CN109943584A - A kind of recombinant vector and restructuring yeast strains and its construction method and application for producing sabinene - Google Patents

Abstract

The present invention relates to gene engineering technology field, a kind of recombinant vector for producing sabinene and restructuring yeast strains and its construction method and application are disclosed.Recombinant plasmid of the present invention includes the sequence ERG20 of the ERG20 of coding F96W and N127W double mutations^ww, and from the sabinene synthase coding sequence of Salvia pomifera and/or Citrus jambhiri.The present invention selects the sequence ERG20 of the ERG20 of suitable double mutations^wwAnd external source sabinene synthase gene carries out matched combined, it constructs the recombinant vector for being able to produce sabinene and is transformed into yeast strain using the carrier, it is remarkably improved the ability of bacterial strain production sabinene, continues Optimizing Reconstruction on this basis, can further improve the yield of bacterial strain production sabinene.

Description

A kind of recombinant vector and restructuring yeast strains and its construction method for producing sabinene And application

Technical field

It is more particularly to a kind of for producing the recombinant vector of sabinene the present invention relates to gene engineering technology field And restructuring yeast strains and its construction method and application.

Background technique

Sabinene (bicyclic [3.1.0] hexane of Sabinene, 1- isopropyl -4- methylene, C₁₀H₁₆, molecular weight 136.23) It is a kind of naturally occurring bicyclic unsaturated monoterpene.Sabinene is used as always perfume additive, fine chemicals for a long time, simultaneously Because it is applied to pharmaceutical industry with good anti-inflammatory and antifungal activity.Researcher is developing it as biology combustion at present Material: sabinene chemical structure is more more compact than common hydrocarbon, so having very high density and the combustion heat, can be used as jet The important composition ingredient of formula aircraft fuel combination.

Currently, the main source of sabinene is to be extracted from plants, but extractible sabinene content is very low in plant, it is difficult full Foot industry needs, and needs to consume a large amount of natural resources.Therefore, Microbe synthesis is then with low cost, high yield and product peace Full property is considered as most promising production method.In the research of Microbe synthesis sabinene, used host is mainly big Enterobacteria and saccharomyces cerevisiae.2014, salty desert of Chinese Scientists et al. constructed the heterologous pathway for producing sabinene in Escherichia coli: Firstly, Escherichia coli have natural MEP approach, precursor substance GPP abundant can be provided for the synthesis of sabinene.In addition, mistake The natural gene IspA (coding farnesyl diphosphate synthase) in Escherichia coli is expressed, to increase the content of GPP；It introduces Mevalonate pathway present in saccharomyces cerevisiae, further to enrich precursor substance.Finally, passing through the screening to GPP synzyme And overexpression, sabinene synthase is introduced, has obtained the coli strain of high yield sabinene, shake flask fermentation is up to 82.18mg/L, fermentation Tank yield reaches 2.65g/L；2017, they obtained one plant of shake flask fermentation yield up to 150mg/ further through substrate MVA feeding patterns The Escherichia coli of L.2017, the system that Bowie et al. devises external synthesis monoterpene, this system introduced 27 kinds of enzymes and is used to Monoterpene is converted glucose into, and synthesizes terpenes using two kinds of confactor NAD (P) H and ATP, has finally synthesized 15.9g/L's Sabinene.

As generally acknowledged safe mode microorganism, genetic background understands saccharomyces cerevisiae, genetic manipulation is simple, can be advised greatly Mould fermenting and producing, and saccharomyces cerevisiae also possesses more powerful protein expression and posttranslational modification system and complete internal membrane system System, is more suitable for the expression of P450 albumen.Compared with Escherichia coli, its thallus vitamin, protein content is high, can make edible medicine With and fodder yeast；Compared with external synthesis, system is simple, at low cost, and in system enzyme stability it is high.Therefore, it realizes High yield of the sabinene in saccharomyces cerevisiae is particularly important.However, the utilization saccharomyces cerevisiae for disclosing report up to now synthesizes Chinese juniper The report of alkene is few.

Summary of the invention

In view of this, the purpose of the present invention is to provide a kind of for producing the recombinant vector and restructuring yeast strains of sabinene And its construction method and application, so that the recombinant vector, which is transformed into yeast strain, can significantly improve bacterial strain production sabinene Ability.

For achieving the above object, the invention provides the following technical scheme:

It is a kind of for producing the recombinant vector of sabinene, comprising encode F96W and N127W double mutations ERG20 sequence ERG20^ww, and from the sabinene synthase coding sequence of Salvia pomifera and/or Citrus jambhiri.

For existing production sabinene restructuring yeast strains, the present invention selects the sequence of the ERG20 of suitable double mutations ERG20^wwAnd external source sabinene synthase gene carries out matched combined, constructs the recombinant vector for being able to produce sabinene and utilizes the load Body is transformed into yeast strain, is remarkably improved the ability of bacterial strain production sabinene.

Preferably, the ERG20^wwWith sabinene synthase coding sequence according to following any one or two or more forms Netic module is embedded into the recombinant vector:

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

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

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

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

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

(6) promoter-sabinene synthase coding sequence-ERG20^wwTerminator；

(7) terminator-ERG20^wwSabinene synthase coding sequence-reverse starting；

(8) terminator-sabinene synthase coding sequence-ERG20^wwReverse starting；

(9) terminator-bidirectional promoter-ERG20^wwWith fusion protein coded sequence-terminator of sabinene synthase；

(10) terminator-ERG20^wwWith fusion protein coded sequence-bidirectional promoter-terminator of sabinene synthase；

(11) promoter-ERG20^wwWith fusion protein coded sequence-terminator of sabinene synthase；

(12) terminator-ERG20^wwWith fusion protein coded sequence-reverse starting of sabinene synthase；

Wherein, the bidirectional promoter is preferably GAL1&GAL10 promoter；The terminator preferably is selected from ADH1t terminator With TDH2t terminator, under general scenario, the multiple terminators and promoter in above-mentioned each netic module are different.

