CN108118007A - A kind of method and its genetic engineering bacterium of genetic engineering bacterium production beta carotene - Google Patents

Abstract

The invention discloses the methods and its genetic engineering bacterium of a kind of genetic engineering bacterium production bata-carotene.The genetic engineering bacterium is a kind of containing the Ye Luowei of carRA genes and carB genes solution fat yeast (Yarrowia lipotica) genetic engineering bacteriums.The present invention solves fat yeast to Ye Luowei by gene engineering method and introduces from Blakeslea trispora (Blakeslea trispora) carRA genes and carB genes, further enhances double fragrant youngster's base pyrophosphate synthases (GGS) and 3 hydroxyl, 3 methyl vinyl CoA-reductase (HMG CoA^cataReductase expression), knock out 3 phosphoglyceride dehydrogenase (Gut2) and peroxidase (Pox2), obtain the Ye Luowei solution fat yeast strains of heterogenous expression bata-carotene, fermentation condition is simple, bata-carotene yield is higher, has important productive value.

Description

A kind of method and its genetic engineering bacterium of genetic engineering bacterium production beta carotene

Technical field

The present invention relates to biological technical fields, and in particular to a kind of method of genetic engineering bacterium production beta carotene and its Genetic engineering bacterium.

Background technology

Beta carotene is a kind of fat-soluble carotenoid of crocus, is FAO (Food and Agriculture Organization of the United Nation) and world health group Knit the nutraceutical additive of the A classes of food additives joint specialist committee identification.In human body, beta carotene is dimension life The precursor of plain A；The in vivo free radical of antioxidant for clearing can be used as, reduces the risk of Aged Heart Failure；Excitation is immune System has the function of anti-cancer and anticancer.In addition beta carotene can be also used for food colour, animal feed additive, health care In product, cosmetics and drug.The market demand of beta carotene is continuously increased at present, its application field also turns from food color To three big field of nutraceutical, health products and drug.

Beta carotene prepares production, and there are three types of methods：Chemical synthesis；Natural extraction method；Microbe fermentation method.Using change It is almost trans isomer form to learn the artificial synthesized beta carotene of synthetic method, does not possess many natural beta-carotins Physiological function, it is impossible to be absorbed by the body completely, long-term use can generate a degree of toxic side effect to human body, with people couple The beta carotene of the attention chemical synthesis of green natural food is not received by most of Food Science men.It is extracted from plant Natural beta-carotin is limited by conditions such as material content, weather, the places of production, and complex process, it is difficult to mass production, using also receiving To limitation.

Microbe fermentation method is easily cultivated because of the influence from conditions such as environment, and yield is high and is subject to the country extensively Outer attention is the best method for producing beta carotene.The common bacterial strain that ferments is broadly divided into three classes：Algae, yeast and true Bacterium.The relatively low requirement for being unable to reach industrialized production of the yield of algae and yeast.

Blakeslea trispora (Blakeslea trispora) is the preferred bacterial strain of current production beta carotene, but with three The medium component of the mould producing beta-carotene by fermentation of spore Bradley is complicated, the cost is relatively high.Blakeslea trispora is mycelium anastomosis Bacterium, the mode for carrying out zoogamy are heterothallism, i.e. the origin source "+" "-" bacterial strain different with property could be merged mutually, into Row zoogamy.Zygosperm usually carries out meiosis when generating, and then generates the sporangium containing haplospore.Induction The hormone of suspensor becomes trisporic acid, and the major function of trisporic acid is induction zoogamy, promotes the biology conjunction of beta carotene Into.In the industrial production, it is often required to consider the proportioning of "+" "-" bacterial strain, fermentation condition is relative complex, this also gives industrial production band Carry out many unnecessary difficulties.

The content of the invention

The present invention is directed to the relatively low deficiency of Blakeslea trispora producing beta-carotene by fermentation yield in the prior art, A kind of method and its genetic engineering bacterium of genetic engineering bacterium production beta carotene, genetic engineering bacterium production β-Hu are provided Radish element yield is higher, and fermentation condition is simple.

The technical solution that the present invention solves above-mentioned technical problem is as follows：

One of technical scheme is：One kind containing Lycopene synthase and lycopene cyclase (carRA) gene/ Ye Luowei solution fat yeast (Yarrowia lipotica) genetic engineering bacteriums of phytoene dehydrogenase (carB) gene.

Wherein, Lycopene synthase/lycopene cyclase (carRA) gene is conventional, it is preferred that source In carRA (Lycopene synthase and lycopene cyclase) gene of Blakeslea trispora beta carotene route of synthesis, more preferably, It is after doing corresponding optimization according to the codon preference of Ye Luowei solution fat yeast, its optimal nucleotide is shown in sequence table Shown in SEQ ID NO.3.

Wherein, the phytoene dehydrogenase (carB) gene is conventional, it is preferred that from Blakeslea trispora β- CarB (phytoene dehydrogenase) gene of carotene synthetic pathway；More preferably, be according to Ye Luowei solve fat yeast codon it is inclined After good property does corresponding optimization, its optimal nucleotide sequence is as shown in SEQ ID NO.4 in sequence table.

Wherein, the preparation method of Ye Luowei solutions fat yeast gene engineering bacteria is conventional preferably to use gene work Journey means build and obtain, and more preferably comprise the following steps：CarRA genes will be contained and carB gene recombined vectors import Ye Luowei Solve fat yeast.

Preferably, the Ye Luowei solution fat yeast gene engineering bacteria is also further containing double fragrant youngster's base pyrophosphate synthases (GGS) gene.

Wherein, described double fragrant youngster's base pyrophosphate synthase (GGS) genes are conventional, preferably solve fat from Ye Luowei Yeast ATCC20362, more preferably its nucleotide sequence are as shown in sequence table SEQ ID NO.2.

Wherein, the preparation method of Ye Luowei solutions fat yeast gene engineering bacteria is conventional preferably to use gene work Journey means build and obtain, and more preferably comprise the following steps：By the recombinant vector containing carRA genes, carB genes and GGS genes Import Ye Luowei solution fat yeast.

Wherein, the recombinant vector containing carRA genes, carB genes and GGS genes, preferably, by include with The method of lower step builds and obtains：CarRA gene, carB gene of the Clone Origin in Blakeslea trispora, at its upstream and downstream Connection becomes complete Expression element from the promoter and terminator of Ye Luowei solution fat yeast, and with GGS genes Expression element is integrated on a plasmid, that is, the recombinant vector obtained.The recombinant vector can be disposably by gene integration to sieve On dimension solution fat Yeast genome.Preferably, the recombinant vector is plasmid pS-B-RA-GGS-F-U (L).

Further more preferably, the carB genes, carRA genes and GGS genes solve fat yeast in the Ye Luowei Copy number in genetic engineering bacterium is more than 1, is preferably 2-100 or even 1000.

Wherein, the Ye Luowei solution fat yeast genes works of the carB genes of high copy number, carRA genes and GGS genes are obtained The method of journey bacterium is conventional, can preferably import the recombinant expression carrier of multicopy number foreign gene or with more The recombinant expression carrier in whole site imports foreign gene.

Further more preferably, Ye Luowei solutions fat yeast gene engineering bacteria also further contains HMG-CoA reductase Active site gene (HMG^cata)。

Wherein, the HMG-CoA^cataReductase is conventional, the preferably HMG-CoA from Ye Luowei solution fat yeast The catalytic site of reductase, more preferably HMG-CoA reductase.

Wherein, the preparation method of Ye Luowei solutions fat yeast gene engineering bacteria is with routine, preferably, using genetic engineering Means build and obtain, and more preferably, comprise the following steps：With containing HMG-CoA reductase active site gene (HMG^cata) weight Group expression vector imports Ye Luowei solution fat yeast.Wherein, the recombinant expression carrier is preferably plasmid pSK-Hmg (y)-Ura3-IntB, nucleotide sequence is as shown in SEQ ID NO.6 in sequence table.

Further more preferably, Ye Luowei solutions fat yeast gene engineering bacteria has also further knocked out glycerol 3-phosphate and has taken off Hydrogen enzyme (Gut2) gene.The knockout technique is conventional, preferably, comprising the following steps：Using plasmid pS-B-RA- GGS-Gut2-U (L) is knocked out, and the nucleotide sequence of the plasmid is as shown in SEQ ID NO.7 in sequence table.

Further more preferably, Ye Luowei solutions fat yeast gene engineering bacteria has also further knocked out peroxidase (Pox2) gene.The knockout technique is conventional, is preferably comprised the following steps：Using plasmid pS-B-RA-GGS- Pox2-U (L) is knocked out, and the nucleotide sequence of the plasmid is as shown in SEQ ID NO.8 in sequence table.

In the present invention, Ye Luowei solution fat yeast is conventional.

A kind of the two of technical scheme, method of genetic engineering bacterium production beta carotene, comprise the following steps：

1) prepare containing carRA (Lycopene synthase and lycopene cyclase) genes and carB (phytoene dehydrogenase) base The Ye Luowei solution fat yeast gene engineering bacterias of cause；

2) the Ye Luowei solution fat yeast gene engineering bacterias obtained by fermented and cultured step 1), obtain β-carrot from zymotic fluid Element.

Ye Luowei solution fat yeast gene engineering bacterias described in step 1) of the present invention are as described above.

Fermented and cultured described in step 2) of the present invention it is rapid 1) obtained by Ye Luowei solution fat yeast gene engineering bacterias method Conventional Ye Luowei solution fat yeast fermentation process ferments in conventional Ye Luowei solution fat yeast fermentation broths, fermentation condition It is conventional with parameter.Preferably it is included in YPD fluid nutrient mediums Ye Luowei described in 28 DEG C of shake cultures and solves fat yeast genes Engineering bacteria.The method that beta carotene is obtained in slave zymotic fluid described in step 2) is also conventional.

On the basis of common knowledge of the art, above-mentioned each optimum condition can be combined to get each preferable reality of the present invention Example.

The reagents and materials used in the present invention are commercially available.

The positive effect of the present invention is：The present invention solves fat yeast by gene engineering method to Ye Luowei (Yarrowia lipotica) introduces red from Blakeslea trispora (Blakeslea trispora) beta carotene gene eggplant Plain synthase and lycopene cyclase (carRA) and phytoene dehydrogenase (carB) gene further enhance double fragrant youngster's base coke phosphorus Acid synthase (GGS) and 3- hydroxyl 3- methyl vinyl CoA-reductases (HMG-CoA^cataReductase expression), further Glycerol-3-phosphate (Gut2) and peroxidase (Pox2) are knocked out, obtains heterogenous expression beta carotene Ye Luowei solutions Fat yeast strain.It is expressive host that the present invention, which select Ye Luowei to solve fat yeast, β-carrot of the heterogenous expression from Blakeslea trispora Plain route of synthesis, fermentation condition is simple, and beta carotene yield is higher, has important productive value.

Description of the drawings

It is further illustrated the present invention below with attached drawing.

Fig. 1 shows beta carotene route of synthesis.It is the in vivo route of synthesis of yeast wherein in box, is external source outside box The route of synthesis of expression.

Fig. 2 is pSK-HMG^cata- URA3.5N-3N plasmid maps.

Fig. 3 is pS-B-RA-GGS-F-U (L) plasmid map.

Fig. 4 is pSK-Hmg (y)-Ura3-IntB plasmid maps.

Fig. 5 is pS-B-RA-GGS-Gut2-Ura3 (L) plasmid map.

Fig. 6 is pS-B-RA-GGS-Pox2-Ura3 (L) plasmid map.

Fig. 7 is pS-B-RA-GGS-rDNA-Ura3 (L) plasmid map.

Specific embodiment

The present inventor by Clone Origin in Blakeslea trispora beta carotene route of synthesis carRA (Lycopene synthase with Lycopene cyclase) and carB (phytoene dehydrogenase) gene, at its upstream fat ferment is solved from Ye Luowei with downstream connection Female promoter and terminator, become complete Expression element, and are integrated into a plasmid with the Expression element of GGS genes On, disposably by gene integration to Ye Luowei solution fat Yeast genomes, obtain the bacterial strain of heterologous production beta carotene.Then gram Grand expression HMG-CoA^cata, enhance mevalonic acid metabolism, the yield of beta carotene made to be increased dramatically.It is sweet to knock out 3- phosphoric acid Oily dehydrogenase (Gut2) reduces the generation of glycerol 3-phosphate and the β oxygen of peroxide respectively with peroxidase (Pox2) gene Change, obtain the high yield Ye Luowei solution fat yeast strains of beta carotene.

It is further illustrated the present invention below by the mode of embodiment, but does not therefore limit the present invention to the reality It applies among a scope.The experimental method of actual conditions is not specified in the following example, according to conventional methods and conditions or according to business Product specification selects.

1st, material and method

It is well known in the art using standard recombinant dna in embodiment and molecule clone technology, associated description can join See：(1) by Cold Spring Harbor Laboratory；Cold Spring Harbor, NY (1989) publish Sambroo, J.Fritsch, E.F. and Maniatis, the written Molecular Cloning of T.:A Laboratory Manual；(2) by Cold Spring Harbor Laboratory:Cold Spring Harbor, NY (1984) are published The written Experiments with Gene Fusions of T.J.Silhavy.M.L.Bennan and L.W.Enquist；(3) by It is written that Greene Publishing Assoc. and Wiley Interscience (1987) publish Ausubel, F.M et al. Current Protocols in Molecular Biology。

Material and method suitable for maintenance and the growth of microculture are well known in the art.Suitable for following implementations The technology of example can be found in American Society for Microbiology:Published by Washington, D.C. (1994) Manual of Methods for General Bacterioloy or Thomas D.Brock are in Sinauer Associates:Second edition Biotechnology published by Sunderland, MA (1989):A Textbook of Related content described in Industrial Microbiology.The operation of Ye Luowei solution fat yeast referring to Barth G, Gaillardin C.1996.Yarrowia lipotica,p 313–388.In Wolf KE(ed),Nonconventional yeasts in biotechnology。

2nd, Ye Luowei solves the culture of fat yeast (Yarrowia lipotica)

Ye Luowei solution fat yeast (Yarrowia lipotica) bacterial strains are generally cultivated at 28 DEG C, the culture medium of use It is mainly the following, solid medium adds the agar of 20g/L.

YPD culture mediums：10g/L yeast extracts (OXOID), 20g/L peptones (OXOID), 20g/L glucose (state Medicine).

MM culture mediums：20g/L glucose, 6.7g/L YNB culture mediums (no amino acid).

YNB culture mediums are purchased from BIOSHARP.

MM+5-FOA culture mediums：20g/L glucose, 6.7g/L YNB culture mediums (no amino acid), 1g/L 5-FOA (5- fluorine Orotic acid), 0.075g/L uracils, 0.075g/L uridines.

Yeast single bacterium colony is inoculated on YPD inclined-planes, 28 DEG C of culture 72h；The yeast colony on inclined-plane is inoculated in 20ml YPD In fluid nutrient medium (pH 5.5), 28 DEG C, 200rpm cultures 72h；Take 1ml culture solutions, 12000rpm centrifugations 1min；It abandons and resets and add Enter 1ml acetone, 12000rpm is centrifuged after ultrasonic disruption 30min；Supernatant is taken to be detected for liquid phase.

3rd, the HPLC detections of beta carotene

Detection method is referring to Shao Z, Zhao H, Zhao H (2009) DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways.Nucleic Acid Res 37:e16.Using 5 μ l sample sizes of syringe, detection column is Agilent Diamonsil SB-C18column, in 472nm Place's acquisition.Acetonitrile and tetrahydrofuran elution, specific elution program are shown in Table 1.

Table 1.HPLC elution programs

A：Acetonitrile；

B：Tetrahydrofuran.

1 Ye Luowei of embodiment solves the importing of fat yeast genes

The embodiment mainly describes the gene transformation method of Ye Luowei solution fat yeast.

Competent cell preparation process

1. inoculation Ye Luowei solution fat yeast single bacteriums drop down onto 5ml YPD fluid nutrient medium (citric acid-sodium citrate buffer solution tune PH is saved in 4.0), 28 DEG C, 220rpm shaken cultivations 10-12h；

2. the above-mentioned culture solutions of 50-100 μ L are transferred in 10ml YPD fluid nutrient mediums (pH4.0), 28 DEG C, 220rpm mistakes Night, culture was until cell OD₆₀₀About 5.5 or so；

3. 28 DEG C of culture solution, 3500rpm centrifugations 5min；

4. abandon supernatant, with 10ml 1 × TE wash buffers cell 2 times；

5. with 20ml or so 0.1M LiAc (pH6.0) dilute suspension, cell concentration is made to reach 5 × 10⁷/ml；

6. for suspension at 28 DEG C, 100rpm is softly incubated 1h；

7. 28 DEG C of culture solution, 3500rpm abandons supernatant after centrifuging 5min, is suspended again with 2ml 100mM LiAc (pH6.0), Cell concentration is made to reach 5 × 10⁸/ml；

Note：Competent cell can preserve one week at 4 DEG C, and centrifugation is needed to be resuspended after abandoning supernatant before use；It preserves for a long time 25% glycerine need to be added to be preserved at -80 DEG C, need to be melted when taking on ice, be resuspended using preceding rinsed with 0.1M LiAc.

Conversion process

1. taking 5 μ L carrier DNA, (carrier DNA are salmon sperm dna, and 10mg/ml is dissolved in 50mM Tris, 5mM In EDTA solution, length is about 500bp) 100 DEG C be incubated 5min and be put on ice for, 1 μ g linearized vectors are placed in 2mL EP tube bottom Portion, 60 DEG C be incubated 5min after be placed on ice, then by the two mixing；

2. by 100 μ L competent cells and above-mentioned DNA mixings, 28 DEG C, 100rpm is incubated 15min, then adds 700 μ L 40%PEG4000 (dissolves, filtration sterilization) mixing with 0.1M LiAc, and 28 DEG C, 100rpm is incubated 1h；

Then 3. it is flat in screening to add in 1.2mL 100mM LiAc, 200ml suspensions by 39 DEG C of heat shock 10min of cell On plate upper flat plate, treat that bacterium colony grows (sufficient O after 28 DEG C of cultures 3~4₂)。

The acquisition of embodiment 2URA3 gene-deleted strains

HMG-CoA reductase is rate-limiting enzyme important in mevalonate pathway, and catalysis HMG-CoA's to mevalonic acid turns Change.In order to increase the yield of beta carotene, we make carbon metabolism flow by the catalytic site of clonal expression HMG-CoA reductase To Isoprenoid pathway.The HMG-CoA reductase gene from Ye Luowei solution fat yeast is cloned, common 3000bp intercepts it and urges Change position 1500-3000bp, and in starting position addition ATG initiation codon.In order to make the sequence normal expression, at its upstream It adds Exp1 promoters and Aco terminators respectively with downstream, obtains complete clonal expression element Exp1-HMG-CoA^cata- Then Aco integrates the upstream and downstream homologous fragment of URA3 genes, obtains plasmid pSK-HMG^cata- URA3.5N-3N, the sequence of the plasmid Row are shown in SEQ ID NO1 (referring to table 2, Fig. 2).The plasmid conversion of PmeI/SphI linearisations is solved into fat yeast into Ye Luowei ATCC20362 (is purchased from ATCC), obtains bacterial strain strain Y2224., so through obtaining Y2224 using URA3 homology arms, import simultaneously Catalytic site.

2. plasmid pSK-HMG of table^cataThe description of-URA3.5N-3N

The expression of 3 Blakeslea trispora beta carotene gene of embodiment

The embodiment mainly describes structure and expression of the Blakeslea trispora beta carotene in Ye Luowei solution fat yeast strains (see Fig. 1).

The integrated plasmid pS-B-RA-GGS-F-U (L) of structure mainly includes the carB (Lycopenes from Blakeslea trispora Dehydrogenase), carRA (Lycopene synthase and lycopene cyclase) and GGS genes (the double spiceleaf base synthase of pyrophosphoric acid).

GGS gene clonings are shown in SEQ ID NO.2 in Ye Luowei solution fat yeast ATCC20362, GGS gene particular sequences.

CarRA gene clonings are done corresponding excellent from Blakeslea trispora according to the codon preference of Ye Luowei solution fat yeast Change, the CarRA gene orders after optimization are shown in SEQ ID NO.3.

