CN113403334A - Plasmid kit for saccharomyces cerevisiae multi-copy integration - Google Patents

Abstract

The invention discloses a group of plasmid kits for saccharomyces cerevisiae multi-copy integration, and belongs to the field of gene and metabolic engineering. The multicopy integrative plasmid toolkit constructed and verified by the invention can realize multicopy, stable and integrative expression of a plurality of exogenous genes on a saccharomyces cerevisiae genome through one-time transformation. And the exogenous gene can be stably and integrally expressed in multiple batches and multiple genes by selecting different screening labels at different Ty sites.

Description

Plasmid kit for saccharomyces cerevisiae multi-copy integration

Technical Field

The invention relates to a group of plasmid kits for saccharomyces cerevisiae multi-copy integration, and belongs to the field of gene and metabolic engineering.

Background

Saccharomyces cerevisiae, a model strain of eukaryotes, is commonly used for introducing heterologous pathways to synthesize high value-added products. These approaches typically involve several to tens of genes, and multiple co-expression of multiple genes in s.cerevisiae requires multiple integrations, which is inefficient, and the integration sites and selection tags do not satisfy the subsequent integration procedures. Therefore, there is a need for a highly efficient, high copy and stable insertion site for integrating genes requiring overexpression into these sites to achieve efficient expression of heterologous genes.

There are a number of Long Terminal Repeats (LTRs) in the Saccharomyces cerevisiae genome, one type of LTR being transposons, which in yeast are designated as Ty transposons. There are five types of the Ty transposon sequence matching, including Ty1Cons, Ty2Cons, Ty3Cons, Ty4Cons and Ty5Cons, and the sequence matching between transposons is also low, the copy number of the five Ty transposons on the genome is about 20-50, and the transposons are distributed uniformly on the genome, and homologous recombination rarely occurs, and the structure is stable, so these Ty sites are ideal foreign gene insertion sites (Maury, J.; Germann, S.M.; Ballalobsen, S.A., et al., EasyClonemulti: A Set of Vectors for simple and Multiple genetic Integrations in Saccharomyces cerevisiae [ J ]. PloS One, 2016 (3), e 0194.). However, the current integrated expression based on the Ty locus has various defects, such as insufficient screening tag and low copy number of the obtained transformant; the integrative expression frame uses the same promoter, the same terminator and other expression elements, so that the homologous recombination probability is increased; the application of such integration sites is limited due to the unknown ability of the different Ty sites to coordinate integration.

To further refine the Ty integration protocol, a high copy integration plasmid kit was constructed in this study. In this high copy integration package, the screening tag classes were first expanded, including 5 auxotrophic tags: uracil-deficient tags (KlURA3), leucine-deficient tags (KlLEU2), histidine-deficient tags (SpHIS5), tryptophan-deficient tags (sctp 1), and methionine-deficient tags (ScMET 15); 5 antibiotic screening tags: nolsemicin resistance tag (Nourethericin resistance, natMX), Hygromycin resistance tag (Hygromycin resistance, hpHMX), Bialaphos resistance tag (Bialaphos resistance, patMX) (Goldstein A L, McCusker J H.three and new dominant drug resistance cassettes for gene delivery in Saccharomyces cerevisiae [ J ]. Yeast (Chichester, England)1999,15(14),1541-53.), Geneticin resistance tag (genetic in resistance, KanmX) (Wach A, Brachat A, Pohlmann R, Nuet al, New hematology modules for PCR-base gene delivery [ J ] (PCR-nucleotide sequences J.) (nucleotide J.) (PCR-nucleotide sequences J.) (PCR-nucleotide sequences J.) (nucleotide J.) (III J.) (nucleotide J.),93, nucleotide J.), 30(6), e 23.). Meanwhile, when a screening gene expression frame is constructed, a weak promoter is used for expressing a related screening gene, and a Degradation tag (deg) is added to the C end of the screening gene (Gilon T, Chomsky O, Kulka R G. grading signals for ubiquitin system protein [ J ]. EMBO J1998, 17(10),2759-66.), the expression level of the gene is reduced at the transcription level and the protein level, and the screening pressure is strengthened, so that the integrated copy number of the foreign gene is increased. Finally, all elements such as promoters and terminators are not identical in order to reduce the probability of homologous recombination of foreign genes. Connecting the constructed expression package with a reporter gene green fluorescent protein EGFP, respectively transforming the expression package into a saccharomyces cerevisiae strain, and determining the integration difficulty and the distribution condition of the integrated copy number of the corresponding labels and sites by detecting the number of the obtained transformants and the intensity distribution of the green fluorescent protein of the transformants.

After obtaining the high-copy integration tool and verifying the integration performance of the high-copy integration tool, the taxifolin synthetic pathway genes are integrated on different Ty sites of a saccharomyces cerevisiae genome by using different screening labels and are used for verifying the multi-copy integration performance.

Disclosure of Invention

The Saccharomyces cerevisiae includes model strain S288c and its derivative strains. The plasmids can realize multi-copy integration in a saccharomyces cerevisiae genome through one-time transformation, and the integration sites are mutually independent and have good stability, so that the plasmids can be used for over-expression of genes.

The invention provides a saccharomyces cerevisiae multicopy integrated gene expression cassette, which consists of a weak promoter sequence, a screening gene sequence with a degradation label deg and a terminator; the weak promoter comprises P_ADE6、P_LEU2、P_URA3、P_PMA1、P_ZWF1、P_ARO7、P_PYC1、P_ADE3、P_YEF3、P_ERG1The screening gene comprises ScTRP1, KlLEU2, KlURA3, ScMET15, SpHIS5, natMX, hpHMX, kanMX, patMX and bleMX.

In one embodiment, the terminator is T_TDH3The nucleotide sequence is SEQ ID NO. 43.

In one embodiment, the weak promoter P_ADE6、P_LEU2、P_URA3、P_PMA1、P_ZWF1、P_ARO7、P_PYC1、P_ADE3、P_YEF3、P_ERG1The nucleotide is shown in SEQ ID NO. 1-10 respectively; the screening gene is provided with a degradation label which is deg, and the nucleotide sequence is shown in SEQ ID NO. 46; the nucleotide sequences formed by the screened gene and the degradation label are respectively shown in SEQ ID NO. 11-20.

In one embodiment, the weak Promoter is disclosed in the literature Promoter-library-based pathway optimization for efficacy (2S) -naringenin production from p-homologous acid in Saccharomyces cerevisiae (published in 2020).

The invention provides a saccharomyces cerevisiae multicopy integration plasmid, which consists of a gene expression frame and a pre-integration expression frame; the pre-integration expression frame consists of an upstream and downstream homologous arm sequence of a Ty transposon, a terminator sequence and a green fluorescent protein expression frame.

In one embodiment, the Ty transposon sequences comprise Ty1Cons, Ty2Cons, Ty3Cons, Ty4Cons, and Ty5 Cons; the terminator comprises T_RFC5-T_POL30、T_SEC13-T_PNP1、T_MTD1-T_RPF2、T_LEU2-T_NFS1、T_DSF1-T_HXT13、T_TIM21-T_GSC2、T_RRP12-T_TAF3、T_RNA14-T_BUB2、T_ADH1And T_CYC1。

In one embodiment, the saccharomyces cerevisiae multicopy integration plasmid is ligated in the order of upstream homology arm, green fluorescent protein expression cassette, terminator sequence, gene expression cassette, downstream homology arm.

In one embodiment, the upstream and downstream homology arms of Ty1Con1 are shown in SEQ ID No.21 and SEQ ID No.22, respectively; the upstream and downstream homology arms of Ty1Cons2 are shown in SEQ ID NO.23 and SEQ ID NO.24 respectively; the upstream and downstream homology arms of Ty2 are shown in SEQ ID NO.25 and SEQ ID NO.26 respectively; the upstream and downstream homology arms of Ty3 are shown in SEQ ID NO.27 and SEQ ID NO.28, respectively; the upstream and downstream homology arms of Ty4 are shown in SEQ ID NO.29 and SEQ ID NO.30 respectively; the upstream and downstream sequences of all Ty elements have been disclosed in Maury, j; germann, s.m.; baallil Jacobsen, S.A., et al, easy CloneMulti A Set of Vectors for Simultaneous and Multiple Genomic Integrations in Saccharomyces cerevisiae [ J ]. Plous One 2016,11(3), e0150394 (published in 2016)

In one embodiment, the terminator T is_RFC5-T_POL30、T_SEC13-T_PNP1、T_MTD1-T_RPF2、T_LEU2-T_NFS1、T_DSF1-T_HXT13、T_TIM21-T_GSC2、T_RRP12-T_TAF3、T_RNA14-T_BUB2、T_ADH1And T_CYC1The sequences of (A) are respectively shown in SEQ ID NO. 31-40; the terminator sequences are disclosed in the literature, Promoter-library-based optimization for efficacy (2S) -naringenin production from p-homologous acid in Saccharomyces cerevisiae (published in 2020).