In the specific embodiment of the invention, the present invention uses ADH1t-ERG20^ww- GAL1&GAL10- sabinene synthase coding The netic module of sequence-TDH2t form；The recombinant vector is basic plasmid with YGG415 plasmid, and YGG415 plasmid map is shown in Fig. 1, plasmid complete genome sequence is as shown in SEQ ID NO:1；

The present invention chooses the sabinene synthase in the source of 7 kinds of separate sources, prepares under the premise of remaining condition is identical different heavy Group carrier, source includes Salvia pomifera, Salvia officinalis, Picea sitchensis, Thuja Plicata, Murraya koenigii, Citrus jambhiri and Litsea cubeba be successively abbreviated as SpSabS1, SoSabS1, PsSabS1, TpSabS1, MkSabS1, CjSabS1 and LcSabS1；The recombination yeast of different recombinant vectors will be converted Bacterial strain and blank yeast strain are by shake flask fermentation detection sabinene, the results show that conversion has containing SpSabS1 and CjSabS1 weight The bacterial strain of group carrier can output sabinene, and remaining bacterial strain shows that recombinant vector of the present invention can be mentioned significantly without sabinene output The sabinene production capacity of high bacterial strain.

In addition, the present invention compares different truncated sabinene synthase coding sequences, truncation mode is respectively to remove Chinese juniper The truncation sabinene synthase coding sequence of alkene synthase N-terminal 1-43 amino acids residue, to remove sabinene synthase N-terminal 1-34 amino acids residual The truncation sabinene synthase coding sequence of base, the truncation sabinene synthase code sequence for removing sabinene synthase N-terminal 1-52 amino acids residue The truncation sabinene synthase coding sequence for arranging, removing sabinene synthase 423-449 amino acids residue compiles each truncated sabinene synthase Code sequence different recombinant vectors are prepared under the premise of remaining condition is identical, by the restructuring yeast strains for converting different recombinant vectors with And restructuring yeast strains of the conversion containing complete sabinene synthase coding sequence detect sabinene by shake flask fermentation, the results show that with Conversion is compared containing the restructuring yeast strains of complete sabinene synthase coding sequence, and conversion, which contains, removes complete sabinene synthase N-terminal 1- Sabinene output increased nearly 5 times of the restructuring yeast strains of the truncation sabinene synthase coding sequence of 34 amino acids residues, and its Remaining truncation mode is without sabinene output.

According to above-mentioned technical effect, sabinene synthase coding sequence of the present invention is either complete sabinene synthase code sequence Column are also possible to remove the truncation sabinene synthase coding sequence of sabinene synthase N-terminal 1-34 amino acids residue.

Preferably, the ERG20^wwIt is carried out with the fusion protein of sabinene synthase by rigidity linker or flexibility linker Forward direction fusion or reversed fusion；Wherein, the rigidity linker amino acid sequence is GGGGS, the flexibility linker amino acid Sequence is PAPAP；The forward direction is fused to ERG20^wwRigid/flexible linker coded sequence-sabinene synthase coding sequence, institute It states and is reversely fused to sabinene synthase coding sequence-rigid/flexible linker coded sequence-ERG20^ww；

Preferably, recombinant vector of the present invention can also additional multilist up to 1 part or 2 parts or more of ERG20^ww, i.e. institute Stating further includes 1 or 2 or more the additional ERG20 copied on recombinant vector^ww.For easy consideration, described 1 or 2 with The additional ERG20 of upper copy^wwFor the promoter in aforementioned 12 kinds of netic modules and between terminator, inserted mode is logical for insertion position It crosses and designs identical restriction enzyme site realization.More specifically, described 1 or 2 or more the additional ERG20 copied^wwIt is inserted in aforementioned (9) or (10) plant the promoter in netic module and between terminators.

The recombinant vector is transformed into the sabinene production effect after yeast strain according to the present invention, and the invention proposes described Recombinant vector is preparing the application in the restructuring yeast strains for producing sabinene；Preferably, the yeast strain is preferably Wine brewing yeast strain, more preferably CEN.PK series Wine brewing yeast strain or BY series Wine brewing yeast strain.

According to the application of the recombinant vector, the present invention also correspondence provides a kind of for producing the recombinant yeast of sabinene Strain, conversion have the yeast strain of recombinant vector of the present invention.On this basis, the yeast strain endogenous ERG20 is opened Mover can be replaced weak promoter, and the weak promoter refers to that the original promoter relative to the endogenous ERG20 of yeast strain is weak opens Mover.In the specific embodiment of the invention, the weak promoter is HXT1p promoter.

Further improvement scheme to the restructuring yeast strains further includes that conversion has the carrier for expressing heterologous efflux protein. Wherein, the heterologous efflux protein is from sac fungus Grosmannia clavigera and/or from Yarrowia lipolytica The efflux protein of Yarrowia lipolytica；In the specific embodiment of the invention, the load of the heterologous efflux protein of expression Body is basic plasmid with PRS424, is embedded in promoter-efflux protein coded sequence-terminator netic module；More specifically, institute Stating promoter-efflux protein coded sequence-terminator netic module is GAL7- efflux protein coded sequence-GPDt；In the present invention In specific embodiment, the efflux protein coded sequence of the pathogen Grosmannia clavigera from pine is such as Shown in SEQ ID NO:2, it is abbreviated as SC-GcABCG1；It is described from Yarrowia lipolytica Yarrowia lipolytica's Efflux protein coded sequence is abbreviated as SC-YL-ABC2, SC-YL-ABC3 as shown in SEQ ID NO:3 or 4；

Meanwhile the sabinene shake flask fermentation test result based on recombinant vector of the present invention, the invention also provides described Application of the restructuring yeast strains in production sabinene.The method of specific production sabinene, comprising:

Step 1 activates restructuring yeast strains access culture medium, prepares seed liquor；

Seed liquor is inoculated in culture medium and is added isopropyl myristate progress two-phase culture by step 2, is collected after culture Culture medium upper organic phase isolates sabinene.

Wherein, the culture medium 40g/L glucose, 6.7g/LYNB, 2g/Ldrop-out remove the amino acid-mixed of leucine It closes object (SC-leu culture medium).