CarB gene clonings are done corresponding excellent from Blakeslea trispora according to the codon preference of Ye Luowei solution fat yeast Change, the CarB gene orders after optimization are shown in SEQ ID NO.4.By said gene addition promoter and terminator constructing function group Then part integrates IntF homologous fragments, obtains pS-B-RA-GGS-F-U (L) plasmid (being shown in Table 3, Fig. 3), the sequence of the plasmid is shown in SEQ ID NO.5.The homologous fragment of the plasmid is IntF genes, and the plasmid integration to Ye Luowei solves fat yeast IntF sites.

The description of 3. plasmid pS-B-RA-GGS-F-U (L) of table

By plasmid pS-B-RA-GGS-F-U (L) AscI/SphI digestions, the linear fragment of 2 sequence of table is included, according to reality The segment is transformed into Ye Luowei solution fat yeast strain Y2224 (Ura3 by the method applied in example 1^-).Transformed cells are coated on MM trainings It supports on base tablet, 28 DEG C of culture 48-72h obtain transformant YH202.The yellow single bacterium colony YH202 grown is cultivated again in MM It rules on base, is inoculated in after cultivating 48-72h on YPD inclined-planes.After growing 72h after inclined-plane, 20ml YPD fluid nutrient mediums are transferred to In, 28 DEG C, liquid phase detects after 200rpm shake cultures 72h, obtains the bacterial strain beta carotene yield as 4.35mg/L.

The clone of embodiment 4HMG-CoA reductase catalytic sites and expression

HMG-CoA reductase is rate-limiting enzyme important in mevalonate pathway, and catalysis HMG-CoA's to mevalonic acid turns Change.In order to increase the yield of beta carotene, we make carbon metabolism flow by the catalytic site of clonal expression HMG-CoA reductase To Isoprenoid pathway.The HMG-CoA reductase gene from Ye Luowei solution fat yeast is cloned, common 3000bp intercepts it and urges Change position 1500-3000bp, and in starting position addition ATG initiation codon.In order to make the sequence normal expression, at its upstream It adds Exp1 promoters and Aco terminators respectively with downstream, obtains complete clonal expression element Exp1-HMG-CoA^cata- Aco.The element is connected on expression vector, obtains plasmid pSK-Hmg (y)-Ura3-IntB (table 4, Fig. 4).The plasmid integration To Ye Luowei solution fat yeast Int B sites.The plasmid particular sequence is shown in SEQ ID NO.6.

The description of table 4. plasmid pSK-Hmg (y)-Ura3-IntB

By plasmid pSK-Hmg (y)-Ura3-IntB PmeI/SphI digestions, the segment containing above-mentioned sequence is recycled, according to reality The segment is transformed into Ye Luowei solution fat yeast strain YH202 (Ura3 by the method applied in example 1^-) in.Transformed cells are coated on MM On culture medium flat plate, 28 DEG C of culture 48-72h obtain transformant YH306.By the yellow single bacterium colony grown again again on MM culture mediums Line is inoculated in after cultivating 48-72h on YPD inclined-planes.After growing 72h after inclined-plane, it is transferred in 20ml YPD fluid nutrient mediums, 28 DEG C, liquid phase detects after 200rpm shake cultures 72h.YH306 yield is 7.13mg/L, illustrates to be overexpressed HMG-CoA^cataIt can Enhance Isoprenoid pathway, increase the yield of beta carotene.Then YH306 single bacterium colonies are inoculated in 20ml YPD culture mediums Middle culture for 24 hours, is coated on MM+5-FOA tablets, and the bacterial strain YH306 (Ura3 of URA3 missings are obtained after cultivating 48-72h^-)。

Embodiment 5, which knocks out Gut2 genes, increases beta carotene yield

The integrated plasmid pS-B-RA-GGS-Gut2-U (L) (table 5, Fig. 5) of structure.Mainly include carB (Lycopene dehydrogenations Enzyme), carRA (Lycopene synthase and lycopene cyclase) and GGS genes (the double spiceleaf base synthase of pyrophosphoric acid).The plasmid Homology arm is the upstream and downstream segment of Gut2 genes, it is therefore an objective to knock out Gut2 genes, and fill into second carB (Lycopene dehydrogenation Enzyme), carRA (Lycopene synthase and lycopene cyclase) and GGS genes (the double spiceleaf base synthase of pyrophosphoric acid).The plasmid has Body sequence is shown in SEQ ID NO.7.

The description of 5. plasmid pS-B-RA-GGS-Gut2-U (L) of table

By plasmid pS-B-RA-GGS-Gut2-U (L) AscI/SphI digestions, the linear fragment of 5 sequence of table is included, according to The segment is transformed into Ye Luowei solution fat yeast strain YH306 (Ura3 by method for transformation^-) in.Transformed cells are coated on MM culture mediums On tablet, 28 DEG C of culture 48-72h obtain transformant YH413.The yellow single bacterium colony grown is rule on MM culture mediums again again, It is inoculated in after culture 48-72h on YPD inclined-planes.After growing 72h after inclined-plane, it is transferred in 20ml YPD fluid nutrient mediums, 28 DEG C, Liquid phase detects after 200rpm shake cultures 72h.The yield of the beta carotene of YH413 is 37.4mg/L.Illustrating to knock out Gut2 can be with The generation of glycerol 3-phosphate is reduced, promotes the yield of acetyl-CoA, increases the yield of beta carotene.Then by YH413 single bacterium colonies It is inoculated in 20ml YPD culture mediums and cultivates for 24 hours, be coated on MM+5-FOA tablets, URA3 missings are obtained after cultivating 48-72h Bacterial strain YH413 (Ura3^-)。

Embodiment 6, which knocks out Pox2 genes, increases beta carotene yield

The integrated plasmid pS-B-RA-GGS-Pox2-U (L) (table 6, Fig. 6) of structure.Mainly include carB (Lycopene dehydrogenations Enzyme), carRA (Lycopene synthase and lycopene cyclase) and GGS genes (the double spiceleaf base synthase of pyrophosphoric acid).The plasmid Homology arm is the upstream and downstream segment of Pox2 genes, it is therefore an objective to knock out Pox2 genes, and fill into second carB (Lycopene dehydrogenation Enzyme), carRA (Lycopene synthase and lycopene cyclase) and GGS genes (the double spiceleaf base synthase of pyrophosphoric acid).The plasmid has Body sequence is shown in SEQ ID NO.8.

The description of 6. plasmid pS-B-RA-GGS-Pox2-U (L) of table

By plasmid pS-B-RA-GGS-Pox2-U (L) AscI/SphI digestions, the linear fragment of 6 sequence of table is included, according to The segment is transformed into Ye Luowei solution fat yeast strain YH413 (Ura3 by the method in embodiment 1^-) in.Transformed cells are coated on On MM culture medium flat plates, 28 DEG C of culture 48-72h obtain transformant YC511.The MM culture mediums again again by the yellow single bacterium colony grown Upper line is inoculated in after cultivating 48-72h on YPD inclined-planes.After growing 72h after inclined-plane, 20ml YPD fluid nutrient mediums are transferred to In, 28 DEG C, liquid phase detects after 200rpm shake cultures 72h.The yield of the beta carotene of bacterial strain YC511 be 53.0mg/L, explanation Peroxide can be reduced by knocking out Pox2, promoted the yield of acetyl-CoA, increased the yield of beta carotene.Then it is YH511 is mono- Colony inoculation is cultivated for 24 hours in 20ml YPD culture mediums, is coated on MM+5-FOA tablets, and obtaining URA3 after culture 48-72h lacks Bacterial strain YH511 (the Ura3 of mistake^-)。

Embodiment 7, which disposably fills into multiple genes, increases beta carotene yield

The present embodiment mainly describes disposably to fill into multiple carB (phytoene dehydrogenase), carRA (Lycopene synthase and kind Lycopene cyclase) and influence of the GGS genes (pyrophosphoric acid double spiceleaf base synthase) to beta carotene yield.Yeast genome RDNA have multiple 100-200 repetitive units, as integration site improve integrate copy number application strategy in yeast Often it is employed.Integration dependent on rDNA can disposably import several or even tens copies, so as to fulfill high efficient expression. Integrated plasmid pS-B-RA-GGS-rDNA-U (L) (table 7, Fig. 7) particular sequence of structure is shown in SEQ ID NO.9.

The description of 7. plasmid pS-B-RA-GGS-rDNA-U (L) of table

By plasmid pS-B-RA-GGS-rDNA-U (L) AscI/SphI digestions, method in embodiment 1 is by the piece Section is transformed into Ye Luowei solution fat yeast strain YH504 (Ura3^-) in.Transformed cells are coated on MM culture medium flat plates, 28 DEG C of trainings Foster 48-72h obtains YC607.The yellow single bacterium colony grown is rule on MM culture mediums again again, is inoculated in after cultivating 48-72h On YPD inclined-planes.After growing 72h after inclined-plane, it is transferred in 20mlYPD fluid nutrient mediums, 28 DEG C, after 200rpm shake cultures 72h Liquid phase detects.The yield of the beta carotene of YC607 is 72.9mg/L.

It is to be understood that after the above of the present invention has been read, those skilled in the art can make the present invention various Change or modification, these equivalent forms also fall within the scope of the appended claims of the present application.

<110>Shanghai Institute of Pharmaceutical Industry, China State Institute of Pharmaceutical Industry

<120>A kind of method and its genetic engineering bacterium of genetic engineering bacterium production beta carotene