In one embodiment, the green fluorescent protein expression cassette comprises a promoter P_GAL7Promoter P_GAL7Upstream of (2) comprises a terminator sequence T_GAL10With a terminator T_GAL10Promoter P of (1)_GAL7Nucleotide sequence ofIs SEQ ID NO.41, and the nucleotide sequence of the green fluorescent protein is SEQ ID NO. 42.

In one embodiment, the selection gene is preferably KlLEU2, KlURA3, SpHIS5, hpmx, natMX.

The invention provides a strain for expressing a target protein, wherein the strain expresses the target protein through a saccharomyces cerevisiae multicopy integration plasmid.

In one embodiment, the protein of interest is inserted upstream of the green fluorescent expression cassette.

The invention provides a strain for producing taxifolin, which expresses relevant genes for producing taxifolin through a saccharomyces cerevisiae multicopy integration plasmid, wherein the genes are expressed through the following three modules:

a first module: overexpression of the relevant genes from tyrosine or phenylalanine to p-coumaric acid synthesis:

from tyrosine to p-coumaric acid genes include ARO4^fbr、ARO7^fbr、FjTAL、EcaroL；

The genes from phenylalanine to p-coumaric acid include ARO4^fbr、ARO7^fbr、FjTAL、EcaroL、SmPAL、SmC4H；

And a second module: overexpresses genes related to p-coumaric acid to naringenin production, including Pc4CL, PhCHS, and MsCHI;

and a third module: naringenin-to-taxifolin synthesis related genes including SmF 3' H, SmCPR and SmF 3H.

In one embodiment, the strain further expresses a malonyl-coa pathway-associated gene ScACC1^{S659A ,S1157A}、SeACS2^S641PAnd SpHIS 5.

In one embodiment, C800 is used as the starting strain, which is disclosed in patent document No. CN 111424020A.

The invention provides a method for producing taxifolin, which takes the bacterial strain for producing taxifolin and takes glucose or ethanol as a substrate to synthesize taxifolin from the beginning.

In one embodiment, the strain is culturedTo OD₆₀₀The method comprises the steps of inoculating 20-30 seed liquid into YPD culture medium according to the proportion of 1-5 mL/100mL, culturing at 30-35 ℃ and 200-250 rpm, fermenting for 72 hours, and adding 95% ethanol or glucose at 12h, 24h, 36h and 48h respectively.

In one embodiment, the 95% ethanol is added at a concentration of 0.5% (0.5mL/100mL), the glucose concentration is 500g/L, and the amount added is 0.5% (0.5mL/100 mL).

In one embodiment, the temperature is controlled to 30 ± 0.1 ℃.

In one embodiment, the strain is cultured to obtain the OD₆₀₀The method comprises the steps of inoculating 20-30 seed liquid into YPD culture medium according to the proportion of 1-5 mL/100mL, culturing at 30-35 ℃, 500-600 rpm and the aeration rate of 1-3 vvn, supplementing fed-batch culture medium at the flow rate of 5.0mL/L when the glucose concentration in a reaction system is reduced to 0g/L, controlling the pH to be 5.5 +/-0.1, and fermenting for 72 hours.

The invention provides an application of the saccharomyces cerevisiae multicopy integration gene expression cassette or the saccharomyces cerevisiae multicopy integration plasmid in production of target protein.

The invention provides application of the strain in producing taxifolin.

In one embodiment of the invention, the microbial cell is Saccharomyces cerevisiae strain C800(CEN. PK2-1D, MAT. alpha.; ura 3-52; leu2-3,112; trp 1-289; his 3. delta.1; MAL 2-8)^C(ii) a SUC 2; gal80: KanMX), said strain C800 being disclosed in the patent document with publication number CN 111424020A. .

The invention provides a recombinant bacterium for producing taxifolin and a production method thereof, wherein the method comprises the steps of selecting the multi-copy carrier, integrating a plurality of copies of taxifolin synthetic genes on 3 different Ty sites, and fermenting by using the recombinant bacterium to produce taxifolin.

The invention has the beneficial effects that: the multicopy integrative plasmid toolkit constructed and verified by the invention can realize multicopy, stable and integrative expression of a plurality of exogenous genes on a saccharomyces cerevisiae genome through one-time transformation. And the exogenous gene can be stably and integrally expressed in multiple batches and multiple genes by selecting different screening labels at different Ty sites, so that the high-efficiency expression of the exogenous gene is realized.

Drawings

FIG. 1 is a diagram showing the genotypes of a multicopy plasmid kit (A: 10 combinations of screening gene expression cassettes for multicopy integration; B: 5 pre-integration expression cassettes; C-G: 50 genotypes of a plasmid kit for multicopy integration of Saccharomyces cerevisiae, where the positions and the order of all elements are the positions and the order in an actual plasmid map).

FIG. 2 is a graph showing the distribution of the number of transformants obtained after one transformation (the abscissa is the selection tag and the ordinate is the number of transformants obtained after one transformation.

FIG. 3 shows the fluorescence intensity distribution of multicopy recombinant strains (transformants were obtained by integrating 50 combination expression cassettes of the expression package into the genome of Saccharomyces cerevisiae, 10-30 transformants were randomly selected, the fluorescence intensity distribution was examined, and a box graph was drawn alongside which the fluorescence intensity distribution of each transformant was simultaneously drawn, Panel A: Blank is the fluorescence intensity distribution of strain C800 in YNB medium, and only one copy of EGFP is present on the genome of C887. Panel B-Panel H is the fluorescence intensity distribution of transformants obtained when the selection tags are, in the order of ScTRP1deg, KlURA3deg, ScMET15deg, hphMXdeg, KanMXdeg, patMXdeg, and bleg, when the selection tags are integrated in the order of Ty1Cons1, Ty1Cons2, Ty2Cons, Ty3Cons and Ty4Cons, when the selection tags are integrated in the order of transformants Ty1 Ty 2deg, Ty3Con 3 Ty and Ty4Con, and when the fluorescence intensity distribution of transformants are not obtained when the selection tags are integrated in the order of KlLUE2 deg.C.C.C.S selection tag, when the selection tag is replaced by KlMX 2, and the selection tags are not obtained when the selection tags are replaced by the combination of KlMX 3, and the selection tag is replaced by the selection tag 2.

FIG. 4 is a schematic diagram of the pathway for the synthesis of taxifolin in Saccharomyces cerevisiae (the taxifolin synthesis pathway is divided into three modules; module one: p-coumaric acid synthesis pathway (over-expression of ARO4/ARO7/TAL or ARO4/ARO 7/PAL/C4H); module two: naringenin synthesis pathway (over-expression of 4 CL/CHS/CHI); and module three: taxifolin synthesis pathway (over-expression of F3' H/CPR/F3H)).

FIG. 5 shows the effect of expression of genes at single Ty site and double Ty sites on the integration pathway.

FIG. 6 is a graph showing the effect of expression of a triple Ty site integration pathway gene; in Panel A, the number in parentheses is the fluorescence intensity.

FIG. 7 shows the copy number of the integrated gene at different sites.

FIG. 8 is a graph showing the yield of the de novo synthesized taxin in the 5-L fermenter.

Detailed Description

YNB medium: 0.72g/L yeast nitrogen source basic culture medium and 20g/L glucose.

YPD medium: 10g/L yeast powder, 20g/L peptone and 20g/L glucose.

Leucine, tryptophan, histidine or uracil were added to YNB medium to a final concentration of 50mg/L, or 50mg/L, as required.

Adding various antibiotics into a non-scalding culture medium in a mother liquor form, wherein the concentration of the nourseothricin mother liquor is 100mg/L, and the working concentration is 100 mu g/L; the concentration of the hygromycin mother liquor is 300mg/L, and the working concentration is 300 mug/L; the concentration of the glufosinate-ammonium mother liquor is 800mg/L, and the working concentration is 800 mug/L (if bialaphos is used, the concentration of the mother liquor is 200mg/L, and the working concentration is 200 mug/L); the concentration of the geneticin mother liquor is 200mg/L, and the working concentration is 200 mug/L; the concentration of the mother liquor of bleomycin is 100mg/L, and the working concentration is 100 mug/L. Transformants using the amino acid-deficient selection tag and the glufosinate antibiotic selection tag were plated on YNB plates, and transformants using the selection tags of the other four antibiotics were plated on YPD plates.

20g/L agar powder is added into the solid culture medium.

