme cutting site and the BsaI enzyme cutting site as an example. FIG. 2 shows that the recognition sequences of two BsmBI restriction sites or BsaI restriction sites are connected back to back, because the recognition sequences and the cleavage sequences of the two BsmBI restriction sites and BsaI restriction sites are not the same sequence, they are all cleaved with a base after the recognition sequences, so that the cleaved nick sequences can be designed artificially, the nicks designed by the present invention are catt and taaa, and the foreign gene (such as ERG 20) is accessed^wwAnd sabinene synthase coding sequence) can be artificially designed into BsmBI or BsaI enzyme cutting sites at the front end and the rear end of the exogenous gene, the cut is designed into aatg and ttta, and the cut is complementarily matched with the cut on the expression cassette and connected to the expression cassette.

With ADH1t-ERG20^ww-GAL1&GAL 10-sabinene synthase coding sequence-TDH 2t gene module and YGG415 plasmid are used as examples to illustrate the construction process of the recombinant vector of the present invention:

GAL1& GAL10 promoter, ADH1t and TDH2t terminator were amplified from BY4741 s.cerevisiae genomic DNA, and two back-to-back BsmBI cleavage sites and two back-to-back BsaI cleavage sites were designed. Assembling PCR products by OE-PCR according to a required sequence to obtain an expression cassette of NotI enzyme cutting sites, ADH1t, back-to-back BsmBI enzyme cutting sites, GAL1& GAL10, back-to-back BsaI enzyme cutting sites, TDH2t-NotI enzyme cutting sites, a NotI enzyme cutting expression cassette and YGG415 plasmid, and inoculating the expression cassette into a vector;

then at ERG20^wwBsmBI enzyme cutting sites are designed at both ends (BsaI enzyme cutting sites can also be designed, as long as the artificially designed cuts of BsaI and BsmBI are consistent, the expression cassette is cut by BsmBI enzyme, ERG20^wwBsaI enzyme digestion can also be used for accessing the expression cassette, so that all gene sequences needing to be introduced can be uniformly designed into BsaI enzyme digestion sites more conveniently), BsaI enzyme digestion sites are designed at two ends of a sabinene synthase coding sequence, and the BsaI enzyme digestion sites are connected to ADH1 t-back BsaI enzyme digestion sites-GAL 1 through BsmBI enzyme digestion and BsaI enzyme digestion&GAL 10-back-to-back BsaI cleavage site-TDH 2t expression cassette, thereby completing the construction of the recombinant vector.

Preferably, the construction method further comprises the step of inoculating 1 or more than 2 copies of additional ERG20 into the recombinant vector^wwMore preferably, 1 or more than 2 copies of additional ERG20 are ligated into the expression cassette^wwThe specific construction mode can be realized by constructing the same enzyme cutting site.

The construction method of the recombinant yeast strain for producing the sabinene directly transforms the recombinant vector into the yeast strain to obtain the recombinant yeast strain.

Preferably, the process of constructing the recombinant strain further comprises:

constructing a gene module of upstream homologous sequence-weak promoter-yeast strain endogenous ERG20 original promoter and downstream homologous sequence of yeast strain endogenous ERG20 original promoter, or constructing a gene module of upstream homologous sequence-weak promoter-yeast strain endogenous ERG20 original promoter and downstream homologous sequence-yeast strain endogenous ERG 20-yeast strain endogenous ERG 20;

the gene module replaces the original promoter of the yeast strain ERG20 to be a weak promoter by utilizing a yeast homologous recombination mechanism.

Preferably, the process of constructing the recombinant strain further comprises:

respectively designing homologous sequences at two ends of a promoter, a coding sequence of the heterologous efflux protein and a terminator, then transforming the homologous sequences and the vector after enzyme digestion into the yeast strain, and carrying out homologous recombination to obtain a vector for expressing the heterologous efflux protein; wherein, two adjacent gene elements have a homologous region.

As can be seen from the above technical scheme, the invention selects the proper sequence ERG20 of the ERG20 with double mutation^wwAnd the exogenous sabinene synthase gene is matched and combined to construct a recombinant vector capable of producing sabinene and the recombinant vector is transformed into a yeast strain, so that the capability of the strain for producing sabinene can be obviously improved, and the yield of the sabinene produced by the strain can be further improved by continuously optimizing and transforming on the basis.

Drawings

FIG. 1 shows a map of YGG415 plasmid;

FIG. 2 is a schematic diagram showing a BsmBI cleavage site back-to-back mode and a BsaI cleavage site back-to-back mode; a is BsmBI enzyme cutting site, B is BsaI enzyme cutting site; the arrow points to the position of the cut, and the shadow position is a back-to-back identification sequence;

FIG. 3 is a schematic representation of a recombinant vector according to the present invention;

FIG. 4 is a schematic representation of a recombinant vector according to the present invention;

FIG. 5 is a schematic diagram showing replacement of the original promoter of endogenous ERG20 of the strain with a weak promoter;

FIG. 6 shows the shake flask fermentation yields of recombinant Saccharomyces cerevisiae expressing sabinene synthase from different sources; the abscissa indicates the name of each strain and the corresponding abbreviation in the table below;

FIG. 7 shows the shake flask fermentation yields of recombinant Saccharomyces cerevisiae expressing different truncated SpSabS1 sabinene synthases; the abscissa indicates the name of each strain and the corresponding abbreviation in the table below;

FIG. 8 is a map of the YGG416 plasmid;

FIG. 9 shows expression of ERG20^wwAnd sabinene synthase fusion protein and expression of one more ERG20^wwThe shake flask fermentation yield of the recombinant saccharomyces cerevisiae; the abscissa represents the name of each strain;

FIG. 10 shows shake flask fermentation yields of recombinant Saccharomyces cerevisiae expressing different heterologous efflux proteins; the abscissa indicates the name of each strain and the corresponding abbreviation in the table below;

FIG. 11 shows shake flask fermentation yield of recombinant Saccharomyces cerevisiae with down-regulated expression of endogenous ERG 20;

FIG. 12 shows the shake flask fermentation yield of recombinant Saccharomyces cerevisiae with a combination of different advantageous transformation strategies according to the present invention.

Detailed Description

The invention discloses a recombinant vector and a recombinant yeast strain for producing sabinene, and a construction method and application thereof. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. The recombinant vectors, strains, and methods of construction and use of the present invention have been described in terms of preferred embodiments, and it will be apparent to those of ordinary skill in the art that variations or appropriate modifications and combinations of the recombinant vectors, strains, and methods of construction and use described herein can be made to implement and use the techniques of the present invention without departing from the spirit, scope, and spirit of the invention.

The sequences appearing in the invention are optimized by a saccharomyces cerevisiae codon and the common restriction enzyme cutting sites are properly avoided;

the ADH1t sequence is shown in SEQ ID NO. 5;

ERG20^wwthe sequence is shown as SEQ ID NO. 6;

GAL1& GAL10 is shown as SEQ ID NO. 7;

the sequence of TDH2t is shown in SEQ ID NO. 8;

the coding sequence of the Salvia pomifera sabinene synthase is shown as SEQ ID NO. 9;

the coding sequence of the Salvia officinalis sabinene synthase is shown as SEQ ID NO. 10;

the coding sequence of the Picea sidetchensis sabinene synthase is shown as SEQ ID NO. 11;

the coding sequence of the Thuja plicata sabinene synthase is shown in SEQ ID NO 12;

the Murraya koenigii sabinene synthase coding sequence is shown in SEQ ID NO 13;

the coding sequence of the Citrus jamshiri sabinene synthase is shown as SEQ ID NO. 14;

the Litsea cubeba sabinene synthase coding sequence is shown in SEQ ID NO. 15;

the HXT1p sequence is shown in SEQ ID NO: 16;

GAL7 is shown in SEQ ID NO 17;

the sequence of GPDt is shown in SEQ ID NO 18;

the sequence of GPM1t is shown in SEQ ID NO. 19;

the sequence of the BsmBI enzyme cutting site is shown as SEQ ID NO. 20;

the back-to-back BsaI restriction enzyme site sequence is shown as SEQ ID NO: 21;

the above gene element sequences can be connected in sequence according to the gene module or expression cassette of the invention, namely the sequences of the corresponding gene modules or expression cassettes.

The invention is further illustrated by the following examples.

Example 1: construction of the recombinant vector of the present invention

1. Construction of expression cassettes and ligation vector procedures

GAL1& GAL10 promoter, ADH1t and TDH2t terminator were amplified from BY4741 s.cerevisiae genomic DNA, and two back-to-back BsmBI cleavage sites and two back-to-back BsaI cleavage sites were designed. Assembling PCR products by OE-PCR according to a required sequence to obtain an expression cassette of NotI enzyme cutting sites, ADH1t, back-to-back BsmBI enzyme cutting sites, GAL1& GAL10, back-to-back BsaI enzyme cutting sites, TDH2t-NotI enzyme cutting sites, a NotI enzyme cutting expression cassette and YGG415 plasmid, and inoculating the expression cassette into a vector;

2. introduction of foreign Gene

(1)ERG20^wwAnd sabinene synthase coding sequence into expression box separately

At ERG20^wwBsmBI enzyme sites are designed at both ends, BsaI enzyme sites are designed at both ends of the sabinene synthase coding sequence, and the BsmBI enzyme sites and the BsaI enzyme sites are connected to ADH1 t-back-to-back BsmBI enzyme sites-GAL 1&GAL 10-Back-to-Back BsaI restriction enzyme site-TDH 2t expression cassette, thereby completing the construction of recombinant vector and obtaining the recombinant vector YGG415-ADH1t-ERG20^ww-GAL1&10-sabinene synthase coding sequence-TDH 2 t; display deviceFIG. 3 is intended;

(2)ERG20^wwintroduction of coding sequence of sabinene synthase fusion protein into expression box

ERG20^wwAnd sabinene synthase coding sequence, adopting coding sequence of rigid linker GGGGS or coding sequence of flexible linker PAP to make forward or reverse fusion connection, designing BsaI enzyme cutting site at two ends of fusion protein sequence, utilizing BsaI enzyme cutting to connect ADH1 t-back BsmBI enzyme cutting site-GAL 1&GAL 10-Back-to-Back BsaI restriction enzyme site-TDH 2t expression cassette, thereby completing the construction of recombinant vector and obtaining the recombinant vector YGG415-ADH1t-GAL1&10-ERG20^wwAnd the coding sequence of the juniperylene synthase fusion protein-TDH 2 t; the schematic view is shown in FIG. 4;

(3) one more ERG20^wwIs constructed by

On the basis of (2), at ERG20^wwBsmBI enzyme cutting sites are designed at both ends, and are connected to ADH1 t-back BsmBI enzyme cutting sites-GAL 1 through BsmBI enzyme cutting&GAL10-ERG20^wwAnd the coding sequence of the sabinene synthase fusion protein-TDH 2t expression cassette, thereby completing the construction of the recombinant vector and obtaining the recombinant vector YGG415-ADH1t-ERG20^ww-GAL1&10-ERG20^wwAnd the coding sequence of the juniperylene synthase fusion protein-TDH 2 t;

wherein the sabinene synthase coding sequence is a sabinene synthase coding sequence derived from Salvia pomifera and/or Citrus jamshiri; or a truncated sabinene synthase coding sequence in which the N-stretch coding sequence of amino acid residues 1 to 43 is removed from the sabinene synthase coding sequence derived from Salvia pomifera and/or Citrus jamshiri.

Example 2: construction of the recombinant Saccharomyces cerevisiae Strain of the invention

1. Chassis strains

The chassis strain is selected as YJGZ1, which is based on a commercial model strain CEN. PK2-1C, and overexpresses precursor genes IDI1 and tHMGR1 while knocking out GAL80, namely, the supply of precursor GPP is increased, so that substrates required by sabinene synthesis are provided more. The specific modification method comprises the following steps:

amplification of P from BY4741 genomic DNA_GAL1,10Promoter, T_ADH1And T_TDH2Terminator and HIS3 marker and the top and bottom 400bp homology arms of Gal80 p. The PCR products were assembled by OE-PCR in the desired order, obtained as Gal80up-T_ADH1-P_GAL1,10-T_TDH2-His-Gal80down expression cassette. The expression cassette was then cut with NotI-HF and inserted into the same site of pRS415K to generate plasmid pJGZ3, which was at T_ADH1And P_GAL1,10With two back-to-back BsmBI sites, P between_GAL1,10And T_TDH2There are two back-to-back BsaI sites in between. The IDI1 amplified from BY4741 genomic DNA was digested with BsmBI and ligated into pJGZ3 at the same cleavage site to produce pJGZ 4. Cleavage of fragment Gal80up-T from pJGZ4 by NotI-HF_ADH1-IDI1-P_GAL1,10-T_TDH2His-Gal80down and inserted into a reconstituted pEASY-Blunt vector not containing the kanamycin resistance gene and the BsaI site to generate plasmid pJGZ 5. Subsequently, tHMGR amplified from BY4741 genomic DNA was inserted into pJGZ5 through BsaI restriction sites to obtain pJGZ 6. Finally, the NotI-HF cleaved integration fragment from pJGZ6 Gal80up-T_ADH1-IDI1-P_GAL1,10-tHMGR-T_TDH2His-Gal80down was transformed into CEN. PK2-1C to give YJGZ 1.