In the specific embodiment of the invention, the step 1 are as follows:

The restructuring yeast strains are chosen single colonie to be inoculated in 3mL SC-leu culture medium, 30 DEG C, 220rpm culture For 24 hours, primary seed solution is obtained；First order seed is transferred in 5mL SC-leu culture medium with initial OD 600=0.2,30 DEG C, 220rpm cultivates 12~18h, obtains secondary seed solution；

In the specific embodiment of the invention, the step 2 are as follows:

Secondary seed solution is transferred in 50mL SC-leu culture medium with initial OD 600=0.1, while being added 20% Isopropyl myristate carries out two-phase culture (220rpm, cultivates 96h by 30 DEG C).

In addition, the present invention also provides the construction method of the recombinant vector, constructed first containing promoter and terminator Expression cassette designs restriction enzyme site at the expression cassette both ends and between promoter and terminator, passes through digestion expression cassette two The restriction enzyme site at end and the carrier for having identical restriction enzyme site, expression cassette is linked into carrier；

Respectively in ERG20^wwAnd the both ends of sabinene synthase coding sequence are designed and the enzyme between the promoter and terminator The identical restriction enzyme site of enzyme site, or first construct ERG20^wwWith the coded sequence of the fusion protein of sabinene synthase, in ERG20^wwWith The both ends of sabinene synthase coding sequence are designed and the identical restriction enzyme site of restriction enzyme site between the promoter and terminator, lead to It crosses digestion mode to be linked into expression cassette, be thus connected in carrier, obtain recombinant vector.

Preferably, the-the second restriction enzyme site of the first restriction enzyme site-terminator-bidirectional promoter-third digestion position is first constructed The-the first restriction enzyme site of point-terminator, the-the second restriction enzyme site of the first restriction enzyme site-promoter-terminator-promoter-third enzyme The-the first restriction enzyme site of enzyme site-terminator, the-the second restriction enzyme site of the first restriction enzyme site-promoter-third restriction enzyme site-termination Sub-the first restriction enzyme site or the-the second restriction enzyme site of the first restriction enzyme site-terminator-third restriction enzyme site-reverse starting- First restriction enzyme site expression cassette；

By the first restriction enzyme site in digestion expression cassette both ends and carrier, expression cassette is linked into carrier；

In ERG20^wwThe second restriction enzyme site is designed at both ends, designs third digestion position at the both ends of sabinene synthase coding sequence Point, or in ERG20^wwThird restriction enzyme site is designed at both ends, designs the second restriction enzyme site at the both ends of sabinene synthase coding sequence, It is linked into expression cassette, is thus connected in carrier by digestion mode, obtain recombinant vector.

Wherein, described first any suitable restriction enzyme site, the first restriction enzyme site root be can choose to third restriction enzyme site Depending on the carrier to be accessed；In the specific embodiment of the invention, second restriction enzyme site and third restriction enzyme site are equal Selected from BsmBI restriction enzyme site and BsaI restriction enzyme site, second restriction enzyme site or third restriction enzyme site are back-to-back mode, That is two the second restriction enzyme sites or two third restriction enzyme sites are connected directly in the form of back-to-back, with BsmBI restriction enzyme site and For BsaI restriction enzyme site, schematic diagram can be found in Fig. 2.The knowledge of two BsmBI restriction enzyme sites or BsaI restriction enzyme site is shown in Fig. 2 Other sequence connects back-to-back, since the two restriction enzyme sites of BsmBI and BsaI identification sequence and cutting sequence are not same section of sequences Column, they are to cut after identifying sequence every a base, in this way can be taking human as the notch sequence after design cutting, the present invention The notch of design is catt and taaa, in access foreign gene (such as ERG20^wwWith sabinene synthase coding sequence) when can will be outer Source gene rear and front end is artificially designed as the restriction enzyme site of BsmBI or BsaI, and design notch is on aatg and ttta, with expression cassette Notch complementary pairing, be connected on expression cassette.

With ADH1t-ERG20^ww- GAL1&GAL10- sabinene synthase coding sequence-TDH2t netic module and YGG415 plasmid For illustrate the building process of recombinant vector of the present invention:

From BY4741 saccharomyces cerevisiae genome DNA cloning GAL1&GAL10 promoter, ADH1t and TDH2t terminator, simultaneously Design two back-to-back BsmBI restriction enzyme sites and two back-to-back BsaI restriction enzyme sites.By OE-PCR according to required suitable Sequence assembles PCR product, and the back-to-back BsmBI restriction enzyme site-GAL1&GAL10- of NotI restriction enzyme site-ADH1t- obtained is back-to-back The expression cassette of BsaI restriction enzyme site-TDH2t-NotI restriction enzyme site, NotI digestion expression cassette and YGG415 plasmid, expression cassette is connect Enter into carrier；

Then in ERG20^wwDesign BsmBI restriction enzyme site in both ends (can also design BsaI restriction enzyme site, as long as guaranteeing BsaI It is consistent with the notch that BsmBI people is design, expression cassette BsmBI digestion, ERG20^wwIt also can access expression cassette with BsaI digestion, this Sample the gene order Uniting of importing in need can be easier at BsaI restriction enzyme site), in sabinene synthase coding sequence Both ends design BsaI restriction enzyme site, ADH1t- back-to-back BsmBI digestion position is connected to by BsmBI digestion and BsaI digestion In the back-to-back BsaI restriction enzyme site-TDH2t expression cassette of point-GAL1&GAL10-, to complete the building of recombinant vector.

Preferably, further including accessing 1 or 2 or more the volume copied in the recombinant vector in the construction method Outer ERG20^ww, 1 or 2 or more the additional ERG20 copied is accessed more preferably on the expression cassette^ww, specific building side Formula can be realized by constructing identical restriction enzyme site.

The present invention is used to produce the construction method of the restructuring yeast strains of sabinene, directly converts the recombinant vector to ferment In mother strains, the restructuring yeast strains are obtained.