Feed medium composition: 400g/L glucose, KH₂PO₄ 18g/L，MgSO₄·7H₂O 10.24g/L，K₂SO₄7g/L，Na₂SO₄0.56g/L, 20 mL. L of mother liquor of metal salt ^-124 mL. L vitamin mother liquor^-1If necessary, 1g/L of each amino acid was added. Metal salt mother liquor: ZnSO₄·7H₂O 5.75g/L，MnCl₂·4H₂O 0.32g/L，CoCl₂·6H₂O 0.47g/L，NaMoO₄·2H₂O 0.48g/L，CaCl₂·2H₂O 2.9g/L，FeSO₄·7H₂O2.8 g/L, 80mL of 0.5M EDTA (pH 8.0). Vitamin mother liquor: 0.05g/L Biotin (Biotin), 1g/L Calcium pantothenate (Calcium panthenate), 1g/L Nicotinic acid (Nicotinic acid), 25g/L myo-Inositol (myo-Inositol), 1g/L Thiamine hydrochloride (Thiamine HCl), 1g/L Pyridoxal hydrochloride (Pyridoxal HCl), 0.02g/L p-Aminobenzoic acid (p-Aminobenzoic acid)

Saccharomyces cerevisiae CEN. PK2-1D (MAT. alpha.; ura 3-52; leu2-3,112; trp 1-289; his 3. delta.1; MAL2-8^C(ii) a SUC2) for gene expression.

Coli JM109 was used for molecular cloning.

Plasmids pcfB2989, pcfB2988, pcfB2797, pcfB2990, pcfB2796 and pcfB2803(Addgene plasma #63636, #63638, #63639, #63645, #63641, #63646) were given by Irina Borodina & Jeromeo Maury (Maury J, Germann S M, Baallial Jacobsen S A, et al.

Plasmids pMDT-SmPAL and pMDT-SmC4H are Silybum marianum-derived Phenylalanine Ammonia Lyase (PAL) and cinnamate hydroxylase (C4H) obtained by tBLASTN from Silybum marianum transcriptome identification and reverse transcription corresponding to Contig 5930 and Contig265(Lv Y, Gao S, Xu S, et al spatial organization of simple biosyntheses in mile host [ Silybum marianum (L.) Gaertn ] [ J ]. Plant J2017, 92(6), 995) 1004 ], respectively, and the corresponding gene DNA sequences are in Table 1, with the nucleotide sequences being SEQ ID NO.44 and SEQ ID NO.45 in that order.

P-coumaric acid, naringenin, eriodictyol, and taxifolin were purchased from Sigma-Aldrich (st.

Nolsethricin (CAS: 96736-11-7) was purchased from Solobio (N9210).

Hygromycin (Hygromycin B Solution, CAS: 3128-04-9) was purchased from Biotechnology engineering (Shanghai) Inc. (B540725).

Glufosinate Ammonium (Ammonium glufosinate, CAS:77182-82-2) was purchased from Ark Pharm (AGZ 938).

Geneticin (G418 Sulfate, CAS:108321-42-2) was purchased from Biotechnology engineering (Shanghai) Inc. (A100859).

Bleomycin (Bleomycin, CAS:11006-33-0) was purchased from Biotechnology engineering (Shanghai) Inc. (A620212).

The detection method comprises the following steps: taking 100 mu L of fermentation liquor, mixing with 900 mu L of methanol, whirling, shaking, uniformly mixing for 30s, centrifuging at 13500rpm for 5min, and filtering supernatant for later use. Shimadzu high performance liquid detection was used. A chromatographic column: ZORBAX SB-C18(4.6 mm. times.150 mm, Shimadzu). Mobile phase A: 100% acetonitrile, mobile phase B: 100 percent of water, adding 1 per mill (V/V) of trifluoroacetic acid into all mobile phases, filtering, and removing bubbles by ultrasonic waves for later use. Flow rate of mobile phase: 1mL/min, column temperature: 30 ℃, sample introduction: 10 μ L, detection wavelength: 290 nm. Liquid phase procedure: 0-10 min, 10-40% of mobile phase A; 10-15 min, 40-60% of mobile phase A; and (3) distinguishing various substances according to different peak emergence times of different substances by using 60-10% of the mobile phase A for 15-18 min.

Example 1: construction of multicopy plasmid expression kits

In the present invention, Gibson assembly technology (Gibson assembly) is used as an assembly means, and the assembly of all sequences is seamless cloning. Corresponding nucleotide sequences are amplified through PCR, homologous arms of about 20-30bp are contained among the sequences, and all plasmid expression kits can be obtained through Gibbson assembly.

(1) Construction of multiple copy integrated screening Gene expression cassette combinations

Selecting 10 combinations of screening gene expression cassettes (Marker genes, shown as pT 0-pT 9 in Table 2 and FIG. 1) for multi-copy integration, including 10 weak promoter sequences, 10 screening gene sequences with degradation tag deg, and terminator T_TDH3The nucleotide sequence is SEQ ID NO. 43.

10 weak promoters are respectively P_ADE6、P_LEU2、P_URA3、P_PMA1、P_ZWF1、P_ARO7、P_PYC1、P_ADE3、P_YEF3、P_ERG1Corresponding coreThe nucleotide sequence is SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10(Gao, S.; Zhou, H.; Zhou, J., et al, Promoter-library-based optimization for efficacy (2S) -naringenin production from p-cortex acid in Saccharomyces cerevisiae [ J].J Agric Food Chem 2020,68(25),6884-6891.)。

10 screening genes with degradation label deg are ScTRP1-deg, KlLEU2-deg, KlURA3-deg, ScMET15-deg, SpHIS5-deg, natMX-deg, hpHphMX-deg, kanMX-deg, patMX-deg and bleMX-deg, and the corresponding nucleotide sequences are SEQ ID No.11, SEQ ID No.12, SEQ ID No.13, SEQ ID No.14, SEQ ID No.15, SEQ ID No.16, SEQ ID No.17, SEQ ID No.18, SEQ ID No.19 and SEQ ID No. 20.

(2) Construction of Pre-integration expression cassette

According to the integration site of the saccharomyces cerevisiae, 5 pre-integration expression frames are constructed, including upstream and downstream homologous arm sequences of 5 sets of Ty sequences, 8 bidirectional terminator sequences, 2 unidirectional terminator sequences and a green fluorescent protein expression frame.

The 5 groups of Ty sequences are Ty1Cons1, Ty1Cons2, Ty2, Ty3 and Ty4 in sequence, the upstream/downstream homology arm of Ty1Con1, the upstream/downstream homology arm of Ty1Con2, the upstream/downstream homology arm of Ty2, the upstream/downstream homology arm of Ty3 and the upstream/downstream homology arm of Ty4, and the nucleotide sequences corresponding to the upstream and downstream homology arms of the 5 groups are SEQ ID NO.21/SEQ ID NO.22, SEQ ID NO.23/SEQ ID NO.24, SEQ ID NO.25/SEQ ID NO.26, SEQ ID NO.27/SEQ ID NO.28 and SEQ ID NO.29/SEQ ID NO.30 in sequence.

8 bidirectional terminators are T in sequence_RFC5-T_POL30、T_SEC13-T_PNP1、T_MTD1-T_RPF2、T_LEU2-T_NFS1、T_DSF1-T_HXT13、T_TIM21-T_GSC2、T_RRP12-T_TAF3、T_RNA14-T _BUB22 unidirectional terminators are in turn T_ADH1And T_CYC1The nucleotide sequence corresponding to the bidirectional terminator is SEQ ID NO.31, SEQ ID NO.32, SEQ ID NO.33 and SEQ ID NO.32 in sequenceThe sequence corresponding to the unidirectional terminator is SEQ ID NO.39 and SEQ ID NO.40 in sequence.

The expression frame of the green fluorescent protein comprises a promoter P_GAL7Promoter P_GAL7Upstream of (2) comprises a terminator sequence T_GAL10The green fluorescent protein is EGFP with terminator T_GAL10Promoter P of (1)_GAL7The nucleotide sequence of the EGFP gene is SEQ ID NO.41, and the nucleotide sequence of the green fluorescent protein is SEQ ID NO. 42. A terminator sequence T is also used in the present invention_TDH3The nucleotide sequence is SEQ ID NO. 43. The invention uses two silybum marianum-derived genes SmPAL and SmC4H, and the nucleotide sequences are SEQ ID NO.44 and SEQ ID NO.45 in sequence.