<130> P1611257C

<160> 9

<170> PatentIn version 3.5

<210> 1

<211> 7201

<212> DNA

<213> Artificial Sequence

<220>

<223> pSK-HMGcata-URA3.5N-3N

<400> 1

cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag 60

ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac 120

cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga 180

ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc 240

accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg 300

gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa 360

gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac 420

caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct 480

gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa 540

agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg 600

ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat tggagctcgc 660

tgagctggcg cgcctctaga aacgtacgtt gtaactattt aaattccttg gtgtttaaac 720

ctttggagta cgactccaac tatgagtgtg cttggatcac tttgacgata cattcttcgt 780

tggaggctgt gggtctgaca gctgcgtttt cggcgcggtt ggccgacaac aatatcagct 840

gcaacgtcat tgctggcttt catcatgatc acatttttgt cggcaaaggc gacgcccaga 900

gagccattga cgttctttct aatttggacc gatagccgta tagtccagtc tatctataag 960

ttcaactaac tcgtaactat taccataaca tatacttcac tgccccagat aaggttccga 1020

taaaaagttc tgcagactaa atttatttca gtctcctctt caccaccaaa atgccctcct 1080

acgaagctcg agctaacgtc cacaagtccg cctttgccgc tcgagtgctc aagctcgtgg 1140

cagccaagaa aaccaacctg tgtgcttctc tggatgttac caccaccaag gagctcattg 1200

agcttgccga taaggtcgga ccttatgtgt gcatgatcaa aacccatatc gacatcattg 1260

acgacttcac ctacgccggc actgtgctcc ccctcaagga acttgctctt aagcacggtt 1320

tcttcctgtt cgaggacaga aagttcgcag atattggcaa cactgtcaag caccagtacc 1380

ggtgtcaccg aatcgccgag tggtccgata tcaccaacgc ccacggtatc gatacgcgag 1440

agaattcatg ctgttcatcg tggttaatgc tgctgtgtgc tgtgtgtgtg tgttgtttgg 1500

cgctcattgt tgcgttatgc agcgtacacc acaatattgg aagcttatta gcctttctat 1560

tttttcgttt gcaaggctta acaacattgc tgtggagagg gatggggata tggaggccgc 1620

tggagggagt cggagaggcg ttttggagcg gcttggcctg gcgcccagct cgcgaaacgc 1680

acctaggacc ctttggcacg ccgaaatgtg ccacttttca gtctagtaac gccttaccta 1740

cgtcattcca tgcgtgcatg tttgcgcctt ttttcccttg cccttgatcg ccacacagta 1800

cagtgcactg tacagtggag gttttggggg ggtcttagat gggagctaaa agcggcctag 1860

cggtacacta gtgggattgt atggagtggc atggagccta ggtggagcct gacaggacgc 1920

acgaccggct agcccgtgac agacgatggg tggctcctgt tgtccaccgc gtacaaatgt 1980

ttgggccaaa gtcttgtcag ccttgcttgc gaacctaatt cccaattttg tcacttcgca 2040

cccccattga tcgagcccta acccctgccc atcaggcaat ccaattaagc tcgcattgtc 2100

tgccttgttt agtttggctc ctgcccgttt cggcgtccac ttgcacaaac acaaacaagc 2160

attatatata aggctcgtct ctccctccca accacactca cttttttgcc cgtcttccct 2220

tgctaacaca aaagtcaaga acacaaacaa ccaccccaac ccccttacac acaagacata 2280

tctacagcaa tggccatggc cacccagtct gtgaaggtgg ttgagaagca cgttcctatc 2340

gtcattgaga agcccagcga gaaggaggag gacacctctt ctgaagactc cattgagctg 2400

actgtcggaa agcagcccaa gcccgtgacc gagacccgtt ctctggacga cctagaggct 2460

atcatgaagg caggtaagac caagcttctg gaggaccacg aggttgtcaa gctctctctc 2520

gagggcaagc ttcctttgta tgctcttgag aagcagcttg gtgacaacac ccgagctgtt 2580

ggcatccgac gatctatcat ctcccagcag tctaatacca agactttaga gacctcaaag 2640

cttccttacc tgcactacga ctacgaccgt gtttttggag cctgttgcga gaacgttatt 2700

ggttacatgc ctctccccgt tggtgttgct ggccccatga acattgatgg caagaactac 2760

cacattccta tggccaccac tgagggttgt cttgttgcct caaccatgcg aggttgcaag 2820

gccatcaacg ccggtggcgg tgttaccact gtgcttactc aggacggtat gacacgaggt 2880

ccttgtgttt ccttcccctc tctcaagcgg gctggagccg ctaagatctg gcttgattcc 2940

gaggagggtc tcaagtccat gcgaaaggcc ttcaactcca cctctcgatt tgctcgtctc 3000

cagtctcttc actctaccct tgctggtaac ctgctgttta ttcgattccg aaccaccact 3060

ggtgatgcca tgggcatgaa catgatctcc aagggcgtcg aacactctct ggccgtcatg 3120

gtcaaggagt acggcttccc tgatatggac attgtgtctg tctcgggtaa ctactgcact 3180

gacaagaagc ccgcagcgat caactggatc gaaggccgag gcaagagtgt tgttgccgaa 3240

gccaccatcc ctgctcacat tgtcaagtct gttctcaaaa gtgaggttga cgctcttgtt 3300

gagctcaaca tcagcaagaa tctgatcggt agtgccatgg ctggctctgt gggaggtttc 3360

aatgcacacg ccgcaaacct ggtgaccgcc atctaccttg ccactggcca ggatcctgct 3420

cagaatgtcg agtcttccaa ctgcatcacg ctgatgagca acgtcgacgg taacctgctc 3480

atctccgttt ccatgccttc tatcgaggtc ggtaccattg gtggaggtac tattttggag 3540

ccccaggggg ctatgctgga gatgcttggc gtgcgaggtc ctcacatcga gacccccggt 3600

gccaacgccc aacagcttgc tcgcatcatt gcttctggag ttcttgcagc ggagctttcg 3660

ctgtgttctg ctcttgctgc cggccatctt gtgcaaagtc atatgaccca caaccggtcc 3720

caggctccta ctccggccaa gcagtctcag gccgatctgc agcgtctaca aaacggttcg 3780

aatatttgca tacggtcata ggcggccgca tggagcgtgt gttctgagtc gatgttttct 3840

atggagttgt gagtgttagt agacatgatg ggtttatata tgatgaatga atagatgtga 3900

ttttgatttg cacgatggaa ttgagaactt tgtaaacgta catgggaatg tatgaatgtg 3960

ggggttttgt gactggataa ctgacggtca gtggacgccg ttgttcaaat atccaagaga 4020

tgcgagaaac tttgggtcaa gtgaacatgt cctctctgtt caagtaaacc atcaactatg 4080

ggtagtatat ttagtaagga caagagttga gattctttgg agtcctagaa acgtattttc 4140

gcgttccaag atcaaattag tagagtaata cgggcacggg aatccattca tagtctcaat 4200

cctgcaggtg agttaattaa aggaaactgt ctctgaacag aagaaggagg acgtctctga 4260

ctacgagaac tcccagtaca aggagttcct agtcccctct cccaacgaga agctggccag 4320

aggtctgctc atgctggccg agctgtcttg caagggctct ctggccactg gcgagtactc 4380

caagcagacc attgagcttg cccgatccga ccccgagttt gtggttggct tcattgccca 4440

gaaccgacct aagggcgact ctgaggactg gcttattctg acccccgggg tgggtcttga 4500

cgacaaggga gacgctctcg gacagcagta ccgaactgtt gaggatgtca tgtctaccgg 4560

aacggatatc ataattgtcg gccgaggtct gtacggccag aaccgagatc ctattgagga 4620

ggccaagcga taccagaagg ctggctggga ggcttaccag aagattaact gttagaggtt 4680

agactatgga tatgtaattt aactgtgtat atagagagcg tgcaagtatg gagcgcttgt 4740

tcagcttgta tgatggtcag acgacctgtc tgatcgagta tgtatgatac tgcacaacct 4800

gtgtatccgc atgatctgtc caatggggca tgttgttgtg tttctcgata cggagatgct 4860

gggtacagtg ctaatacgtt gaactactta tacttatatg aggctcgaag aaagctgact 4920

tgtgtatgac gcatgcgacg ccacccatgg actctaagcg gccgcatgga cctgtcgact 4980

tggctcgagg gggggcccgg taccagcttt tgttcccttt agtgagggtt aatttcgagc 5040

ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca 5100

cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa 5160

ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag 5220

ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc 5280

gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 5340

cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg 5400

tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 5460

cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 5520

aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 5580

cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 5640

gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 5700

ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 5760

cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 5820

aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 5880

tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc 5940

ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 6000

tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 6060

ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 6120

agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 6180

atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca 6240

cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag 6300

ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac 6360

ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc 6420

agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct 6480

agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc 6540

gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg 6600

cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc 6660

gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat 6720

tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag 6780

tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat 6840

aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg 6900

cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca 6960

cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga 7020

aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc 7080

ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata 7140

tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg 7200

c 7201

<210> 2

<211> 984

<212> DNA

<213> Yarrowia lipotica

<220>

<223>Double perfume youngster's base pyrophosphate synthase genes, GGS

<400> 2

atggattata acagcgcgga tttcaaggag atatggggca aggccgccga caccgcgctg 60

ctgggaccgt acaactacct cgccaacaac cggggccaca acatcagaga acacttgatc 120

gcagcgttcg gagcggttat caaggtggac aagagcgatc tcgagaccat ttcgcacatc 180

accaagattt tgcataactc gtcgctgctt gttgatgacg tggaagacaa ctcgatgctc 240

cgacgaggcc tgccggcagc ccattgtctg tttggagtcc cccaaaccat caactccgcc 300

aactacatgt actttgtggc tctgcaggag gtgctcaagc tcaagtctta tgatgccgtc 360

tccattttca ccgaggaaat gatcaacttg catagaggtc agggtatgga tctctactgg 420

agagaaacac tcacttgccc ctcggaagac gagtatctgg agatggtggt gcacaagacc 480

ggtggactgt ttcggctggc tctgagactt atgctgtcgg tggcatcgaa acaggaggac 540

catgaaaaga tcaactttga tctcacacac cttaccgaca cactgggagt catttaccag 600

attctggatg attacctcaa cctgcagtcc acggaattga ccgagaacaa gggattctgc 660

gaagatatca gcgaaggaaa gttttcgttt ccgctgattc acagcatacg caccaacccg 720

gataaccacg agattctcaa cattctcaaa cagcgaacaa gcgacgcttc actcaaaaag 780

tacgccgtgg actacatgag aacagaaacc aagagtttcg actactgcct caagaggata 840

caggccatgt cactcaaggc aagttcgtac attgatgatc tagcagcagc tggccacgat 900

gtctccaagc tacgagccat tttgcattat tttgtgtcca cctctgactg tgaggagaga 960

aagtactttg aggatgcgca gtga 984

<210> 3

<211> 1829

<212> DNA

<213> Blakeslea trispora

<220>

<223>CarRA, Lycopene synthase and lycopene cyclase gene

<400> 3

atggcttcca ttctcaccta tctggagttt catctctact acacactgcc tgtccttgcg 60

gcattgtgtt ggctgctgaa gccgtttcac tcgcagcaag acaatctcaa gtacaaattt 120

ctgatgttga tggccgcctc gaccgcatcg atttgggaca attacatcgt ttatcatcga 180

gcttggtggt actgtcctac ctgtgttgtg gctgtcattg gctacgtgcc tctggaagag 240

tacatgttct ttatcatcat gactctgatg actgtcgcgt tctcgaactt tgttatgcgt 300

tggcacttgc acaccttctt tattagaccc aacacctcgt ggaagcagac actgctggtg 360

cgacttgtgc ctgtttctgc tctgttggca atcacctatc acgcttggca cttgacactg 420

ccaaataaat cctcgtttta cggttcgtgc atcctttggt atgcttgtcc tgtgttggct 480

attctttggc tgggtgctgg cgagtacatc ttgcgtcgac ctgtggctgt ccttttgtcg 540

attgttatcc cttccgtgta cctgtgttgg gctgacatcg tcgctatttc cgctggcaca 600

tggcacattt ctcttagaac aagcactggc aaaatggtgg tgcccgatct gcctgtggaa 660

gagtgcctgt tttttacctt gatcaacaca gtcttggttt ttgctacctg tgctattgac 720

cgagcccagg ccatcctcca tctgtacaaa tcgtctgttc agaatcagaa ccctaaacaa 780

gccatttccc ttttccagca cgtcaaagag ctggcatggg ccttctgtct tcctgaccag 840

atgctcaaca atgagttgtt tgatgatctt accatcagct gggacattct gcgtaaagcc 900

tcgaagtcgt tctacactgc atctgccgtt tttccatcct atgtgcgtca agacttgggt 960

gttctctatg ctttctgtag agctaccgat gacctgtgcg atgatgagtc caaatctgtt 1020

caagagagaa gagaccagct ggatcttact cgacagtttg ttcgtgatct ctttagccag 1080

aagacctccg cgcctattgt gattgactgg gagttgtatc agaaccagct tcctgcttcg 1140

tgtatttctg cctttagagc ctttactcga cttcgacatg tccttgaagt ggaccctgtg 1200

gaggagctgc tggatggtta caaatgggat cttgagcgtc gtcctatcct tgatgaacaa 1260

gacttggagg catactccgc ttgtgtggcc tcctccgtgg gtgaaatgtg cacacgtgtg 1320

attcttgctc aagaccagaa ggagaatgat gcttggatta ttgaccgtgc acgtgagatg 1380

ggcctggtgc tgcaatacgt taacattgct cgtgacattg tgaccgacag cgagactctg 1440

ggtcgatgtt atctgcctca gcagtggctt agaaaagaag agacagaaca gattcagcaa 1500

ggcaacgccc gtagcctggg tgatcagaga ctgttgggct tgtctctgaa gctcgtggga 1560

aaggcagacg ctatcatggt gagagctaag aagggcattg acaagttgcc ggcaaactgt 1620

caaggcggtg tgcgagctgc ttgccaagtg tacgctgcaa ttggatctgt gctcaagcag 1680

cagaagacaa catatcctac aagagcccat ctgaaaggaa gcgaacgtgc caagattgct 1740

ctgttgtccg tgtacaacct ctatcagtct gaagacaagc ctgtggctct ccgtcaagct 1800

agaaagatta agtccttttt tgttgacta 1829

<210> 4

<211> 1752

<212> DNA

<213> Blakeslea trispora

<220>

<223>CarB, phytoene dehydrogenase gene

<400> 4

atggcttccg atcagaagaa gcacattgtt gtcattggtg ccggtattgg cggaactgct 60

actgctgctc gtcttgctcg tgaaggtttt cgagttactg ttgttgagaa gaacgacttt 120

tccggtggcc gttgttcttt cattcatcac gacggtcatc gatttgatca gggtccctct 180

ctctacttga tgcctaagct ctttgaagac gcatttgctg acttggacga gcgtattggt 240

gatcacttgg acttgctgcg atgtgacaat aactataagg ttcactttga cgacggtgat 300

gccgtgcagc tctcttccga tctgaccaag atgaagggcg agttggaccg tattgagggt 360

ccccttggat ttggtagatt cttggacttc atgaaggaga cacatgtcca ctatgaacaa 420

ggtacattta ttgctatcaa gcgaaacttt gagaccattt gggatttgat tcgtctccag 480

tatgtgcctg agatctttcg attgcacttg tttggtaaga tctatgaccg agcctctaaa 540

tacttccaga ccaagaaaat gcgtatggct tttacctttc agacaatgta catgggtatg 600

tcgccttacg atgctccagc agtttactct ttgctccagt acaccgagtt tgctgaaggt 660

atctggtatc ctcgtggtgg tttcaacatg gttgttcaga agctggagtc tatcgcctcc 720

aaaaagtacg gtgctgagtt cagataccag tcgcctgttg ctaaaattaa cactgtcgac 780

aaagacaagc gtgtgaccgg tgtcaccttg gagagcggag aagtcattga agccgatgca 840

gtcgtgtgta atgcggatct tgtttacgct tatcaccatc tgctgcctcc ttgcaattgg 900

acaaagaaga cactggcctc taagaaactc acctcttctt ctatttcgtt ttactggtcc 960

atgtctacaa aggtgcctca gctggacgtg cacaatatct tcttggctga agcctacaag 1020

gagtcttttg acgagatttt caacgacttc ggtttgccct ctgaagcgtc tttctatgtc 1080

aacgttccat ctcgaattga tgagtctgcc gcacctccca acaaggactc cattattgtg 1140

ttggttccaa ttggccacat gaagtctaag acaggaaact ctgctgaaga gaattatcct 1200

gagttggtga accgtgcacg aaagatggtt ctggaagtta tcgagcgtcg tttgggagtg 1260

aacaactttg ctaatttgat tgaacatgaa gaagtgaatg atccttctgt ttggcagagc 1320

aagtttaacc tctggagagg ttctattctt ggtctgtctc atgacgtgtt ccaagttctc 1380

tggttcagac cttctaccaa ggactccaca aaccgttatg acaatctgtt ctttgtcgga 1440

gcttctacac atccaggtac tggtgttcct atcgttcttg ctggatctaa gctgacctcc 1500

gaccaagtct gtaagagctt tggccagaat cccctgccaa gaaagctgca agacagccag 1560

aagaagtacg ctcctgagca gactcgaaag accgagagcc actggatcta ctactgtctt 1620

gcttgttact ttgttacctt cctctttttc tatttcttcc caagagatga caccacaact 1680

cctgcttctt tcattaacca actgctgcct aacgttttcc aagtgcagaa cagcaacgac 1740

attcgaattt ag 1752

<210> 5

<211> 16313

<212> DNA

<213> Artificial Sequence

<220>

<223> pS-B-RA-GGS-F-Ura3(L)