Preferably, in the recombinant bacterial strain building process further include:

It is endogenous to construct yeast strain endogenous ERG20 original promoter upstream homologous sequence-weak promoter-yeast strain The netic module of ERG20 original promoter downstream homologous sequence, or building yeast strain endogenous ERG20 original promoter upstream are same The netic module of the endogenous downstream the ERG20 homologous sequence of the endogenous ERG20- yeast strain of source sequence-weak promoter-yeast strain；

It is weak that the netic module, which is replaced the endogenous ERG20 original promoter of yeast strain using yeast homologous recombination mechanism, Promoter.

Preferably, in the recombinant bacterial strain building process further include:

Separately design the homologous sequence of promoter, the coded sequence of heterologous efflux protein and terminator both ends, then together with Carrier after digestion is transformed into the yeast strain, and homologous recombination is the carrier for expressing heterologous efflux protein；Wherein, adjacent There is homology region between two Genetic elements.

From the above technical scheme, the present invention selects the sequence ERG20 of the ERG20 of suitable double mutations^wwAnd it is outer Source sabinene synthase gene carries out matched combined, constructs the recombinant vector for being able to produce sabinene and is transformed into yeast using the carrier In bacterial strain, it is remarkably improved the ability of bacterial strain production sabinene, continues Optimizing Reconstruction on this basis, it is raw to can further improve bacterial strain Produce the yield of sabinene.

Detailed description of the invention

Fig. 1 show YGG415 plasmid map；

Fig. 2 show the schematic diagram of the back-to-back mode of BsmBI restriction enzyme site and the back-to-back mode of BsaI restriction enzyme site；A is BsmBI restriction enzyme site, B are BsaI restriction enzyme site；Arrow meaning is incision site, and shadow positions are back-to-back identification sequence；

Fig. 3 show the schematic diagram of recombinant vector of the present invention；

Fig. 4 show the schematic diagram of recombinant vector of the present invention；

Fig. 5 show the schematic diagram that the replacement endogenous ERG20 original promoter of bacterial strain is weak promoter；

Fig. 6 show the shake flask fermentation yield of the recombinant Saccharomyces cerevisiae of the sabinene synthase of expression separate sources；Abscissa table Show corresponding abbreviation in the title and lower section table of each bacterial strain；

Fig. 7 show the shake flask fermentation yield of the recombinant Saccharomyces cerevisiae of the different truncated SpSabS1 sabinene synthase of expression；It is horizontal Corresponding abbreviation in the title and lower section table of each bacterial strain of coordinate representation；

Fig. 8 show YGG416 plasmid map；

Fig. 9 show expression ERG20^wwPortion ERG20 is reached with sabinene synthase fusion protein and multilist^wwRecombination wine brewing The shake flask fermentation yield of yeast；Abscissa indicates the title of each bacterial strain；

Figure 10 show the shake flask fermentation yield of the recombinant Saccharomyces cerevisiae of the different heterologous efflux proteins of expression；Abscissa indicates Corresponding abbreviation in the title and lower section table of each bacterial strain；

Figure 11 show the shake flask fermentation yield for lowering the recombinant Saccharomyces cerevisiae of endogenous ERG20 expression；

Figure 12 show the shake flask fermentation yield of the recombinant Saccharomyces cerevisiae of different advantage Reconstruc-tion policy combinations of the invention.

Specific embodiment

The recombinant vector and restructuring yeast strains and its construction method and answer that the invention discloses a kind of for producing sabinene With those skilled in the art can use for reference present disclosure, be suitably modified realization of process parameters.In particular, it should be pointed out that all Similar replacement and change is apparent to those skilled in the art, they are considered as being included in the present invention.This It invents the recombinant vector, bacterial strain and construction method and application is described by preferred embodiment, related personnel Obviously can not depart from the content of present invention, in spirit and scope to recombinant vector as described herein, bacterial strain and construction method and Using being modified or appropriate changes and combinations, carry out implementation and application the technology of the present invention.

Sequence appeared in the present invention preferably through saccharomyces cerevisiae codon optimization and suitably evades common restriction enzyme Enzyme site；

ADH1t sequence is as shown in SEQ ID NO:5；

ERG20^wwSequence is as shown in SEQ ID NO:6；

GAL1&GAL10 sequence is as shown in SEQ ID NO:7；

TDH2t sequence is as shown in SEQ ID NO:8；

Salvia pomifera sabinene synthase coding sequence is as shown in SEQ ID NO:9；

Salvia officinalis sabinene synthase coding sequence is as shown in SEQ ID NO:10；

Picea sitchensis sabinene synthase coding sequence is as shown in SEQ ID NO:11；

Thuja plicata sabinene synthase coding sequence is as shown in SEQ ID NO:12；

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

Citrus jambhiri sabinene synthase coding sequence is as shown in SEQ ID NO:14；

Litsea cubeba sabinene synthase coding sequence is as shown in SEQ ID NO:15；

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

GAL7 sequence is as shown in SEQ ID NO:17；

GPDt sequence is as shown in SEQ ID NO:18；

GPM1t sequence is as shown in SEQ ID NO:19；

Shown in back-to-back BsmBI restriction enzyme site sequence SEQ ID NO:20；

Shown in back-to-back BsaI restriction enzyme site sequence SEQ ID NO:21；

Above-mentioned each Genetic elements sequence can be linked in sequence according to netic module of the present invention or expression cassette situation, i.e., For the sequence of corresponding netic module or expression cassette.

Below with reference to embodiment, the present invention is further explained.