The YJGZ1 selected in the embodiment is a strain obtained in earlier systematic scientific research work, and is directly adopted for convenience, but is not necessarily used, and in subsequent comparison strains, the strain is also adopted as a blank strain for comparison, and sabinene cannot be generated.

2. Transforming the recombinant vector

The recombinant vector is transferred into YJGZ1 by a lithium acetate conversion method, an SC-LEU solid plate (6.7 g/L of synthetic yeast nitrogen source YNB, 20g/L of glucose, 2g/L of leucine-deficient mixed amino acid powder and 2% of agar powder) is adopted for screening after conversion, the obtained transformant is subjected to streak purification culture, yeast plasmid is extracted for PCR verification, and the correctly verified recombinant strain is stored to be the recombinant saccharomyces cerevisiae.

3. Expression of heterologous efflux protein genes

And (3) transforming a recombinant vector capable of expressing the efflux protein on the basis of the recombinant saccharomyces cerevisiae in the step (2). The construction method of the recombinant vector capable of expressing the efflux protein comprises the following steps:

first, the vector PRS424 was double-digested with Not I and BamH I s. Then, designing primers to divide an expression cassette GPM1t-GAL7-GPDt (obtained by earlier stage system scientific research of the invention and directly adopted for convenience) into GPM1t-GAL7 and GPD for PCR, respectively designing homologous sequences with efflux genes SC-GcABCG1, SC-YL-ABC2 and SC-YL-ABC3, enabling homologous regions of 20-40 bp to be reserved between two adjacent gene elements, splicing a vector PRS424 by using yeast homologous recombination, then transforming the homologous regions into yeast strains, screening an SC-LEU-TRP (synthetic yeast nitrogen source YNB 6.7g/L, glucose 20g/L, mixed amino acid powder of tryptophan deficiency and leucine 2g/L and agar powder of 2%) solid plate according to a screening label carried by the PRS424, extracting yeast plasmid for PCR verification after scribing and pure culture of obtained transformants, obtaining the recombinant saccharomyces cerevisiae.

4. Down-regulating expression of endogenous ERG20 of YJGZ1

On the basis of the recombinant saccharomyces cerevisiae obtained in 2 or 3, primers are designed, the whole genome DNA of the yeast YJGZ1 is used as a template to amplify a fragment HXT1p and an endogenous ERG20 (which can also be obtained by other amplification methods), a URA3 nutritional label loxP-URA3-loxP fragment is selected, a homologous region of 20-40 bp is arranged between two adjacent fragments, then the fragment loxP-URA3-loxP-HXT1p-ERG20 is obtained by overlapping extension PCR to replace ERG20p-ERG20 on the YJGZ genome, a schematic diagram is shown in figure 5, the transformed yeast is subjected to streak purification culture by using an SC-URA solid plate (synthetic yeast nitrogen source YNB 6.7g/L, glucose 20g/L, uracil-lacking mixed amino acid powder 2g/L, 2% agar powder) to obtain transformants, the transformants are subjected to PCR verification, and the nutritional label URA3 can be recovered by a 5-fluoorotic acid plate, obtaining the recombinant saccharomyces cerevisiae.

Example 3: shake flask fermentation test of different recombinant Saccharomyces cerevisiae strains

(1) Sabinene synthase coding sequence from different sources

According to "(1) ERG20 in example 1^wwAnd hinokeneThe method for respectively introducing enzyme coding sequences into expression cassettes "is used for constructing recombinant vectors, the sabinene synthase coding sequences are respectively derived from Salvia pomifera, Salvia officinalis, Picea aspergilli, Thuja plicata, Murraya koenigii, Citrus jamhiri and Litsea cubeba and are abbreviated as SpSabS1, SoSabS1, PsSabS1, TpSabs1, MkSabS1, CjSabS1 and LcSabS1 in sequence, 7 recombinant vectors are transformed into the same YJGZ1 strain, and a blank control strain YJGZ1 is arranged, and the method comprises the following steps:

SyBE_Sc04100001：YGG415-ADH1t-ERG20^ww-GAL1&10-SpSabS1-TDH2t；

SyBE_Sc04100002：YGG415-ADH1t-ERG20^ww-GAL1&10-SoSabS1-TDH2t；

SyBE_Sc04100003：YGG415-ADH1t-ERG20^ww-GAL1&10-PsSabS1-TDH2t；

SyBE_Sc04100004：YGG415-ADH1t-ERG20^ww-GAL1&10-TpSabS1-TDH2t；

SyBE_Sc04100005：YGG415-ADH1t-ERG20^ww-GAL1&10-MkSabS1-TDH2t

SyBE_Sc04100006：YGG415-ADH1t-ERG20^ww-GAL1&10-CjSabS1-TDH2t；

SyBE_Sc04100007：YGG415-ADH1t-ERG20^ww-GAL1&10-LcSabS1-TDH2t；

shake flask fermentations were performed for SyBE _ Sc04100001-SyBE _ Sc04100007 and the blank control strain for the Chassis strain YJGZ 1.

Seed culture medium: 40g/L glucose; 6.7 g/LYNB; 2g/Ldrop-out of leucine.

Fermentation medium: 40g/L glucose; 6.7 g/LYNB; 2g/Ldrop-out of leucine.

A single colony of the strain is inoculated into 3mL of SC-leu culture medium and cultured for 24h at 30 ℃ and 220 rpm. The primary seeds were transferred to 5mL SC-leu medium at initial OD600 ═ 0.2, and cultured at 30 ℃ and 220rpm for 12-18 h. The secondary seeds were transferred to 50mL of SC-leu medium at initial OD600 ═ 0.1, and two-phase culture was carried out by adding 20% isopropyl myristate at the same time (220rpm, 30 ℃ c., 96 h).

Sabinene quantification method: fermentation end1mL of the organic phase on the upper layer of the fermentation medium is spotted, and anhydrous sodium sulfate is added to stand in a four-degree refrigerator for dewatering for 4-5 hours. After removal of water the organic phase was filtered using a 2 μm organic filter. Then using gas chromatography to determine: the boiling point of sabinene is 164 deg.C at 760mmHg, so the detector temperature is set at 260 deg.C, the sample inlet is 250 deg.C, and the column temperature is 250 deg.C. The chromatographic condition is DB-5 silica gel capillary chromatographic column, and the sample injection amount is 1 mu L; keeping the initial temperature of the column temperature at 50 ℃ for 4 min; then at 5 ℃ min^-1Heating to 100 deg.C, and maintaining for 1 min; finally at 25 ℃ min^-1Heating to 250 deg.C, and maintaining for 5 min;

the results are shown in fig. 6, no sabinene chromatographic peak was detected in the blank control strain YJGZ1, chromatographic peaks at 12.098min were detected in the strains SyBE _ Sc04100001 and SyBE _ Sc04100006 at the same peak-off time as the standards, and no sabinene chromatographic peak was detected in the remaining strains, demonstrating that the strains SyBE _ Sc04100001 and SyBE _ Sc0410000 achieved sabinene synthesis with a yield of 0.519mg/L for SyBE _ Sc04100001 higher than for the strain SyBE _ Sc 04100006. Therefore, the coding sequences of the SbS1 and CjSabS1 derived sabinene synthase can obviously improve the sabinene synthesis capability of the strain, and the SpSbS1 source is the best.

(2) Sabinene synthase coding sequences in different truncation modes

Based on the SpSbS 1-derived sabinene synthase coding sequence, the following four truncation operations were performed to obtain different truncated sabinene synthase coding sequences:

t1SpSabS 1: a truncated sabinene synthase coding sequence with the 1-43 amino acid residue from the N-terminus of sabinene synthase removed;

t2SpSabS 1: a truncated sabinene synthase coding sequence with 1-34 amino acid residues from the N-terminus of sabinene synthase removed;

t3SpSabS 1: a truncated sabinene synthase coding sequence with the 1-52 amino acid residue from the N-terminal of the sabinene synthase removed;

t4SpSabS 1: truncated sabinene synthase coding sequence with the amino acid residue at position 423 and 499 of sabinene synthase removed;

each of the truncated sabinene synthase-encoding sequences described above was individually prepared according to "(1) ERG20 in example 1^wwAnd the sabinene synthase coding sequence was introduced into the expression cassette "to obtain 4 different recombinant vectors, which were transferred to YJGZ1Strains SyBE _ Sc04100008, SyBE _ Sc04100009, SyBE _ Sc04100010 and SyBE _ Sc04100011 were obtained in sequence, and then shake flask fermentation experiments were performed together with the previously obtained SyBE _ Sc 04100001;

the result is shown in figure 7, the recombined brewing strain SyBE _ Sc04100009 obtained by the truncation mode of only 1-34 amino acids improves the yield of sabinene by nearly five times, and the yield of the sabinene reaches 2.57mg/L, which is 4.95 times of the yield of the control strain SyBE _ Sc 04100001. Strains SyBE _ Sc04100008, SyBE _ Sc04100010 and SyBE _ Sc04100011 obtained by the other three truncation modes have no sabinene synthesis.

(3)ERG20^wwFusion expression of sabinene synthase coding sequence and multiple expression of one ERG20^ww

ERG20ww and t2SpSabS1 are respectively subjected to forward and reverse fusion by coding sequences of flexible linker (GGGGS, shown in SEQ ID NO: 22) and rigid linker (PAPAP, shown in SEQ ID NO: 23), different recombinant vectors are constructed and transferred into YJGZ1 by referring to a method of introducing the coding sequences of ERG20ww and sabinene synthase fusion protein into an expression cassette in example 1 (2), the transformed yeast is screened by adopting an SC-LEU solid plate, and after the obtained transformant is subjected to streak purification culture, yeast plasmids are extracted for PCR verification, and glycerol bacteria are stored for the forward fusion flexible linker connecting strain, the reverse fusion flexible linker connecting strain, the forward fusion rigid linker connecting strain and the reverse fusion rigid linker connecting strain which are verified to be correct and are sequentially named as SyBE _ Sc04100012, SyBE _ 041Sc 00013, SyBE _ 041Sc 00014 and SyBE _ Sc 00015;

reference example 1 "(3) expressing one more ERG20^wwThe method of the construction process of (1) is constructed by expressing one ERG20 on the basis of SyBE _ Sc04100012, SyBE _ Sc04100013, SyBE _ Sc04100014 and SyBE _ Sc04100015^wwThe recombinant strains of (1) are sequentially named as SyBE _ Sc04100016, SyBE _ Sc04100017, SyBE _ Sc04100018 and SyBE _ Sc 04100019;

to exclude the expression of more ERG20^wwThe copy number difference is brought, a positive control strain is constructed, namely, the strain SyBE _ Sc04100009 is used as an original strain, and one more ERG20 is expressed^wwConstructed on the vector YGG416 (plasmid map is shown in FIG. 8, the whole gene sequence is shown in SEQ ID NO:24It is similar to YGG 415), the specific construction method: the constructed YGG415-ADH1t-ERG20 was digested simultaneously with SalI and BamHI^ww-GAL1&10-TDH2t (obtained during construction of recombinant vector in example 1) to obtain fragment ADH1t-ERG20^ww-GAL1&10-TDH2t ligated into vector YGG416 digested with SalI and BamI to obtain YGG416-ADH1t-ERG20^ww-GAL1&10-TDH2t, then transforming into a strain SyBE _ Sc04100009, screening by using an SC-LEU-URA solid plate, carrying out streak purification culture on the obtained transformant, extracting yeast plasmid for PCR verification, and storing a glycerol strain named SyBE _ Sc04100020 for a recombinant strain with correct verification.

The above SyBE _ Sc04100009, SyBE _ Sc04100012-SyBE _ Sc04100020 were subjected to shake flask fermentation experiments, and the results in FIG. 9 show that the control strain SyBE _ Sc04100009 produces 2.23 mg/L. For flexible linker (GGGGS), the yield of the forward fusion strain SyBE _ Sc04100012 is 1.94mg/L, and the yield of the reverse fusion strain SyBE _ Sc04100013 is 2.56mg/L, so that the reverse fusion effect is better than that of the forward fusion, and one part of ERG20 is added in more than the reverse fusion^wwThe effect of (5) was even better, i.e., the yield of the strain SyBE _ Sc04100017 was 3.18 mg/L. The reason is presumed to be that the N-terminal of t2SpSabS1 is positively charged and attracts another copy of ERG20 which is negatively charged^wwThus, the precursor can be fully utilized to accelerate the reaction. And ERG20^wwPositive fusion of/tSpSabS 1 would expose a negative charge, with another copy of ERG20^wwThe charges are the same.