Construction of 50 plasmid kits for multiple copy integration of s.cerevisiae: the 10 screening gene expression cassettes (Marker genes) are sequentially and respectively inserted into the middle of two double terminators in 5 pre-integration expression cassettes (Gibson assembly technology is used, no redundant base insertion exists, and both insertion are traceless insertion), and then a group of saccharomyces cerevisiae multi-copy integration kit containing 50 plasmids is obtained. Wherein the Ty1Cons1 pre-integrated expression cassette is combined with 10 screening genes to obtain 10 plasmids of pcT111, pcT112, pcT113, pcT114, pcT115, pcT116, pcT117, pcT118, pcT119 and pcT110 in total in sequence. Wherein the Ty1Cons2 pre-integration expression frame is combined with 10 screening genes, and a total of 10 plasmids of pcT121, pcT122, pcT123, pcT124, pcT125, pcT126, pcT127, pcT128, pcT129 and pcT120 are obtained in sequence. The Ty2 pre-integrated expression frame is combined with 10 screening genes, and pcT21, pcT22, pcT23, pcT24, pcT25, pcT26, pcT27, pcT28, pcT29 and pcT20 total 10 plasmids are obtained in sequence. The Ty3 pre-integrated expression frame is combined with 10 screening genes, and pcT31, pcT32, pcT33, pcT34, pcT35, pcT36, pcT37, pcT38, pcT39 and pcT30 total 10 plasmids are obtained in sequence. Wherein the Ty4 pre-integrated expression frame is combined with 10 screening genes to obtain 10 plasmids of pcT41, pcT42, pcT43, pcT44, pcT45, pcT46, pcT47, pcT48, pcT49 and pcT40 in total.

The actual positions of all the genes, promoters, terminators, etc. in the 10 combinations of the screening gene expression cassettes for multicopy integration, 5 pre-integration expression cassettes, and 50 plasmid kits for multicopy integration of s.cerevisiae are shown in FIG. 1.

Gene ScACC1^S659A,S1157A、SeACS^S641P、SmF3′H^D284N、SmCPR^I453V、ARO4^fbr、ARO7^fbrFjTAL, EcaroL and SmF3H, and the sequentially corresponding nucleotide sequences are SEQ ID NO. 47-SEQ ID NO. 55. Other promoters P mentioned_GAL1And P_GAL10The corresponding nucleotide sequences are SEQ ID NO.56 and SEQ ID NO.57 in sequence.

All nucleotide sequences of the present invention are shown in Table 1. The genotypes of the plasmids and strains are shown in table 2. All key primer sequences are shown in table 3.

Table 1 all nucleotide sequences

TABLE 2 genotypes of plasmids and strains

TABLE 3 Key primer sequences

Example 2: validation of integration Capacity of multicopy integrating plasmid tool

Plasmids pcT110 to pcT119 were amplified respectively using primers Ty11-inte-up/down, plasmids pcT120 to pcT129 were amplified respectively using primers Ty12-inte-up/down, plasmids pcT20 to pcT29 were amplified respectively using primers Ty2-inte-up/down, plasmids pcT30 to pcT39 were amplified respectively using primers Ty3-inte-up/down, plasmids pcT40 to pcT49 were amplified respectively using primers Ty4-inte-up/down, and integrated expression frame portions of the vectors were obtained respectively. PCR products are recovered and purified, and then integrated into a Saccharomyces cerevisiae strain by a Saccharomyces cerevisiae efficient transformation method (see Gietz, R.D.; Schiestl, R.H., High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method [ J ]. Nat Protoc 2007,2(1), 31-4.).

When the screening tags are TRP1, LEU2, URA3, HIS5, natMX, hphMX, patMX and bleMX, the strain is transformed into the strain C800. When the screening tag was MET15, strain C815 was selected.

When the selection tag was KanMX, strain C850 was selected. The transformed cells were plated on corresponding screening plates (when the screening gene was TRP1, they were plated on YNB-containing TRP)^-Plate coated with screening gene LEU2 containing YNB-LEU^-Plate coated with YNB-URA when the selection gene is URA3^-Plate, screening gene is applied to YNB-HIS containing HIS5^-Plate coated with YNB-MET when the screening gene is MET15^-Plates were plated on YPD plates containing nourseothricin when the selection gene was natMX, and plated on hphMXYPD plates containing hygromycin, YPD plates containing geneticin coated with the selection gene KanMX, YPD plates containing glufosinate-ammonium coated with the selection gene patMX, and YPD plates containing bleomycin coated with the selection gene bleMX) were cultured at 30 ℃ for 3-5 days until single colonies were obtained. And counting the obtained single colonies, inoculating the single colonies for culture, and detecting the fluorescence intensity. The integration efficiency of each combination in the kit was determined from the number of transformants obtained and the distribution of fluorescence intensity of the transformants. The number distribution of transformants obtained for each combination after one transformation is shown in FIG. 2. The fluorescence intensity distribution range of the multicopy recombinant strains obtained for each combination is shown in FIG. 3.

As can be seen from FIG. 2, about 100-500 transformants can be obtained after a large part of plasmids in the Ty expression package are transformed once, but pcT122, pcT22, pcT32 and pcT 42; pcT25, pcT35, pcT45 and pcT116, pcT26, pcT46 did not obtain transformants after multiple transformations, whereas pcT114, pcT124, pcT24, pcT34 and pcT44 using ScMET15deg as a selection tag obtained more than 1 ten thousand transformants, however, the transformants had almost no fluorescence (fig. 3), and it was found that ScMET15deg was not suitable for high copy integration here.

As can be seen from FIG. 3, the fluorescence intensity values of C887, a blank strain and a strain having only one copy of EGFP, were about 8000 and 25000 (FIG. 3A). When ScMET15deg, KanMXdeg, patMXdeg and BleMXdeg were used as selection tags, the obtained transformants had a weak fluorescence intensity, and most of them had only one copy (FIG. 3D, FIG. 3F, FIG. 3G and FIG. 3H), and when ScTRR1deg was used as selection tags, most of the transformants had a weak fluorescence intensity (FIG. 3B). While the transformants with high probability of obtaining strong fluorescence signals by using KlURA3deg, KlLEU2deg, SpHIS5deg, natMXdeg and hphMXdeg (FIG. 3C, FIG. 3E and FIG. 3I) have the average fluorescence intensity of 7.9-16.5 times that of the control group, and the strongest one reaches 48.2 times that of the control group. These multicopy integrative plasmids can thus be used to increase the copy number in s.cerevisiae.

Example 3: use of multicopy integrative plasmid kit

The process of synthesis of taxifolin involves the introduction and overexpression of multiple genes and is therefore used to validate the performance of a multicopy integrative plasmid kit. The synthesis route of taxifolin is shown in fig. 4, wherein the module I is glucose to p-coumaric acid, the module II is p-coumaric acid to naringenin, and the module III is naringenin to taxifolin. The multicopy integrative plasmid kit with significantly increased copy number in example 2 was picked for use in the production of taxifolin or related intermediates.

1. Construction of single Ty site integrated recombinant bacterium

Primers Ty4-inte-up/Ty4-inte-down are used for amplifying pcfB4-47LL and pcfB4-P05m4 respectively, PCR products are efficiently transformed by saccharomyces cerevisiae and integrated to Ty4 sites of the saccharomyces cerevisiae strain C800 respectively, and strains C8011 and C805 are obtained respectively. Strain C8011 integrates the genes related to the synthesis of p-coumaric acid in module I, and can synthesize p-coumaric acid from head, and strain C805 integrates the genes related to the synthesis of naringenin to taxifolin in module III, and can synthesize taxifolin from naringenin.

2. Construction of double Ty site integrated recombinant bacteria

And (3) using a primer Ty3-inte-up/Ty3-inte-down amplification plasmid pcfB3-P03, purifying and refining the obtained PCR product, and efficiently converting and integrating the purified PCR product into a Ty3 site of a strain C8011 genome to obtain a strain C824. And (3) using a primer Ty4-inte-up/Ty4-inte-down amplification plasmid pcfB4-P05m4, purifying and refining the obtained PCR product, and efficiently converting and integrating the purified PCR product into a Ty4 site of a genome of a strain C803 through saccharomyces cerevisiae to obtain a strain C857. Strain C824 integrates related genes from glucose to naringenin synthesis in module I and module II, and can synthesize naringenin from head, and strain C857 integrates related genes from p-coumaric acid to taxifolin synthesis in module II and module III, and can synthesize taxifolin from p-coumaric acid.

pT820 was amplified using primers Pf-gal80/Pf-gal80D, and after recovery of PCR products, the PCR products were transformed into strain C803 and strain C857, respectively, by the Saccharomyces cerevisiae high efficiency transformation method, to obtain strain C823 and strain C877, respectively. Strain C823 and strain C877 enhanced the malonyl-CoA pathway on the basis of the starting strains C803 and C857, respectively.

3. Construction of triple Ty site integration recombinant bacteria

Integrating a module-related gene on the basis of the strain C877, and respectively obtaining a strain C900 and a strain C901 according to the expression formula by taking tyrosine or phenylalanine as a precursor: primers Ty2-inte-up/Ty2-inte-down are used for amplifying pcT21-LL and pcT21-LHL, PCR products are recovered and then are transformed to Ty2 sites in a strain C877 through a saccharomyces cerevisiae high-efficiency transformation method, and strains C900 and C901 are obtained respectively. Both the strain C900 and the strain C901 can synthesize taxifolin from the head, wherein the strain C900 takes tyrosine as a precursor and obtains p-coumaric acid through TAL approach to synthesize taxifolin, and the strain C901 takes phenylalanine as a precursor and obtains the p-coumaric acid through PAL/C4H/CPR approach to obtain the taxifolin.