<400> 5

cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag 60

ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac 120

cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga 180

ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc 240

accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg 300

gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa 360

gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac 420

caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct 480

gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa 540

agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg 600

ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat tggagctcgc 660

tgagctggcg cgccgccaaa gaacccatag cctctttaga acaatttgca caagcctctc 720

tataatcttt tcttcatctt cacatgctca aagcgtctgt tctgttgtaa ttatgttcga 780

gacctctcgt gacaaacaag cgacgagggg aaacatgtgc agagagccgg caagaaggaa 840

cagatataca cgagacaggg cggagatacc ggttacggaa taataaatga aggctgcaac 900

cgcttgggga caccccccca tagtcctttt taagcaatgg tgttatattt cttcttgtag 960

ccagctctct tagctcattc caattgccct cgacatcgcg ctctcttgtt tcgccctttt 1020

gtcttgacag aatacaattt ttttgtctct cgactgccta cagtagggtt tcaaccagcc 1080

ttaggttaga gttgagtgcg gggtatggct gtgcatacac caaaaagcca gaagtccaga 1140

tggtgtgcaa ctccaggttt ccatgggcta atctccttcc tgcgacacaa gcaagctgtc 1200

tcagagtcgg aatcgggtgt aacctacttg tagggtagac atcgactttc ggctttaccg 1260

agctccagag cgggtcgatc gtgtttcaga gactgaaaaa aggagaaaaa aaaagacttc 1320

caaggggcct attccgggaa tccgagagac attggatatg gacttgcaaa caacatctgg 1380

gctaataatg ggcctcagat ttagcggata tttcgtagct cattttgaac gtctactgat 1440

aaccaaattt ctactgtaaa gctgctcttc cttgaagatg tagtcgtctt tcgcttggag 1500

ggagaggtac catgttggta gtatatacac acggcatgta actgaggccc gatgagcctg 1560

tctgctgctc tgtctgcact ccaggttcgt tattgtcact cttctggcga tgatgcaact 1620

cgtattgaag ccctcagacg cgttccacat cagagttttg tatcaccatt attattgccg 1680

agtctccgat ttaacgcaaa tagactaagt tgagtgtcgt atatccacaa cgatcgacac 1740

tacccctcat accaatggct tttgttcgtg ttgttgttgg atgaggacct gaaacgtacg 1800

ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac 1860

gccaagctcg attaattaac tcacctgcag gattgagact atgaatggat tcccgtgccc 1920

gtattactct actaatttga tcttggaacg cgaaaatacg tttctaggac tccaaagaat 1980

ctcaactctt gtccttacta aatatactac ccatagttga tggtttactt gaacagagag 2040

gacatgttca cttgacccaa agtttctcgc atctcttgga tatttgaaca acggcgtcca 2100

ctgaccgtca gttatccagt cacaaaaccc ccacattcat acattcccat gtacgtttac 2160

aaagttctca attccatcgt gcaaatcaaa atcacatcta ttcattcatc atatataaac 2220

ccatcatgtc tactaacact cacaactcca tagaaaacat cgactcagaa cacacgctcc 2280

atgcggccgc tcactgcgca tcctcaaagt actttctctc ctcacagtca gaggtggaca 2340

caaaataatg caaaatggct cgtagcttgg agacatcgtg gccagctgct gctagatcat 2400

caatgtacga acttgccttg agtgacatgg cctgtatcct cttgaggcag tagtcgaaac 2460

tcttggtttc tgttctcatg tagtccacgg cgtacttttt gagtgaagcg tcgcttgttc 2520

gctgtttgag aatgttgaga atctcgtggt tatccgggtt ggtgcgtatg ctgtgaatca 2580

gcggaaacga aaactttcct tcgctgatat cttcgcagaa tcccttgttc tcggtcaatt 2640

ccgtggactg caggttgagg taatcatcca gaatctggta aatgactccc agtgtgtcgg 2700

taaggtgtgt gagatcaaag ttgatctttt catggtcctc ctgtttcgat gccaccgaca 2760

gcataagtct cagagccagc cgaaacagtc caccggtctt gtgcaccacc atctccagat 2820

actcgtcttc cgaggggcaa gtgagtgttt ctctccagta gagatccata ccctgacctc 2880

tatgcaagtt gatcatttcc tcggtgaaaa tggagacggc atcataagac ttgagcttga 2940

gcacctcctg cagagccaca aagtacatgt agttggcgga gttgatggtt tgggggactc 3000

caaacagaca atgggctgcc ggcaggcctc gtcggagcat cgagttgtct tccacgtcat 3060

caacaagcag cgacgagtta tgcagaatct tggtgatgtg cgaaatggtc tcgagatcgc 3120

tcttgtccac cttgataacc gctccgaacg ctgcgatcaa gtgttctctg atgttgtggc 3180

cccggttgtt ggcgaggtag ttgtacggtc ccagcagcgc ggtgtcggcg gccttgcccc 3240

atatctcctt gaaatccgcg ctgttataat ccatggttgt gaattagggt ggtgagaatg 3300

gttggttgta gggaagaatc aaaggccggt ctcgggatcc gtgggtatat atatatatat 3360

atatatatac gatccttcgt tacctccctg ttctcaaaac tgtggttttt cgtttttcgt 3420

tttttgcttt ttttgatttt tttagggcca actaagcttc cagatttcgc taatcacctt 3480

tgtactaatt acaagaaagg aagaagctga ttagagttgg gctttttatg caactgtgct 3540

actccttatc tctgatatga aagtgtagac ccaatcacat catgtcattt agagttggta 3600

atactgggag gatagataag gcacgaaaac gagccatagc agacatgctg ggtgtagcca 3660

agcagaagaa agtagatggg agccaattga cgagcgaggg agctacgcca atccgacata 3720

cgacacgctg agatcgtctt ggccgggggg tacctacaga tgtccaaggg taagtgcttg 3780

actgtaattg tatgtctgag gacaaatatg tagtcagccg tataaagtca taccaggcac 3840

cagtgccatc atcgaaccac taactctcta tgatacatgc ctccggtatt attgtaccat 3900

gcgtcgcttt gttacatacg tatcttgcct ttttctctca gaaactccag actttggcta 3960

ttggtcgaga taagcccgga ccatagtgag tctttcacac tctacatttc tcccttgctc 4020

caactattta aatcgaggaa gaggacaagc ggctgcttct taagtttgtg acatcagtat 4080

ccaaggcacc attgcaagga ttcaaggctt tgaacccgtc atttgccatt cgtaacgctg 4140

gtagacaggt tgatcggttc cctacggcct ccacctgtgt caatcttctc aagctgcctg 4200

actatcagga cattgatcaa cttcggaaga aacttttgta tgccattcga tcacatgctg 4260

gtttcgattt gtcttagagg aacgcatata cagtaatcat agagaataaa cgatattcat 4320

ttattaaagt agatagttga ggtagaagtt gtaaagagtg ataaatagcg gccgctagtc 4380

aacaaaaaag gacttaatct ttctagcttg acggagagcc acaggcttgt cttcagactg 4440

atagaggttg tacacggaca acagagcaat cttggcacgt tcgcttcctt tcagatgggc 4500

tcttgtagga tatgttgtct tctgctgctt gagcacagat ccaattgcag cgtacacttg 4560

gcaagcagct cgcacaccgc cttgacagtt tgccggcaac ttgtcaatgc ccttcttagc 4620

tctcaccatg atagcgtctg cctttcccac gagcttcaga gacaagccca acagtctctg 4680

atcacccagg ctacgggcgt tgccttgctg aatctgttct gtctcttctt ttctaagcca 4740

ctgctgaggc agataacatc gacccagagt ctcgctgtcg gtcacaatgt cacgagcaat 4800

gttaacgtat tgcagcacca ggcccatctc acgtgcacgg tcaataatcc aagcatcatt 4860

ctccttctgg tcttgagcaa gaatcacacg tgtgcacatt tcacccacgg aggaggccac 4920

acaagcggag tatgcctcca agtcttgttc atcaaggata ggacgacgct caagatccca 4980

tttgtaacca tccagcagct cctccacagg gtccacttca aggacatgtc gaagtcgagt 5040

aaaggctcta aaggcagaaa tacacgaagc aggaagctgg ttctgataca actcccagtc 5100

aatcacaata ggcgcggagg tcttctggct aaagagatca cgaacaaact gtcgagtaag 5160

atccagctgg tctcttctct cttgaacaga tttggactca tcatcgcaca ggtcatcggt 5220

agctctacag aaagcataga gaacacccaa gtcttgacgc acataggatg gaaaaacggc 5280

agatgcagtg tagaacgact tcgaggcttt acgcagaatg tcccagctga tggtaagatc 5340

atcaaacaac tcattgttga gcatctggtc aggaagacag aaggcccatg ccagctcttt 5400

gacgtgctgg aaaagggaaa tggcttgttt agggttctga ttctgaacag acgatttgta 5460

cagatggagg atggcctggg ctcggtcaat agcacaggta gcaaaaacca agactgtgtt 5520

gatcaaggta aaaaacaggc actcttccac aggcagatcg ggcaccacca ttttgccagt 5580

gcttgttcta agagaaatgt gccatgtgcc agcggaaata gcgacgatgt cagcccaaca 5640

caggtacacg gaagggataa caatcgacaa aaggacagcc acaggtcgac gcaagatgta 5700

ctcgccagca cccagccaaa gaatagccaa cacaggacaa gcataccaaa ggatgcacga 5760

accgtaaaac gaggatttat ttggcagtgt caagtgccaa gcgtgatagg tgattgccaa 5820

cagagcagaa acaggcacaa gtcgcaccag cagtgtctgc ttccacgagg tgttgggtct 5880

aataaagaag gtgtgcaagt gccaacgcat aacaaagttc gagaacgcga cagtcatcag 5940

agtcatgatg ataaagaaca tgtactcttc cagaggcacg tagccaatga cagccacaac 6000

acaggtagga cagtaccacc aagctcgatg ataaacgatg taattgtccc aaatcgatgc 6060

ggtcgaggcg gccatcaaca tcagaaattt gtacttgaga ttgtcttgct gcgagtgaaa 6120

cggcttcagc agccaacaca atgccgcaag gacaggcagt gtgtagtaga gatgaaactc 6180

cagataggtg agaatggaag ccatggttga tgtgtgttta attcaagaat gaatatagag 6240

aagagaagaa gaaaaaagat tcaattgagc cggcgatgca gacccttata taaatgttgc 6300

cttggacaga cggagcaagc ccgcccaaac ctacgttcgg tataatatgt taagcttttt 6360

aacacaaagg tttggcttgg ggtaacctga tgtggtgcaa aagaccgggc gttggcgagc 6420

cattgcgcgg gcgaatgggg ccgtgactcg tctcaaattc gagggcgtgc ctcaattcgt 6480

gcccccgtgg ctttttcccg ccgtttccgc cccgtttgca ccactgcagc cgcttctttg 6540

gttcggacac cttgctgcga gctaggtgcc ttgtgctact taaaaagtgg cctcccaaca 6600

ccaacatgac atgagtgcgt gggccaagac acgttggcgg ggtcgcagtc ggctcaatgg 6660

cccggaaaaa acgctgctgg agctggttcg gacgcagtcc gccgcggcgt atggatatcc 6720

gcaaggttcc atagcgccat tgccctccgt cggcgtctat cccgcaacct ctaaatagag 6780

cgggaatata acccaagctt cttttttttc ctttaacacg cacaccccca actatcatgt 6840

tgctgctgct gtttgactct actctgtgga ggggtgctcc cacccaaccc aacctacagg 6900

tggatccggc gctgtgattg gctgataagt ctcctatccg gactaattct gaccaatggg 6960

acatgcgcgc aggacccaaa tgccgcaatt acgtaacccc aacgaaatgc ctacccctct 7020

ttggagccca gcggccccaa atccccccaa gcagcccggt tctaccggct tccatctcca 7080

agcacaagca gcccggttct accggcttcc atctccaagc acccctttct ccacacccca 7140

caaaaagacc cgtgcaggac atcctactgc gtgtttaaac accactaaaa ccccacaaaa 7200

tatatcttac cgaatataca gatctactat agaggaacaa ttgccccgga gaagacggcc 7260

aggccgccta gatgacaaat tcaacaactc acagctgact ttctgccatt gccactaggg 7320

gggggccttt ttatatggcc aagccaagct ctccacgtcg gttgggctgc acccaacaat 7380

aaatgggtag ggttgcacca acaaagggat gggatggggg gtagaagata cgaggataac 7440

ggggctcaat ggcacaaata agaacgaata ctgccattaa gactcgtgat ccagcgactg 7500

acaccattgc atcatctaag ggcctcaaaa ctacctcgga actgctgcgc tgatctggac 7560

accacagagg ttccgagcac tttaggttgc accaaatgtc ccaccaggtg caggcagaaa 7620

acgctggaac agcgtgtaca gtttgtctta acaaaaagtg agggcgctga ggtcgagcag 7680

ggtggtgtga cttgttatag cctttagagc tgcgaaagcg cgtatggatt tggctcatca 7740

ggccagattg agggtctgtg gacacatgtc atgttagtgt acttcaatcg ccccctggat 7800

atagccccga caataggccg tggcctcatt tttttgcctt ccgcacattt ccattgctcg 7860

gtacccacac cttgcttctc ctgcacttgc caaccttaat actggtttac attgaccaac 7920

atcttacaag cggggggctt gtctagggta tatataaaca gtggctctcc caatcggttg 7980

ccagtctctt ttttcctttc tttccccaca gattcgaaat ctaaactaca catcacacaa 8040

tgcctgttac tgacgtcctt aagcgaaagt ccggtgtcat cgtcggcgac gatgtccgag 8100

ccgtgagtat ccacgacaag atcagtgtcg agacgacgcg ttttgtgtaa tgacacaatc 8160

cgaaagtcgc tagcaacaca cactctctac acaaactaac ccagctctcc atggcctcca 8220

agaaacacat tgtcattatc ggtgctggcg tgggtggcac ggctacagct gctcgattgg 8280

cccgagaagg cttcaaggtc actgtggtgg agaaaaacga ctttggtggc ggccgatgct 8340

ccttgatcca tcaccagggc caccgatttg atcagggccc ctcgctctac ctgatgccca 8400

agtactttga ggacgccttt gccgatctgg acgagcgaat ccaagaccac ctggagctgc 8460

tgcgatgcga caacaactac aaggtgcact ttgacgacgg tgagtcgatc cagctgtcgt 8520

ctgacttgac acgaatgaag gctgaattgg accgagtgga gggccccctt ggttttggcc 8580

gattcctgga tttcatgaaa gagacacaca tccactacga aagcggcacc ctgattgcgc 8640

tcaagaagaa tttcgaatcc atctgggacc tgattcgaat caagtacgct cctgagatct 8700

ttcgattgca cctgtttggc aagatctacg accgagcttc caagtacttc aagaccaaga 8760

agatgcgaat ggcattcacg tttcagacca tgtacatggg catgtcgccc tacgatgcgc 8820

ctgctgtcta cagcctgttg cagtacaccg agttcgctga aggcatctgg tacccccgag 8880

gcggcttcaa catggtggtt cagaagctgg aggcgattgc aaagcaaaag tacgatgccg 8940

agtttatcta caatgcgcct gttgccaaga ttaacaccga tgatgccacc aaacaagtga 9000

caggtgtgac cttggaaaat ggccacatca tcgacgccga tgcggttgtg tgtaacgcag 9060

atctggtcta cgcttaccac aatctgttgc ctccctgccg atggacgcaa aacacactgg 9120

cttccaagaa attgacgtct tcttccattt ccttctactg gtccatgtcc accaaggtgc 9180

ctcaattgga cgtgcacaac atctttttgg ccgaggctta ccaggagagc tttgacgaaa 9240

tcttcaagga ctttggcctg ccttctgaag cctccttcta cgtcaatgtg ccctctcgaa 9300

tcgacccttc tgctgctccc gacggcaagg actctgtcat tgtcttggtg cctattggtc 9360

atatgaagag caagacgggc gatgcttcca ccgagaacta ccccgctatg gtggacaagg 9420

cacgaaagat ggtgctggct gtgattgagc gacgactggg catgtcgaat ttcgccgact 9480

tgattgagca tgagcaagtc aatgatcccg ctgtgtggca gagcaagttc aatctgtgga 9540

gaggctctat tctgggtttg tctcatgatg tgcttcaggt gctgtggttc cgacccagca 9600

caaaggattc taccggtcga tacgataacc tgttctttgt gggtgcaagc acgcatcccg 9660

gaactggtgt tcccattgtc cttgcaggaa gcaagctcac ctctgaccaa gttgtcaaga 9720

gcttggaaag acgcccaagc ctagaaagat cgagatggag aacacgcaag cacctttgga 9780

ggagcctgat gctgaatcga cattccctgt gtggttctgg ttgcgagctg ccttttgggt 9840

catgtttatg ttcttttact tcttccctca atccaatggc caaacgcccg catcttttat 9900

caataatttg ctgcctgaag tgttccgagt tcataactct aatgtcattt aggcggccgc 9960

atgagaagat aaatatataa atacattgag atattaaatg cgctagatta gagagcctca 10020

tactgctcgg agagaagcca agacgagtac tcaaagggga ttacaccatc catatccaca 10080

gacacaagct ggggaaaggt tctatataca ctttccggaa taccgtagtt tccgatgtta 10140

tcaatggggg cagccaggat ttcaggcact tcggtgtctc ggggtgaaat ggcgttcttg 10200

gcctccatca agtcgtacca tgtcttcatt tgcctgtcaa agtaaaacag aagcagatga 10260

agaatgaact tgaagtgaag gagaattcag gaggacgaga aggtcaacaa gggtgaggcc 10320

tggagagagt tctacaaggg tccccgggat cggctgtaca gaactggaga tccaggcaga 10380

caaggtatag ggcggcgcct acaatccatg ccaacccgtt ccatgtgctc gccgaggcgg 10440

cataaatcgc cgtgacgatc agcggtccag tgatcgaagt taggctggta agagccgcga 10500

gcgatccttg aagctgtccc tgatggtcgt catctacctg cctggacagc atggcctgca 10560

acgcgggcat cccgatgccg ccggaagcga gaagaatcat aatggggaag gccatccagc 10620

ctcgcgtcgc gaacgccagc aagacgtagc ccagcgcgtc ggccgccatg ccggcgataa 10680

tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg 10740

cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc 10800

ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa 10860

agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga 10920

ctgggttgaa ggctctcaag ggcatcggtc gacgctctcc cttatgcgac tcctgcatta 10980

ggaagcagcc cagtagattt ctggtggtat tgtgactggg gatgtagttg agaataagtc 11040

atacacaagt cagctttctt caagcctcat ataagtataa gtagttcaat cgtattagct 11100

acttgtaccc agcatctccg tatcgagaaa cacaacaaca tgccccattg gacagatcat 11160

gcggatacac aggttgtgca gtatcataca tactcgatca gacaggtcgt ctgaccatca 11220

tacaagctga acaagcgctc catacttgca cgctctctat atacacagtt aaatgacata 11280

tccatagtct aacctctaac agttaatctt ctggtaagcc tcccagccag ccttctggta 11340

tcgcttggcc tcctcaatag gatctcggtt ctggccgtac agacctcggc cgacaattat 11400

gatatccgtt ccggtagaca tgacatcctc aacagttcgg tactgctgtc cgagagcgtc 11460

tcccttgtcg tcaagaccca ccccgggggt cagaataagc cagtcctcag agtcgccctt 11520

aggtcggttc tgggcaatga agccaaccac aaactcgggg tcggatcggg caagctcaat 11580

ggtctgcttg gagtactcgc cagtggccag agagcccttg caagacagct cggccagcat 11640

gagcagacct ctggccagct tctcgttggg agaggggacc aggaactcct tgtactggga 11700

gttctcgtag tcagagacgt cctccttctt ctgttcagag acagtttcct cggcaccagc 11760

tcgcaggcca gcaatgattc cggttccggg tacaccgtgg gcgttggtga tatcggacca 11820

ctcggcgatt cggtagacac cgttcttgta ctggtgcttg acagtgttgc caatatctgc 11880

gaactttctg tcctcgaaca ggaagaaacc gtgcttaaga gcaagttcct tgagggggag 11940

cacagtgccg gcgtaggtga agtcgtcaat gatgtcgata tgggtcttga tcatgcacac 12000

ataaggtccg accttatcgg caagctcaat gagctccttg gtggtggtaa catccagaga 12060

agcacacagg ttggttttct tggctgccac gagcttgagc actcgagcgg caaaggcgga 12120

cttgtggacg ttagctcgag cttcgtagga gggcattttg gtggtgaaga ggagactgaa 12180

ataaatttag tctgcagaac tttttatcgg aaccttatct ggggcagtga agtatatgtt 12240

atggtaatag ttacgagtta gttgaactta tagatagact ggactatacg gctatcggtc 12300

caaattagaa agaacgtcaa tggctctctg ggcgtcgcct ttgccgacaa aaatgtgatc 12360

atgatgaaag ccagcaatga cgttgcagct gatattgttg tcggccaacc gcgccgaaaa 12420

cgcagctgtc agacccacag cctccaacga agaatgtatc gtcaaagtga tccaagcaca 12480

ctcatagttg gagtcgtact ccaaaggcgg caatgacgag tcagacagga ggcagacaag 12540

gtatagggcg gcgcctacaa tccatgccaa cccgttccat gtgctcgccg aggcggcata 12600

aatcgccgtg acgatcagcg gtccagtgat cgaagttagg ctggtaagag ccgcgagcga 12660

tccttgaagc tgtccctgat ggtcgtcatc tacctgcctg gacagcatgg cctgcaacgc 12720

gggcatcccg atgccgccgg aagcgagaag aatcataatg gggaaggcca tccagcctcg 12780

cgtcgcgaac gccagcaaga cgtagcccag cgcgtcggcc gccatgccgg cgataatggc 12840

ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg 12900

caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc 12960

ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa 13020

gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg 13080

gttgaaggct ctcaagggca tcggtcgacg ctctccctta tgcgactcct gcattaggaa 13140

gcagcccagt atcgatagag acggagtcac tgccttaatt aatgatagca accttcgtcc 13200

gacgaacaca acaatcaaat taaataaaaa aatgcgagga caatgcaggg ttggtatgaa 13260

agagccgcgt gcagttttgt agtcggaggt gcagctcttg aagaagggaa aaagagccaa 13320

agctggtaaa gtacataaaa atatgatgtg tgccgtttgt ggcatgtcac gaaaacgact 13380

gtaccaggac cggatgtagg cacagtgggg gggtgtaagt gagccggcag ccgaggttcc 13440

ttttgagact gtctgaaagg atcagatatg agctctcaca tttcccggac cctgtacaag 13500

cacgttacag gtggttttgc cgatatccac tacactttcg ttctcctacc aagtctattt 13560

tgtgggtgtg tctagttatc gctccgacaa ggttcaggcg gaatacgacg actcttgctt 13620

ctttgttctg cagaataaca gaaaaagggg aaatacgaaa actgacggac aagggatgag 13680

aaacaagggc gagagtgtga cacacatagg aggtgatttg tcaactatgg acaatgaccg 13740

tgccacacaa aatgtgacac aatgacagga tgcttgtgaa agggaatggt tagtgggaat 13800

gtgataactg gagtgagtcg tgtgagtggg atggagtagc gagaatgtga ggtattacac 13860

aagcagagac ggtaccgttg ttgagcattg cacacggcac agttacaagt aactctcggg 13920

tcacggtaag aagtgctttt cagattgttc ccctttccgt cgatcctaaa gatacaaatg 13980

agccactttt acgatgatca ttatacggta tgtattttgt agatgtagct tggagggcgg 14040

ccgcatgcga cgccacccat ggactctaag cggccgcatg gacctgtcga cttggctcga 14100

gggggggccc ggtaccagct tttgttccct ttagtgaggg ttaatttcga gcttggcgta 14160

atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat 14220

acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt 14280

aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta 14340

atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc 14400

gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa 14460

ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa 14520

aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct 14580

ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac 14640

aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc 14700

gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc 14760

tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg 14820

tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga 14880

gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag 14940

cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta 15000

cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag 15060

agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg 15120

caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac 15180

ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc 15240

aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag 15300

tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc 15360

agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac 15420

gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc 15480

accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg 15540

tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag 15600

tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc 15660

acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac 15720

atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag 15780

aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac 15840

tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg 15900

agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc 15960

gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact 16020

ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg 16080

atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa 16140

tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt 16200

tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg 16260

tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgc 16313

<210> 6

<211> 10429

<212> DNA

<213> Artificial Sequence

<220>

<223> pSK-Hmg(y)-Ura-IntB.5N-3N

<400> 6

cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag 60

ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac 120

cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga 180

ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc 240

accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg 300

gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa 360

gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac 420

caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct 480

gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa 540

agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg 600

ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat tggagctcgc 660

tgagctggcg cgcctctaga aacgtacgtt gtaactattt aaattccttg gtgtttaaac 720

tacgtggagt agctggtgtt agattagttt tgtagaaatc ggtcatattc atcctagtat 780

tcaatacttc cctgttccac tgaaacgtgt tcgaaacttg ttttctagac cgacttgtta 840

ttgaaatgag cgtaaagaag gtgaaattcg gcgacgtttg cgcacagcca ttcaagtgga 900

atattgttga ctagagtcat gagcgtaggc atttataggt gcttcagaca taacaaatgt 960

ctaaggtata acggaataca actttcaaga gccacgagtc accagactga gcttctcacg 1020

accccatgca tcgaaatttc cctctagtga atgccacggc actacagtat gtacatacat 1080

acaggatata ctcaagatac ggcattggct tattgatatc ttttttgcca acatgttttc 1140

ccatagggtc accacccaca gttctcactc agatcatgga gactctaacc ttgagacatc 1200

aattatcagc tctcgaggat aatgttagtg cagttccagg actcattgtg caactgtcac 1260

cacggcattt tgggtctgtt cttttgaagt acagaaatat cctcattgtt ggtatacttt 1320

gggacttttt cttgttacag aagaataaaa aaacctcgac tgatgtacta attacatggt 1380

taacatcccc aaggtcaaag tacagatatt gtaccgactt ctgaaatttg tgggatccac 1440

acacggacct ctgcgatgat acaatatcat ggttccatgg tctcttgaat cacaccactc 1500

aataataaac accagctctt tcaatccaac ttagctttct tccacttgaa cataggccat 1560

ccccccttct ttctatttta cataaatagc aagatcctta cccctacatg tctcataaca 1620

caatctcaac ttgacttccc ataagaagtt cactacccta aggactctgg cagtatttac 1680

tgctctcgta tcgatactgg gctgcttcct aatgcaggag tcgcataagg gagagcgtcg 1740

accgatgccc ttgagagcct tcaacccagt cagctccttc cggtgggcgc ggggcatgac 1800

tatcgtcgcc gcacttatga ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc 1860