For the rigid linker (PAPAP), the forward fusion strain SyBE _ Sc04100014 produced 0.146mg/L and the reverse fusion strain SyBE _ Sc04100015 produced 2.17mg/L, with lower yields compared to the control strain SyBE _ Sc04100009, but one copy of ERG20 for more expression^wwThe yield of the strains SyBE _ Sc041000018 and SyBE _ Sc041000019 is 4.39mg/L and 3.31mg/L respectively. This indicates that the effect of fusion is not very apparent for the use of a rigid linker (PAPAP), in contrast to expressing one more portion of ERG20^wwHas the effect of increasing yield.

Meanwhile, compared with SyBE _ Sc04100009 and SyBE _ Sc04100020, the yield of the SyBE _ Sc04100020 is 2.33mg/L, which is not much different from that of the control strain by 2.23mg/L, and the ERG20 with more expressed parts is excluded^wwResulting in copy number differences.

Example 4: effect of heterologous efflux Gene expression

Constructing recombinant yeast strains expressing three different heterologous efflux proteins by taking a strain SyBE _ Sc04100009 as a starting strain and referring to the method in the step 3 of expressing heterologous efflux protein genes in the example 2, sequentially naming the recombinant yeast strains as SyBE _ Sc04100021(SC-GcABCG1), SyBE _ Sc04100022(SC-YL-ABC2) and SyBE _ Sc04100023(SC-YL-ABC3), and performing a shake flask fermentation test, wherein the results are shown in a figure 9;

FIG. 10 shows that the control strain SyBE _ Sc04100009 produced 2.23mg/L, the strain SyBE _ Sc04100021 produced 4.57mg/L, the strain SyBE _ Sc04100022 produced 3.70mg/L, and the strain SyBE _ Sc04100023 produced 2.54 mg/L. Compared with SC-YL-ABC2, SC-YL-ABC3 and SC-GcABCG1, SC-GcABCG1 achieves a good efflux effect, and the yield of sabinene is improved by about one time.

Example 5: effect of Down-regulating expression of endogenous ERG20

Referring to the method in example 2 '4, for down-regulating the expression of endogenous ERG20 of YJGZ 1', a recombinant Saccharomyces cerevisiae strain SyBE _ Sc041000025 is constructed and subjected to a shake flask fermentation test, and the recombinant vector YGG415-ADH1t-ERG20ww-GAL1&10-t2SpSabS1-TDH2t is transformed in the SyBE _ Sc 041000025;

FIG. 11 shows that the control strain SyBE _ Sc04100009 (transformed with the recombinant vector YGG415-ADH1t-ERG20ww-GAL1&10-t2SpSabS1-TDH2t) produced 2.03mg/L and the strain SyBE _ Sc04100025 produced 4.19mg/L, indicating that down-regulation of endogenous ERG20 can effectively reduce GPP conversion to FPP and reduce downstream branch metabolism, so that more GPP is used to synthesize sabinene.

Example 6: dominantly integrated recombinant saccharomyces cerevisiae strains

The dominant transformation strategies in examples 1 and 2 were integrated and recombinant s.cerevisiae was constructed in the combinatorial manner in table 1, still using YJGZ1 as the chassis strain.

TABLE 1

The constructed SyBE _ Sc04100026-SyBE _ Sc04100029 strain is subjected to a shake flask fermentation test, and the result is shown in FIG. 12, and the yield of the strain SyBE _ Sc04100029 is the highest and reaches 17.57mg/L after fermentation for 96 hours.

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 decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Tianjin university

<120> recombinant vector and recombinant yeast strain for producing sabinene, and construction method and application thereof

<130> MP1900919

<160> 24

<170> SIPOSequenceListing 1.0

<210> 1

<211> 6929

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt gtcacagctt 60

gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg 120

ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata 180

tcgactacgt cgtaaggccg tttctgacag agtaaaattc ttgagggaac tttcaccatt 240

atgggaaatg gttcaagaag gtattgactt aaactccatc aaatggtcag gtcattgagt 300

gttttttatt tgttgtattt tttttttttt agagaaaatc ctccaatatc aaattaggaa 360

tcgtagtttc atgattttct gttacaccta actttttgtg tggtgccctc ctccttgtca 420

atattaatgt taaagtgcaa ttctttttcc ttatcacgtt gagccattag tatcaatttg 480

cttacctgta ttcctttact atcctccttt ttctccttct tgataaatgt atgtagattg 540

cgtatatagt ttcgtctacc ctatgaacat attccatttt gtaatttcgt gtcgtttcta 600

ttatgaattt catttataaa gtttatgtac aaatatcata aaaaaagaga atctttttaa 660

gcaaggattt tcttaacttc ttcggcgaca gcatcaccga cttcggtggt actgttggaa 720

ccacctaaat caccagttct gatacctgca tccaaaacct ttttaactgc atcttcaatg 780

gccttacctt cttcaggcaa gttcaatgac aatttcaaca tcattgcagc agacaagata 840

gtggcgatag ggtcaacctt attctttggc aaatctggag cagaaccgtg gcatggttcg 900

tacaaaccaa atgcggtgtt cttgtctggc aaagaggcca aggacgcaga tggcaacaaa 960

cccaaggaac ctgggataac ggaggcttca tcggagatga tatcaccaaa catgttgctg 1020

gtgattataa taccatttag gtgggttggg ttcttaacta ggatcatggc ggcagaatca 1080

atcaattgat gttgaacctt caatgtaggg aattcgttct tgatggtttc ctccacagtt 1140

tttctccata atcttgaaga ggccaaaaca ttagctttat ccaaggacca aataggcaat 1200

ggtggctcat gttgtagggc catgaaagcg gccattcttg tgattctttg cacttctgga 1260

acggtgtatt gttcactatc ccaagcgaca ccatcaccat cgtcttcctt tctcttacca 1320

aagtaaatac ctcccactaa ttctctgaca acaacgaagt cagtaccttt agcaaattgt 1380

ggcttgattg gagataagtc taaaagagag tcggatgcaa agttacatgg tcttaagttg 1440

gcgtacaatt gaagttcttt acggattttt agtaaacctt gttcaggtct aacactaccg 1500

gtaccccatt taggaccacc cacagcacct aacaaaacgg catcaacctt cttggaggct 1560

tccagcgcct catctggaag tgggacacct gtagcatcga tagcagcacc accaattaaa 1620

tgattttcga aatcgaactt gacattggaa cgaacatcag aaatagcttt aagaacctta 1680

atggcttcgg ctgtgatttc ttgaccaacg tggtcacctg gcaaaacgac gatcttctta 1740

ggggcagaca taggggcaga cattagaatg gtatatcctt gaaatatata tatatattgc 1800

tgaaatgtaa aaggtaagaa aagttagaaa gtaagacgat tgctaaccac ctattggaaa 1860

aaacaatagg tccttaaata atattgtcaa cttcaagtat tgtgatgcaa gcatttagtc 1920

atgaacgctt ctctattcta tatgaaaagc cggttccggc ctctcacctt tcctttttct 1980

cccaattttt cagttgaaaa aggtatatgc gtcaggcgac ctctgaaatt aacaaaaaat 2040

ttccagtcat cgaatttgat tctgtgcgat agcgcccctg tgtgttctcg ttatgttgag 2100

gaaaaaaata atggttgcta agagattcga actcttgcat cttacgatac ctgagtattc 2160

ccacagttaa ctgcggtcaa gatatttctt gaatcaggcg ccttagaccg ctcggccaaa 2220

caaccaatta cttgttgaga aatagagtat aattatccta taaatataac gtttttgaac 2280

acacatgaac aaggaagtac aggacaattg attttgaaga gaatgtggat tttgatgtaa 2340

ttgttgggat tccattttta ataaggcaat aatattaggt atgtggatat actagaagtt 2400

ctcctcgacc gtcgatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg 2460

catcaggaaa ttgtaaacgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc 2520

agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag 2580

accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg 2640

gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca 2700

tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa 2760

gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg 2820

aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta 2880

accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcgcgcca ttcgccattc 2940

aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg 3000

gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca 3060

cgacgttgta aaacgacggc cagtgagcgc gcgtaatacg actcactata gggcgaattg 3120

ggtaccgggc cccccctcga ggtcgacggt atcgataagc ttgatatcga attcctgcag 3180

cccccagtag agaccgcctg gctctagtag cgatctacac tagcactatc agcgttatta 3240

agcaccggtg gagtgacgac cttcagcacg ttcgtactgt tcaacgatgg tgtagtcttc 3300

gttgtgggag gtgatgtcca gtttgatgtc ggttttgtaa gcacccggca gctgaaccgg 3360

ttttttagcc atgtaggtgg ttttaacttc agcgtcgtag tgaccaccgt ctttcagttt 3420

cagacgcatt ttgatttcac ctttcagagc accgtcttcc gggtacatac gttcggtgga 3480

agcttcccaa cccatggttt ttttctgcat aaccggaccg tcggacggga agttggtacc 3540

acgcagttta actttgtaga tgaactcacc gtcttgcagg gaggagtcct gggtaacggt 3600

aacaacacca ccgtcttcga agttcataac acgttcccat ttgaaacctt ccgggaagga 3660

cagtttcagg tagtccggga tgtcagccgg gtgtttaacg taagctttgg aaccgtactg 3720

gaactgcggg gacaggatgt cccaagcgaa cggcagcgga ccacctttgg taactttcag 3780

tttagcggtc tgggtacctt cgtacggacg accttcacct tcaccttcga tttcgaactc 3840

gtgaccgtta acggaacctt ccatacgaac tttgaaacgc atgaactctt tgataacgtc 3900

ttcggaggaa gccatctagt atttctcctc tttctctagt atgtgtgaaa ttgttatccg 3960

ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa 4020

tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac 4080

ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt 4140

gggtctcatt ttggggatcc actagttcta gagcggccgc caccgcggtg gagctccagc 4200

ttttgttccc tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt 4260

cctgtgtgaa attgttatcc gctcacaatt ccacacaaca taggagccgg aagcataaag 4320

tgtaaagcct ggggtgccta atgagtgagg taactcacat taattgcgtt gcgctcactg 4380

cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg 4440

gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc 4500

tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc 4560

acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg 4620

aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat 4680

cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag 4740

gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga 4800

tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg 4860

tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt 4920

cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac 4980

gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc 5040

ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt 5100

ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc 5160

ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc 5220

agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg 5280

aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag 5340

atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg 5400

tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt 5460

tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca 5520

tctggcccca gtgctgcaat gataccgcga gatccacgct caccggctcc agatttatca 5580

gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc 5640

tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt 5700

ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg 5760

gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc 5820

aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg 5880

ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga 5940

tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga 6000

ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta 6060

aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg 6120

ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact 6180

ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata 6240

agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt 6300

tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa 6360

ataggggttc cgcgcacatt tccccgaaaa gtgccacctg ggtccttttc atcacgtgct 6420

ataaaaataa ttataattta aattttttaa tataaatata taaattaaaa atagaaagta 6480

aaaaaagaaa ttaaagaaaa aatagttttt gttttccgaa gatgtaaaag actctagggg 6540

gatcgccaac aaatactacc ttttatcttg ctcttcctgc tctcaggtat taatgccgaa 6600

ttgtttcatc ttgtctgtgt agaagaccac acacgaaaat cctgtgattt tacattttac 6660

ttatcgttaa tcgaatgtat atctatttaa tctgcttttc ttgtctaata aatatatatg 6720

taaagtacgc tttttgttga aattttttaa acctttgttt attttttttt cttcattccg 6780

taactcttct accttcttta tttactttct aaaatccaaa tacaaaacat aaaaataaat 6840

aaacacagag taaattccca aattattcca tcattaaaag atacgaggcg cgtgtaagtt 6900

acaggcaagc gatccgtcct aagaaacca 6929

<210> 2

<211> 4380

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atggaaacag actcaaaaag tgtagaaagt ggtgaaactg ctgctatgcc aggtcaacaa 60