4. RNA expression level of taxifolin synthesis related gene in recombinant strain (detection of integrated copy number of different Ty sites and verification of stability direction)

(1) Copy number detection

Strain culture: single colonies of the strain C900 and the strain C901 which are constructed in the above way are selected and inoculated in a 250mL shake flask containing 5mL YNB medium, and cultured for 24 hours at 30 ℃ and 220rpm, so as to obtain a seed medium. Seed cultures were adjusted to starting OD₆₀₀0.1 to a 250mL shake flask in 50mL YNB liquid medium. Incubated at 30 ℃ and 220rpm for 6-8 hours to early log phase. The fermentation broth was centrifuged at 13500rpm for 3 min. Removing supernatant and collecting yeast cells.

RT-PCR procedure: yeast total RNA was extracted using the RNAprep pure Plant Kit (TIANGEN, Beijing, China)). Using the total RNA as a template, the DNA was removed using a PrimeScript RT kit, and then reverse transcription was performed to obtain cDNA. RT-PCR was performed using SYBR Premix Ex Taq (Tli RNAseH Plus) kit and RT-PCR was performed using cDNA as a template. The internal reference gene ACT1 for homogenization was subjected to RT-PCR using the primer pair ACT1-F/ACT 1-R. RT-PCR was performed on a LightCycler 480II instrument. All results were normalized according to the reference gene ACT 1. The expression intensity of the gene was determined according to 2^-ΔΔCtMethod (Livak K J, Schmitgen T D. analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCT method[J]Methods 2001,25(4), 402-8). The integrated copy number of the genes of the modules I, II and III is determined by using primers qEGFP-F/R, qCHS-F/R and qF3H-F/R respectivelyAmount of the compound (A).

According to RT-PCR verification, the module one, the module two and the module three genes in the strain C900 are respectively integrated at the Ty2 site, the Ty3 site and the Ty4 site, and the copy numbers are 4, 5 and 7 in sequence, while the copy numbers of the strain C901 at the Ty2 site, the Ty3 site and the Ty4 site are 8, 5 and 7 in sequence (FIG. 7).

(2) Stability verification

Inoculating strains C900 and C901 into 250mL shake flasks containing 20mL of liquid YPD medium, culturing at 30 ℃ and 220rpm, inoculating 1% (mL/100mL) of the strains into 250mL shake flasks containing fresh 20mL of liquid YPD medium every 12h of culture, carrying out passage for 60 passages, respectively inoculating the two strains obtained after passage and the corresponding starting strains into 250mL shake flasks containing 20mL of liquid YPD medium after 30 days of total passage, respectively, culturing at 30 ℃ and 220rpm for 14-16h, inoculating the strains into 250mL shake flasks containing 20mL of YPD medium according to an inoculation ratio of 1mL/100mL, culturing at 30 ℃ and 220rpm, fermenting for 72h, and respectively adding 0.5% (0.5mL/100mL) of 95% ethanol at 12h, 24h, 36h and 48h, and measuring the yield of the taxifolin after the completion. The results showed that the yields of taxifolin of the starting strains C900 and C901 were 50.93mg/L and 33.83mg/L, respectively, and the yields of taxifolin of the strains after passage of strains C900 and C901 were 52.34mg/L and 33.48mg/L, respectively, and it was apparent that the ability of the strains after passage to produce taxifolin did not significantly change, thereby demonstrating that the stability of the three-site integration strain based on the Ty transposon is good.

5. Recombinant bacterium fermentation production of taxifolin

(1) The strain C8011, the strain C803, the strain C805, the strain C824, the strain C857 and the strain C877 which are obtained by construction are utilized: respectively selecting single colony, inoculating into 250mL shake flask of 20mL YPD medium, controlling shaking table at 30 deg.C and 220rpm, culturing for 14-16h to strain OD₆₀₀Seed solutions were obtained around 25, when the strain was in mid-log phase. The mixture was inoculated into a 250mL shake flask containing 20mL YPD medium at an inoculation ratio of 1mL/100mL, cultured at 30 ℃ and 220rpm, and 0.5% (0.5mL/100mL) of 500g/L glucose solution or 0.5% (0.5mL/100mL) of 95% ethanol was added at 12h, 24h, 36h and 48h, respectively, as required. Sampling after fermentation, detecting the end product andcontent of intermediate product.

Strain C8011 has integrated the genes involved in module one, and it was possible to synthesize 544.01mg/L of p-coumaric acid starting from glucose by fermentation in a 250mL shake flask for 72h (FIG. 5A). Strain C803 integrated the gene related to module two, and fermented in a 250mL shake flask for 72h could synthesize 623.60mg/L naringenin from 1000mg/L p-coumaric acid (FIG. 5B). Strain C805, which incorporates the genes related to Module three, was fermented in 250mL for 72h to synthesize 603.90mg/L of taxifolin from 1000mg/L of naringenin, while 392.30mg/L of eriodictyol was accumulated, and the remainder was 83.80mg/L of naringenin (FIG. 5A). Constitutive strains with stable gene phenotypes can be obtained based on single-site integration of the Ty transposon.

The strain C824 is a strain C8011 integrated with the related gene of module II, and can synthesize naringenin from glucose as a substrate, and synthesize 264.05mg/L naringenin by 72h fermentation in a 250mL shake flask (FIG. 5A). Strain C857 is a strain C803 integrated with related genes of module two, can synthesize taxifolin from p-coumaric acid as a substrate, can synthesize 346.43mg/L taxifolin from 1000mg/L p-coumaric acid after 72h of fermentation in a 250mL shake flask, meanwhile, the accumulation amount of intermediate products of naringenin and eriodictyol is 66.32mg/L and 142.66mg/L, and the rest of substrate p-coumaric acid is 76.52mg/L (FIG. 5C). The double-site integration based on the Ty transposon can still obtain a constitutive strain with good stability.

(2) In order to verify the relationship between the synthesis capacity of the taxifolin and the copy number of a module gene, 10 strains with different fluorescence intensities are selected from a C900 and C901 transformant library, and fermentation verification is carried out by using a 48-depth orifice plate, and the result proves that the higher the fluorescence intensity is, the higher the accumulation amount of the taxifolin and other flavone related compounds is (FIG. 6A, the number in the horizontal coordinate bracket is the fluorescence intensity). Wherein the strain C901 using phenylalanine as a precursor obtains the highest accumulated amount of taxifolin of 20.66mg/L, and the accumulated amounts of coumaric acid, naringenin and eriodictyol are respectively 56.67, 31.24 and 19.01 mg/L. The highest accumulation of taxifolin obtained by the strain C900 taking tyrosine as a precursor is 19.77mg/L, and the accumulation of p-coumaric acid, naringenin and eriodictyol are 31.42, 23.67 and 15.56mg/L respectively.

Selecting single colonies of C900 and C901, inoculating into 250mL shake flask of 20mL YPD medium, controlling shaking table at 30 deg.C and 220rpm, culturing for 14-16h to strain OD₆₀₀Seed solutions were obtained around 25, when the strain was in mid-log phase. The seed solution was inoculated into a 250mL shake flask containing 20mL YPD medium at an inoculation ratio of 1mL/100mL, cultured at 30 ℃ and 220rpm, fermented for 72 hours, and 0.5% (0.5mL/100mL) of 95% ethanol was added at 12h, 24h, 36h, and 48h, respectively. The transformants with the highest accumulation of taxifolin obtained were named C900 and C901. The optimal fermentation carbon source was then checked at the shake flask level by rescreening and adding 500g/L glucose solution at 0.5% (0.5mL/100mL) or 95% ethanol at 0.5% (0.5mL/100mL) at 12h, 24h, 36h and 48h, respectively. At 72h, the amounts of p-coumaric acid, naringenin, eriodictyol and taxifolin accumulated by fermenting strain C900 with glucose/ethanol as carbon source were 11.88/8.78, 13.35/10.85, 22.72/13.40 and 50.93/33.83mg/L, respectively; the amounts of p-coumaric acid, naringenin, eriodictyol and taxifolin accumulated by fermentation of strain C901 using glucose/ethanol as a carbon source were 15.97/13.27, 24.40/20.72, 45.43/24.56 and 70.54/49.47mg/L, respectively (FIG. 6B).