agcgctctgg gtcattttcg gcgaggaccg ctttcgctgg agcgcgacga tgatcggcct 1920

gtcgcttgcg gtattcggaa tcttgcacgc cctcgctcaa gccttcgtca ctggtcccgc 1980

caccaaacgt ttcggcgaga agcaggccat tatcgccggc atggcggccg acgcgctggg 2040

ctacgtcttg ctggcgttcg cgacgcgagg ctggatggcc ttccccatta tgattcttct 2100

cgcttccggc ggcatcggga tgcccgcgtt gcaggccatg ctgtccaggc aggtagatga 2160

cgaccatcag ggacagcttc aaggatcgct cgcggctctt accagcctaa cttcgatcac 2220

tggaccgctg atcgtcacgg cgatttatgc cgcctcggcg agcacatgga acgggttggc 2280

atggattgta ggcgccgccc tataccttgt ctgcctcctg tctgactcgt cattgccgcc 2340

tttggagtac gactccaact atgagtgtgc ttggatcact ttgacgatac attcttcgtt 2400

ggaggctgtg ggtctgacag ctgcgttttc ggcgcggttg gccgacaaca atatcagctg 2460

caacgtcatt gctggctttc atcatgatca catttttgtc ggcaaaggcg acgcccagag 2520

agccattgac gttctttcta atttggaccg atagccgtat agtccagtct atctataagt 2580

tcaactaact cgtaactatt accataacat atacttcact gccccagata aggttccgat 2640

aaaaagttct gcagactaaa tttatttcag tctcctcttc accaccaaaa tgccctccta 2700

cgaagctcga gctaacgtcc acaagtccgc ctttgccgct cgagtgctca agctcgtggc 2760

agccaagaaa accaacctgt gtgcttctct ggatgttacc accaccaagg agctcattga 2820

gcttgccgat aaggtcggac cttatgtgtg catgatcaag acccatatcg acatcattga 2880

cgacttcacc tacgccggca ctgtgctccc cctcaaggaa cttgctctta agcacggttt 2940

cttcctgttc gaggacagaa agttcgcaga tattggcaac actgtcaagc accagtacaa 3000

gaacggtgtc taccgaatcg ccgagtggtc cgatatcacc aacgcccacg gtgtacccgg 3060

aaccggaatc attgctggcc tgcgagctgg tgccgaggaa actgtctctg aacagaagaa 3120

ggaggacgtc tctgactacg agaactccca gtacaaggag ttcctggtcc cctctcccaa 3180

cgagaagctg gccagaggtc tgctcatgct ggccgagctg tcttgcaagg gctctctggc 3240

cactggcgag tactccaagc agaccattga gcttgcccga tccgaccccg agtttgtggt 3300

tggcttcatt gcccagaacc gacctaaggg cgactctgag gactggctta ttctgacccc 3360

cggggtgggt cttgacgaca agggagacgc tctcggacag cagtaccgaa ctgttgagga 3420

tgtcatgtct accggaacgg atatcataat tgtcggccga ggtctgtacg gccagaaccg 3480

agatcctatt gaggaggcca agcgatacca gaaggctggc tgggaggctt accagaagat 3540

taactgttag aggttagact atggatatgt catttaactg tgtatataga gagcgtgcaa 3600

gtatggagcg cttgttcagc ttgtatgatg gtcagacgac ctgtctgatc gagtatgtat 3660

gatactgcac aacctgtgta tccgcatgat ctgtccaatg gggcatgttg ttgtgtttct 3720

cgatacggag atgctgggta caagtagcta atacgattga actacttata cttatatgag 3780

gcttgaagaa agctgacttg tgtatgactt attctcaact acatccccag tcacaatacc 3840

accagaaatc tactgggctg cttcctaatg caggagtcgc ataagggaga gcgtcgaccg 3900

atgcccttga gagccttcaa cccagtcagc tccttccggt gggcgcgggg catgactatc 3960

gtcgccgcac ttatgactgt cttctttatc atgcaactcg taggacaggt gccggcagcg 4020

ctctgggtca ttttcggcga ggaccgcttt cgctggagcg cgacgatgat cggcctgtcg 4080

cttgcggtat tcggaatctt gcacgccctc gctcaagcct tcgtcactgg tcccgccacc 4140

aaacgtttcg gcgagaagca ggccattatc gccggcatgg cggccgacgc gctgggctac 4200

gtcttgctgg cgttcgcgac gcgaggctgg atggccttcc ccattatgat tcttctcgct 4260

tccggcggca tcgggatgcc cgcgttgcag gccatgctgt ccaggcaggt agatgacgac 4320

catcagggac agcttcaagg atcgctcgcg gctcttacca gcctaacttc gatcactgga 4380

ccgctgatcg tcacggcgat ttatgccgcc tcggcgagca catggaacgg gttggcatgg 4440

attgtaggcg ccgccctata ccttgtctgc ctggatctcc agttctgtac agccgatccc 4500

ggggaccctt gtagaactct ctccaggcct cacccttgtt gaccttctcg tcctcctgaa 4560

ttcatgctgt tcatcgtggt taatgctgct gtgtgctgtg tgtgtgtgtt gtttggcgct 4620

cattgttgcg ttatgcagcg tacaccacaa tattggaagc ttattagcct ttctattttt 4680

tcgtttgcaa ggcttaacaa cattgctgtg gagagggatg gggatatgga ggccgctgga 4740

gggagtcgga gaggcgtttt ggagcggctt ggcctggcgc ccagctcgcg aaacgcacct 4800

aggacccttt ggcacgccga aatgtgccac ttttcagtct agtaacgcct tacctacgtc 4860

attccatgcg tgcatgtttg cgcctttttt cccttgccct tgatcgccac acagtacagt 4920

gcactgtaca gtggaggttt tgggggggtc ttagatggga gctaaaagcg gcctagcggt 4980

acactagtgg gattgtatgg agtggcatgg agcctaggtg gagcctgaca ggacgcacga 5040

ccggctagcc cgtgacagac gatgggtggc tcctgttgtc caccgcgtac aaatgtttgg 5100

gccaaagtct tgtcagcctt gcttgcgaac ctaattccca attttgtcac ttcgcacccc 5160

cattgatcga gccctaaccc ctgcccatca ggcaatccaa ttaagctcgc attgtctgcc 5220

ttgtttagtt tggctcctgc ccgtttcggc gtccacttgc acaaacacaa acaagcatta 5280

tatataaggc tcgtctctcc ctcccaacca cactcacttt tttgcccgtc ttcccttgct 5340

aacacaaaag tcaagaacac aaacaaccac cccaaccccc ttacacacaa gacatatcta 5400

cagcaatggc catggccacc cagtctgtga aggtggttga gaagcacgtt cctatcgtca 5460

ttgagaagcc cagcgagaag gaggaggaca cctcttctga agactccatt gagctgactg 5520

tcggaaagca gcccaagccc gtgaccgaga cccgttctct ggacgaccta gaggctatca 5580

tgaaggcagg taagaccaag cttctggagg accacgaggt tgtcaagctc tctctcgagg 5640

gcaagcttcc tttgtatgct cttgagaagc agcttggtga caacacccga gctgttggca 5700

tccgacgatc tatcatctcc cagcagtcta ataccaagac tttagagacc tcaaagcttc 5760

cttacctgca ctacgactac gaccgtgttt ttggagcctg ttgcgagaac gttattggtt 5820

acatgcctct ccccgttggt gttgctggcc ccatgaacat tgatggcaag aactaccaca 5880

ttcctatggc caccactgag ggttgtcttg ttgcctcaac catgcgaggt tgcaaggcca 5940

tcaacgccgg tggcggtgtt accactgtgc ttactcagga cggtatgaca cgaggtcctt 6000

gtgtttcctt cccctctctc aagcgggctg gagccgctaa gatctggctt gattccgagg 6060

agggtctcaa gtccatgcga aaggccttca actccacctc tcgatttgct cgtctccagt 6120

ctcttcactc tacccttgct ggtaacctgc tgtttattcg attccgaacc accactggtg 6180

atgccatggg catgaacatg atctccaagg gcgtcgaaca ctctctggcc gtcatggtca 6240

aggagtacgg cttccctgat atggacattg tgtctgtctc gggtaactac tgcactgaca 6300

agaagcccgc agcgatcaac tggatcgaag gccgaggcaa gagtgttgtt gccgaagcca 6360

ccatccctgc tcacattgtc aagtctgttc tcaaaagtga ggttgacgct cttgttgagc 6420

tcaacatcag caagaatctg atcggtagtg ccatggctgg ctctgtggga ggtttcaatg 6480

cacacgccgc aaacctggtg accgccatct accttgccac tggccaggat cctgctcaga 6540

atgtcgagtc ttccaactgc atcacgctga tgagcaacgt cgacggtaac ctgctcatct 6600

ccgtttccat gccttctatc gaggtcggta ccattggtgg aggtactatt ttggagcccc 6660

agggggctat gctggagatg cttggcgtgc gaggtcctca catcgagacc cccggtgcca 6720

acgcccaaca gcttgctcgc atcattgctt ctggagttct tgcagcggag ctttcgctgt 6780

gttctgctct tgctgccggc catcttgtgc aaagtcatat gacccacaac cggtcccagg 6840

ctcctactcc ggccaagcag tctcaggccg atctgcagcg tctacaaaac ggttcgaata 6900

tttgcatacg gtcataggcg gccgcatgga gcgtgtgttc tgagtcgatg ttttctatgg 6960

agttgtgagt gttagtagac atgatgggtt tatatatgat gaatgaatag atgtgatttt 7020

gatttgcacg atggaattga gaactttgta aacgtacatg ggaatgtatg aatgtggggg 7080

ttttgtgact ggataactga cggtcagtgg acgccgttgt tcaaatatcc aagagatgcg 7140

agaaactttg ggtcaagtga acatgtcctc tctgttcaag taaaccatca actatgggta 7200

gtatatttag taaggacaag agttgagatt ctttggagtc ctagaaacgt attttcgcgt 7260

tccaagatca aattagtaga gtaatacggg cacgggaatc cattcatagt ctcaatcctg 7320

caggtgagtt aattaaacct gctcctgcac ctaagttcgt caaaatcggt tttactcaga 7380

gaatcaacaa ccctaccaac tgtacatact gctaaccctg attctttgaa taaccccaat 7440

aaggctcctg ctgacccttc tgccgttcta ggaaaccagc ttgttgagta cgctgtgaac 7500

atcactcttg gtatacccgg tcagcccttc tcagttcaga ttgacacagg ctcgtctgac 7560

ttgtgggtga agagtgacgg ctcctccggt gcattcaaca agaaggcttc ttctactttt 7620

caggaggacg ttcccaacgg ctttgcaatt gcccacggag acaaaacctc tgccattgga 7680

gattgggtca aagataccat caatatcggt ggtgttagta ttgaccagta tagtattgac 7740

cagtatgagt tcgccatggc tactcagaca aatactgacc cggtttttgg tatcggctac 7800

ccgagcaacg aggcgtctta tgtcaataac cttggaaatg gggaccagtt ccagtacgac 7860

aatctcccca ttcgtctagc caaggacgga ttcatcaaca cacctgctta ttctctgtac 7920

ctggattctc tccagtccca aactgactct cttctttttg gtgctgtgga tacttcgaag 7980

ttcactggag gtcttgcact cctccctttt atcaaggatg agccctctga cccctctcca 8040

cgggagtttc aagtcactct cgcttctgtc gatattaata atgcaggatc taccactaac 8100

gctctcggca ctcctcgttt ggctgttttg gattctggaa cgaccctctc ccttgttcgc 8160

atgcgacgcc acccatggac tctaagcggc cgcatggacc tgtcgacttg gctcgagggg 8220

gggcccggta ccagcttttg ttccctttag tgagggttaa tttcgagctt ggcgtaatca 8280

tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga 8340

gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt 8400

gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga 8460

atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc 8520

actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg 8580

gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc 8640

cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc 8700

ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga 8760

ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc 8820

ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat 8880

agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg 8940

cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc 9000

aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga 9060

gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact 9120

agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt 9180

ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag 9240

cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg 9300

tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa 9360

aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata 9420

tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg 9480

atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata 9540

cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg 9600

gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct 9660

gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt 9720

tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc 9780

tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga 9840

tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt 9900

aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc 9960

atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa 10020

tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca 10080

catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca 10140

aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct 10200

tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc 10260

gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa 10320

tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt 10380

tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgc 10429

<210> 7

<211> 16371

<212> DNA

<213> Artificial Sequence

<220>

<223> pS-B-RA-GGS-Gut2-Ura3(L)