caaatctcat caaatgccca aggtttaatt catgcttact caatggaatt ggttagatca 120

tcttctagag caacaggtgg tggtggtgct ccaggtagaa atccattcac tggtacatct 180

aatgatccag cattagatcc acattctaaa gcttttgatg ctagaagatg ggctcaagct 240

gttttacatt caacaggtga aggtccagat cattgtccaa gaccaacagc tggtgttgct 300

tacagaaatt tgagagttca tggttacggt tctccaacag attatcaaaa agatgttttt 360

aatgttttgt tacaagcacc attagaagca gctcaatatt ttatgtcttc aagaagaggt 420

agagaagttc caattttgag agatggtttt gatggtttag ttagatcagg tgaaatgtta 480

ttagttttgg gtagaccagg ttcaggtgtt actacattgt taaaaacagt tgcaggtgaa 540

actaatggtt tgcaagttga tgcagaagct tttatttctt accaaggtat tccaatgcaa 600

gcaattcaaa agagattcag aggtgaagtt gtttaccaag ctgaaactga tgttcatttt 660

ccacaattga cagttggtca aactttgtta tttgctgcaa aagcaagaac accacaaatg 720

aggccagatg gtgttactag agcacaatac gctaaacata ttagagatgt tgttatggca 780

gtttttggta tttctcatac agttaataca agagttggtt cagatttggt tagaggtgtt 840

tcaggtggtg aaagaaaaag agtttctatt gctgaagttg ctttatctgg ttcagctttg 900

caatgttggg ataattctac aagaggtttg gattctgctt cagctttatc ttttgctaat 960

acattaagat tatcaactga attggcaggt actacagctt tggttgctat gtatcaagct 1020

tcagaagctg cttacgaaac ttttggtaaa gtttgtttat tgtacgaagg tagacaaatt 1080

ttctttggtc cagctaatga agcaaaagca tttttcgttg atatgggtta tgaatgtcca 1140

gatagacaaa caacagctga tttcttgact tctttaacta atccaggtga aagagttgtt 1200

agaccaggtt ttgaaaatag agttccaaga acaccagatg attttgttgc ttactggaaa 1260

gcatcagcta ctagagcttc tttattgcaa gatattgctg aatttgatca agaacatcca 1320

atggatggta caccaattga agcaatggca acagttagaa aagctcatca agcaccattg 1380

acaccaaata agtcaccatt cactttatca tttccacaac aagttgcttt atgtatgact 1440

agaggttacg aaagaacaat gggtgacaaa acatttttca ttgttactgt tggtggtaat 1500

ttggttattt cattggtttt aggttcagtt ttctatcaat tgtcaccaga tgcttcatct 1560

attacttcaa gatgtatttt attgtttttc gctattttgt ttaatgcatt atcttcttca 1620

ttagaaattt tatcattgta tgctcaaaga ccaattgttg aaaaacatgc tagatatgca 1680

ttgtacacac catctgctga agcagtttct tctgcatttt gtgaattgcc atctaaaatt 1740

ttctctgcaa ttgcttttaa tattccatta tactttatgg cagatttgag acatggtgca 1800

ggtcatttct ttttcttttt attgtttgct tttacttgta ctttgacaat gtcttttatt 1860

ttgagaacaa ttggtcaagc atctagaact gttcaagaag ctttgacacc agctgctgtt 1920

tttattattt ctttggttat ttatactggt tttgttattc cagttaaatc aatgcaaggt 1980

tggatgagat ggattaatta cttgaatcca attgcttacg cttatgaatc attattagtt 2040

aatgaattgt caggtagaaa ttttccatgt gcatcttttg ttccagcata cccaaatttg 2100

tcttcatctg aacatacatg ttctacagca ggtgcagctc caggtgcaga ttttgttgtt 2160

ggtgacacta ttttgaattc atcatatgaa tactaccatg cacataaatg gagaaatttg 2220

ggtattttaa ttggtttctt gattgcattt ttctttgctt atttggttgc atctgaatac 2280

attacagctg aacaatctaa aggtgaagtt ttagttttta gaagaggtca taaagaatca 2340

gctgttgttg aaagaaaaac tgctacatct gatgattcag atggtgaaaa aggtcatcaa 2400

actgaacaaa aagatatttg tcattggaga aatgtttgtt atgatattac aattaaaggt 2460

caaggtagaa gattgttaga tcatgttgat ggttgggtta aaccaggtac attgacatgt 2520

ttaatgggtg tttctggtgc aggtaaaaca actttgttag atgttttggc taatagagtt 2580

actatgggtg ttgttacagg tgacatgtta gttaatggtt caccaagaga ttcttcattt 2640

caaagaaaaa ctggttatgt tcaacaacaa gatgttcatt tggaaacatc tactgttaga 2700

gaagctttga gattttctgc acaattgaga caaccaacta cagtttctac tcaagataaa 2760

tacatttttg ttgaagaagt tattgaatta ttggaaatgg atgaatatgc agatgcaatt 2820

gttggtgttc caggtactgg tttaaatgtt gaacaaagaa aaagattgac tattggtgtt 2880

gaattagctg caaaaccaga tttgttattg tttttggatg aaccaacatc aggtttagat 2940

tcacaaacag catggtcagt tgctgcattg attagaaaat tgtctgctag aggtcaagct 3000

gttttgtgta caattcatca accatctgca ttattgtacc aacaatttga tagaatttta 3060

ttgttggcag ctggtggtag aactgtttat tttggtgaca ttggtccaaa tgcagaaact 3120

attatttctt actttgaaag aaatggtgct gaaccatgtg gtcaagatga aaatccagct 3180

gaatggatgt tatcagttat tggtgcaggt ccaggtggtg ttgctaaaca agattgggtt 3240

tctatttgga gaaattcaga tgaatactct gctgttcaag ctgaattaga taatttggct 3300

aaaagaaaag atactatggc ttcatctggt gctacagatg cagctgctgt tactacatat 3360

gctactccat ttttctttca attgtatatg tgttctaaaa gagtttttga acaatactgg 3420

agaacaccat cttacattta tgcaaaaatg attttatgtt ttgcagtttc attgtttatt 3480

ggtttgtctt ttagaaaagc tccattgtct gaacaaggtt tgcaaaatca aatgttttct 3540

atttttatgt tattggttat ttttgcattt ttggcttatc aaactatgcc acattttatt 3600

agacaaagag aattgtatga aattagagaa agagcttcaa gaacatactc atggtatgtt 3660

tttatgttgg caaatattat tgttgaattg ccatggaata ctattgcatc attattggtt 3720

ttcttgccat tttattatat tgttggtatg aatcataatg ctgaagcaac tcattcagtt 3780

tctgaaagag gtggtttgat gtttttgtta gtttgggttt tcttagtttt tgaatctact 3840

tttactgata tggttgttgc tggttctcca actgctgaat tgggtgcaac aatggctttg 3900

ttattatttg cttttacttt aattttctgt ggtgttatgg ttggtaaaga tcaattgcca 3960

ggtttttgga tttttatgta tagagtttct ccattgactt atttggttgg tggtttgttg 4020

gctactggtg ttggtcatca tgaagttaca tgtactgcta gagaattgtt atcatttcaa 4080

ccagttggta atcaaacatg tttagaatat atgactccat atatgaaatt agcaggtggt 4140

aaagttatta atccaaatgc tgttgcacca gcatcatgtg aattttgtac attagcaaat 4200

acagatgctt tcttggcttc tattaatgtt tcatatgatc aaagatggag agattttggt 4260

ttgatgtggg catatgttgt ttttaatgtt tttggtgctt tgtttatgta ttggttggtt 4320

agagccccaa agggtgactt aaaggcaaga ttgttcaaat tagtaggtaa aactgcttaa 4380

<210> 3

<211> 4410

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atggaacaaa ctccaccaga ttacacaggt ttagataaga acatcgatgc agaagttaga 60

tcaattgctg aatcaatgta ccaaacaaga agagaaggtg ttaacgattc agatactgat 120

gaagaattgc aaagaactaa cacaatccaa ccaaatttga acgttaaccc atttttggat 180

acatctgatc cacaattaga tccattgtct aaggaattca attcaagaaa gtggatcaag 240

actatcttgg gtttgaaggc tagattcggt aactcaagat caatctcagc aggtgtttct 300

tttaaaaatt tgtcagcttt cggttatggt ggtggtaacg attaccaaaa gacttttact 360

aactcagtta tggctattgg tccaatgatt aagaaagttt tgggtggtaa taagggttct 420

gaagttcaaa ttttaagaca tttcgatggt ttggttagag ctggtgaaac atgtgttgtt 480

ttaggtagac caggttctgg ttgtactaca tttttgaagt cagttgcatg tgaaacttac 540

ggtttccatt tgggtgaaaa atctgaatgg aattaccaag gtgttccaag agatgttatg 600

caaaagaatg caagaggtga aatcgtttac aacgctgaag ttgatgttca ttttccacat 660

ttgactgttg gtgacacatt gttatttgct gcattagcta gaacaccaca aaatagattg 720

gaaggtgttt caagagaaca acatgcaact catgttagag atgtttctat ggctatgtta 780

ggtttgactc atacaatgga tacaaaggtt ggtaacgatt tcgttagagg tgtttcaggt 840

ggtgaaagaa aaagagtttc tattgcagaa tcagttgttt gtggtgctcc attacaatgt 900

tgggataatt ctacaagagg tttggatgct gcaaatgcta ctgaattcat tagatcattg 960

agattgtcag cagaaatgac tgatgcttct atgtttgttt cattgtacca agcatctcaa 1020

gaagcttacg atatgttcga taaggtttgt gttttgtacg aaggtagaca aatatatttt 1080

ggtaaaacta cagaagctaa gcaatttttc ttggatttgg gtttcgattg tgcagataga 1140

caaactactg gtgacttttt gacttcattg acaaacccaa tcgaaagaat catcagacca 1200

ggttgggaat ctagagttcc aagaactcca gatgatttcg aaaagtgttg gttggaatct 1260

gaagctagac aattgttatt gcaagatatc gatgaattca ataacgaatt cgttttaggt 1320

ggtccagcat tggataactt catgggtttg agaaaggatg ctcaagcaaa acatactaga 1380

gttcaatctc catacacaat ctcatggcca atgcaaacta gattatgttt gtggagaggt 1440

ttcttgagaa ttaaaggtga catgtctact gatatcgcaa cagttttcgg taacttcgtt 1500

atggctttgg ttttgtcttc aatgttctac aacatgccac aaactacaga atctttcttt 1560

tctagaggtg cattattgtt tttcgctatc ttgattaatg ctttcgcatc tatcttggaa 1620

attttatcat tgtacgaaca aagaccaatc gttgataagc aaaacagata cgcaatgtac 1680

catccagctg ctgatgcttt ggctgcaatc atcactacat tcccaactaa gacattaact 1740

ttggtttcag ttaatttgac tatctatttc atgacaaatt tgagaagaga agttggtcca 1800

tttttcattt tctttttgtt ttctttattg tgtacaatgg ctatgtcaat gatttttaga 1860

actatcggtt ctgttacaaa gactttggaa caagctttgg caccagcttc tatcatcatc 1920

ttggctttgg ttatatatac tggtttttct ttaccaattt catacatgca tggttgggca 1980

agatggatta attggttgaa tccagttgca tatggttttg aagctgttat ggttaacgaa 2040

ttcagaaaca gagaatacga atgttctatg ttcgttccat caggtggtgc ttacgaaaat 2100

gtttctttgg attacagatc atgtgctgct gttggtgcag aaccaggttt gagattcgtt 2160

aacggtgacg cttttattaa ccaatcttac gaatactaca acgcacattt gtggagaaac 2220

atgggtattt tgtttggttt tattattttc tttggtgcat tctatttgtt cgctgttgaa 2280

tacatccaag gtgctaagtc aaagggtgaa gttttggttt ttagaaagga acatattaag 2340

aaacaaagaa aggaaaagaa tggtgacatc gaatctggtg ttacaatggc tggtgaaaaa 2400

ggtactcaag aatctgaatc ttcaaacaca tcaattaatt tgcaagcaca aagaggtatc 2460

tatcaatgga aggatgtttg ttacgatatc aaggttaagg atggtgaaag aagattattg 2520

gatcatgttg atggtttcgt taaaccaggt acattaactg ctttgatggg tgcatcaggt 2580

gctggtaaaa ctacattgtt ggatgttttg gcagatagaa aatctactgg tgttgttaca 2640

ggtgaaatgt tggttaatgg tgaacataga gatggttcat tccaaagaaa gactggttac 2700

gttcaacaac aagatttgca tacagcaact gctacagtta gagaatcttt ggaattttca 2760

gctttattga gacaaccatc ttcaatccca gaatctgaaa agttggcata cgttgatgaa 2820

gttattagaa ttttggaaat ggaaacatac gcagatgctg ttgttggtgt tccaggtgaa 2880

ggtttaaacg ttgaacaaag aaagagatta actattggtg ttgaattggc tgcaaagcca 2940

gaattgttgt tgtttttgga tgaaccaaca tctggtttgg attcacaaac tgcttggtct 3000

atcgttaagt tgttgaagaa attagctgca aatggtcaag caattttgtg tacaatccat 3060

caaccatcag ctatcttgtt ccaagaattc gatagattgt tatttttggc ttctggtggt 3120

agaacagttt attacggtga cattggtcca caatcttcaa tcttgactga atacttcgaa 3180

agaaatggtg cagatccatg tccaaaacaa ggtaatccag ctgaatggat gttggaagtt 3240

attggtgctg caccaggttc aactgcaaaa agagattggc cagttgtttg ggctgaatct 3300

ccagaaagag ctgcaaaaag agaagaattg gatgaaatgg ctagaactgt tgaaagagtt 3360

caaacaaaca ctacagaaag agattcaaca ggttattctg attcagatca atttgctgtt 3420

ggttggtgga ctcaattcaa aattgtttct aaaagacaat ttcaagcatt gtggagaaca 3480

ccatcttact tatggtcaaa agttttcttg tgtgctgcat ctgcaatttt cattggtttt 3540

tctttcttta aggctccaaa cgatatgcaa ggtttacaaa ataagatgtt ctctttcttt 3600

atgttatttt tgattttcaa tacagttgtt gaacaaatca tcccacaatt cgataagatg 3660

agagaattgt acgaagctag agaaagatca tcaaagactt actcttggca agtttttatg 3720

ggttcaaaca tggtcgttga attgatctgg caatttttca tgggtgttat tgttttcttt 3780

tgtttttact acccagttgg tttccaatgg actgctgaat ataatgattc agttcatgaa 3840

agaggtggtt tgtttttctt gtacgtctta ttgttgtttt tatacaattc tacatttgca 3900

catatgttga tcgctggtat cgataataag gatactgctg cacaaattgg tactttgttg 3960

tttacattga tgttgttgtt ttgtggtgtt ttggctacaa aagaacaaat gccaggtttt 4020

tgggttttta tgtacagagt ttctccatta acttacttcg ttggtggtat gatggcaact 4080

ggtatgggta gagctccagt tacttgttca ccacatgaat tggttagatt tccagctgtt 4140

cctggtaaat cttgtggtga atacatggat ggttttattt ctgcattagg tgactcagct 4200

ggttatttgg tttcttcatc tgctgatatg tgtgaatact gtccaatgaa gtcatctgat 4260

caattcttgg attctgttga tatctcatac actcaaagat ggagaaactg gggtatttta 4320

tgggcttacc cattgtttaa tatcttcgct gcattcgcat tgtactattt ctttagagtt 4380

ccaaagaaat ctaaagcaca aaaagcttaa 4410

<210> 4

<211> 4458

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atgactgatc cagttccaat tacacaagat ccaactatct attcttcaca acaagatgct 60