Embodiment 1: recombinant vector of the present invention is constructed

1, the building and connection carrier process of expression cassette

From BY4741 saccharomyces cerevisiae genome DNA cloning GAL1&GAL10 promoter, ADH1t and TDH2t terminator, simultaneously Design two back-to-back BsmBI restriction enzyme sites and two back-to-back BsaI restriction enzyme sites.By OE-PCR according to required suitable Sequence assembles PCR product, and the back-to-back BsmBI restriction enzyme site-GAL1&GAL10- of NotI restriction enzyme site-ADH1t- obtained is back-to-back The expression cassette of BsaI restriction enzyme site-TDH2t-NotI restriction enzyme site, NotI digestion expression cassette and YGG415 plasmid, expression cassette is connect Enter into carrier；

2, the importing of foreign gene

(1)ERG20^wwExpression cassette is directed respectively into sabinene synthase coding sequence

In ERG20^wwBsmBI restriction enzyme site is designed at both ends, designs BsaI digestion position at the both ends of sabinene synthase coding sequence It is back-to-back to be connected to the back-to-back BsmBI restriction enzyme site-GAL1&GAL10- of ADH1t- by BsmBI digestion and BsaI digestion for point In BsaI restriction enzyme site-TDH2t expression cassette, to complete the building of recombinant vector, recombinant vector of the present invention is obtained YGG415-ADH1t-ERG20^ww- GAL1&10- sabinene synthase coding sequence-TDH2t；Schematic diagram is shown in Fig. 3；

(2)ERG20^wwExpression cassette is imported with the coded sequence of sabinene synthase fusion protein

By ERG20^wwWith sabinene synthase coding sequence, using the coded sequence or flexibility linker of rigid linker GGGGS The coded sequence of PAPAP carries out fusion connection forward or backwards, designs BsaI restriction enzyme site at fusion protein sequence both ends, passes through BsaI digestion is connected to the back-to-back BsaI restriction enzyme site-TDH2t table of the back-to-back BsmBI restriction enzyme site-GAL1&GAL10- of ADH1t- Up in box, to complete the building of recombinant vector, recombinant vector YGG415-ADH1t-GAL1&10- of the present invention is obtained ERG20^wwWith the coded sequence-TDH2t of sabinene synthase fusion protein；Schematic diagram is shown in Fig. 4；

(3) multilist reaches portion ERG20^wwBuilding process

On the basis of (2), in ERG20^wwBsmBI restriction enzyme site is designed at both ends, is connected to ADH1t- by BsmBI digestion Back-to-back BsmBI restriction enzyme site-GAL1&GAL10-ERG20^wwWith the coded sequence-TDH2t expression cassette of sabinene synthase fusion protein In, to complete the building of recombinant vector, obtain recombinant vector YGG415-ADH1t-ERG20 of the present invention^ww-GAL1&10- ERG20^wwWith the coded sequence-TDH2t of sabinene synthase fusion protein；

Wherein, sabinene synthase coding sequence is the Chinese juniper from Salvia pomifera and/or Citrus jambhiri Alkene synthase coding sequence；Or to be compiled in the sabinene synthase from Salvia pomifera and/or Citrus jambhiri In code sequence basis, the truncation sabinene synthase coding sequence of N sections of 1-43 amino acids residue coded sequences is removed.

Embodiment 2: recombinant Saccharomyces cerevisiae bacterial strain of the present invention is constructed

1, chassis bacterial strain

Chassis bacterial strain is selected as YJGZ1, is to pass through knockout on the basis of commercially available type strain CEN.PK2-1C It has been overexpressed precursor-gene IDI1 and tHMGR1 while GAL80, that is, has increased the supply of precursor GPP, more to provide Chinese juniper The substrate that alkene synthesis needs.Specific remodeling method is as follows:

From BY4741 genomic DNA amplification P_GAL1,10Promoter, T_ADH1And T_TDH2Terminator and HIS3 label and Gal80p 400bp homology arm up and down.PCR product is assembled according to required sequence by OE-PCR, is obtained as Gal80up-T_ADH1-P_GAL1,10- T_TDH2The expression cassette of-His-Gal80down.Then expression cassette with NotI-HF digestion and is inserted into the identical bits of pRS415K Point generates plasmid pJGZ3, in T_ADH1And P_GAL1,10Between tool there are two the back-to-back site BsmBI, P_GAL1,10And T_TDH2It Between there are two the back-to-back site BsaI.With BsmBI digestion from the IDI1 of BY4741 genomic DNA amplification, and it is connected to identical To generate pJGZ4 in the pJGZ3 of restriction enzyme site.Segment Gal80up-T is cut from pJGZ4 by NotI-HF_ADH1-IDI1- P_GAL1,10-T_TDH2- His-Gal80down and the pEASY- without kalamycin resistance gene and the site BsaI for being inserted into reconstruction To generate plasmid pJGZ5 in Blunt carrier.It then, will be restricted by BsaI from the tHMGR of BY4741 genomic DNA amplification Site is inserted into pJGZ5 to obtain pJGZ6.Finally, by the integration segment Gal80up- of the NotI-HF cutting from pJGZ6 T_ADH1-IDI1-P_GAL1,10-tHMGR-T_TDH2- His-Gal80down is transformed into CEN.PK2-1C, obtains YJGZ1.

YJGZ1 selected by the present embodiment is the acquired bacterial strain of systematicness early period research work, is directly adopted for convenience With, it is not necessary to it uses, in subsequent comparison bacterial strain, the present invention will be carried out equally pair using this kind of bacterial strain as blank bacterial strain Than itself can't generate sabinene.

2, the recombinant vector is converted

Recombinant vector of the present invention is transferred in YJGZ1 using lithium acetate transformation method, SC-LEU solid is used after conversion (synthetic yeast nitrogen source YNB 6.7g/L, glucose 20g/L lack the kilnitamin powder 2g/L of leucine, 2% agar to plate Powder) it is screened, obtained transformant carries out extracting yeast plasmid progress PCR verifying after scribing line divides pure culture, correct to verifying Recombinant bacterial strain save, be recombinant Saccharomyces cerevisiae of the present invention.