(3) Strain C901 was fermented in a 5L fermentor: the seed medium was transferred at 1% to a 5L fermenter containing 2.5L YPD medium as above. The temperature was controlled at 30. + -. 0.1 ℃, the speed 600rpm, and the aeration rate 3 vvn. Starting a feed pump when the glucose concentration is reduced to 0g/L, setting the speed of feeding the culture medium to be 5.0mL/L, controlling the pH to be 5.5 +/-0.1 at the same time, and measuring the concentration of various substances in the fermentation liquid every 12h during the fermentation process till the end of 72h of fermentation. As a result, as shown in FIG. 8, 135.83mg/L of taxifolin could be synthesized de novo within 72 hours while p-coumaric acid, naringenin and eriodictyol were accumulated in amounts of 40.37, 10.32 and 41.10 mg/L.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

<110> university of south of the Yangtze river

<120> a set of plasmid kits for saccharomyces cerevisiae multicopy integration

<130> BAA210539A

<160> 57

<170> PatentIn version 3.3

<210> 1

<211> 508

<212> DNA

<213> Artificial sequence

<400> 1

ctgaacgtat cgagactcgg ttgtgtcgtt atgctagcaa tgtcctcaca ggctccattc 60

cttctttcgc tctattggat atcatcacag ctattctccc tggtgcaaaa tatcatatta 120

aattggattt atccttacca acgatggtga agctgacgca tagataggat atgtaattct 180

acatcagctt gtaaataaac aaaaatgact ttcaatatcc ttcaaccgtt cctgactctt 240

tcctgctgac ccgtttttcc aaatttctcg tcgaacttga aattgaaaaa aaaaaaaaaa 300

aattgaatga ggactcatta aacagatgat gccgtaataa atgcaatata tcttgctatt 360

taactctttc tttctttgaa aaccttgaca tacgtattta aataattggc tgtccctgcc 420

tcgaagtata tttctcttct acttttatct tagcgatatc cctaagagtt taatcctccc 480

aggtccataa caaaagaagt caagttca 508

<210> 2

<211> 1031

<212> DNA

<213> Artificial sequence

<400> 2

gagctcgctg tgaagatccc agcaaaggct tacaaagtgt tatctctttt gagacttgtt 60

gagttgaaca ctggtgtttt catcaaactt accaaggacg tgtacccatt gttgaaactt 120

gtatcaccat atattgttat cggacaacct tcacttgcat ctatccgttc tttaatccaa 180

aagagatcta gaataatgtg gcaaaggcca gaagataaag aaccaaaaga gataatcttg 240

aatgacaaca atatcgttga agagaaatta ggtgatgaag gtgtcatttg tatcgaggat 300

atcatccatg agatttcgac gttgggcgaa aatttctcga aatgtacttt cttcctatta 360

ccattcaaat tgaacagaga agtcagtgga ttcggtgcca tctcccgttt gaataaactg 420

aaaatgcgcg aacaaaacaa ggagactcgt caaatttcaa acgctgccac ggctccagtt 480

atccaagtag atatcgactc aatgatttcc aagttgaatt gattaactat aaaaggaaaa 540

tatctgtaca atagacatcg ggctcccatt ggccctaccc acatatgtag aaatacatta 600

ctctattcac tactgcattt agttatgttt aacatttgat atagcagact accgccaggc 660

acaatatatt ccccttccct cttgccattc gctgtacttg tggtggattc caattcagcg 720

cagtcacgtg ctagtaatca ccgcattttt ttcttttcct ttcaggctaa aaccggttcc 780

gggcctgatc cctgcactca ttttctaacg gaaaaccttc agaagcataa ctacccattc 840

cagtttagag tcatgacagg ttcaacatca gatgcttcat atacttttat atattgaatt 900

atataaatat atctatgtac tctaagtaag tacatctgct ttaacgcatt cctacatttg 960

cttcgattta tttttattgt tgatacctat ttgaagaagt aaaaagtatc ccacactaca 1020

cagattatac c 1031

<210> 3

<211> 493

<212> DNA

<213> Artificial sequence

<400> 3

ttttatttag gttctatcga ggagaaaaag cgacaagaag agatagacca tggataaact 60

gattatgttc taaacactcc tcagaagctc atcgaactgt catcctgcgt gaagattaaa 120

atccaactta gaaatttcga gcttacggag acaatcatat gggagaagca attggaagat 180

agaaaaaagg tactcggtac ataaatatat gtgattctgg gtagaagatc ggtctgcatt 240

ggatggtggt aacgcatttt tttacacaca ttacttgcct cgagcatcaa atggtggtta 300

ttcgtggatc tatatcacgt gatttgctta agaattgtcg ttcatggtga cacttttagc 360

tttgacatga ttaagctcat ctcaattgat gttatctaaa gtcatttcaa ctatctaaga 420

tgtggttgtg attgggccat tttgtgaaag ccagtacgcc agcgtcaata cactcccgtc 480

aattagttgc acc 493

<210> 4

<211> 545

<212> DNA

<213> Artificial sequence

<400> 4

ccctcgttca cagaaagtct gaagaagcta tagtagaact atgagctttt tttgtttctg 60

ttttcctttt tttttttttt acctctgtgg aaattgttac tctcacactc tttagttcgt 120

ttgtttgttt tgtttattcc aattatgacc ggtgacgaaa cgtggtcgat ggtgggtacc 180

gcttatgctc ccctccatta gtttcgatta tataaaaagg ccaaatattg tattattttc 240

aaatgtccta tcattatcgt ctaacatcta atttctctta aattttttct ctttctttcc 300

tataacacca atagtgaaaa tctttttttc ttctatatct acaaaaactt tttttttcta 360

tcaacctcgt tgataaattt tttctttaac aatcgttaat aattaattaa ttggaaaata 420

accatttttt ctctctttta tacacacatt caaaagaaag aaaaaaaata taccccagct 480

agttaaagaa aatcattgaa aagaataaga agataagaaa gatttaatta tcaaacaata 540

tcaat 545

<210> 5

<211> 600

<212> DNA

<213> Artificial sequence

<400> 5

gccgtcgaaa aggatctcgt ctctgttggg agcacctggt aagtaaggtg tagttttgca 60

cccgtgtaca taagcgtgaa atcaccacaa actgtgtgta tcaagtacat agtgacattt 120

aaataatagc aagaacaaca ataatagtag cgctactgga agcaccacgt aatagtggaa 180

aagaactgga aaaaccgcta taagatgcat actccggcgg tcttacgcgg agatacaagc 240

ttccaacggt gctaaaagcc cggtttcggc tcggccggag gaggaagaga gacgaaaaaa 300

aaaaaaatga ctaaaaaaaa aatggaatat tattaatgtg ggatttttgg ctcaaggtgt 360

ggtggcccct tttctaaggg tggcgaattc ttcaatgtac ggaaaactcg ccaaggctat 420

cccatatata agcaaactgt gggttcatct atataccgac acataacacc taaagtggct 480

tcctcctgcc cctctctccc ttttctccac tcacccctcc ttctccccct tccccctctc 540

caattggctg tatagacaga aagagtaaat ccaatagaat agaaaaccac ataaggcaag 600

<210> 6

<211> 520

<212> DNA

<213> Artificial sequence

<400> 6

tggattacat ttgattcagt catacacgaa ttatggtctt gatactgaca aattttccag 60

attgaggcgg ttcttatggt ttagaacttg gggactttac aagtcgaaag aggatttaga 120

tagagaagcc aagatcaatg aagaaatgat acgcaaactg aaagcagcta aatgaaatca 180

cctattgcgc cgctcgcgga atacaattac taaattttat atatattctt taaaaatgca 240

tctatacatt cgtttttcca cgtataccaa attcgaaaaa agttgttaaa ccatcgtttt 300

cacgtttttt aatttttttt tggttctctt tttttttttt tttcaatatc aacttttttt 360

caaacttcgt gttgcatttc ctttatcgta aattttcaat ggatctctat aatcttcgaa 420

gttcgaagaa aagaagaaaa aaagtattga aaagttgaaa catcgattcc gttttgctaa 480

caaatagcac tcagcatcct gcataaaatt ggtataagat 520

<210> 7

<211> 520

<212> DNA

<213> Artificial sequence

<400> 7

cgtatccaag ccgaaacggc gctcgcctca tccccacggg aataaggcag ccgacaaaag 60

aaaaacgacc gaaaaggaac cagaaagaaa aaagagggtg ggcgcgccgc ggacgtgtaa 120

aaagatatgc atccagcttc tatatcgctt taactttacc gttttgggca tcgggaacgt 180

atgtaacatt gatctcctct tgggaacggt gagtgcaacg aatgcgatat agcaccgacc 240

atgtgggcaa attcgtaata aattcggggt gagggggatt caagacaagc aaccttgtta 300