gaaatcagat cattggcaga atctatccat tcacaacatt ctaacaactc aaacaactct 120

acagaattga ctaatccata cgttgatact tcagatccag aattagatcc atggtctggt 180

caattcaatt caagaaagtg gtctagaact atcttgggtt taaaaagaag atacggtaca 240

tcaaaggaaa tcactgctgg tgtttctttt aaaaatttgg gtgcttacgg ttacggtggt 300

ggtgctgatt accaaaagac tgttgctaat gcagttttgg gtttagaagg tgttgttaga 360

acattgttcc atttggaaaa gaaagaagat aaggttcaaa ttttgtctga ttttaatggt 420

gttttatggc caggtgaaac ttgtgttgtt ttgggtagac caggttcagg ttgtactaca 480

ttgttgaagt ctatcgcttg tgaaacatac ggtttccaat tggataagga aactgaatgg 540

aactaccaag gtattccaag aaagatcatg caaaagactt gtagaggtga aatcgtttac 600

aacgctgaag ttgatgttca ttttccacat ttgacagttg gtgacacttt gatgtttgca 660

tctttagcta gaacaccaca aaatagattt gatggtgtta ctagagaaca atacgctaaa 720

catacaagag atgttactat ggcatcattg ggtttatctc atacattgga tactaaggtt 780

ggtaacgatt ttgttagagg tgtttctggt ggtgaaagaa agagagtttc aatcgctgaa 840

tctatcgttt gtggttcacc attgcaatgt tgggataatt ctactagagg tttagatgct 900

gcaactgcaa cagaattttt gagatggtta agacattcag cagaattgac aggtgcttca 960

atgtttgttt ctttgtacca agcatctcaa gaagcttacg aattgttcga taaggttact 1020

gttttgtacg aaggtcaaca aatatatttt ggtccaggtg aacaagctaa gcaatacttc 1080

gaagaaatgg gtttcgaatg tccacataga caaactactg gtgacttttt gacatcaatt 1140

acttctccag cagaaagaat tgttgctcca ggttttgaag gtaaaacacc aagaactgca 1200

tcagaatttg ctgaaagatg gagacaatct caagcttacg caaatttgca agaagaaatc 1260

gaaagattca atactgaatt tccagttggt ggtaacagag ttgctgatat catggaattg 1320

aagcaagaaa agcaatcaga tcatatcaaa gtttcttcac catacactat ctctatccca 1380

atgcaggtta agttgtgttt gacaagaggt ttccaaagat tgagaggtga cttgtcaatg 1440

gctttgacta cagttttggg taacttcgtt gttgcattga tcttgtcttc aatgttctac 1500

aacatgccag aagatacttc ttctttcttt tctagaggtg cattgttgtt tttcgctatg 1560

ttgatgaatg caatgtcttc agttttggaa atcatcgttt tgtacgaatt aagaccaatc 1620

gttgaaaagc atcaaagata cgctatgtac catccattct gtgaagcttt ggcatcaatc 1680

atctgtgatt tcccaacaaa gttcttgact atgttatgtg ttaatgttac attgtacttc 1740

atgtctaatt tgagaagaga agctggtcca tttttcattt tctttttgtt tactttgttg 1800

tgtgttttgg caatgtctat gatttttaga acaatcgctg ctgttactaa gacattgcaa 1860

caagctttag caccagctgc tgttattatc ttggctttga tcatctatac aggttttact 1920

ttgccaattt cttacatgag aggttgggca agatggatca actacatcga tccaatcgct 1980

tacggttttg aagcagttat ggttaacgaa ttcagaaaca gagaattccc atgtgctttg 2040

tttattccac aacaatcaac ttacgatcaa ttaggttctc cataccaagg ttgtatggca 2100

gttggtgcta aaccaggtga aagattcgtt aacggtgaca gatatttgga aatggctttt 2160

gattactctc aagcacattt gtggagaaat ttgggtatta tgtttggttt tattttgttt 2220

ttcgctttta catatttgac tgctgttgaa ttcattcaat ctgctaagtc taagggtgaa 2280

gttttggttt tcttgagatc atctttgaag cagagaaaga aaagagctca tttgatggat 2340

gttgaagcta atgcagaaaa agttggtgct gcacaagata gagaaatttt ggttcaacaa 2400

gaagaaggtc aacaagaaga aacatcttca tgtactccat ctgattcaac tccaaaggat 2460

atcttccaat ggaaggatgt ttgttacgat atcaaggtta aaggtggtga aaagagattg 2520

ttggataacg ttgatggttg ggttaaacca ggtactttga cagctttaat gggttgttct 2580

ggtgcaggta aaactacatt gttggatgtt ttggcagata gaaaagctac aggtgttatt 2640

actggtgaca tgagagttaa tggtcaaaag agagatgctt cattccaaag aaagactggt 2700

tacgttcaac aacaagattt gcatactgca acatctactg ttagagaagc tttagaattt 2760

tcagcattgt taagacaacc atctaatgtt ccaaaagcag aaaagattgc ttacgttgat 2820

gaagttattg atatcttgga aatgcaagct tacgcagatg ctgttgttgg tgttccaggt 2880

gaaggtttaa acgttgaaca aagaaagaga ttgactatcg gtgttgaatt agctgcaaag 2940

ccagaattgt tgttgttttt ggatgaacca acatcaggtt tagattctca aactgcttgg 3000

tcaatcatct gtttgttgaa gaaattggct aacagaggtc aagcaatctt atgtacaatc 3060

catcaaccat ctgctatctt gttccaagaa ttcgatagat tattgtttat gacattaggt 3120

ggtaaaactg tttactacgg tgacattggt gcaaactctt cagctttgat taattacttc 3180

gaatctaaag gtgctgatcc atgtccagaa gaagcaaatc cagctgaatg gatgttagct 3240

gcaattggtg ctgcaccagg ttcaattgct aaacatgatt gggcagttgt ttggaatgaa 3300

tctgaagaaa gagctagaga aagagatttg ttggataaaa tggcagaaga attggctgca 3360

caatcaacac atgatgaaaa gaatgaattg gttacttcta agtcagttgg ttcttcacaa 3420

acatcttcat cttcatactc tgctaagtct caatacgcaa catctcaagc tactcaattg 3480

tactacttaa caaagagatt gtggacttat tactggagat caccaagata catctggtct 3540

aagttgttga tgtcaatcgc atctgctttg tttattggtt tttcatacta caaggcttct 3600

caagatatcc aaggtttaca aaaccaaatg tttgctttct ttatgttgtt tttaatcttc 3660

gttatcatca tggttcaaat tttgccacat ttcgttgctc aaagagaatt gtacgaagca 3720

agagaaagat catcaatggc ttactcatgg caagctttta tgggttctaa catcttggtt 3780

gaattaccat ggcaaacttt ggttgctgtt ttggttttct tttgtttcta ctacccaatc 3840

ggtttgcaaa acaatgctac aggtcatttg ggtgaaagag gtgctttgtt tttcttgttg 3900

ttgtggtcat tctatgttta caattctact tttgctcata tgatgggtgc tgcattcgaa 3960

aataaggaaa acgctgcaac aatcggttat ttgttgttcg ctttgtgttt gattttctgt 4020

ggtgttttgg caactaagga agatatgcca catttctgga tttttatgta cagagtttct 4080

ccattgacat acttaatctc aggtttgttg tctgctggtg ttggtgaaac aagagttgaa 4140

tgtactgata acgaattggt tttgtttaaa ccaatgaacg gtactaactg tggtaaatac 4200

atgcatcctt ttatggaagg tttgggtcat acagatatgc caatgggtta tttggttgat 4260

ccatcagcta ctgatatgtg tggttactgt ccaatctcta acacaaacgg ttatttggat 4320

caaatcgatg ttaagtactc acaaagatgg agaaactacg gtattttatt cgcataccca 4380

gcttttaatg tttttatggc attcgctttc tattacattt ttagagttcc aaagaaatct 4440

agaaaacaaa aagcttaa 4458

<210> 5

<211> 327

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

catgccggta gaggtgtggt caataagagc gacctcatgc tatacctgag aaagcaacct 60

gacctacagg aaagagttac tcaagaataa gaattttcgt tttaaaacct aagagtcact 120

ttaaaatttg tatacactta ttttttttat aacttattta ataataaaaa tcataaatca 180

taagaaattc gcttatttag aagtgtcaac aacgtatcta ccaacgattt gacccttttc 240

catcttttcg taaatttctg gcaaggtaga caagccgaca accttgattg gagacttgac 300

caaacctctg gcgaagaagt ccaaagc 327

<210> 6

<211> 1059

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

atggcttcag aaaaagaaat taggagagag agattcttga acgttttccc taaattagta 60

gaggaattga acgcatcgct tttggcttac ggtatgccta aggaagcatg tgactggtat 120

gcccactcat tgaactacaa cactccaggc ggtaagctaa atagaggttt gtccgttgtg 180

gacacgtatg ctattctctc caacaagacc gttgaacaat tggggcaaga agaatacgaa 240

aaggttgcca ttctaggttg gtgcattgag ttgttgcagg cttactggtt ggtcgccgat 300

gatatgatgg acaagtccat taccagaaga ggccaaccat gttggtacaa ggttcctgaa 360

gttggggaaa ttgccatctg ggacgcattc atgttagagg ctgctatcta caagcttttg 420

aaatctcact tcagaaacga aaaatactac atagatatca ccgaattgtt ccatgaggtc 480

accttccaaa ccgaattggg ccaattgatg gacttaatca ctgcacctga agacaaagtc 540

gacttgagta agttctccct aaagaagcac tccttcatag ttactttcaa gactgcttac 600

tattctttct acttgcctgt cgcattggcc atgtacgttg ccggtatcac ggatgaaaag 660

gatttgaaac aagccagaga tgtcttgatt ccattgggtg aatacttcca aattcaagat 720

gactacttag actgcttcgg taccccagaa cagatcggta agatcggtac agatatccaa 780

gataacaaat gttcttgggt aatcaacaag gcattggaac ttgcttccgc agaacaaaga 840

aagactttag acgaaaatta cggtaagaag gactcagtcg cagaagccaa atgcaaaaag 900

attttcaatg acttgaaaat tgaacagcta taccacgaat atgaagagtc tattgccaag 960

gatttgaagg ccaaaatttc tcaggtcgat gagtctcgtg gcttcaaagc tgatgtctta 1020

actgcgttct tgaacaaagt ttacaagaga agcaaataa 1059

<210> 7

<211> 667

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ttatattgaa ttttcaaaaa ttcttacttt ttttttggat ggacgcaaag aagtttaata 60