3, the expression of heterologous efflux protein gene

On the basis of the recombinant Saccharomyces cerevisiae described in 2, conversion can express the recombinant vector of efflux protein.It can express outer The construction of recombinant vector method for arranging albumen is as follows:

Firstly, using Not I and BamH I s double digestion carrier PRS424.Then, design primer is by expression cassette GPM1t- GAL7-GPDt (system early period scientific research of the invention has obtained, and directlys adopt for convenience) is divided into GPM1t-GAL7 and two sections of GPD PCR is carried out, and separately designs homologous sequence between outlet gene SC-GcABCG1, SC-YL-ABC2, SC-YL-ABC3, makes phase Possess the homology region of 20~40bp between two adjacent Genetic elements, to utilize yeast homologous recombination splicing carrier Then PRS424 is transformed into yeast strain, use SC-LEU-TRP (synthetic yeast nitrogen according to the screening label of PRS424 institute band Source YNB 6.7g/L, glucose 20g/L lack the kilnitamin powder 2g/L of tryptophan and leucine, 2% agar powder) it is solid Body plate is screened, and obtained transformant carries out extracting yeast plasmid progress PCR verifying after scribing line divides pure culture, obtains the present invention The recombinant Saccharomyces cerevisiae.

4, the expression of the endogenous ERG20 of YJGZ1 is lowered

On the basis of the recombinant Saccharomyces cerevisiae obtained in 2 or 3, design primer is using yeast YJGZ1 complete genome DNA as template Amplified fragments HXT1p and endogenous ERG20 (can also expand acquisition by other means) selects URA3 nutritional labeling loxP-URA3- LoxP segment possesses the homology region of 20~40bp between two neighboring segment, then obtains segment by Overlap extension PCR LoxP-URA3-loxP-HXT1p-ERG20 replaces the ERG20p-ERG20 on YJGZ genome, and schematic diagram is shown in Fig. 5, after conversion Using SC-URA solid panel, (synthetic yeast nitrogen source YNB 6.7g/L, glucose 20g/L lack the kilnitamin of uracil to yeast Powder 2g/L, 2% agar powder) screened, obtained transformant carry out scribing line divide after pure culture extract Yeast genome into Row PCR verifying, it is subsequent URA3 nutritional labeling to be recycled by 5- fluororotic acid plate, obtain recombination wine brewing ferment of the present invention It is female.

Embodiment 3: the shake flask fermentation test of different recombinant Saccharomyces cerevisiae bacterial strains

(1) the sabinene synthase coding sequence of separate sources

According to " (1) ERG20 in embodiment 1^wwBe directed respectively into expression cassette with sabinene synthase coding sequence " method building weight Group carrier, sabinene synthase coding sequence are respectively derived from Salvia pomifera, Salvia officinalis, Picea Sitchensis, Thuja plicata, Murraya koenigii, Citrus jambhiri and Litsea cubeba are successively It is abbreviated as SpSabS1, SoSabS1, PsSabS1, TpSabS1, MkSabS1, CjSabS1 and LcSabS1, by 7 kinds of recombinant vectors It is transformed into identical YJGZ1 bacterial strain, while setting blank control bacterial strain YJGZ1, as follows:

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；

It is that chassis bacterium YJGZ1 carries out shaking flask to SyBE_Sc04100001-SyBE_Sc04100007 and blank control bacterial strain Fermentation.

Seed culture medium: 40g/L glucose；6.7g/LYNB；2g/Ldrop-out removes the amino acid mixing of leucine Object.

Fermentation medium: 40g/L glucose；6.7g/LYNB；2g/Ldrop-out removes the amino acid mixing of leucine Object.

Above-mentioned bacterial strains are chosen single colonie to be inoculated in 3mL SC-leu culture medium, 30 DEG C, 220rpm is cultivated for 24 hours.By level-one Seed is transferred in 5mL SC-leu culture medium with initial OD 600=0.2, and 30 DEG C, 220rpm cultivates 12~18h.By second level kind Son is transferred in 50mL SC-leu culture medium with initial OD 600=0.1, while 20% isopropyl myristate progress two is added Mutually culture (220rpm, cultivates 96h by 30 DEG C).

Sabinene quantitative approach: fermentation termination takes fermentation medium upper organic phase 1mL, and anhydrous sodium sulfate is added and is statically placed in four Refrigerator is spent to remove water 4~5 hours.Organic phase is filtered using 2 μm of organic filter membranes after water removal.Then gas Chromatographic Determination: Chinese juniper is used The boiling point of alkene is 164 DEG C of at 760mmHg, so detector temperature is set as 260 DEG C, 250 DEG C of injection port, 250 DEG C of column temperature.Color Spectral condition is DB-5 silica gel capillary chromatographic column, 1 μ L of sample volume；50 DEG C of column temperature initial temperature, keep 4min；Then with 5 DEG C min^-1100 DEG C are risen to, 1min is kept；Finally with 25 DEG C of min^-1250 DEG C are risen to, 5min is kept；

As a result see Fig. 6, be not detected sabinene chromatographic peak in blank control bacterial strain YJGZ1, bacterial strain SyBE_Sc04100001 and SyBE_Sc04100006 detects that the chromatographic peak of appearance time identical as standard items, remaining bacterial strain are not detected in 12.098min Sabinene chromatographic peak, it was demonstrated that bacterial strain SyBE_Sc04100001 and SyBE_Sc0410000 realize the synthesis of sabinene, and SyBE_ Sc04100001 yield is 0.519mg/L, is higher than bacterial strain SyBE_Sc04100006.Therefore the Chinese juniper in the source SpSbS1 and CjSabS1 Alkene synthase coding sequence is remarkably improved the sabinene synthesis capability of bacterial strain, wherein being best with the source SpSbS1.