gtcagctcaa acagcgattt aacggttgag taacacatca aaacaccgtt cgaggtcaag 360

cctggcgtgt ttaacaagtt cttgatatca tatataaatg taataagaag tttggtaata 420

ttcaattcga agtgttcagt cttttacttc tcttgtttta tagaagaaaa aacatcaaga 480

aacatcttta acatacacaa acacatacta tcagaataca 520

<210> 8

<211> 500

<212> DNA

<213> Artificial sequence

<400> 8

acgtgagcta aagcacagat tgttggaaaa gcaagcggac ggccgcggag cgctgaacgt 60

gattttccat tgtatgttat cgcagcagcg cggaccgtct gcagcgatgc tgctgcttcg 120

gtcgcttgac acggcggaac tttctcgctg tcgtctatgg gtgttgcgtg gggggttctc 180

gcgctggcaa tccgtatacg gtgacgacga gagcgttacg gcgggttacc tacccgatct 240

gtggcgttga caggtttaca caatcgcacg tgatcatata tttgccatga ctcctcccag 300

tgacaatttt gttctttttt cctctttatc gctttcgtac tatggtcagt cattcattca 360

ttatatacgc gctctccata acccgtaact ttttattata tatagactcg tttacaatac 420

aacgatagcg ataccattca attgaagttg tgagaccagg taacgagacg aacacaactt 480

tacaagtcaa ataagaaatc 500

<210> 9

<211> 720

<212> DNA

<213> Artificial sequence

<400> 9

aataagcgcc actatcaggg aatagcaact ttcccttctg tttcaatctt tttacctatt 60

cctttttaaa agatatatat acattaaact ccttctacaa gtatatattt tatacatatc 120

tacagggcgt atatatacat aacattttaa gataagcaag tgaatgttga ttcccgtttc 180

ttagtcaaca cttctttcta ttttacccgg tcgttaccct attaaaaaaa caacttacaa 240

tcattgttcg ccccttccat acttactgcc actcgcaaaa gggcccaacc agggcaatta 300

cgtatcaaaa aatcatgaca ggctgggtaa taaatattcg tgaagaaaga agaaattaaa 360

aaaagaaacg aagaagcaaa aaaaagaaaa gactccgttt aatcactttc aaccgcggtt 420

tatccggccc cacccatgca taaccctaaa ttattagatc acttagcacg tgaaaaagaa 480

acgtttttaa tgtttttttt ttttttttct ttttcttttt ttgcgttggt gaaaattttt 540

tcgcttcctc gagtataatt atctcatctc atctttcata taagataaga agttttataa 600

aaaccttttg catcaaaatt ttgtagaata tctctttttc ttacgctctc tttctttcct 660

taattgtttt ctaaagaacc gtgtattttt ctagttcgaa tccatcgata acattaaaag 720

<210> 10

<211> 723

<212> DNA

<213> Artificial sequence

<400> 10

atgtcggtga taaactttac aggaagtagt ggacccttgg tgaaggtctg cggattacag 60

agtacagagg cggcagagtg tgctctagat agtgatgccg acctactagg aataatatgc 120

gttcccaaca ggaagaggac aatagacccc gtgatagcta ggaagataag ttcactagtc 180

aaagcctaca agaacagttc aggaacaccc aagtacctag taggagtttt taggaaccag 240

cccaaggaag acgtactagc gctagtgaat gattatggaa tagatatagt tcaattacat 300

ggcgatgagt catggcaaga atatcaggag ttcttaggac ttcccgtcat aaagcggttg 360

gtatttccca aggattgtaa tatcctcctt tccgccgcaa gtcaaaagcc ccactcattc 420

atacccctct tcgacagtga ggccggagga acaggagagc ttctagactg gaacagtata 480

tcggattggg taggacgaca agagagtccc gagagtcttc atttcatgct agctggagga 540

ctaacacccg agaacgtcgg agatgcccta cgactaaatg gagtaatagg agtagacgtt 600

agtggagggg tagagacaaa tggagtgaag gacagtaata aaatagctaa tttcgtaaaa 660

aatgcaaaga aggcttgtaa aaattggttc tcttctttgt ctcatttcgt tattcatttg 720

taa 723

<210> 11

<211> 723

<212> DNA

<213> Artificial sequence

<400> 11

atgtcggtga taaactttac aggaagtagt ggacccttgg tgaaggtctg cggattacag 60

agtacagagg cggcagagtg tgctctagat agtgatgccg acctactagg aataatatgc 120

gttcccaaca ggaagaggac aatagacccc gtgatagcta ggaagataag ttcactagtc 180

aaagcctaca agaacagttc aggaacaccc aagtacctag taggagtttt taggaaccag 240

cccaaggaag acgtactagc gctagtgaat gattatggaa tagatatagt tcaattacat 300

ggcgatgagt catggcaaga atatcaggag ttcttaggac ttcccgtcat aaagcggttg 360

gtatttccca aggattgtaa tatcctcctt tccgccgcaa gtcaaaagcc ccactcattc 420

atacccctct tcgacagtga ggccggagga acaggagagc ttctagactg gaacagtata 480

tcggattggg taggacgaca agagagtccc gagagtcttc atttcatgct agctggagga 540

ctaacacccg agaacgtcgg agatgcccta cgactaaatg gagtaatagg agtagacgtt 600

agtggagggg tagagacaaa tggagtgaag gacagtaata aaatagctaa tttcgtaaaa 660

aatgcaaaga aggcttgtaa aaattggttc tcttctttgt ctcatttcgt tattcatttg 720

taa 723

<210> 12

<211> 1136

<212> DNA

<213> Artificial sequence

<400> 12

atgtctaaga atatcgttgt cctaccgggt gatcacgtcg gtaaagaagt tactgacgaa 60

gctattaagg tcttgaatgc cattgctgaa gtccgtccag aaattaagtt caatttccaa 120

catcacttga tcgggggtgc tgccatcgat gccactggca ctcctttacc agatgaagct 180

ctagaagcct ctaagaaagc cgatgctgtc ttactaggtg ctgttggtgg tccaaaatgg 240

ggtacgggcg cagttagacc agaacaaggt ctattgaaga tcagaaagga attgggtcta 300

tacgccaact taagaccatg taactttgct tctgattctt tactagatct ttctcctttg 360

aagcctgaat atgcaaaggg taccgatttc gtcgtcgtta gagaattggt tggtggtatc 420

tactttggtg aaagaaaaga agatgaaggt gacggagttg cttgggactc tgagaaatac 480

agtgttcctg aagttcaaag aattacaaga atggctgctt tcttggcatt gcaacaaaac 540

ccaccattac caatctggtc acttgacaag gctaacgtgc ttgcctcttc cagattgtgg 600

agaaagactg ttgaagaaac catcaagact gagttcccac aattaactgt tcagcaccaa 660

ttgatcgact ctgctgctat gattttggtt aaatcaccaa ctaagctaaa cggtgttgtt 720

attaccaaca acatgtttgg tgatattatc tccgatgaag cctctgttat tccaggttct 780

ttgggtttat taccttctgc atctctagct tccctacctg acactaacaa ggcattcggt 840

ttgtacgaac catgtcatgg ttctgcccca gatttaccag caaacaaggt taacccaatt 900

gctaccatct tatctgcagc tatgatgttg aagttatcct tggatttggt tgaagaaggt 960

agggctcttg aagaagctgt tagaaatgtc ttggatgcag gtgtcagaac cggtgacctt 1020

ggtggttcta actctaccac tgaggttggc gatgctatcg ccaaggctgt caaggaaatc 1080

ttggcgcttg taaaaattgg ttctcttctt tgtctcattt cgttattcat ttgtaa 1136

<210> 13

<211> 852

<212> DNA

<213> Artificial sequence

<400> 13

atgtccacaa aatcatatac cagtagagct gagactcatg caagtccggt tgcatcgaaa 60

cttttacgtt taatggatga aaagaaaacc aatttgtgtg cttctcttga cgttcgttcg 120

actgatgagc tattgaaact tgttgaaacg ttgggtccat acatttgcct tttgaaaaca 180

cacgttgata tcttggatga tttcagttat gagggtactg tcgttccatt gaaagcattg 240

gcagagaaat acaagttctt gatatttgag gacagaaaat tcgccgatat cggtaacaca 300

gtcaaattac aatatacatc gggcgtttac cgtatcgcag aatggtctga tatcaccaac 360

gcccacgggg ttactggtgc tggtattgtt gctggcttga aacaaggtgc gcaagaggtc 420

accaaagaac caaggggatt attgatgctt gctgaattat cttccaaggg ttctctagca 480

cacggtgaat atactaaggg taccgttgat attgcaaaga gtgataaaga tttcgttatt 540

gggttcattg ctcagaacga tatgggagga agagaagaag ggtttgattg gctaatcatg 600

accccaggtg taggtttaga cgacaaaggc gatgcattgg gtcagcagta cagaaccgtc 660

gacgaagttg taagtggtgg atcagatatc atcattgttg gcagaggact tttcgccaag 720

ggtagagatc ctaaggttga aggtgaaaga tacagaaatg ctggatggga agcgtaccaa 780

aagagaatca gcgctcccca tgcttgtaaa aattggttct cttctttgtc tcatttcgtt 840

attcatttgt aa 852

<210> 14

<211> 1383