atcatattac atggcaatac caccatatac atatccatat ctaatcttac ttatatgttg 120

tggaaatgta aagagcccca ttatcttagc ctaaaaaaac cttctctttg gaactttcag 180

taatacgctt aactgctcat tgctatattg aagtacggat tagaagccgc cgagcgggcg 240

acagccctcc gacggaagac tctcctccgt gcgtcctggt cttcaccggt cgcgttcctg 300

aaacgcagat gtgcctcgcg ccgcactgct ccgaacaata aagattctac aatactagct 360

tttatggtta tgaagaggaa aaattggcag taacctggcc ccacaaacct tcaaatcaac 420

gaatcaaatt aacaaccata ggataataat gcgattagtt ttttagcctt atttctgggg 480

taattaatca gcgaagcgat gatttttgat ctattaacag atatataaat gcaaaagctg 540

cataaccact ttaactaata ctttcaacat tttcggtttg tattacttct tattcaaatg 600

tcataaaagt atcaacaaaa aattgttaat atacctctat actttaacgt caaggagaaa 660

aaactat 667

<210> 8

<211> 400

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

atttaactcc ttaagttact ttaatgattt agtttttatt attaataatt catgctcatg 60

acatctcata tacacgttta taaaacttaa atagattgaa aatgtattaa agattcctca 120

gggattcgat ttttttggaa gtttttgttt ttttttcctt gagatgctgt agtatttggg 180

aacaattata caatcgaaag atatatgctt acattcgacc gttttagccg tgatcattat 240

cctatagtaa cataacctga agcataactg acactactat catcaatact tgtcacatga 300

gaactctgtg aataattagg ccactgaaat ttgatgcctg aaggaccggc atcacggatt 360

ttcgataaag cacttagtat cacactaatt ggcttttcgc 400

<210> 9

<211> 1746

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

atgcccttga actctttgca taacttggag aggaaaccat ctaaagcttg gtctacttct 60

tgtactgctc cagctgctag attgcaagct tctttctctt tgcaacagga ggaaccaaga 120

caaattagaa ggtccggtga ctatcaacca tctttgtggg actttaacta cattcagtcc 180

ttgaacactc catacaaaga gcagagatac gttaataggc aggctgagtt gattatgcaa 240

gtcaggatgt tgttgaaggt taagatggag gctattcaac agttggagtt gattgatgac 300

ttgcagtatt tgggcttgtc ttactttttc ccagacgaga tcaagcagat tttgtcctct 360

attcacaatg agcacaggta cttccataac aacgacttgt acttgactgc tttgggtttt 420

aggattttga ggcagcatgg ttttaatgtc tctgaagacg tcttcgattg tttcaagact 480

gagaagtgct ctgatttcaa cgctaatttg gcccaggata ctaaaggtat gttgcaattg 540

tacgaggctt ctttcttgtt gagagaaggt gaggatactt tggaattggc tagaaggttc 600

tctactagat ctttgagaga gaagttggat gaagacggtg atgagattga tgaagacttg 660

tcttcttgga ttaggcattc tttggacttg ccattgcatt ggagaattca aggtttggaa 720

gctagatggt tcttggatgc ttatgctaga aggccagata tgaacccatt gattttcaag 780

ttggccaagt tgaacttcaa cattgtccag gctacttatc aggaggaatt gaaggatgtt 840

tctaggtggt ggaattcttc ttgcttggct gaaaagttgc catttgttcg cgataggatt 900

gttgagtgtt tcttctgggc tattggtgct tttgaaccac atcagtactc ttaccagaga 960

aagatggccg ctattattat taccttcgtc accattattg acgacgttta cgacgtctat 1020

ggtactttgg aggagttgga attgttcact gacatgatta gaaggtggga caacatttct 1080

atttcccagt tgccatatta catgcaggtt tgctacttgg ctttgtataa ctttgtctct 1140

gaaagggctt acgacatttt gaaagaccag cacttcaact ctattccata cttgcagaga 1200

tcttgggttt ctttggttga gggttacttg aaagaagctt actggtacta caatggttac 1260

aagccatctt tggaggagta tttgaacaac gctaagatct ctatctctgc ccccactatt 1320

atttctcagt tgtacttcac tttggccaac tctactgacg agactgtcat tgaatctttg 1380

tacgagtacc acaacatttt gtacttgtct ggcactattt tgagattggc tgacgatttg 1440

ggtacttctc aacatgaatt ggagagaggt gatgttccaa aagctattca gtgctacatg 1500

aaggatacta acgcctctga aagagaagct gttgaacacg tcaaattctt gattcgcgag 1560

acttggaaag agatgaacac tgtcactact gcttctgatt gcccatttac tgatgacttg 1620

gttgctgttg ctactaattt ggctagagct gcccagttta tttacttgga cggtgatggt 1680

catggtgttc aacactctga aattcatcag caaatgggcg gtttgttgtt tcaaccatac 1740

gtctaa 1746

<210> 10

<211> 1773

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

atgtcttcaa tctcaattaa tatcgctatg ccattgaact ctttgcataa cttcgaaaga 60

aagccatcaa aggcttggtc aacttcttgt acagcaccag ctgcaagatt gagagcttct 120

tcatctttac aacaagaaaa gccacatcaa atcagaagat caggtgacta tcaaccatct 180

ttgtgggatt tcaactacat ccaatctttg aacacaccat acaaggaaca aagacatttc 240

aacagacaag cagaattgat catgcaagtt agaatgttgt tgaaagttaa gatggaagct 300

atccaacaat tggaattgat cgatgatttg caatatttgg gtttatctta tttctttcaa 360

gatgaaatta aacaaatttt atcatctatc cataatgaac caagatattt tcataataat 420

gatttgtact tcacagcatt gggttttaga attttaagac aacatggttt taatgtttct 480

gaagatgttt tcgattgttt caagatcgaa aagtgttcag atttcaacgc taatttggca 540

caagatacta agggcatgtt gcaattgtac gaagcatcat ttttgttgag agaaggtgaa 600

gatacattgg aattggctag aagattttca actagatcat tgagagaaaa gttcgatgaa 660

ggtggtgacg aaatcgatga agatttgtca tcttggatta gacattcttt ggatttgcca 720

ttgcattgga gagttcaagg tttggaagct agatggtttt tagatgctta tgcaagaaga 780

ccagatatga acccattgat ttttaagttg gcaaagttga acttcaacat tgttcaagct 840

acttaccaag aagaattgaa ggatatctct agatggtgga actcatcttg tttagcagaa 900

aagttgccat tcgttagaga tagaatcgtt gaatgtttct tttgggctat tgctgcattt 960

gaaccacatc aatactcata ccaaagaaag atggctgctg ttattatcac ttttattaca 1020

atcatcgatg atgtttacga tgtttacggt actatcgaag aattggaatt gttgacagat 1080

atgatcagaa gatgggataa taagtcaatc tctcaattgc catactacat gcaagtttgt 1140

tatttggcat tgtacaactt tgtttctgaa agagcttacg atatcttgaa ggatcaacat 1200

ttcaattcaa ttccatactt acaaagatca tgggtttctt tggttgaagg ttatttgaag 1260

gaagcttact ggtactacaa cggttacaag ccatctttag aagaatactt gaacaacgct 1320

aaaatttcaa tctctgcacc aactatcatc tcacaattgt acttcacatt ggcaaactca 1380

atcgatgaaa ctgctatcga atctttgtac caataccata acatcttgta cttgtctggt 1440

acaattttaa gattggcaga tgatttgggt acttcacaac atgaattaga aagaggtgac 1500

gttccaaagg ctatccaatg ttacatgaac gatacaaatg cttctgaaag agaagcagtt 1560

gaacatgtta agttcttgat cagagaagca tggaaagaaa tgaatacagt tactacagct 1620

tcagattgtc cttttactga tgatttggtt gctgctgctg ctaatttggc tagagctgca 1680

caattcatat atttggatgg tgacggtcat ggtgttcaac attctgaaat ccatcaacaa 1740

atgggtggtt tgttatttca accatacgtt taa 1773

<210> 11

<211> 1884

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

atgtcagtta tttctatcgt tccattggct tctaactcat gtttgtacaa gtctttgatg 60

tcttcaactc atgaattgaa agctttatgt agaccaattg caacattggg catgtgtaga 120

cgtggtaaat cagttatggc ttctatgtca acttctttaa ctacagcagt ttctgatgat 180

ggtgttcaaa gaagaatcgg tcatcatcat tcaaatttgt gggatgataa cttcatccaa 240

tctttgtctt caccatacgg tgcttcttca tatgctgaat ctgctaagaa attgattggt 300

gaagttaagg aaatttttaa ttcattgtct atggctgctg gtggtttgat gtcaccagtt 360

gatgatttgt tacaacattt gtctatggtt gataacgttg aaagattagg tatcgataga 420

cattttcaaa ctgaaattaa agtttcattg gattacgttt actcatactg gtctgaaaaa 480

ggtattggtt ctggtagaga tatcgtttgt actgatttga acactacagc tttgggtttt 540

agaattttga gattacatgg ttacacagtt tttccagatg ttttcgaaca tttcaaggat 600

caaatgggta gaatcgcttg ttctgcaaat catacagaaa gacaaatttc ttcaatcttg 660

aatttgttta gagcttcatt gatcgcattc ccaggtgaaa aagttatgga agaagctgaa 720

attttctctg caacttattt gaaggaagct ttacaaacaa ttccagtttc ttcattgtca 780

caagaaatgc aatacgtttt ggattacaga tggcattcta atttgccaag attagaaact 840

agaacataca tcgatatctt gggtgaaact acaattaatc aaatgcaaga tgttaatatt 900

caaaaattgt tagaattggc aaagttggaa ttcaatatct tccattcaat tcaacaaaat 960

gaattaaaat gtatttctag atggtggaaa gaatcaggtt ctccagaatt gacttttatt 1020

agacatagac atatcgaatt ctacacattg gcttctggta tcgatatgga accaaaacat 1080

tcagctttta gattgtcttt cgttaagatg tgtcatttga ttactgtttt agatgatatc 1140

tatgatactt ttggtacaat ggatgaattg agattgttta cttcagctgt taagagatgg 1200

gatagatcag aaatcgaatg tttgccagaa tacatgaagg gtgtttacat catcttgtat 1260

gaaactgtta atgaaatggc tagagaagca agaaagtcac aaggtagaga tacattgaac 1320

tacgctagat tggcattgga agaatacatc ggtgcttatt tgaaggaagc agaatggatc 1380

tctatgggtt acttaccaac tttcgaagaa tacttcaaga acggtaaagt ttcttcaggt 1440

catagaatcg ctactttaca accaatcttg acattggata tcccattccc acatcatatc 1500

ttgcaagaaa tcgatttccc atcaaagttt aatgaattgg catgttctat cttgagatta 1560

agaggtgaca caagatgtta ccaagctgat agagatagag gtgaaaaagc atcatgtatc 1620

tcttgttaca tgaaggataa tccaggttct actgaagaag atgctttgaa ccatatcaac 1680

ggtatgatcg aagatacaat taaacaattg aactgggaat tgttaagacc agataacaac 1740

gttccaatct cttctaagaa acattcattc gatatctcta gagcattcca tcatttgtat 1800

agatacagag atggttacac tgtttcttca aacgaaacta aaaatttggt tgttagaact 1860

gttttggaac cattaccaat gtaa 1884

<210> 12

<211> 1815

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

atggctttgt tttctgcatc aacatctgtt ttgtcttcat gtttgaaatc tccaccaaac 60

catcatgtta agttgtttaa taagaattca caatctttgt ctagaagaag attgaaccct 120

tttatttcta aggcttcaac tacaactgtt gaaacaccaa ctagaagaac aggtaatcat 180

catccaaatt tgtgggatga tggtttgatt ggtactattc aagaacaacc atatgatgat 240

tcacattgta tggaaagagc tgaaagattg atcggtgaaa ttaaagatat gttcaacgaa 300

tctggtaaat tctgtggtga aaacgctttc gaaagattgg ttatggttga taaggttcaa 360

agattagcaa tcgatagaca tttccaaaac gaaatcgctc aagcattgga ttacgtttac 420

agatactggt ctgattgttc aagagatttg aattctgctg cattgggttt aagaattttg 480

agattaaaca gatacccagt ttcttcagat gttttgagac atttcaaggg taacgatggt 540

caattcttgt gtccatctgc tcaatcagaa gaagaaaaga ttggttctat cttgaatttg 600

tacagagctt cattaatcgc attcccagaa gaaaacatca tggatgaagc taaggcattc 660

gctacaactt atttgaacca agttttgcaa aacaacaata tttcttctca tttgtcaaaa 720

gaaattaaat acaatttgga atatggttgg catacaaatt tgccaagagt tgaagctaga 780

aactacatgg atatctatgg tgaaaataga tcatggactg aaatgggtgg taacatgcaa 840

attttgaatt tggcaaagtt ggattttaat attatgcaat ctgttcatag attggaattg 900

gaatcaatct tgaagtggtg gaaggattct aatttggata aggttgattt cgctagacat 960

agacatgttg aatactttgc attagcttgt gcatactgta tcgatgctaa gtactacgca 1020

tacagaagag attttgcaaa attatgtgct ttggcaacaa tcgttgatga tatctatgat 1080

acatatggta ctatcgaaga aattaaattg tttaatgaag ctgttaagat gtgggattct 1140

tcattaccaa actcattgcc agaaaacatc aagatcgctt acaaggcatt ccatatggct 1200

gttaacgaat ctgctgaagc tgctaagaaa actcaaggta gagatatttt gccatacgct 1260

agaaaggttt gggaacatta tttgatcggt ttgactaagg aagctgaatg gttagcaaac 1320

ggttacatcc catctttaga agaatatttg gaaaacggtg ctccatcttc aggttacaga 1380

gttactatgt tacaaccaac attgacttta gatgcattgt tgccagataa catcttgttg 1440

gaaatggatt acccatctag attcaatgaa ttgttgtgtt tgtctttaag attgaaaggt 1500

gacacaagaa cttttaaagc tgaagcaaat agaggtgaat tggtttctgg tatctcatgt 1560

tacatcaagg atcatccagg ttcttcagaa gaagaagctt tggattattt gaaagatttg 1620

ttgcaaaaga gattgaagga attggatcaa gaatacttaa agccaaacaa cgttccagca 1680

atctcaaagg atcatgctta caacatcgca agatcatacc aattgttata taaagaaaga 1740

gatggtttta caaactctaa taaggatatc aaagatttgg ttactcaaat tttgttggaa 1800

ccaattccat tataa 1815

<210> 13

<211> 1809

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

atggcattaa atttgttatc ttcattgcca gctacttgta acttcacaag attgtcattg 60

ccattgtctt caaacgttaa cggtttagtt ccaccaatta ctagagttca atatagaatg 120

gctgcatcaa ctacaacttc tactattaaa ccagcagatc aaacaatcat cagaagatca 180

gctgattaca gaccaacaat ctggtctttc gattacttgc aatcattgga ttctaagtac 240