(2) the sabinene synthase coding sequence of different truncation modes

Based on the sabinene synthase coding sequence in the source SpSbS1, carries out following four kinds of truncations operation and obtain different truncations Sabinene synthase coding sequence:

T1SpSabS1: remove the truncation sabinene synthase coding sequence of sabinene synthase N-terminal 1-43 amino acids residue；

T2SpSabS1: remove the truncation sabinene synthase coding sequence of sabinene synthase N-terminal 1-34 amino acids residue；

T3SpSabS1: remove the truncation sabinene synthase coding sequence of sabinene synthase N-terminal 1-52 amino acids residue；

T4SpSabS1: remove the truncation sabinene synthase coding sequence of sabinene synthase 423-499 amino acids residue；

By above-mentioned each truncated sabinene synthase coding sequence respectively according to " (1) ERG20 in embodiment 1^wwIt is compiled with sabinene synthase Code sequence is directed respectively into expression cassette " method obtains 4 kinds of different recombinant vectors, and is transferred in YJGZ1, bacterial strain is obtained in order SyBE_Sc04100008, SyBE_Sc04100009, SyBE_Sc04100010 and SyBE_Sc04100011, then together with before The SyBE_Sc04100001 of acquisition carries out shake flask fermentation test；

As a result see Fig. 7, the recombination wine brewing bacterial strain SyBE_Sc04100009 that only the truncation mode of 1~34 amino acids obtains Make nearly five times of sabinene output increased, sabinene yield has reached 2.57mg/L, is control strain SyBE_Sc04100001 yield 4.95 again.Its excess-three kind truncates bacterial strain SyBE_Sc04100008, SyBE_Sc04100010 and SyBE_ that mode obtains Sc04100011 does not have sabinene synthesis.

(3)ERG20^wwPortion ERG20 is reached with sabinene synthase coding sequence amalgamation and expression and multilist^ww

Respectively with flexible linker (shown in GGGGS, SEQ ID NO:22) and rigidity linker (PAPAP, SEQ ID NO: Shown in 23) coded sequence ERG20ww be subjected to forward and reverse with t2SpSabS1 respectively merge, referring to embodiment 1 " (2) The method of the coded sequence of ERG20ww and sabinene synthase fusion protein importing expression cassette " constructs different recombinant vectors and is transferred to In YJGZ1, yeast is screened using SC-LEU solid panel after conversion, and obtained transformant extract after scribing line divides pure culture Yeast plasmid carries out PCR verifying, correctly positive to verifying to merge flexibility linker connection bacterial strain, reversed fusion flexibility linker Connection bacterial strain, positive fusion rigidity linker connection bacterial strain and reversely merge rigidity linker connect bacterial strain preservation glycerol stock and according to It is secondary to be named as SyBE_Sc04100012, SyBE_Sc04100013, SyBE_Sc04100014, SyBE_Sc04100015；

Referring to embodiment 1, " (3) multilist reaches portion ERG20^wwBuilding process " method construct in SyBE_ Multilist reaches portion on the basis of Sc04100012, SyBE_Sc04100013, SyBE_Sc04100014, SyBE_Sc04100015 ERG20^wwRecombinant bacterial strain, be successively named as SyBE_Sc04100016, SyBE_Sc04100017, SyBE_Sc04100018, SyBE_Sc04100019；

Portion ERG20 is reached to exclude multilist^wwBring copy number difference, constructs positive control strain, i.e., with bacterial strain SyBE_Sc04100009 is starting strain, expresses a ERG20 more^wwIn carrier YGG416, (plasmid map is shown in Fig. 8, entirely for building Gene order is similar with YGG415 as shown in SEQ ID NO:24) on, specific construction method: use Sal I and Bam H I pair The YGG415-ADH1t-ERG20 that digestion has constructed^ww(embodiment 1 can obtain-GAL1&10-TDH2t during constructing recombinant vector ) obtain segment ADH1t-ERG20^ww- GAL1&10-TDH2t is connected into using the carrier YGG416 after I double digestion of Sal I and Bam, Obtain YGG416-ADH1t-ERG20^wwThen-GAL1&10-TDH2t is transformed into bacterial strain SyBE_Sc04100009, using SC- LEU-URA solid panel is screened, and obtained transformant carries out extracting yeast plasmid progress PCR verifying after scribing line divides pure culture, Glycerol stock name SyBE_Sc04100020 is saved to correct recombinant bacterial strain is verified.

Above-mentioned SyBE_Sc04100009, SyBE_Sc04100012-SyBE_Sc04100020 are subjected to shake flask fermentation examination It tests, Fig. 9 is the results show that control strain SyBE_Sc04100009 yield is 2.23mg/L.For flexible linker (GGGGS), just It is 1.94mg/L to fusant bacterial strain SyBE_Sc04100012 yield, reversed fusant bacterial strain SyBE_Sc04100013 yield is 2.56mg/L so reversed fusion ratio forward direction syncretizing effect will be got well, and adds a ERG20 in reversely fusion Quito^wwEffect more Good, i.e., bacterial strain SyBE_Sc04100017 yield is 3.18mg/L.Speculate that the N-terminal that reason is t2SpSabS1 has positive charge, inhales Leader tape has the ERG20 of another copy of negative electrical charge^ww, thus precursor can be made full use of to accelerate reaction.And ERG20^ww/ The positive fusion of tSpSabS1 can expose negative electrical charge, the ERG20 with another copy^wwInstitute is electrically charged identical.

For rigid joint (PAPAP), positive fusant bacterial strain SyBE_Sc04100014 yield 0.146mg/L is reversed to merge Bacterial strain SyBE_Sc04100015 yield is 2.17mg/L, and yield wants low compared with control strain SyBE_Sc04100009, but right Portion ERG20 is reached in multilist^wwBacterial strain SyBE_Sc041000018, SyBE_Sc041000019, yield is respectively 4.39mg/L And 3.31mg/L.This shows that the effect of fusion is not it is obvious that in comparison, multilist reaches for using rigid joint (PAPAP) A ERG20^wwMore propose production effect.

Meanwhile SyBE_Sc04100009 and SyBE_Sc04100020 is compared, SyBE_Sc04100020 yield is 2.33mg/L, it is little with control strain 2.23mg/L difference, multilist is eliminated up to portion ERG20^wwBring copy number difference.