<400> 7

cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag 60

ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac 120

cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga 180

ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc 240

accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg 300

gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa 360

gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac 420

caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct 480

gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa 540

agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg 600

ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat tggagctcgc 660

tgagctggcg cgcctgccaa cggctattca acggtgacaa cggatgatgc tgtcacatga 720

cgccattttt tatgttgtat ccaacagcac ggtactaaaa caggccattt gtaaaggcct 780

cactcagctc acacacgctc aacggtcacg ataaggtcgc actagaggcg ttagttggtt 840

tcaagaatag tggttattgg tcttgggata cggggttgga caatatacaa atgggctcgc 900

gtacacttat acagtcctac cattctgtcg ccctctgatt ctccgccaca tcagccacgc 960

cgcaacgtct cctcctcatc cccctcctgc tcttccactc gcaaaacgtc caaactcaat 1020

tgtgtcaaaa ttggaggttc tcttcgtttg agcctaccat tttcaatttt ttagttgcga 1080

cagcggcccg gtcagaggtt cacaacaagg tctagagaca ctttgtcatg gggccgagaa 1140

ggaccataaa aaccaaacga tggtcacgtc aggtcaatta ctgaccagtc tcacatccga 1200

cccctcgcgt gctcgaccgg aggatttctc tgcactcgtc cttgcatacc tcggctagcg 1260

ggatttattc accaatcaca cagccgagag tttttccgga cccttcatcc aacagcttag 1320

agttgcatga gtcagtagca acgtagactt tgagcctttg tgacagatgt ccaagtgcag 1380

cacgttgtag gaaaataagg tgaaggattg gccaatgtga acagaggcga caagagtccg 1440

tctggagggc ttgttgtagt caattgcccg cgcaattgat tgacctcatc gtttctgccg 1500

gaaccccccc acaagcccgg ataaatagac acgccccaca agccgttcgt ctggtctgct 1560

cacagcacac ttccatttaa aattcaaaca aagcgcacca ccgcaaagca tacttaaccc 1620

actcaatgta gacgtcgcgg aacttctctt tcctacccac gtacgttgtg tggaattgtg 1680

agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa gctcgattaa 1740

ttaactcacc tgcaggattg agactatgaa tggattcccg tgcccgtatt actctactaa 1800

tttgatcttg gaacgcgaaa atacgtttct aggactccaa agaatctcaa ctcttgtcct 1860

tactaaatat actacccata gttgatggtt tacttgaaca gagaggacat gttcacttga 1920

cccaaagttt ctcgcatctc ttggatattt gaacaacggc gtccactgac cgtcagttat 1980

ccagtcacaa aacccccaca ttcatacatt cccatgtacg tttacaaagt tctcaattcc 2040

atcgtgcaaa tcaaaatcac atctattcat tcatcatata taaacccatc atgtctacta 2100

acactcacaa ctccatagaa aacatcgact cagaacacac gctccatgcg gccgctcact 2160

gcgcatcctc aaagtacttt ctctcctcac agtcagaggt ggacacaaaa taatgcaaaa 2220

tggctcgtag cttggagaca tcgtggccag ctgctgctag atcatcaatg tacgaacttg 2280

ccttgagtga catggcctgt atcctcttga ggcagtagtc gaaactcttg gtttctgttc 2340

tcatgtagtc cacggcgtac tttttgagtg aagcgtcgct tgttcgctgt ttgagaatgt 2400

tgagaatctc gtggttatcc gggttggtgc gtatgctgtg aatcagcgga aacgaaaact 2460

ttccttcgct gatatcttcg cagaatccct tgttctcggt caattccgtg gactgcaggt 2520

tgaggtaatc atccagaatc tggtaaatga ctcccagtgt gtcggtaagg tgtgtgagat 2580

caaagttgat cttttcatgg tcctcctgtt tcgatgccac cgacagcata agtctcagag 2640

ccagccgaaa cagtccaccg gtcttgtgca ccaccatctc cagatactcg tcttccgagg 2700

ggcaagtgag tgtttctctc cagtagagat ccataccctg acctctatgc aagttgatca 2760

tttcctcggt gaaaatggag acggcatcat aagacttgag cttgagcacc tcctgcagag 2820

ccacaaagta catgtagttg gcggagttga tggtttgggg gactccaaac agacaatggg 2880

ctgccggcag gcctcgtcgg agcatcgagt tgtcttccac gtcatcaaca agcagcgacg 2940

agttatgcag aatcttggtg atgtgcgaaa tggtctcgag atcgctcttg tccaccttga 3000

taaccgctcc gaacgctgcg atcaagtgtt ctctgatgtt gtggccccgg ttgttggcga 3060

ggtagttgta cggtcccagc agcgcggtgt cggcggcctt gccccatatc tccttgaaat 3120

ccgcgctgtt ataatccatg gttgtgaatt agggtggtga gaatggttgg ttgtagggaa 3180

gaatcaaagg ccggtctcgg gatccgtggg tatatatata tatatatata tatacgatcc 3240

ttcgttacct ccctgttctc aaaactgtgg tttttcgttt ttcgtttttt gctttttttg 3300

atttttttag ggccaactaa gcttccagat ttcgctaatc acctttgtac taattacaag 3360

aaaggaagaa gctgattaga gttgggcttt ttatgcaact gtgctactcc ttatctctga 3420

tatgaaagtg tagacccaat cacatcatgt catttagagt tggtaatact gggaggatag 3480

ataaggcacg aaaacgagcc atagcagaca tgctgggtgt agccaagcag aagaaagtag 3540

atgggagcca attgacgagc gagggagcta cgccaatccg acatacgaca cgctgagatc 3600

gtcttggccg gggggtacct acagatgtcc aagggtaagt gcttgactgt aattgtatgt 3660

ctgaggacaa atatgtagtc agccgtataa agtcatacca ggcaccagtg ccatcatcga 3720

accactaact ctctatgata catgcctccg gtattattgt accatgcgtc gctttgttac 3780

atacgtatct tgcctttttc tctcagaaac tccagacttt ggctattggt cgagataagc 3840

ccggaccata gtgagtcttt cacactctac atttctccct tgctccaact atttaaatcg 3900

aggaagagga caagcggctg cttcttaagt ttgtgacatc agtatccaag gcaccattgc 3960

aaggattcaa ggctttgaac ccgtcatttg ccattcgtaa cgctggtaga caggttgatc 4020

ggttccctac ggcctccacc tgtgtcaatc ttctcaagct gcctgactat caggacattg 4080

atcaacttcg gaagaaactt ttgtatgcca ttcgatcaca tgctggtttc gatttgtctt 4140

agaggaacgc atatacagta atcatagaga ataaacgata ttcatttatt aaagtagata 4200

gttgaggtag aagttgtaaa gagtgataaa tagcggccgc tagtcaacaa aaaaggactt 4260

aatctttcta gcttgacgga gagccacagg cttgtcttca gactgataga ggttgtacac 4320

ggacaacaga gcaatcttgg cacgttcgct tcctttcaga tgggctcttg taggatatgt 4380

tgtcttctgc tgcttgagca cagatccaat tgcagcgtac acttggcaag cagctcgcac 4440

accgccttga cagtttgccg gcaacttgtc aatgcccttc ttagctctca ccatgatagc 4500

gtctgccttt cccacgagct tcagagacaa gcccaacagt ctctgatcac ccaggctacg 4560

ggcgttgcct tgctgaatct gttctgtctc ttcttttcta agccactgct gaggcagata 4620

acatcgaccc agagtctcgc tgtcggtcac aatgtcacga gcaatgttaa cgtattgcag 4680

caccaggccc atctcacgtg cacggtcaat aatccaagca tcattctcct tctggtcttg 4740

agcaagaatc acacgtgtgc acatttcacc cacggaggag gccacacaag cggagtatgc 4800

ctccaagtct tgttcatcaa ggataggacg acgctcaaga tcccatttgt aaccatccag 4860

cagctcctcc acagggtcca cttcaaggac atgtcgaagt cgagtaaagg ctctaaaggc 4920

agaaatacac gaagcaggaa gctggttctg atacaactcc cagtcaatca caataggcgc 4980

ggaggtcttc tggctaaaga gatcacgaac aaactgtcga gtaagatcca gctggtctct 5040

tctctcttga acagatttgg actcatcatc gcacaggtca tcggtagctc tacagaaagc 5100

atagagaaca cccaagtctt gacgcacata ggatggaaaa acggcagatg cagtgtagaa 5160

cgacttcgag gctttacgca gaatgtccca gctgatggta agatcatcaa acaactcatt 5220

gttgagcatc tggtcaggaa gacagaaggc ccatgccagc tctttgacgt gctggaaaag 5280

ggaaatggct tgtttagggt tctgattctg aacagacgat ttgtacagat ggaggatggc 5340

ctgggctcgg tcaatagcac aggtagcaaa aaccaagact gtgttgatca aggtaaaaaa 5400

caggcactct tccacaggca gatcgggcac caccattttg ccagtgcttg ttctaagaga 5460

aatgtgccat gtgccagcgg aaatagcgac gatgtcagcc caacacaggt acacggaagg 5520

gataacaatc gacaaaagga cagccacagg tcgacgcaag atgtactcgc cagcacccag 5580

ccaaagaata gccaacacag gacaagcata ccaaaggatg cacgaaccgt aaaacgagga 5640

tttatttggc agtgtcaagt gccaagcgtg ataggtgatt gccaacagag cagaaacagg 5700

cacaagtcgc accagcagtg tctgcttcca cgaggtgttg ggtctaataa agaaggtgtg 5760

caagtgccaa cgcataacaa agttcgagaa cgcgacagtc atcagagtca tgatgataaa 5820

gaacatgtac tcttccagag gcacgtagcc aatgacagcc acaacacagg taggacagta 5880

ccaccaagct cgatgataaa cgatgtaatt gtcccaaatc gatgcggtcg aggcggccat 5940

caacatcaga aatttgtact tgagattgtc ttgctgcgag tgaaacggct tcagcagcca 6000

acacaatgcc gcaaggacag gcagtgtgta gtagagatga aactccagat aggtgagaat 6060

ggaagccatg gttgatgtgt gtttaattca agaatgaata tagagaagag aagaagaaaa 6120

aagattcaat tgagccggcg atgcagaccc ttatataaat gttgccttgg acagacggag 6180

caagcccgcc caaacctacg ttcggtataa tatgttaagc tttttaacac aaaggtttgg 6240

cttggggtaa cctgatgtgg tgcaaaagac cgggcgttgg cgagccattg cgcgggcgaa 6300

tggggccgtg actcgtctca aattcgaggg cgtgcctcaa ttcgtgcccc cgtggctttt 6360

tcccgccgtt tccgccccgt ttgcaccact gcagccgctt ctttggttcg gacaccttgc 6420

tgcgagctag gtgccttgtg ctacttaaaa agtggcctcc caacaccaac atgacatgag 6480

tgcgtgggcc aagacacgtt ggcggggtcg cagtcggctc aatggcccgg aaaaaacgct 6540

gctggagctg gttcggacgc agtccgccgc ggcgtatgga tatccgcaag gttccatagc 6600

gccattgccc tccgtcggcg tctatcccgc aacctctaaa tagagcggga atataaccca 6660

agcttctttt ttttccttta acacgcacac ccccaactat catgttgctg ctgctgtttg 6720

actctactct gtggaggggt gctcccaccc aacccaacct acaggtggat ccggcgctgt 6780

gattggctga taagtctcct atccggacta attctgacca atgggacatg cgcgcaggac 6840

ccaaatgccg caattacgta accccaacga aatgcctacc cctctttgga gcccagcggc 6900

cccaaatccc cccaagcagc ccggttctac cggcttccat ctccaagcac aagcagcccg 6960

gttctaccgg cttccatctc caagcacccc tttctccaca ccccacaaaa agacccgtgc 7020

aggacatcct actgcgtgtt taaacaacaa ttgccccgga gaagacggcc aggccgccta 7080

gatgacaaat tcaacaactc acagctgact ttctgccatt gccactaggg gggggccttt 7140

ttatatggcc aagccaagct ctccacgtcg gttgggctgc acccaacaat aaatgggtag 7200

ggttgcacca acaaagggat gggatggggg gtagaagata cgaggataac ggggctcaat 7260

ggcacaaata agaacgaata ctgccattaa gactcgtgat ccagcgactg acaccattgc 7320

atcatctaag ggcctcaaaa ctacctcgga actgctgcgc tgatctggac accacagagg 7380

ttccgagcac tttaggttgc accaaatgtc ccaccaggtg caggcagaaa acgctggaac 7440

agcgtgtaca gtttgtctta gcaaaaagtg aaggcgctga ggtcgagcag ggtggtgtga 7500

cttgttatag cctttagagc tgcgaaagcg cgtatggatt tggctcatca ggccagattg 7560

agggtctgtg gacacatgtc atgttagtgt acttcaatcg ccccctggat atagccccga 7620

caataggccg tggcctcatt tttttgcctt ccgcacattt ccattgctcg gtacccacac 7680

cttgcttctc ctgcacttgc caaccttaat actggtttac attgaccaac atcttacaag 7740

cggggggctt gtctagggta tatataaaca gtggctctcc caatcggttg ccagtctctt 7800

ttttcctttc tttccccaca gattcgaaat ctaaactaca catcacacaa tgcctgttac 7860

tgacgtcctt aagcgaaagt ccggtgtcat cgtcggcgac gatgtccgag ccgtgagtat 7920

ccacgacaag atcagtgtcg agacgacgcg ttttgtgtaa tgacacaatc cgaaagtcgc 7980

tagcaacaca cactctctac acaaactaac ccagctctat gtccgatcag aagaagcaca 8040

ttgttgtcat tggtgccggt attggcggaa ctgctactgc tgctcgtctt gctcgtgaag 8100

gttttcgagt tactgttgtt gagaagaacg acttttccgg tggccgttgt tctttcattc 8160

atcacgacgg tcatcgattt gatcagggtc cctctctcta cttgatgcct aagctctttg 8220

aagacgcatt tgctgacttg gacgagcgta ttggtgatca cttggacttg ctgcgatgtg 8280

acaataacta taaggttcac tttgacgacg gtgatgccgt gcagctctct tccgatctga 8340

ccaagatgaa gggcgagttg gaccgtattg agggtcccct tggatttggt agattcttgg 8400

acttcatgaa ggagacacat gtccactatg aacaaggtac atttattgct atcaagcgaa 8460

actttgagac catttgggat ttgattcgtc tccagtatgt gcctgagatc tttcgattgc 8520

acttgtttgg taagatctat gaccgagcct ctaaatactt ccagaccaag aaaatgcgta 8580

tggcttttac ctttcagaca atgtacatgg gtatgtcgcc ttacgatgct ccagcagttt 8640

actctttgct ccagtacacc gagtttgctg aaggtatctg gtatcctcgt ggtggtttca 8700

acatggttgt tcagaagctg gagtctatcg cctccaaaaa gtacggtgct gagttcagat 8760

accagtcgcc tgttgctaaa attaacactg tcgacaaaga caagcgtgtg accggtgtca 8820

ccttggagag cggagaagtc attgaagccg atgcagtcgt gtgtaatgcg gatcttgttt 8880

acgcttatca ccatctgctg cctccttgca attggacaaa gaagacactg gcctctaaga 8940

aactcacctc ttcttctatt tcgttttact ggtccatgtc tacaaaggtg cctcagctgg 9000

acgtgcacaa tatcttcttg gctgaagcct acaaggagtc ttttgacgag attttcaacg 9060

acttcggttt gccctctgaa gcgtctttct atgtcaacgt tccatctcga attgatgagt 9120

ctgccgcacc tcccaacaag gactccatta ttgtgttggt tccaattggc cacatgaagt 9180

ctaagacagg aaactctgct gaagagaatt atcctgagtt ggtgaaccgt gcacgaaaga 9240

tggttctgga agttatcgag cgtcgtttgg gagtgaacaa ctttgctaat ttgattgaac 9300

atgaagaagt gaatgatcct tctgtttggc agagcaagtt taacctctgg agaggttcta 9360

ttcttggtct gtctcatgac gtgttccaag ttctctggtt cagaccttct accaaggact 9420

ccacaaaccg ttatgacaat ctgttctttg tcggagcttc tacacatcca ggtactggtg 9480

ttcctatcgt tcttgctgga tctaagctga cctccgacca agtctgtaag agctttggcc 9540

agaatcccct gccaagaaag ctgcaagaca gccagaagaa gtacgctcct gagcagactc 9600

gaaagaccga gagccactgg atctactact gtcttgcttg ttactttgtt accttcctct 9660

ttttctattt cttcccaaga gatgacacca caactcctgc ttctttcatt aaccaactgc 9720

tgcctaacgt tttccaagtg cagaacagca acgacattcg aatttagtat ttatcactct 9780

ttacaacttc tacctcaact atctacttta ataaatgaat atcgtttatt ctctatgatt 9840

actgtatatg cgttcctcta agacaaatcg aaaccagcat gtgatcgaat ggcatacaaa 9900

agtttcttcc gaagttgatc aatgtcctga tagtcaggca gcttgagaag attgacacag 9960

gtggaggccg tagggaaccg atcaacctgt ctaccagcgt tacgaatggc aaatgacggg 10020

ttcaaagcct tgaatccttg caatggtgcc ttggatactg atgtcacaaa cttaagaagc 10080

agccgcttgt cctcttcctc ggaattcagg aggacgagaa ggtcaacaag ggtgaggcct 10140

ggagagagtt ctacaagggt ccccgggatc ggctgtacag aactggagat ccaggcagac 10200

aaggtatagg gcggcgccta caatccatgc caacccgttc catgtgctcg ccgaggcggc 10260

ataaatcgcc gtgacgatca gcggtccagt gatcgaagtt aggctggtaa gagccgcgag 10320

cgatccttga agctgtccct gatggtcgtc atctacctgc ctggacagca tggcctgcaa 10380

cgcgggcatc ccgatgccgc cggaagcgag aagaatcata atggggaagg ccatccagcc 10440

tcgcgtcgcg aacgccagca agacgtagcc cagcgcgtcg gccgccatgc cggcgataat 10500

ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc 10560

gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg 10620

gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa 10680

gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac 10740

tgggttgaag gctctcaagg gcatcggtcg acgctctccc ttatgcgact cctgcattag 10800

gaagcagccc agtagatttc tggtggtatt gtgactgggg atgtagttga gaataagtca 10860

tacacaagtc agctttcttc aagcctcata taagtataag tagttcaatc gtattagcta 10920

cttgtaccca gcatctccgt atcgagaaac acaacaacat gccccattgg acagatcatg 10980

cggatacaca ggttgtgcag tatcatacat actcgatcag acaggtcgtc tgaccatcat 11040

acaagctgaa caagcgctcc atacttgcac gctctctata tacacagtta aatgacatat 11100

ccatagtcta acctctaaca gttaatcttc tggtaagcct cccagccagc cttctggtat 11160

cgcttggcct cctcaatagg atctcggttc tggccgtaca gacctcggcc gacaattatg 11220

atatccgttc cggtagacat gacatcctca acagttcggt actgctgtcc gagagcgtct 11280

cccttgtcgt caagacccac cccgggggtc agaataagcc agtcctcaga gtcgccctta 11340

ggtcggttct gggcaatgaa gccaaccaca aactcggggt cggatcgggc aagctcaatg 11400

gtctgcttgg agtactcgcc agtggccaga gagcccttgc aagacagctc ggccagcatg 11460

agcagacctc tggccagctt ctcgttggga gaggggacca ggaactcctt gtactgggag 11520

ttctcgtagt cagagacgtc ctccttcttc tgttcagaga cagtttcctc ggcaccagct 11580

cgcaggccag caatgattcc ggttccgggt acaccgtggg cgttggtgat atcggaccac 11640

tcggcgattc ggtagacacc gttcttgtac tggtgcttga cagtgttgcc aatatctgcg 11700

aactttctgt cctcgaacag gaagaaaccg tgcttaagag caagttcctt gagggggagc 11760

acagtgccgg cgtaggtgaa gtcgtcaatg atgtcgatat gggtcttgat catgcacaca 11820

taaggtccga ccttatcggc aagctcaatg agctccttgg tggtggtaac atccagagaa 11880

gcacacaggt tggttttctt ggctgccacg agcttgagca ctcgagcggc aaaggcggac 11940

ttgtggacgt tagctcgagc ttcgtaggag ggcattttgg tggtgaagag gagactgaaa 12000

taaatttagt ctgcagaact ttttatcgga accttatctg gggcagtgaa gtatatgtta 12060

tggtaatagt tacgagttag ttgaacttat agatagactg gactatacgg ctatcggtcc 12120

aaattagaaa gaacgtcaat ggctctctgg gcgtcgcctt tgccgacaaa aatgtgatca 12180

tgatgaaagc cagcaatgac gttgcagctg atattgttgt cggccaaccg cgccgaaaac 12240

gcagctgtca gacccacagc ctccaacgaa gaatgtatcg tcaaagtgat ccaagcacac 12300

tcatagttgg agtcgtactc caaaggcggc aatgacgagt cagacaggag gcagacaagg 12360

tatagggcgg cgcctacaat ccatgccaac ccgttccatg tgctcgccga ggcggcataa 12420

atcgccgtga cgatcagcgg tccagtgatc gaagttaggc tggtaagagc cgcgagcgat 12480

ccttgaagct gtccctgatg gtcgtcatct acctgcctgg acagcatggc ctgcaacgcg 12540

ggcatcccga tgccgccgga agcgagaaga atcataatgg ggaaggccat ccagcctcgc 12600

gtcgcgaacg ccagcaagac gtagcccagc gcgtcggccg ccatgccggc gataatggcc 12660

tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc 12720

aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc 12780

tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag 12840

acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg 12900

ttgaaggctc tcaagggcat cggtcgacgc tctcccttat gcgactcctg cattaggaag 12960

cagcccagta tcgatagaga cggagtcact gccttaatta accctccaac tcgagggttg 13020

atgtagatct taagaccagc cagaaccaca aggaaacagg cgagaatcat ctgggtgagg 13080

atcttaaagg aggtggagat acctagaatg taccagatgg cgatatcaaa gactccaagc 13140

gcagctctga tgatcttctc ggtttggtcg ttggtctcag tgaaccacag gtagtagaag 13200

tagccggtgc cgatcatgat ggcagccagc agcacgtagt agagcacatt gaggaaggtc 13260

attgtagggt gtttatctgt gcggtcaagt tgccaattgg tgataagtga gactgtcgta 13320

taacttgtaa gtatacagta actctcgctt gttggttgct acttttacct tcattcggtt 13380

aagacttgag tgcatatcgt gtgcatgttt gctgtgtgga gtgcaaggcg gcaatagcaa 13440

caagccgtgt atagttgagg tggtcgcatt ttggggtctt cctgtcatcg tttcattgct 13500

caatctccac tacagtagcc taacttctac tggttagtca aagcacgtac cgactgtgca 13560

ccatacggaa aacttagaac aatgccccac gatgccccac ggagatacac cgaagcgtgt 13620

gatctgcggt aagtaaatac gagtggtggg agtggggtag ttggactttg ttcgtggtgg 13680

tcttgtgacg actactggtt ctgtaagcgg tctagttagc caagctatgt agctactgta 13740

attgtagttt cacttcccat catgtccaaa agcgtcgtat ctaaggaaca atacgacctg 13800

ttcgccaact gttgcgtgtg agacccggtg ccatcacttc aatgaacaac tcgtgttatc 13860

aattcaaacc aagataaatg ataacaaacg atataggtaa gaccacattg atctcatgta 13920

tgctagacct atctaacttc tcactaagac ccctccttgt gtgtccatac cccattattg 13980

taaagcagat actcaacaac tcagcaatag tctctttgct gcggggaaat agtccgttga 14040

agtgctaaac atacgagttg ctgctccttc attcaagtga ttatcttatt ggctatacgc 14100

gcatgcgacg ccacccatgg actctaagcg gccgcatgga cctgtcgact tggctcgagg 14160

gggggcccgg taccagcttt tgttcccttt agtgagggtt aatttcgagc ttggcgtaat 14220

catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac 14280

gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa 14340

ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat 14400

gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc 14460

tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg 14520

cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag 14580

gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc 14640

gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag 14700

gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga 14760

ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc 14820

atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 14880

tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt 14940

ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca 15000

gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca 15060

ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag 15120

ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca 15180

agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg 15240

ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa 15300

aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta 15360

tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag 15420

cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga 15480

tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac 15540

cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc 15600

ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta 15660

gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac 15720

gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat 15780

gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa 15840

gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg 15900

tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag 15960

aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc 16020

cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct 16080

caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat 16140

cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg 16200

ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc 16260

aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta 16320

tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg c 16371

<210> 8

<211> 15837

<212> DNA

<213> Artificial Sequence

<220>

<223> pS-B-RA-GGS-Pox2-Ura3(L)