aagggtgaat cttacgctag acaattggaa aagttgaagg aacaagttaa ggcaatgtta 300

caacaagatt acaaggctgt tgatttggac ccattgcatc aattggaatt gatcgataat 360

ttgcatagat taggtgtttc ataccatttc aaggatgaaa ttaaaagaac tttggatggt 420

atctataata agaacacaaa taagtctttg tacgctgctg ctttgaagtt tagaattttg 480

agacaacatg gttacgatat cccagttaag gaaacttttt caagattcat tgatgataaa 540

ggttctttta aatcttcatc tcatggtgac gattgtagag gcatgttagc tttgtatgaa 600

gcagcttact tgttagttga agaagaatca actattttta gagattcaat ctcttttaca 660

actacatatt tgaaggaatg ggttggtaaa catgattcta ataagcatgg tgacgaatac 720

ttgtgtacat tggttaacca tgctttggaa ttgccattgc attggagaat gagaagattg 780

gaagcaagat ggttcatcga tgtttacgaa tctggtccag atatgaaccc aatcttgttg 840

gaattggcaa agttggattt caacatcgtt caagctgttc atcaagaaga tttgaaatac 900

gtttcaagat ggtggaagaa aactggtttg ggtgaaaagt tgacattttc tagagataga 960

gttgttgaaa atttcttgtg gactgttggt gacatcttcg aaccacaatt cggttattgt 1020

agaagaatgt cagctatggt taactctttg ttgactacaa tcgatgatat ctatgatgtt 1080

tacggtactt tggatgaatt ggaattgttt actgatgcag ttgatagatg ggatgcaact 1140

gctacagaac aattgccata ctacatgaag ttgtgtttcc atgttttgta caacttcgtt 1200

aacgaaatgg cattcgatgc tttgagagat caagaagttg gtatgatcat cccatacttc 1260

aagaaaactt gggcagattt gtgtaaagct tactttgcag aagctaaatg gtacaactca 1320

ggttacatcc caactttcca agaatacatg gaaaacgcat ggatctctat cacagctcca 1380

ttgatgttga tccatgcata cgcttttact gcaaatccaa tcacaaagga agctttggaa 1440

ttcttacaag attctccaaa catcatcaga ttttcatcta tgattgttag attggctgat 1500

gatttgggta cttcatctga tgaattgaaa agaggtgacg ttccaaagtc aatccaatgt 1560

tacatgcatg aaacaggtat ttctgaagat gaagcaagag aacatatcag aaatttgatc 1620

gctgaatcat ggatcaagtt gaactctgca agattcggta acccacatta tttgtctgat 1680

gtttttattg gtatcgctat gaacatggca agagttgctc aatgtatcta tcaatttggt 1740

gacggtcatg gtgacgaaga aaacactaag gatagagttg ttacattgtt tttcgatcca 1800

attccataa 1809

<210> 14

<211> 1809

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

atggcattga atttgttgtc ttcaatccca gctgcatgta acttcactag attgtctttg 60

ccattgtctt caaaggttaa cggttttgtt ccaccaatta caagagttca ataccatgtt 120

gctgcatcaa ctacaccaat taaaccagtt gatcaaacta tcatcagaag atcagctgat 180

tatggtccaa caatctggtc attcgattac atccaatctt tggattcaaa gtacaagggt 240

gaatcttacg caagacaatc agaaaagttg aaggaacaag tttctgctat gttgcaacaa 300

gatgataaag ttgttgattt ggacccattg catcaattgg aattgatcga taatttgcat 360

agattaggtg tttcttacca tttcgaagat gaaattaaaa gaactttaga tagaattcat 420

aataagaata ctaataagtc attgtacgct acagcattga agtttagaat tttgagacaa 480

catggttaca acactccagt taaggaaaca ttttctagat tcatggatga aaagggtatt 540

tttaagttgt cttcacattc agatgattgt aaaggcatgt tagctttgta tgaagctgca 600

tacttgttgg ttgaagaaga atcttcaatt tttagagatg ctacttcttt tactacagca 660

tatttgaagg aatgggttat taaacatgat aacatcaagc atgatgatga acatttgtgt 720

acattggtta accatgcatt ggaattgcca ttgcattgga gaatgccaag attggaagct 780

agatggttta ttgatgttta cgaaaatggt ccagatatgt ctccaatttt gttggaattg 840

gctaaggttg atttcaacat cgttcaagca gttcatcaag aaaatttgaa gtacgcttca 900

agatggtgga agaaaactgg tttgggtgaa aatttgaact tcgttagaga tagaattgtt 960

gaaaatttct tgtggacagt tggtgaaaag ttcgaaccac aattcggtta cttcagaaga 1020

atgtctacta tggttattgc tttgatcaca gcagttgatg atgtttacga tgtttacggt 1080

actttggatg aattggaaat ttttacagat gctgttgaaa gatgggatgc tactgctgtt 1140

gaacaattgc cacattacat gaagttgtgt tttcatgcat tgagaaattc tattaatgaa 1200

atgacttttg atgctttgag agatcaaggt gttgatatcg ttatctcata tttgacaaaa 1260

gcttgggcag atatctgtaa ggcttactta gttgaagcaa agtggtacaa ctctggttac 1320

atcccatcat tgcaagaata catggaaaac gcttggatct ctatcggttc aactgttatt 1380

ttggttcatg cttacacttt tacagcaaac ccaatcacaa aggaaggttt agaattcgtt 1440

aaggattacc caaacatcat cagatggtct tcagttattt tgagattcgc agatgatttg 1500

ggtacttctt cagatgaatt gaaaagaggt gacgttcata agtctatcca atgttacatg 1560

catgaagctg gtgtttcaga aggtgaagca agagaacata tcaacgattt gatcgctcaa 1620

acatggatga agatgaacag agatagattc ggtaacccac attttgtttc tgatgttttc 1680

gttggtatcg ctatgaattt ggcaagaatg tcacaatgta tgtatcaatt tggtgacggt 1740

catggttgtg gtgctcaaga aatcacaaag gcaagagttt tgtctttgtt tattgatcca 1800

attgcttaa 1809

<210> 15

<211> 1743

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

atggcattgc aattgttgac tccatctttt tcattccaac attctccatc accacataga 60

ttgactacat tgagatacac tcatcataca atcagatgta cagcttctgc accatcttat 120

tcagatttgg ttggtagaag atcagctaac tacaagccat ctaagtggga ttcaaacttc 180

gttgaaactt tggaatctga ttataagaaa gaaaatcatg aaatgtacat tgaaaaattg 240

atgggtgacg ttaagcattt gatgaagaaa gttgttaacc caatcgaaaa gatggaattg 300

gttgatacaa tccaaagatt gggtttaggt tatttgttta ataaggaaat taaagaagtt 360

ttgaacacta ttgctacatc aaaggcaact tttaaaacta agaaagattt gcatgctgtt 420

gcattgcaat tcagattgtt aagacaacat ggttatgaag tttctccaga tgcattccat 480

aagtttaaag atgaaaaagg tggttttaaa gaatctttgt gtatggatat caagggcatg 540

ttgtcattgt acgaagcttc tcatttgtca tttcaaggtg aagttgtttt agatgaagca 600

agagaattca cttcaacaca tttgaaggct atcgagggta acatcgatcc agttttgttg 660

aagaaagtta gacattcttt ggaaatgcca ttacattgga gaatgttgag attagaagct 720

agatggtaca tcgaaactta cgatgaagaa gatagaaaga atccatcttt ggctgaattg 780

gcaaagcatg atttcaactc agttcaaaca atctatcaaa gatcattgaa gagaatgtca 840

agatggtgga gagatttggg tttaggtgaa agattggaat tttctagaga tagattagtt 900

gaatgtttct tttggactac aggtgttatt ttcgatccac aattcgaaag atgtagaggt 960

gttttgacta aggttaacca attagtttct acaattgatg atgtttatga tgtttacggt 1020

tcattggaag aattggaatt gtttactgat gcagttgata gatgggatat tagagctatg 1080

gaacaattgc cagaatacat gaaaatttgt tatttggcat tgtacaacac tacaaacgat 1140

atcgcttacg aagcattgaa ggaagaaggt ttagatgtta tcccatattt gaagaaagtt 1200

tggactgatt tgtgtaagtc ttacatcgtt gaagcaagat ggtattcaaa tggttacaag 1260

ccaactttgg aagaatattt ggaaaacgct tggacatcta ttgctggtcc agttgcatta 1320

ggtcatgctt acttctcatt cggtcaaaag atgccattcg aagctttgaa ctactctaac 1380

acttcttcat taattaaatg gtcttcaatg atttttagat tgtgtgatga tttggcaaca 1440

tcttcagatg aagttgctag aggtgacgtt ccaaagtcta tccaatgtta catgtacgaa 1500

gcaggtgttt ctgaatcagt tgctagagat catatcaagt atttgatcga tgaagcatgg 1560

aagaaaatga acgaatgttt ggttccatca actccatttt tgcaaccatt gattaatgct 1620

ggttttaatt tggctagaat ggcacattgt atgtacgaac atggtgacgg tcatggtttt 1680

tctaacgaat tggataagaa aagagttttg ttgttgttgg ctgaaccttt taagtttatg 1740

taa 1743

<210> 16

<211> 1123

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

tgcaggtctc atctggaata taattccccc ctcctgaagc aaatttttcc tttgagccgg 60

aatttttgat attccgagtt ctttttttcc attcgcggag gttattccat tcctaaacga 120

gtggccacaa tgaaacttca attcatatcg accgactatt tttctccgaa ccaaaaaaat 180

agcagggcga gattggagct gcggaaaaaa gaggaaaaaa ttttttcgta gttttcttgt 240

gcaaattagg gtgtaaggtt tctagggctt attggttcaa gcagaagaga caacaattgt 300

aggtcctaaa ttcaaggcgg atgtaaggag tattggtttc gaaagttttt ccgaagcggc 360

atggcaggga ctacttgcgc atgcgctcgg attatcttca tttttgcttg caaaaacgta 420

gaatcatggt aaattacatg aagaattctc tttttttttt tttttttttt ttttttacct 480

ctaaagagtg ttgaccaact gaaaaaaccc ttcttcaaga gagttaaact aagactaacc 540

atcataactt ccaaggaatt aatcgatatc ttgcactcct gatttttctt caaagagaca 600

gcgcaaagga ttatgacact gttgcattga gtcaaaagtt tttccgaagt gacccagtgc 660

tctttttttt tttccgtgaa ggactgacaa atatgcgcac aagatccaat acgtaatgga 720

aattcggaaa aactaggaag aaatgctgca gggcattgcc gtgccgatct tttgtctttc 780

agatatatga gaaaaagaat attcatcaag tgctgataga agaataccac tcatatgacg 840

tgggcagaag acagcaaacg taaacatgag ctgctgcgac atttgatggc ttttatccga 900

caagccagga aactccacca ttatctaatg tagcaaaata tttcttaaca cccgaagttg 960

cgtgtccccc tcacgttttt aatcatttga attagtatat tgaaattata tataaaggca 1020

acaatgtccc cataatcaat tccatctggg gtctcatgtt ctttccccac cttaaaatct 1080

ataaagatat cataatcgtc aactagttga tatacgtaaa atc 1123

<210> 17

<211> 725

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

tttgccagct tactatcctt cttgaaaata tgcactctat atcttttagt tcttaattgc 60

aacacataga tttgctgtat aacgaatttt atgctatttt ttaaatttgg agttcagtga 120

taaaagtgtc acagcgaatt tcctcacatg tagggaccga attgtttaca agttctctgt 180

accaccatgg agacatcaaa aattgaaaat ctatggaaag atatggacgg tagcaacaag 240

aatatagcac gagccgcgga gttcatttcg ttacttttga tatcactcac aactattgcg 300

aagcgcttca gtgaaaaaat cataaggaaa agttgtaaat attattggta gtattcgttt 360

ggtaaagtag agggggtaat ttttcccctt tattttgttc atacattctt aaattgcttt 420

gcctctcctt ttggaaagct atacttcgga gcactgttga gcgaaggctc attagatata 480

ttttctgtca ttttccttaa cccaaaaata agggaaaggg tccaaaaagc gctcggacaa 540

ctgttgaccg tgatccgaag gactggctat acagtgttca caaaatagcc aagctgaaaa 600

taatgtgtag ctatgttcag ttagtttggc tagcaaagat ataaaagcag gtcggaaata 660

tttatgggca ttattatgca gagcatcaac atgataaaaa aaaacagttg aatattccct 720

caaaa 725

<210> 18

<211> 500

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

gtgaatttac tttaaatctt gcatttaaat aaattttctt tttatagctt tatgacttag 60

tttcaattta tatactattt taatgacatt ttcgattcat tgattgaaag ctttgtgttt 120

tttcttgatg cgctattgca ttgttcttgt ctttttcgcc acatgtaata tctgtagtag 180

atacctgata cattgtggat gctgagtgaa attttagtta ataatggagg cgctcttaat 240

aattttgggg atattggctt ttttttttaa agtttacaaa tgaatttttt ccgccaggat 300

aacgattctg aagttactct tagcgttcct atcggtacag ccatcaaatc atgcctataa 360

atcatgccta tatttgcgtg cagtcagtat catctacatg aaaaaaactc ccgcaatttc 420

ttatagaata cgttgaaaat taaatgtacg cgccaagata agataacata tatctagatg 480

cagtaatata cacagattcc 500

<210> 19

<211> 400

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

gtctgaagaa tgaatgattt gatgatttct ttttccctcc atttttctta ctgaatatat 60

caatgatata gacttgtata gtttattatt tcaaattaag tagctatata tagtcaagat 120

aacgtttgtt tgacacgatt acattattcg tcgacatctt ttttcagcct gtcgtggtag 180

caatttgagg agtattatta attgaatagg ttcattttgc gctcgcataa acagttttcg 240

tcagggacag tatgttggaa tgagtggtaa ttaatggtga catgacatgt tatagcaata 300

accttgatgt ttacatcgta gtttaatgta caccccgcga attcgttcaa gtaggagtgc 360

accaattgca aagggaaaag ctgaatgggc agttcgaata 400

<210> 20

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

tttaggagac gcgtctccca tt 22

<210> 21

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

aatgggagac cggtctccta aa 22

<210> 22

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 22

Gly Gly Gly Gly Ser

1 5

<210> 23

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 23

Pro Ala Pro Ala Pro

1 5

<210> 24

<211> 5806

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt gtcacagctt 60

gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg 120

ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata 180

ccacagcttt tcaattcaat tcatcatttt ttttttattc ttttttttga tttcggtttc 240

tttgaaattt ttttgattcg gtaatctccg aacagaagga agaacgaagg aaggagcaca 300