Embodiment 4: the influence of heterologous outlet gene expression

Using bacterial strain SyBE_Sc04100009 as starting strain, referring to embodiment 2 expression of heterologous efflux protein gene " 3, " In method building expression three kinds of heterologous efflux proteins of difference restructuring yeast strains, be successively named as SyBE_Sc04100021 (SC-GcABCG1), SyBE_Sc04100022 (SC-YL-ABC2), SyBE_Sc04100023 (SC-YL-ABC3) carry out shaking flask As a result Fig. 9 is shown in fermentation test；

Figure 10 is the results show that control strain SyBE_Sc04100009 yield is 2.23mg/L, bacterial strain SyBE_ Sc04100021 yield is 4.57mg/L, and bacterial strain SyBE_Sc04100022 yield is 3.70mg/L, bacterial strain SyBE_ Sc04100023 yield is 2.54mg/L.SC-YL-ABC2, SC-YL-ABC3 and SC-GcABCG1 in comparison, SC-GcABCG1 Good outlet effect is reached, sabinene yield improves about one times.

Embodiment 5: the influence of the expression of endogenous ERG20 is lowered

Referring to embodiment 2 " 4, lower the endogenous ERG20 of YJGZ1 expression " in method, construct recombinant Saccharomyces cerevisiae bacterial strain SyBE_Sc041000025 simultaneously carries out shake flask fermentation test, and conversion has recombinant vector of the present invention in SyBE_Sc041000025 YGG415-ADH1t-ERG20ww-GAL1&10-t2SpSabS1-TDH2t；

Figure 11 the results show that control strain SyBE_Sc04100009 (its conversion has recombinant vector YGG415- of the present invention ADH1t-ERG20ww-GAL1&10-t2SpSabS1-TDH2t) yield is 2.03mg/L, bacterial strain SyBE_Sc04100025 yield For 4.19mg/L, shows that the downward of endogenous ERG20 can effectively reduce GPP and be changed into FPP, reduces the shunt metabolism in downstream, To which more GPP are used to synthesize sabinene.

Embodiment 6: the recombinant Saccharomyces cerevisiae bacterial strain of advantage integration

By the advantage Reconstruc-tion policy integration in Examples 1 and 2, recombinant Saccharomyces cerevisiae is constructed according to combination in table 1, still Using YJGZ1 as chassis bacterial strain.

Table 1

Constructed SyBE_Sc04100026-SyBE_Sc04100029 bacterial strain is subjected to shake flask fermentation test, as a result such as Shown in Figure 12, from yield, ferment 96 hours, bacterial strain SyBE_Sc04100029 yield highest has reached 17.57mg/L.

The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered It is considered as protection scope of the present invention.

Sequence table

<110>University Of Tianjin

<120>a kind of recombinant vector and restructuring yeast strains and its construction method and application for producing sabinene

<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 (13)

1. a kind of for producing the recombinant vector of sabinene, which is characterized in that include coding F96W and N127W double mutations The sequence ERG20 of ERG20^ww, and from the sabinene synthase coding of Salvia pomifera and/or Citrus jambhiri Sequence.

2. recombinant vector according to claim 1, which is characterized in that include the base of any one or two or more forms as follows Because of module:

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

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

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

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

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

(6) promoter-sabinene synthase coding sequence-ERG20^wwTerminator；

(7) terminator-ERG20^wwSabinene synthase coding sequence-reverse starting；

(8) terminator-sabinene synthase coding sequence-ERG20^wwReverse starting；

(9) terminator-bidirectional promoter-ERG20^wwWith fusion protein coded sequence-terminator of sabinene synthase；

(10) terminator-ERG20^wwWith fusion protein coded sequence-bidirectional promoter-terminator of sabinene synthase；

(11) promoter-ERG20^wwWith fusion protein coded sequence-terminator of sabinene synthase；

(12) terminator-ERG20^wwWith fusion protein coded sequence-reverse starting of sabinene synthase.

3. recombinant vector according to claim 1 or claim 2, which is characterized in that the sabinene synthase coding sequence is removal sabinene The truncation sabinene synthase coding sequence of synthase N-terminal 1-34 amino acids residue.

4. recombinant vector according to claim 1 to 3, which is characterized in that the recombinant vector further includes 1 or 2 A above additional ERG20 copied^ww。

5. recombinant vector described in claim 1-4 any one is preparing answering in the restructuring yeast strains for producing sabinene With.

6. a kind of for producing the restructuring yeast strains of sabinene, which is characterized in that conversion has the right to require described in 1-4 any one The yeast strain of recombinant vector.

7. restructuring yeast strains according to claim 6, which is characterized in that the promoter of the endogenous ERG20 of yeast strain For weak promoter, the weak promoter is weak promoter relative to the original promoter of the endogenous ERG20 of yeast strain.

8. restructuring yeast strains described according to claim 6 or 7, which is characterized in that further include that conversion has the heterologous outlet egg of expression White carrier.

9. application of the restructuring yeast strains described in claim 6-8 any one in production sabinene.

10. for producing the construction method of the restructuring yeast strains of sabinene described in claim 6, which is characterized in that right It asks recombinant vector described in 1-4 any one to convert into yeast strain, obtains the restructuring yeast strains.

11. construction method according to claim 10, which is characterized in that further include:

It is former to construct the endogenous ERG20 of yeast strain endogenous ERG20 original promoter upstream homologous sequence-weak promoter-yeast strain The netic module of beginning promoter downstream homologous sequence, or building yeast strain endogenous ERG20 original promoter upstream homologous sequence- The netic module of the endogenous downstream the ERG20 homologous sequence of the endogenous ERG20- yeast strain of weak promoter-yeast strain；

It is weak starting that the netic module, which is replaced the endogenous ERG20 original promoter of yeast strain using yeast homologous recombination mechanism, Son.

12. 0 or 11 construction method according to claim 1, which is characterized in that further include:

The homologous sequence for separately designing promoter, the coded sequence of heterologous efflux protein and terminator both ends, then together with digestion Carrier afterwards is transformed into the yeast strain, and homologous recombination is the carrier for expressing heterologous efflux protein；Wherein, adjacent two There is homology region between Genetic elements, it can homologous recombination connection.

13. a kind of method for producing sabinene characterized by comprising

Step 1 activates the access culture medium of restructuring yeast strains described in claim 6-8 any one, prepares seed liquor；

Seed liquor is inoculated in culture medium and is added isopropyl myristate progress two-phase culture by step 2, and culture is collected after culture Epibasal tier organic phase isolates sabinene.