<400> 8

cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag 60

ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac 120

cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga 180

ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc 240

accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg 300

gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa 360

gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac 420

caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct 480

gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa 540

agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg 600

ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat tggagctcgc 660

tgagctggcg cgccaatgta ctgtacctcc tgtactcgta ctcgtacggc actcgtagaa 720

acggggcaat acgggggaga agcgatcgcc cgtctgttca atcgccacaa gtccgagtaa 780

tgctcgagta tcgaagtctt gtacctccct gtcaatcatg gcaccactgg tcttgacttg 840

tctattcata ctggacaagc gccagagtta agcttgtagc gaatttcgcc ctcggacatc 900

accccatacg acggacacac atgcccgaca aacagcctct cttattgtag ctgaaagtat 960

attgaatgtg aacgtgtaca atatcaggta ccagcgggag gttacggcca aggtgatacc 1020

ggaataaccc tggcttggag atggtcggtc cattgtactg aagtgtccgt gtcgtttccg 1080

tcactgcccc aattggacat gtttgttttt ccgatctttc gggcgccctc tccttgtctc 1140

cttgtctgtc tcctggactg ttgctacccc atttctttgg cctccattgg ttcctccccg 1200

tctttcacgt cgtctatggt tgcatggttt cccttatact tttccccaca gtcacatgtt 1260

atggaggggt ctagatggag gcctaatttt gacgtgcaag gggcgaattg gggcgagaaa 1320

cacgtcgtgg acatggtgca aggcccgcag ggttgattcg acgcttttcc gcgaaaaaaa 1380

caagtccaaa tacccccgtt tattctccct cggctctcgg tatttcacat gaaaactata 1440

acctagacta cacgggcaac cttaacccca gagtatactt atataccaaa gggatgggtc 1500

ctcaaaaatc acacaagcaa cgacgcccgt acgttgtgtg gaattgtgag cggataacaa 1560

tttcacacag gaaacagcta tgaccatgat tacgccaagc tcgattaatt aactcacctg 1620

caggattgag actatgaatg gattcccgtg cccgtattac tctactaatt tgatcttgga 1680

acgcgaaaat acgtttctag gactccaaag aatctcaact cttgtcctta ctaaatatac 1740

tacccatagt tgatggttta cttgaacaga gaggacatgt tcacttgacc caaagtttct 1800

cgcatctctt ggatatttga acaacggcgt ccactgaccg tcagttatcc agtcacaaaa 1860

cccccacatt catacattcc catgtacgtt tacaaagttc tcaattccat cgtgcaaatc 1920

aaaatcacat ctattcattc atcatatata aacccatcat gtctactaac actcacaact 1980

ccatagaaaa catcgactca gaacacacgc tccatgcggc cgctcactgc gcatcctcaa 2040

agtactttct ctcctcacag tcagaggtgg acacaaaata atgcaaaatg gctcgtagct 2100

tggagacatc gtggccagct gctgctagat catcaatgta cgaacttgcc ttgagtgaca 2160

tggcctgtat cctcttgagg cagtagtcga aactcttggt ttctgttctc atgtagtcca 2220

cggcgtactt tttgagtgaa gcgtcgcttg ttcgctgttt gagaatgttg agaatctcgt 2280

ggttatccgg gttggtgcgt atgctgtgaa tcagcggaaa cgaaaacttt ccttcgctga 2340

tatcttcgca gaatcccttg ttctcggtca attccgtgga ctgcaggttg aggtaatcat 2400

ccagaatctg gtaaatgact cccagtgtgt cggtaaggtg tgtgagatca aagttgatct 2460

tttcatggtc ctcctgtttc gatgccaccg acagcataag tctcagagcc agccgaaaca 2520

gtccaccggt cttgtgcacc accatctcca gatactcgtc ttccgagggg caagtgagtg 2580

tttctctcca gtagagatcc ataccctgac ctctatgcaa gttgatcatt tcctcggtga 2640

aaatggagac ggcatcataa gacttgagct tgagcacctc ctgcagagcc acaaagtaca 2700

tgtagttggc ggagttgatg gtttggggga ctccaaacag acaatgggct gccggcaggc 2760

ctcgtcggag catcgagttg tcttccacgt catcaacaag cagcgacgag ttatgcagaa 2820

tcttggtgat gtgcgaaatg gtctcgagat cgctcttgtc caccttgata accgctccga 2880

acgctgcgat caagtgttct ctgatgttgt ggccccggtt gttggcgagg tagttgtacg 2940

gtcccagcag cgcggtgtcg gcggccttgc cccatatctc cttgaaatcc gcgctgttat 3000

aatccatggt tgtgaattag ggtggtgaga atggttggtt gtagggaaga atcaaaggcc 3060

ggtctcggga tccgtgggta tatatatata tatatatata tacgatcctt cgttacctcc 3120

ctgttctcaa aactgtggtt tttcgttttt cgttttttgc tttttttgat ttttttaggg 3180

ccaactaagc ttccagattt cgctaatcac ctttgtacta attacaagaa aggaagaagc 3240

tgattagagt tgggcttttt atgcaactgt gctactcctt atctctgata tgaaagtgta 3300

gacccaatca catcatgtca tttagagttg gtaatactgg gaggatagat aaggcacgaa 3360

aacgagccat agcagacatg ctgggtgtag ccaagcagaa gaaagtagat gggagccaat 3420

tgacgagcga gggagctacg ccaatccgac atacgacacg ctgagatcgt cttggccggg 3480

gggtacctac agatgtccaa gggtaagtgc ttgactgtaa ttgtatgtct gaggacaaat 3540

atgtagtcag ccgtataaag tcataccagg caccagtgcc atcatcgaac cactaactct 3600

ctatgataca tgcctccggt attattgtac catgcgtcgc tttgttacat acgtatcttg 3660

cctttttctc tcagaaactc cagactttgg ctattggtcg agataagccc ggaccatagt 3720

gagtctttca cactctacat ttctcccttg ctccaactat ttaaatcgag gaagaggaca 3780

agcggctgct tcttaagttt gtgacatcag tatccaaggc accattgcaa ggattcaagg 3840

ctttgaaccc gtcatttgcc attcgtaacg ctggtagaca ggttgatcgg ttccctacgg 3900

cctccacctg tgtcaatctt ctcaagctgc ctgactatca ggacattgat caacttcgga 3960

agaaactttt gtatgccatt cgatcacatg ctggtttcga tttgtcttag aggaacgcat 4020

atacagtaat catagagaat aaacgatatt catttattaa agtagatagt tgaggtagaa 4080

gttgtaaaga gtgataaata gcggccgcta gtcaacaaaa aaggacttaa tctttctagc 4140

ttgacggaga gccacaggct tgtcttcaga ctgatagagg ttgtacacgg acaacagagc 4200

aatcttggca cgttcgcttc ctttcagatg ggctcttgta ggatatgttg tcttctgctg 4260

cttgagcaca gatccaattg cagcgtacac ttggcaagca gctcgcacac cgccttgaca 4320

gtttgccggc aacttgtcaa tgcccttctt agctctcacc atgatagcgt ctgcctttcc 4380

cacgagcttc agagacaagc ccaacagtct ctgatcaccc aggctacggg cgttgccttg 4440

ctgaatctgt tctgtctctt cttttctaag ccactgctga ggcagataac atcgacccag 4500

agtctcgctg tcggtcacaa tgtcacgagc aatgttaacg tattgcagca ccaggcccat 4560

ctcacgtgca cggtcaataa tccaagcatc attctccttc tggtcttgag caagaatcac 4620

acgtgtgcac atttcaccca cggaggaggc cacacaagcg gagtatgcct ccaagtcttg 4680

ttcatcaagg ataggacgac gctcaagatc ccatttgtaa ccatccagca gctcctccac 4740

agggtccact tcaaggacat gtcgaagtcg agtaaaggct ctaaaggcag aaatacacga 4800

agcaggaagc tggttctgat acaactccca gtcaatcaca ataggcgcgg aggtcttctg 4860

gctaaagaga tcacgaacaa actgtcgagt aagatccagc tggtctcttc tctcttgaac 4920

agatttggac tcatcatcgc acaggtcatc ggtagctcta cagaaagcat agagaacacc 4980

caagtcttga cgcacatagg atggaaaaac ggcagatgca gtgtagaacg acttcgaggc 5040

tttacgcaga atgtcccagc tgatggtaag atcatcaaac aactcattgt tgagcatctg 5100

gtcaggaaga cagaaggccc atgccagctc tttgacgtgc tggaaaaggg aaatggcttg 5160

tttagggttc tgattctgaa cagacgattt gtacagatgg aggatggcct gggctcggtc 5220

aatagcacag gtagcaaaaa ccaagactgt gttgatcaag gtaaaaaaca ggcactcttc 5280

cacaggcaga tcgggcacca ccattttgcc agtgcttgtt ctaagagaaa tgtgccatgt 5340

gccagcggaa atagcgacga tgtcagccca acacaggtac acggaaggga taacaatcga 5400

caaaaggaca gccacaggtc gacgcaagat gtactcgcca gcacccagcc aaagaatagc 5460

caacacagga caagcatacc aaaggatgca cgaaccgtaa aacgaggatt tatttggcag 5520

tgtcaagtgc caagcgtgat aggtgattgc caacagagca gaaacaggca caagtcgcac 5580

cagcagtgtc tgcttccacg aggtgttggg tctaataaag aaggtgtgca agtgccaacg 5640

cataacaaag ttcgagaacg cgacagtcat cagagtcatg atgataaaga acatgtactc 5700

ttccagaggc acgtagccaa tgacagccac aacacaggta ggacagtacc accaagctcg 5760

atgataaacg atgtaattgt cccaaatcga tgcggtcgag gcggccatca acatcagaaa 5820

tttgtacttg agattgtctt gctgcgagtg aaacggcttc agcagccaac acaatgccgc 5880

aaggacaggc agtgtgtagt agagatgaaa ctccagatag gtgagaatgg aagccatggt 5940

tgatgtgtgt ttaattcaag aatgaatata gagaagagaa gaagaaaaaa gattcaattg 6000

agccggcgat gcagaccctt atataaatgt tgccttggac agacggagca agcccgccca 6060

aacctacgtt cggtataata tgttaagctt tttaacacaa aggtttggct tggggtaacc 6120

tgatgtggtg caaaagaccg ggcgttggcg agccattgcg cgggcgaatg gggccgtgac 6180

tcgtctcaaa ttcgagggcg tgcctcaatt cgtgcccccg tggctttttc ccgccgtttc 6240

cgccccgttt gcaccactgc agccgcttct ttggttcgga caccttgctg cgagctaggt 6300

gccttgtgct acttaaaaag tggcctccca acaccaacat gacatgagtg cgtgggccaa 6360

gacacgttgg cggggtcgca gtcggctcaa tggcccggaa aaaacgctgc tggagctggt 6420

tcggacgcag tccgccgcgg cgtatggata tccgcaaggt tccatagcgc cattgccctc 6480

cgtcggcgtc tatcccgcaa cctctaaata gagcgggaat ataacccaag cttctttttt 6540

ttcctttaac acgcacaccc ccaactatca tgttgctgct gctgtttgac tctactctgt 6600

ggaggggtgc tcccacccaa cccaacctac aggtggatcc ggcgctgtga ttggctgata 6660

agtctcctat ccggactaat tctgaccaat gggacatgcg cgcaggaccc aaatgccgca 6720

attacgtaac cccaacgaaa tgcctacccc tctttggagc ccagcggccc caaatccccc 6780

caagcagccc ggttctaccg gcttccatct ccaagcacaa gcagcccggt tctaccggct 6840

tccatctcca agcacccctt tctccacacc ccacaaaaag acccgtgcag gacatcctac 6900

tgcgtgttta aacaacaatt gccccggaga agacggccag gccgcctaga tgacaaattc 6960

aacaactcac agctgacttt ctgccattgc cactaggggg gggccttttt atatggccaa 7020

gccaagctct ccacgtcggt tgggctgcac ccaacaataa atgggtaggg ttgcaccaac 7080

aaagggatgg gatggggggt agaagatacg aggataacgg ggctcaatgg cacaaataag 7140

aacgaatact gccattaaga ctcgtgatcc agcgactgac accattgcat catctaaggg 7200

cctcaaaact acctcggaac tgctgcgctg atctggacac cacagaggtt ccgagcactt 7260

taggttgcac caaatgtccc accaggtgca ggcagaaaac gctggaacag cgtgtacagt 7320

ttgtcttagc aaaaagtgaa ggcgctgagg tcgagcaggg tggtgtgact tgttatagcc 7380

tttagagctg cgaaagcgcg tatggatttg gctcatcagg ccagattgag ggtctgtgga 7440

cacatgtcat gttagtgtac ttcaatcgcc ccctggatat agccccgaca ataggccgtg 7500

gcctcatttt tttgccttcc gcacatttcc attgctcggt acccacacct tgcttctcct 7560

gcacttgcca accttaatac tggtttacat tgaccaacat cttacaagcg gggggcttgt 7620

ctagggtata tataaacagt ggctctccca atcggttgcc agtctctttt ttcctttctt 7680

tccccacaga ttcgaaatct aaactacaca tcacacaatg cctgttactg acgtccttaa 7740

gcgaaagtcc ggtgtcatcg tcggcgacga tgtccgagcc gtgagtatcc acgacaagat 7800

cagtgtcgag acgacgcgtt ttgtgtaatg acacaatccg aaagtcgcta gcaacacaca 7860

ctctctacac aaactaaccc agctctatgt ccgatcagaa gaagcacatt gttgtcattg 7920

gtgccggtat tggcggaact gctactgctg ctcgtcttgc tcgtgaaggt tttcgagtta 7980

ctgttgttga gaagaacgac ttttccggtg gccgttgttc tttcattcat cacgacggtc 8040

atcgatttga tcagggtccc tctctctact tgatgcctaa gctctttgaa gacgcatttg 8100

ctgacttgga cgagcgtatt ggtgatcact tggacttgct gcgatgtgac aataactata 8160

aggttcactt tgacgacggt gatgccgtgc agctctcttc cgatctgacc aagatgaagg 8220

gcgagttgga ccgtattgag ggtccccttg gatttggtag attcttggac ttcatgaagg 8280

agacacatgt ccactatgaa caaggtacat ttattgctat caagcgaaac tttgagacca 8340

tttgggattt gattcgtctc cagtatgtgc ctgagatctt tcgattgcac ttgtttggta 8400

agatctatga ccgagcctct aaatacttcc agaccaagaa aatgcgtatg gcttttacct 8460

ttcagacaat gtacatgggt atgtcgcctt acgatgctcc agcagtttac tctttgctcc 8520

agtacaccga gtttgctgaa ggtatctggt atcctcgtgg tggtttcaac atggttgttc 8580

agaagctgga gtctatcgcc tccaaaaagt acggtgctga gttcagatac cagtcgcctg 8640

ttgctaaaat taacactgtc gacaaagaca agcgtgtgac cggtgtcacc ttggagagcg 8700

gagaagtcat tgaagccgat gcagtcgtgt gtaatgcgga tcttgtttac gcttatcacc 8760

atctgctgcc tccttgcaat tggacaaaga agacactggc ctctaagaaa ctcacctctt 8820

cttctatttc gttttactgg tccatgtcta caaaggtgcc tcagctggac gtgcacaata 8880

tcttcttggc tgaagcctac aaggagtctt ttgacgagat tttcaacgac ttcggtttgc 8940

cctctgaagc gtctttctat gtcaacgttc catctcgaat tgatgagtct gccgcacctc 9000

ccaacaagga ctccattatt gtgttggttc caattggcca catgaagtct aagacaggaa 9060

actctgctga agagaattat cctgagttgg tgaaccgtgc acgaaagatg gttctggaag 9120

ttatcgagcg tcgtttggga gtgaacaact ttgctaattt gattgaacat gaagaagtga 9180

atgatccttc tgtttggcag agcaagttta acctctggag aggttctatt cttggtctgt 9240

ctcatgacgt gttccaagtt ctctggttca gaccttctac caaggactcc acaaaccgtt 9300

atgacaatct gttctttgtc ggagcttcta cacatccagg tactggtgtt cctatcgttc 9360

ttgctggatc taagctgacc tccgaccaag tctgtaagag ctttggccag aatcccctgc 9420

caagaaagct gcaagacagc cagaagaagt acgctcctga gcagactcga aagaccgaga 9480

gccactggat ctactactgt cttgcttgtt actttgttac cttcctcttt ttctatttct 9540

tcccaagaga tgacaccaca actcctgctt ctttcattaa ccaactgctg cctaacgttt 9600

tccaagtgca gaacagcaac gacattcgaa tttagtattt atcactcttt acaacttcta 9660

cctcaactat ctactttaat aaatgaatat cgtttattct ctatgattac tgtatatgcg 9720

ttcctctaag acaaatcgaa accagcatgt gatcgaatgg catacaaaag tttcttccga 9780

agttgatcaa tgtcctgata gtcaggcagc ttgagaagat tgacacaggt ggaggccgta 9840

gggaaccgat caacctgtct accagcgtta cgaatggcaa atgacgggtt caaagccttg 9900

aatccttgca atggtgcctt ggatactgat gtcacaaact taagaagcag ccgcttgtcc 9960

tcttcctcgg aattcaggag gacgagaagg tcaacaaggg tgaggcctgg agagagttct 10020

acaagggtcc ccgggatcgg ctgtacagaa ctggagatcc aggcagacaa ggtatagggc 10080

ggcgcctaca atccatgcca acccgttcca tgtgctcgcc gaggcggcat aaatcgccgt 10140

gacgatcagc ggtccagtga tcgaagttag gctggtaaga gccgcgagcg atccttgaag 10200

ctgtccctga tggtcgtcat ctacctgcct ggacagcatg gcctgcaacg cgggcatccc 10260

gatgccgccg gaagcgagaa gaatcataat ggggaaggcc atccagcctc gcgtcgcgaa 10320

cgccagcaag acgtagccca gcgcgtcggc cgccatgccg gcgataatgg cctgcttctc 10380

gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc 10440

gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa 10500

aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat 10560

aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc 10620

tctcaagggc atcggtcgac gctctccctt atgcgactcc tgcattagga agcagcccag 10680

tagatttctg gtggtattgt gactggggat gtagttgaga ataagtcata cacaagtcag 10740

ctttcttcaa gcctcatata agtataagta gttcaatcgt attagctact tgtacccagc 10800

atctccgtat cgagaaacac aacaacatgc cccattggac agatcatgcg gatacacagg 10860

ttgtgcagta tcatacatac tcgatcagac aggtcgtctg accatcatac aagctgaaca 10920

agcgctccat acttgcacgc tctctatata cacagttaaa tgacatatcc atagtctaac 10980

ctctaacagt taatcttctg gtaagcctcc cagccagcct tctggtatcg cttggcctcc 11040

tcaataggat ctcggttctg gccgtacaga cctcggccga caattatgat atccgttccg 11100

gtagacatga catcctcaac agttcggtac tgctgtccga gagcgtctcc cttgtcgtca 11160

agacccaccc cgggggtcag aataagccag tcctcagagt cgcccttagg tcggttctgg 11220

gcaatgaagc caaccacaaa ctcggggtcg gatcgggcaa gctcaatggt ctgcttggag 11280

tactcgccag tggccagaga gcccttgcaa gacagctcgg ccagcatgag cagacctctg 11340

gccagcttct cgttgggaga ggggaccagg aactccttgt actgggagtt ctcgtagtca 11400

gagacgtcct ccttcttctg ttcagagaca gtttcctcgg caccagctcg caggccagca 11460

atgattccgg ttccgggtac accgtgggcg ttggtgatat cggaccactc ggcgattcgg 11520

tagacaccgt tcttgtactg gtgcttgaca gtgttgccaa tatctgcgaa ctttctgtcc 11580

tcgaacagga agaaaccgtg cttaagagca agttccttga gggggagcac agtgccggcg 11640

taggtgaagt cgtcaatgat gtcgatatgg gtcttgatca tgcacacata aggtccgacc 11700

ttatcggcaa gctcaatgag ctccttggtg gtggtaacat ccagagaagc acacaggttg 11760

gttttcttgg ctgccacgag cttgagcact cgagcggcaa aggcggactt gtggacgtta 11820

gctcgagctt cgtaggaggg cattttggtg gtgaagagga gactgaaata aatttagtct 11880

gcagaacttt ttatcggaac cttatctggg gcagtgaagt atatgttatg gtaatagtta 11940

cgagttagtt gaacttatag atagactgga ctatacggct atcggtccaa attagaaaga 12000

acgtcaatgg ctctctgggc gtcgcctttg ccgacaaaaa tgtgatcatg atgaaagcca 12060

gcaatgacgt tgcagctgat attgttgtcg gccaaccgcg ccgaaaacgc agctgtcaga 12120

cccacagcct ccaacgaaga atgtatcgtc aaagtgatcc aagcacactc atagttggag 12180

tcgtactcca aaggcggcaa tgacgagtca gacaggaggc agacaaggta tagggcggcg 12240

cctacaatcc atgccaaccc gttccatgtg ctcgccgagg cggcataaat cgccgtgacg 12300

atcagcggtc cagtgatcga agttaggctg gtaagagccg cgagcgatcc ttgaagctgt 12360

ccctgatggt cgtcatctac ctgcctggac agcatggcct gcaacgcggg catcccgatg 12420

ccgccggaag cgagaagaat cataatgggg aaggccatcc agcctcgcgt cgcgaacgcc 12480

agcaagacgt agcccagcgc gtcggccgcc atgccggcga taatggcctg cttctcgccg 12540

aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat 12600

accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg 12660

acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt 12720

gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc 12780

aagggcatcg gtcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtatc 12840

gatagagacg gagtcactgc cttaattaac tttgatcttg ctgctaacat ctcttctact 12900

gctacttgtg atgactgtct tgtccacaat ggcttcatcc agtcctacaa caacacctac 12960

aatcagatcg gccccaagct cgactctgtg attgagcagt atcccgacta ccagattgct 13020

gtcaccggtc actctctcgg aggagctgca gcccttctgt tcggaatcaa cctcaaggtt 13080

aacggccacg atcccctcgt tgttactctt ggtcagccca ttgtcggtaa cgctggcttt 13140

gctaactggg tcgataaact cttctttggc caggagaacc ccgatgtctc caaggtgtcc 13200

aaagaccgaa agctctaccg aatcacccac cgaggagata tcgtccctca agtgcccttc 13260

tgggacggtt accagcactg ctctggtgag gtctttattg actggcccct gatccaccct 13320

cctctctcca acgttgtcat gtgccagggc cagagcaata aacagtgctc tgccggtaac 13380

actctgctcc agcaggtcaa tgtgattgga aaccatctgc agtacttcgt caccgagggt 13440

gtctgtggta tctaagctat ttatcactct ttacaacttc tacctcaact atctacttta 13500

ataaatgaat atcgtttatt ctctatgatt actgtatatg cgttcctcta agacaaatcg 13560

aaaccagcat gcgacgccac ccatggactc taagcggccg catggacctg tcgacttggc 13620

tcgagggggg gcccggtacc agcttttgtt ccctttagtg agggttaatt tcgagcttgg 13680

cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca 13740

acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca 13800

cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc 13860

attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt 13920

cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact 13980

caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag 14040

caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata 14100

ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc 14160

cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg 14220

ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc 14280

tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg 14340

gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc 14400

ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga 14460

ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg 14520

gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa 14580

aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg 14640

tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt 14700

ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat 14760

tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct 14820

aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta 14880

tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa 14940

ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac 15000

gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa 15060

gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag 15120

taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg 15180

tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag 15240

ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg 15300

tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc 15360

ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat 15420

tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata 15480

ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa 15540

aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca 15600

actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc 15660

aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc 15720

tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg 15780

aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgc 15837

<210> 9

<211> 15603

<212> DNA

<213> Artificial Sequence

<220>

<223> pS-B-RA-GGS-rDNA-Ura3(L)