gacttagatt ggtatatata cgcatatgta gtgttgaaga aacatgaaat tgcccagtat 360

tcttaaccca actgcacaga acaaaaacct gcaggaaacg aagataaatc atgtcgaaag 420

ctacatataa ggaacgtgct gctactcatc ctagtcctgt tgctgccaag ctatttaata 480

tcatgcacga aaagcaaaca aacttgtgtg cttcattgga tgttcgtacc accaaggaat 540

tactggagtt agttgaagca ttaggtccca aaatttgttt actaaaaaca catgtggata 600

tcttgactga tttttccatg gagggcacag ttaagccgct aaaggcatta tccgccaagt 660

acaatttttt actcttcgaa gacagaaaat ttgctgacat tggtaataca gtcaaattgc 720

agtactctgc gggtgtatac agaatagcag aatgggcaga cattacgaat gcacacggtg 780

tggtgggccc aggtattgtt agcggtttga agcaggcggc agaagaagta acaaaggaac 840

ctagaggcct tttgatgtta gcagaattgt catgcaaggg ctccctatct actggagaat 900

atactaaggg tactgttgac attgcgaaga gcgacaaaga ttttgttatc ggctttattg 960

ctcaaagaga catgggtgga agagatgaag gttacgattg gttgattatg acacccggtg 1020

tgggtttaga tgacaaggga gatgcattgg gtcaacagta tagaaccgtg gatgatgtgg 1080

tgtctacagg atctgacatt attattgttg gaagaggact atttgcaaag ggaagggatg 1140

ctaaggtaga gggtgaacgt tacagaaaag caggctggga agcatatttg agaagatgcg 1200

gccagcaaaa ctaaaaaact gtattataag taaatgcatg tatactaaac tcacaaatta 1260

gagcttcaat ttaattatat cagttattac cctatgcggt gtgaaatacc gcacagatgc 1320

gtaaggagaa aataccgcat caggaaattg taaacgttaa tattttgtta aaattcgcgt 1380

taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt 1440

ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc 1500

cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg 1560

gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac 1620

taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg 1680

tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag 1740

cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt 1800

cgcgccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt 1860

cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc 1920

cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgagcgcgcg taatacgact 1980

cactataggg cgaattgggt accgggcccc ccctcgaggt cgacggtatc gataagcttg 2040

atatcgaatt cctgcagccc ccagtagaga ccgcctggct ctagtagcga tctacactag 2100

cactatcagc gttattaagc accggtggag tgacgacctt cagcacgttc gtactgttca 2160

acgatggtgt agtcttcgtt gtgggaggtg atgtccagtt tgatgtcggt tttgtaagca 2220

cccggcagct gaaccggttt tttagccatg taggtggttt taacttcagc gtcgtagtga 2280

ccaccgtctt tcagtttcag acgcattttg atttcacctt tcagagcacc gtcttccggg 2340

tacatacgtt cggtggaagc ttcccaaccc atggtttttt tctgcataac cggaccgtcg 2400

gacgggaagt tggtaccacg cagtttaact ttgtagatga actcaccgtc ttgcagggag 2460

gagtcctggg taacggtaac aacaccaccg tcttcgaagt tcataacacg ttcccatttg 2520

aaaccttccg ggaaggacag tttcaggtag tccgggatgt cagccgggtg tttaacgtaa 2580

gctttggaac cgtactggaa ctgcggggac aggatgtccc aagcgaacgg cagcggacca 2640

cctttggtaa ctttcagttt agcggtctgg gtaccttcgt acggacgacc ttcaccttca 2700

ccttcgattt cgaactcgtg accgttaacg gaaccttcca tacgaacttt gaaacgcatg 2760

aactctttga taacgtcttc ggaggaagcc atctagtatt tctcctcttt ctctagtatg 2820

tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta 2880

aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg 2940

ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga 3000

gaggcggttt gcgtattggg tctcattttg gggatccact agttctagag cggccgccac 3060

cgcggtggag ctccagcttt tgttcccttt agtgagggtt aattgcgcgc ttggcgtaat 3120

catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatag 3180

gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgaggtaa ctcacattaa 3240

ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat 3300

gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc 3360

tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg 3420

cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag 3480

gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc 3540

gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag 3600

gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga 3660

ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc 3720

atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 3780

tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt 3840

ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca 3900

gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca 3960

ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag 4020

ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca 4080

agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg 4140

ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa 4200

aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta 4260

tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag 4320

cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga 4380

tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagat ccacgctcac 4440

cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc 4500

ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta 4560

gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac 4620

gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat 4680

gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa 4740

gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg 4800

tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag 4860

aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc 4920

cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct 4980

caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat 5040

cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg 5100

ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc 5160

aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta 5220

tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgggt 5280

ccttttcatc acgtgctata aaaataatta taatttaaat tttttaatat aaatatataa 5340

attaaaaata gaaagtaaaa aaagaaatta aagaaaaaat agtttttgtt ttccgaagat 5400

gtaaaagact ctagggggat cgccaacaaa tactaccttt tatcttgctc ttcctgctct 5460

caggtattaa tgccgaattg tttcatcttg tctgtgtaga agaccacaca cgaaaatcct 5520

gtgattttac attttactta tcgttaatcg aatgtatatc tatttaatct gcttttcttg 5580

tctaataaat atatatgtaa agtacgcttt ttgttgaaat tttttaaacc tttgtttatt 5640

tttttttctt cattccgtaa ctcttctacc ttctttattt actttctaaa atccaaatac 5700

aaaacataaa aataaataaa cacagagtaa attcccaaat tattccatca ttaaaagata 5760

cgaggcgcgt gtaagttaca ggcaagcgat ccgtcctaag aaacca 5806

Claims (9)

1. A recombinant Saccharomyces cerevisiae strain for the production of sabinene, transformed with a recombinant vector comprising a sequence encoding ERG20 double mutated at F96W and N127W and a vector expressing a heterologous efflux proteinERG20 ^ww And is derived fromSalvia pomiferaAnd removing 1-34 amino acid residues from the N-terminal of the sabinene synthase,Salvia pomiferathe coding sequence of the sabinene synthase is shown in SEQ ID NO 9;

the heterologous efflux protein coding sequence is shown as SEQ ID NO. 2.

2. The recombinant saccharomyces cerevisiae strain of claim 1, wherein the recombinant vector comprises any one or more of the following forms of gene modules:

(1) terminator-ERG20 ^ww -bidirectional promoter-sabinene synthase coding sequence-terminator;

(2) terminator-sabinene synthase coding sequence-bidirectional promoter- ERG20 ^ww -a terminator;

(3) promoter- ERG20 ^ww -terminator-promoter-sabinene synthase coding sequence-terminator;

(4) promoter-sabinene synthase coding sequence-terminator-promoter-ERG20 ^ww -a terminator;

(5) promoter- ERG20 ^ww -sabinene synthase coding sequence-terminator;

(6) promoter-sabinene synthase coding sequence- ERG20 ^ww -a terminator;

(7) terminator- ERG20 ^ww -sabinene synthase coding sequence-reverse promoter;

(8) terminator-sabinene synthase coding sequence-ERG20 ^ww -a reverse promoter;

(9) terminator-bidirectional promoter-ERG 20^wwAnd a terminator, a fusion protein coding sequence of sabinene synthase;

(10) terminator-ERG 20^wwAnd a fusion protein coding sequence of sabinene synthase-bidirectional promoter-terminator;

(11) promoter-ERG 20^wwAnd a terminator, a fusion protein coding sequence of sabinene synthase;

(12) terminator-ERG 20^wwAnd a fusion protein coding sequence of sabinene synthase, a reverse promoter.

3. The recombinant strain of Saccharomyces cerevisiae according to any one of claims 1-2, wherein the recombinant vector further comprises 1 or more copies of additionalERG20 ^ww 。

4. The recombinant Saccharomyces cerevisiae strain of claim 1, wherein the yeast strain is endogenous to the strainERG20Is a weak promoter, which is endogenous to the yeast strainERG20The original promoter of (a) is a weak promoter.

5. Use of the recombinant strain of saccharomyces cerevisiae according to any one of claims 1-4 for the production of sabinene.

6. The method for constructing a recombinant saccharomyces cerevisiae strain for the production of sabinene according to claim 1, wherein the recombinant vector according to claim 1 and a vector expressing a heterologous efflux protein are transformed into a yeast strain to obtain the recombinant yeast strain.

7. The building method according to claim 6, further comprising:

construction of Yeast Strain endogenesisERG20Upstream homologous sequence of original promoter-Weak promoter-Yeast Strain endogenesisERG20Gene modules of homologous sequences downstream of the original promoter, or constructing yeast strains endogenouslyERG20Upstream homologous sequence of original promoter-Weak promoter-Yeast Strain endogenesisERG20Endogenous to the yeast strainERG20A gene module of a downstream homologous sequence;

replacing the endogenous yeast strain with the gene module by using a yeast homologous recombination mechanismERG20The original promoter is a weak promoter.

8. The construction method according to claim 6 or 7, further comprising:

respectively designing homologous sequences at two ends of a promoter, a coding sequence of the heterologous efflux protein and a terminator, then transforming the homologous sequences and the vector after enzyme digestion into the yeast strain, and carrying out homologous recombination to obtain a vector for expressing the heterologous efflux protein; wherein, two adjacent gene elements have homologous regions and can be connected by homologous recombination.

9. A method of producing sabinene, comprising:

step 1, inoculating the recombinant saccharomyces cerevisiae strain of any one of claims 1-4 into a culture medium for activation, and preparing a seed solution;

and 2, inoculating the seed solution into a culture medium, adding isopropyl myristate to perform two-phase culture, and collecting an upper organic phase of the culture medium after culture to separate sabinene.

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