<400> 9

cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag 60

ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac 120

cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga 180

ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc 240

accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg 300

gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa 360

gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac 420

caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct 480

gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa 540

agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg 600

ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat tggagctcgc 660

tgagctggcg cgcctcgatc ctaaggggtg gcataactgt cgcgtacggc ccgataaggg 720

ccttctccaa aagggaagcc ggttgaaatt ccggcacttg gatgtggatt ctccacggca 780

acgtaactga atgtggggac ggtggcacaa gtcttggaag gagttatctt ttctttttaa 840

cggagtcaac accctggaat tagtttgtct agagataggg tatcgttccg gaagaggggg 900

gcagctttgt cccctccgat gcacttgtga cgccccttga aaacccgcag gaaggaatag 960

ttttcacgcc aagtcgtact gataaccgca gcaggtctcc aaggtgaaca gcctctagtt 1020

gatagaataa tgtagataag ggaagtcggc aaaatagatc cgtaacttcg ggataaggat 1080

tggctctggg ggttggtgga tggaagcgtg ggagacccca agggactggc ggctgggcaa 1140

ctggcagccg gacccgcggc agacactgcg tcgctccgtc cacatcatca accgccccag 1200

aactggtacg gacaagggga atctgactgt ctaattaaaa catagctttg cgatggttgt 1260

aaaacaatgt tgacgcaaag tgatttctgc ccagtgctct gaatgtcaaa gtgaagaaat 1320

tcaacgtacg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga 1380

ccatgattac gccaagctcg attaattaac tcacctgcag gattgagact atgaatggat 1440

tcccgtgccc gtattactct actaatttga tcttggaacg cgaaaatacg tttctaggac 1500

tccaaagaat ctcaactctt gtccttacta aatatactac ccatagttga tggtttactt 1560

gaacagagag gacatgttca cttgacccaa agtttctcgc atctcttgga tatttgaaca 1620

acggcgtcca ctgaccgtca gttatccagt cacaaaaccc ccacattcat acattcccat 1680

gtacgtttac aaagttctca attccatcgt gcaaatcaaa atcacatcta ttcattcatc 1740

atatataaac ccatcatgtc tactaacact cacaactcca tagaaaacat cgactcagaa 1800

cacacgctcc atgcggccgc tcactgcgca tcctcaaagt actttctctc ctcacagtca 1860

gaggtggaca caaaataatg caaaatggct cgtagcttgg agacatcgtg gccagctgct 1920

gctagatcat caatgtacga acttgccttg agtgacatgg cctgtatcct cttgaggcag 1980

tagtcgaaac tcttggtttc tgttctcatg tagtccacgg cgtacttttt gagtgaagcg 2040

tcgcttgttc gctgtttgag aatgttgaga atctcgtggt tatccgggtt ggtgcgtatg 2100

ctgtgaatca gcggaaacga aaactttcct tcgctgatat cttcgcagaa tcccttgttc 2160

tcggtcaatt ccgtggactg caggttgagg taatcatcca gaatctggta aatgactccc 2220

agtgtgtcgg taaggtgtgt gagatcaaag ttgatctttt catggtcctc ctgtttcgat 2280

gccaccgaca gcataagtct cagagccagc cgaaacagtc caccggtctt gtgcaccacc 2340

atctccagat actcgtcttc cgaggggcaa gtgagtgttt ctctccagta gagatccata 2400

ccctgacctc tatgcaagtt gatcatttcc tcggtgaaaa tggagacggc atcataagac 2460

ttgagcttga gcacctcctg cagagccaca aagtacatgt agttggcgga gttgatggtt 2520

tgggggactc caaacagaca atgggctgcc ggcaggcctc gtcggagcat cgagttgtct 2580

tccacgtcat caacaagcag cgacgagtta tgcagaatct tggtgatgtg cgaaatggtc 2640

tcgagatcgc tcttgtccac cttgataacc gctccgaacg ctgcgatcaa gtgttctctg 2700

atgttgtggc cccggttgtt ggcgaggtag ttgtacggtc ccagcagcgc ggtgtcggcg 2760

gccttgcccc atatctcctt gaaatccgcg ctgttataat ccatggttgt gaattagggt 2820

ggtgagaatg gttggttgta gggaagaatc aaaggccggt ctcgggatcc gtgggtatat 2880

atatatatat atatatatac gatccttcgt tacctccctg ttctcaaaac tgtggttttt 2940

cgtttttcgt tttttgcttt ttttgatttt tttagggcca actaagcttc cagatttcgc 3000

taatcacctt tgtactaatt acaagaaagg aagaagctga ttagagttgg gctttttatg 3060

caactgtgct actccttatc tctgatatga aagtgtagac ccaatcacat catgtcattt 3120

agagttggta atactgggag gatagataag gcacgaaaac gagccatagc agacatgctg 3180

ggtgtagcca agcagaagaa agtagatggg agccaattga cgagcgaggg agctacgcca 3240

atccgacata cgacacgctg agatcgtctt ggccgggggg tacctacaga tgtccaaggg 3300

taagtgcttg actgtaattg tatgtctgag gacaaatatg tagtcagccg tataaagtca 3360

taccaggcac cagtgccatc atcgaaccac taactctcta tgatacatgc ctccggtatt 3420

attgtaccat gcgtcgcttt gttacatacg tatcttgcct ttttctctca gaaactccag 3480

actttggcta ttggtcgaga taagcccgga ccatagtgag tctttcacac tctacatttc 3540

tcccttgctc caactattta aatcgaggaa gaggacaagc ggctgcttct taagtttgtg 3600

acatcagtat ccaaggcacc attgcaagga ttcaaggctt tgaacccgtc atttgccatt 3660

cgtaacgctg gtagacaggt tgatcggttc cctacggcct ccacctgtgt caatcttctc 3720

aagctgcctg actatcagga cattgatcaa cttcggaaga aacttttgta tgccattcga 3780

tcacatgctg gtttcgattt gtcttagagg aacgcatata cagtaatcat agagaataaa 3840

cgatattcat ttattaaagt agatagttga ggtagaagtt gtaaagagtg ataaatagcg 3900

gccgctagtc aacaaaaaag gacttaatct ttctagcttg acggagagcc acaggcttgt 3960

cttcagactg atagaggttg tacacggaca acagagcaat cttggcacgt tcgcttcctt 4020

tcagatgggc tcttgtagga tatgttgtct tctgctgctt gagcacagat ccaattgcag 4080

cgtacacttg gcaagcagct cgcacaccgc cttgacagtt tgccggcaac ttgtcaatgc 4140

ccttcttagc tctcaccatg atagcgtctg cctttcccac gagcttcaga gacaagccca 4200

acagtctctg atcacccagg ctacgggcgt tgccttgctg aatctgttct gtctcttctt 4260

ttctaagcca ctgctgaggc agataacatc gacccagagt ctcgctgtcg gtcacaatgt 4320

cacgagcaat gttaacgtat tgcagcacca ggcccatctc acgtgcacgg tcaataatcc 4380

aagcatcatt ctccttctgg tcttgagcaa gaatcacacg tgtgcacatt tcacccacgg 4440

aggaggccac acaagcggag tatgcctcca agtcttgttc atcaaggata ggacgacgct 4500

caagatccca tttgtaacca tccagcagct cctccacagg gtccacttca aggacatgtc 4560

gaagtcgagt aaaggctcta aaggcagaaa tacacgaagc aggaagctgg ttctgataca 4620

actcccagtc aatcacaata ggcgcggagg tcttctggct aaagagatca cgaacaaact 4680

gtcgagtaag atccagctgg tctcttctct cttgaacaga tttggactca tcatcgcaca 4740

ggtcatcggt agctctacag aaagcataga gaacacccaa gtcttgacgc acataggatg 4800

gaaaaacggc agatgcagtg tagaacgact tcgaggcttt acgcagaatg tcccagctga 4860

tggtaagatc atcaaacaac tcattgttga gcatctggtc aggaagacag aaggcccatg 4920

ccagctcttt gacgtgctgg aaaagggaaa tggcttgttt agggttctga ttctgaacag 4980

acgatttgta cagatggagg atggcctggg ctcggtcaat agcacaggta gcaaaaacca 5040

agactgtgtt gatcaaggta aaaaacaggc actcttccac aggcagatcg ggcaccacca 5100

ttttgccagt gcttgttcta agagaaatgt gccatgtgcc agcggaaata gcgacgatgt 5160

cagcccaaca caggtacacg gaagggataa caatcgacaa aaggacagcc acaggtcgac 5220

gcaagatgta ctcgccagca cccagccaaa gaatagccaa cacaggacaa gcataccaaa 5280

ggatgcacga accgtaaaac gaggatttat ttggcagtgt caagtgccaa gcgtgatagg 5340

tgattgccaa cagagcagaa acaggcacaa gtcgcaccag cagtgtctgc ttccacgagg 5400

tgttgggtct aataaagaag gtgtgcaagt gccaacgcat aacaaagttc gagaacgcga 5460

cagtcatcag agtcatgatg ataaagaaca tgtactcttc cagaggcacg tagccaatga 5520

cagccacaac acaggtagga cagtaccacc aagctcgatg ataaacgatg taattgtccc 5580

aaatcgatgc ggtcgaggcg gccatcaaca tcagaaattt gtacttgaga ttgtcttgct 5640

gcgagtgaaa cggcttcagc agccaacaca atgccgcaag gacaggcagt gtgtagtaga 5700

gatgaaactc cagataggtg agaatggaag ccatggttga tgtgtgttta attcaagaat 5760

gaatatagag aagagaagaa gaaaaaagat tcaattgagc cggcgatgca gacccttata 5820

taaatgttgc cttggacaga cggagcaagc ccgcccaaac ctacgttcgg tataatatgt 5880

taagcttttt aacacaaagg tttggcttgg ggtaacctga tgtggtgcaa aagaccgggc 5940

gttggcgagc cattgcgcgg gcgaatgggg ccgtgactcg tctcaaattc gagggcgtgc 6000

ctcaattcgt gcccccgtgg ctttttcccg ccgtttccgc cccgtttgca ccactgcagc 6060

cgcttctttg gttcggacac cttgctgcga gctaggtgcc ttgtgctact taaaaagtgg 6120

cctcccaaca ccaacatgac atgagtgcgt gggccaagac acgttggcgg ggtcgcagtc 6180

ggctcaatgg cccggaaaaa acgctgctgg agctggttcg gacgcagtcc gccgcggcgt 6240

atggatatcc gcaaggttcc atagcgccat tgccctccgt cggcgtctat cccgcaacct 6300

ctaaatagag cgggaatata acccaagctt cttttttttc ctttaacacg cacaccccca 6360

actatcatgt tgctgctgct gtttgactct actctgtgga ggggtgctcc cacccaaccc 6420

aacctacagg tggatccggc gctgtgattg gctgataagt ctcctatccg gactaattct 6480

gaccaatggg acatgcgcgc aggacccaaa tgccgcaatt acgtaacccc aacgaaatgc 6540

ctacccctct ttggagccca gcggccccaa atccccccaa gcagcccggt tctaccggct 6600

tccatctcca agcacaagca gcccggttct accggcttcc atctccaagc acccctttct 6660

ccacacccca caaaaagacc cgtgcaggac atcctactgc gtgtttaaac aacaattgcc 6720

ccggagaaga cggccaggcc gcctagatga caaattcaac aactcacagc tgactttctg 6780

ccattgccac tagggggggg cctttttata tggccaagcc aagctctcca cgtcggttgg 6840

gctgcaccca acaataaatg ggtagggttg caccaacaaa gggatgggat ggggggtaga 6900

agatacgagg ataacggggc tcaatggcac aaataagaac gaatactgcc attaagactc 6960

gtgatccagc gactgacacc attgcatcat ctaagggcct caaaactacc tcggaactgc 7020

tgcgctgatc tggacaccac agaggttccg agcactttag gttgcaccaa atgtcccacc 7080

aggtgcaggc agaaaacgct ggaacagcgt gtacagtttg tcttagcaaa aagtgaaggc 7140

gctgaggtcg agcagggtgg tgtgacttgt tatagccttt agagctgcga aagcgcgtat 7200

ggatttggct catcaggcca gattgagggt ctgtggacac atgtcatgtt agtgtacttc 7260

aatcgccccc tggatatagc cccgacaata ggccgtggcc tcattttttt gccttccgca 7320

catttccatt gctcggtacc cacaccttgc ttctcctgca cttgccaacc ttaatactgg 7380

tttacattga ccaacatctt acaagcgggg ggcttgtcta gggtatatat aaacagtggc 7440

tctcccaatc ggttgccagt ctcttttttc ctttctttcc ccacagattc gaaatctaaa 7500

ctacacatca cacaatgcct gttactgacg tccttaagcg aaagtccggt gtcatcgtcg 7560

gcgacgatgt ccgagccgtg agtatccacg acaagatcag tgtcgagacg acgcgttttg 7620

tgtaatgaca caatccgaaa gtcgctagca acacacactc tctacacaaa ctaacccagc 7680

tctatgtccg atcagaagaa gcacattgtt gtcattggtg ccggtattgg cggaactgct 7740

actgctgctc gtcttgctcg tgaaggtttt cgagttactg ttgttgagaa gaacgacttt 7800

tccggtggcc gttgttcttt cattcatcac gacggtcatc gatttgatca gggtccctct 7860

ctctacttga tgcctaagct ctttgaagac gcatttgctg acttggacga gcgtattggt 7920

gatcacttgg acttgctgcg atgtgacaat aactataagg ttcactttga cgacggtgat 7980

gccgtgcagc tctcttccga tctgaccaag atgaagggcg agttggaccg tattgagggt 8040

ccccttggat ttggtagatt cttggacttc atgaaggaga cacatgtcca ctatgaacaa 8100

ggtacattta ttgctatcaa gcgaaacttt gagaccattt gggatttgat tcgtctccag 8160

tatgtgcctg agatctttcg attgcacttg tttggtaaga tctatgaccg agcctctaaa 8220

tacttccaga ccaagaaaat gcgtatggct tttacctttc agacaatgta catgggtatg 8280

tcgccttacg atgctccagc agtttactct ttgctccagt acaccgagtt tgctgaaggt 8340

atctggtatc ctcgtggtgg tttcaacatg gttgttcaga agctggagtc tatcgcctcc 8400

aaaaagtacg gtgctgagtt cagataccag tcgcctgttg ctaaaattaa cactgtcgac 8460

aaagacaagc gtgtgaccgg tgtcaccttg gagagcggag aagtcattga agccgatgca 8520

gtcgtgtgta atgcggatct tgtttacgct tatcaccatc tgctgcctcc ttgcaattgg 8580

acaaagaaga cactggcctc taagaaactc acctcttctt ctatttcgtt ttactggtcc 8640

atgtctacaa aggtgcctca gctggacgtg cacaatatct tcttggctga agcctacaag 8700

gagtcttttg acgagatttt caacgacttc ggtttgccct ctgaagcgtc tttctatgtc 8760

aacgttccat ctcgaattga tgagtctgcc gcacctccca acaaggactc cattattgtg 8820

ttggttccaa ttggccacat gaagtctaag acaggaaact ctgctgaaga gaattatcct 8880

gagttggtga accgtgcacg aaagatggtt ctggaagtta tcgagcgtcg tttgggagtg 8940

aacaactttg ctaatttgat tgaacatgaa gaagtgaatg atccttctgt ttggcagagc 9000

aagtttaacc tctggagagg ttctattctt ggtctgtctc atgacgtgtt ccaagttctc 9060

tggttcagac cttctaccaa ggactccaca aaccgttatg acaatctgtt ctttgtcgga 9120

gcttctacac atccaggtac tggtgttcct atcgttcttg ctggatctaa gctgacctcc 9180

gaccaagtct gtaagagctt tggccagaat cccctgccaa gaaagctgca agacagccag 9240

aagaagtacg ctcctgagca gactcgaaag accgagagcc actggatcta ctactgtctt 9300

gcttgttact ttgttacctt cctctttttc tatttcttcc caagagatga caccacaact 9360

cctgcttctt tcattaacca actgctgcct aacgttttcc aagtgcagaa cagcaacgac 9420

attcgaattt agtatttatc actctttaca acttctacct caactatcta ctttaataaa 9480

tgaatatcgt ttattctcta tgattactgt atatgcgttc ctctaagaca aatcgaaacc 9540

agcatgtgat cgaatggcat acaaaagttt cttccgaagt tgatcaatgt cctgatagtc 9600

aggcagcttg agaagattga cacaggtgga ggccgtaggg aaccgatcaa cctgtctacc 9660

agcgttacga atggcaaatg acgggttcaa agccttgaat ccttgcaatg gtgccttgga 9720

tactgatgtc acaaacttaa gaagcagccg cttgtcctct tcctcggaat tcaggaggac 9780

gagaaggtca acaagggtga ggcctggaga gagttctaca agggtccccg ggatcggctg 9840

tacagaactg gagatccagg cagacaaggt atagggcggc gcctacaatc catgccaacc 9900

cgttccatgt gctcgccgag gcggcataaa tcgccgtgac gatcagcggt ccagtgatcg 9960

aagttaggct ggtaagagcc gcgagcgatc cttgaagctg tccctgatgg tcgtcatcta 10020

cctgcctgga cagcatggcc tgcaacgcgg gcatcccgat gccgccggaa gcgagaagaa 10080

tcataatggg gaaggccatc cagcctcgcg tcgcgaacgc cagcaagacg tagcccagcg 10140

cgtcggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac 10200

cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga 10260

tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca 10320

cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc 10380

cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgacgct 10440

ctcccttatg cgactcctgc attaggaagc agcccagtag atttctggtg gtattgtgac 10500

tggggatgta gttgagaata agtcatacac aagtcagctt tcttcaagcc tcatataagt 10560

ataagtagtt caatcgtatt agctacttgt acccagcatc tccgtatcga gaaacacaac 10620

aacatgcccc attggacaga tcatgcggat acacaggttg tgcagtatca tacatactcg 10680

atcagacagg tcgtctgacc atcatacaag ctgaacaagc gctccatact tgcacgctct 10740

ctatatacac agttaaatga catatccata gtctaacctc taacagttaa tcttctggta 10800

agcctcccag ccagccttct ggtatcgctt ggcctcctca ataggatctc ggttctggcc 10860

gtacagacct cggccgacaa ttatgatatc cgttccggta gacatgacat cctcaacagt 10920

tcggtactgc tgtccgagag cgtctccctt gtcgtcaaga cccaccccgg gggtcagaat 10980

aagccagtcc tcagagtcgc ccttaggtcg gttctgggca atgaagccaa ccacaaactc 11040

ggggtcggat cgggcaagct caatggtctg cttggagtac tcgccagtgg ccagagagcc 11100

cttgcaagac agctcggcca gcatgagcag acctctggcc agcttctcgt tgggagaggg 11160

gaccaggaac tccttgtact gggagttctc gtagtcagag acgtcctcct tcttctgttc 11220

agagacagtt tcctcggcac cagctcgcag gccagcaatg attccggttc cgggtacacc 11280

gtgggcgttg gtgatatcgg accactcggc gattcggtag acaccgttct tgtactggtg 11340

cttgacagtg ttgccaatat ctgcgaactt tctgtcctcg aacaggaaga aaccgtgctt 11400

aagagcaagt tccttgaggg ggagcacagt gccggcgtag gtgaagtcgt caatgatgtc 11460

gatatgggtc ttgatcatgc acacataagg tccgacctta tcggcaagct caatgagctc 11520

cttggtggtg gtaacatcca gagaagcaca caggttggtt ttcttggctg ccacgagctt 11580

gagcactcga gcggcaaagg cggacttgtg gacgttagct cgagcttcgt aggagggcat 11640

tttggtggtg aagaggagac tgaaataaat ttagtctgca gaacttttta tcggaacctt 11700

atctggggca gtgaagtata tgttatggta atagttacga gttagttgaa cttatagata 11760

gactggacta tacggctatc ggtccaaatt agaaagaacg tcaatggctc tctgggcgtc 11820

gcctttgccg acaaaaatgt gatcatgatg aaagccagca atgacgttgc agctgatatt 11880

gttgtcggcc aaccgcgccg aaaacgcagc tgtcagaccc acagcctcca acgaagaatg 11940

tatcgtcaaa gtgatccaag cacactcata gttggagtcg tactccaaag gcggcaatga 12000

cgagtcagac aggaggcaga caaggtatag ggcggcgcct acaatccatg ccaacccgtt 12060

ccatgtgctc gccgaggcgg cataaatcgc cgtgacgatc agcggtccag tgatcgaagt 12120

taggctggta agagccgcga gcgatccttg aagctgtccc tgatggtcgt catctacctg 12180

cctggacagc atggcctgca acgcgggcat cccgatgccg ccggaagcga gaagaatcat 12240

aatggggaag gccatccagc ctcgcgtcgc gaacgccagc aagacgtagc ccagcgcgtc 12300

ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt 12360

gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat 12420

cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg 12480

tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg 12540

cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gacgctctcc 12600

cttatgcgac tcctgcatta ggaagcagcc cagtatcgat agagacggag tcactgcctt 12660

aattaaaaag tgaagaaatt caaccaagcg cgggtaaacg gcgggagtaa ctatgactct 12720

cttaaggtag ccaaatgcct cgtcatctaa ttagtgacgc gcatgaatgg attaacgaga 12780

ttcccactgt ccctatctac tatctagcga aaccacagcc aagggaacgg gcttggcaga 12840

atcagcgggg aaagaagacc ctgttgagct tgactctagt ttgacattgt gaagagacat 12900

agggggtgta gaataagtgg gagcttcggc gccggtgaaa taccactacc cttatcgttt 12960

ctttacttat ttagtaagtg gaagtggttt aacaaccatt ttctagcatt cctttccagg 13020

ctgaagacat tgtcaggtgg ggagtttggc tggggcggca catctgttaa aagataacgc 13080

agatgtccta agggggactc aatgagaaca gaaatctcat gtagaacaaa agggtaaaag 13140

tccccttgat tttgattttc agtgtgaata caaaccatga aagtgtggcc tatcgatcct 13200

ttagttgttc ggagtttgaa cctagaggtg ccagaaaagt taccacaggg ataactggct 13260

tgtggcagtc aagcgttcat agcgacattg ctttttgatc cttcgatgtc ggctcttcct 13320

atcataccga aggcatgcga cgccacccat ggactctaag cggccgcatg gacctgtcga 13380

cttggctcga gggggggccc ggtaccagct tttgttccct ttagtgaggg ttaatttcga 13440

gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc 13500

cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct 13560

aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc 13620

agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt 13680

ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag 13740

ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca 13800

tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt 13860

tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc 13920

gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct 13980

ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg 14040

tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca 14100

agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact 14160

atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta 14220

acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta 14280

actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct 14340

tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt 14400

tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga 14460

tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca 14520

tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat 14580

caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg 14640

cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt 14700

agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag 14760

acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc 14820

gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag 14880

ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca 14940

tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa 15000

ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga 15060

tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata 15120

attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca 15180

agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg 15240

ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg 15300

ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg 15360

cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag 15420

gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac 15480

tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca 15540

tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag 15600

tgc 15603

Claims (10)

1. a kind of solve fat yeast (Yarrowia lipotica) genetic engineering containing the Ye Luowei of carRA genes and carB genes Bacterium.

2. Ye Luowei as described in claim 1 solves fat yeast gene engineering bacteria, which is characterized in that the carRA genes Nucleotide sequence is as shown in SEQ ID NO.3 in sequence table；SEQ in the nucleotide sequence of the carB genes such as sequence table Shown in ID NO.4.

3. Ye Luowei as described in claim 1 solves fat yeast gene engineering bacteria, which is characterized in that the Ye Luowei solution fat ferment Female genetic engineering bacterium also further contains GGS genes.

4. Ye Luowei as claimed in claim 3 solves fat yeast gene engineering bacteria, which is characterized in that the core of the GGS genes Nucleotide sequence is as shown in sequence table SEQ ID NO.2；Alternatively,

The preparation method of the Ye Luowei solution fat yeast gene engineering bacterias comprises the following steps：It will include using containing carRA The recombinant vector of gene, carB genes and GGS genes imports Ye Luowei solution fat yeast；Preferably, it is described to contain carRA The recombinant vector of gene, carB genes and GGS genes is built by method comprising the following steps and is obtained：Clone Origin is in three spores The mould carRA genes of Bradley, carB genes solve promoter and the end of fat yeast with downstream connection from Ye Luowei at its upstream It is only sub, complete Expression element is become, and is integrated into the Expression element of GGS genes on a plasmid, that is, the weight obtained Group carrier；Preferably, the recombinant vector is plasmid pS-B-RA-GGS-F-U (L)；Alternatively,

The copy of the carB genes, carRA genes and GGS genes in the Ye Luowei solution fat yeast gene engineering bacterias Number is more than 1, is preferably 2-100 or even 1000.

5. the Ye Luowei solution fat yeast gene engineering bacterias as described in claim 1 or 3, which is characterized in that the Ye Luowei solutions The fat yeast gene engineering bacteria also further active site gene containing HMG-CoA reductase, preferably HMG-CoA reductase are urged Change position.

6. Ye Luowei as claimed in claim 5 solves fat yeast gene engineering bacteria, which is characterized in that the Ye Luowei solution fat ferment The preparation method of female genetic engineering bacterium comprises the following steps：With the recombination expression containing HMG-CoA reductase active site gene Vector introduction Ye Luowei solves fat yeast；Preferably, the recombinant expression carrier is plasmid pSK-Hmg (y)-Ura3- IntB, nucleotide sequence is as shown in SEQ ID NO.6 in sequence table.

7. the Ye Luowei solution fat yeast gene engineering bacterias as described in claim 1 or 5, which is characterized in that the Ye Luowei solutions Fat yeast gene engineering bacteria has also further knocked out Gut2 genes；And/or the Ye Luowei solution fat yeast gene engineering bacteria is also Pox2 genes are further knocked out.

8. Ye Luowei as claimed in claim 7 solves fat yeast gene engineering bacteria, which is characterized in that the knockout technique includes Following steps：It is knocked out using plasmid pS-B-RA-GGS-Gut2-U (L), SEQ ID in the nucleotide sequence of the plasmid such as sequence table Shown in NO.7.

9. Ye Luowei as claimed in claim 7 solves fat yeast gene engineering bacteria, which is characterized in that the knockout technique includes Following steps：It is knocked out using plasmid pS-B-RA-GGS-Pox2-U (L), SEQ ID in the nucleotide sequence of the plasmid such as sequence table Shown in NO.8.

A kind of 10. method of genetic engineering bacterium production beta carotene, which is characterized in that comprise the following steps：

1) prepare the Ye Luowei as described in claim 1 containing carRA genes and carB genes and solve fat yeast gene engineering bacteria；

2) the Ye Luowei solution fat yeast gene engineering bacterias obtained by fermented and cultured step 1), obtain beta carotene from zymotic fluid.