<212> DNA

<213> Artificial sequence

<400> 14

atgccatctc atttcgatac tgttcaacta cacgccggcc aagagaaccc tggtgacaat 60

gctcacagat ccagagctgt accaatttac gccaccactt cttatgtttt cgaaaactct 120

aagcatggtt cgcaattgtt tggtctagaa gttccaggtt acgtctattc ccgtttccaa 180

aacccaacca gtaatgtttt ggaagaaaga attgctgctt tagaaggtgg tgctgctgct 240

ttggctgttt cctccggtca agccgctcaa acccttgcca tccaaggttt ggcacacact 300

ggtgacaaca tcgtttccac ttcttactta tacggtggta cttataacca gttcaaaatc 360

tcgttcaaaa gatttggtat cgaggctaga tttgttgaag gtgacaatcc agaagaattc 420

gaaaaggtct ttgatgaaag aaccaaggct gtttatttgg aaaccattgg taatccaaag 480

tacaatgttc cggattttga aaaaattgtt gcaattgctc acaaacacgg tattccagtt 540

gtcgttgaca acacatttgg tgccggtggt tacttctgtc agccaattaa atacggtgct 600

gatattgtaa cacattctgc taccaaatgg attggtggtc atggtactac tatcggtggt 660

attattgttg actctggtaa gttcccatgg aaggactacc cagaaaagtt ccctcaattc 720

tctcaacctg ccgaaggata tcacggtact atctacaatg aagcctacgg taacttggca 780

tacatcgttc atgttagaac tgaactatta agagatttgg gtccattgat gaacccattt 840

gcctctttct tgctactaca aggtgttgaa acattatctt tgagagctga aagacacggt 900

gaaaatgcat tgaagttagc caaatggtta gaacaatccc catacgtatc ttgggtttca 960

taccctggtt tagcatctca ttctcatcat gaaaatgcta agaagtatct atctaacggt 1020

ttcggtggtg tcttatcttt cggtgtaaaa gacttaccaa atgccgacaa ggaaactgac 1080

ccattcaaac tttctggtgc tcaagttgtt gacaatttaa agcttgcctc taacttggcc 1140

aatgttggtg atgccaagac cttagtcatt gctccatact tcactaccca caaacaatta 1200

aatgacaaag aaaagttggc atctggtgtt accaaggact taattcgtgt ctctgttggt 1260

atcgaattta ttgatgacat tattgcagac ttccagcaat cttttgaaac tgttttcgct 1320

ggccaaaaac cagcttgtaa aaattggttc tcttctttgt ctcatttcgt tattcatttg 1380

taa 1383

<210> 15

<211> 747

<212> DNA

<213> Artificial sequence

<400> 15

atggcagaac cagcccaaaa aaagcaaaaa caaactgttc aggagcgcaa ggcgtttatc 60

tcccgtatca ctaatgaaac taaaattcaa atcgctattt cgctgaatgg tggttatatt 120

caaataaaag attcgattct tcctgcaaag aaggatgacg atgtagcttc ccaagctact 180

cagtcacagg tcatcgatat tcacacaggt gttggctttt tggatcatat gatccatgcg 240

ttggcaaaac actctggttg gtctcttatt gttgaatgta ttggtgacct gcacattgac 300

gatcaccata ctaccgaaga ttgcggtatc gcattagggc aagcgttcaa agaagcaatg 360

ggtgctgtcc gtggtgtaaa aagattcggt actgggttcg caccattgga tgaggcgcta 420

tcacgtgccg tagtcgattt atctagtaga ccatttgctg taatcgacct tggattgaag 480

agagagatga ttggtgattt atccactgaa atgattccac actttttgga aagtttcgcg 540

gaggcggcca gaattacttt gcatgttgat tgtctgagag gtttcaacga tcaccacaga 600

agtgagagtg cgttcaaggc tttggctgtt gccataagag aagctatttc tagcaatggc 660

accaatgacg ttccctcaac caaaggtgtt ttgatggctt gtaaaaattg gttctcttct 720

ttgtctcatt tcgttattca tttgtaa 747

<210> 16

<211> 621

<212> DNA

<213> Artificial sequence

<400> 16

atgggtacca ctcttgacga cacggcttac cggtaccgca ccagtgtccc gggggacgcc 60

gaggccatcg aggcactgga tgggtccttc accaccgaca ccgtcttccg cgtcaccgcc 120

accggggacg gcttcaccct gcgggaggtg ccggtggacc cgcccctgac caaggtgttc 180

cccgacgacg aatcggacga cgaatcggac gacggggagg acggcgaccc ggactcccgg 240

acgttcgtcg cgtacgggga cgacggcgac ctggcgggct tcgtggtcgt ctcgtactcc 300

ggctggaacc gccggctgac cgtcgaggac atcgaggtcg ccccggagca ccgggggcac 360

ggggtcgggc gcgcgttgat ggggctcgcg acggagttcg cccgcgagcg gggcgccggg 420

cacctctggc tggaggtcac caacgtcaac gcaccggcga tccacgcgta ccggcggatg 480

gggttcaccc tctgcggcct ggacaccgcc ctgtacgacg gcaccgcctc ggacggcgag 540

caggcgctct acatgagcat gccctgcccc gcttgtaaaa attggttctc ttctttgtct 600

catttcgtta ttcatttgta a 621

<210> 17

<211> 1077

<212> DNA

<213> Artificial sequence

<400> 17

atgggtaaaa agcctgaact caccgcgacg tctgtcgaga agtttctgat cgaaaagttc 60

gacagcgtct ccgacctgat gcagctctcg gagggcgaag aatctcgtgc tttcagcttc 120

gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa 180

gatcgttatg tttatcggca ctttgcatcg gccgcgctcc cgattccgga agtgcttgac 240

attggggaat tcagcgagag cctgacctat tgcatctccc gccgtgcaca gggtgtcacg 300

ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc agccggtcgc ggaggccatg 360

gatgcgatcg ctgcggccga tcttagccag acgagcgggt tcggcccatt cggaccgcaa 420

ggaatcggtc aatacactac atggcgtgat ttcatatgcg cgattgctga tccccatgtg 480

tatcactggc aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat 540

gagctgatgc tttgggccga ggactgcccc gaagtccggc acctcgtgca cgcggatttc 600

ggctccaaca atgtcctgac ggacaatggc cgcataacag cggtcattga ctggagcgag 660

gcgatgttcg gggattccca atacgaggtc gccaacatct tcttctggag gccgtggttg 720

gcttgtatgg agcagcagac gcgctacttc gagcggaggc atccggagct tgcaggatcg 780

ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc aactctatca gagcttggtt 840

gacggcaatt tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc 900

ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt ctggaccgat 960

ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc ccagcactcg tccgagggca 1020

aaggaagctt gtaaaaattg gttctcttct ttgtctcatt tcgttattca tttgtaa 1077

<210> 18

<211> 858

<212> DNA

<213> Artificial sequence

<400> 18

atgggtaagg aaaagactca cgtttcgagg ccgcgattaa attccaacat ggatgctgat 60

ttatatgggt ataaatgggc tcgcgataat gtcgggcaat caggtgcgac aatctatcga 120

ttgtatggga agcccgatgc gccagagttg tttctgaaac atggcaaagg tagcgttgcc 180

aatgatgtta cagatgagat ggtcagacta aactggctga cggaatttat gcctcttccg 240

accatcaagc attttatccg tactcctgat gatgcatggt tactcaccac tgcgatcccc 300

ggcaaaacag cattccaggt attagaagaa tatcctgatt caggtgaaaa tattgttgat 360

gcgctggcag tgttcctgcg ccggttgcat tcgattcctg tttgtaattg tccttttaac 420

agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa tgaataacgg tttggttgat 480

gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg 540

cataagcttt tgccattctc accggattca gtcgtcactc atggtgattt ctcacttgat 600

aaccttattt ttgacgaggg gaaattaata ggttgtattg atgttggacg agtcggaatc 660

gcagaccgat accaggatct tgccatccta tggaactgcc tcggtgagtt ttctccttca 720

ttacagaaac ggctttttca aaaatatggt attgataatc ctgatatgaa taaattgcag 780

tttcatttga tgctcgatga gtttttcgct tgtaaaaatt ggttctcttc tttgtctcat 840

ttcgttattc atttgtaa 858

<210> 19

<211> 603

<212> DNA

<213> Artificial sequence

<400> 19

atgggtagcc cagaacgacg cccggtcgag atccgtcccg ccaccgccgc cgacatggcg 60

gcggtctgcg acatcgtcaa tcactacatc gagacgagca cggtcaactt ccgtacggag 120

ccgcagactc cgcaggagtg gatcgacgac ctggagcgcc tccaggaccg ctacccctgg 180

ctcgtcgccg aggtggaggg cgtcgtcgcc ggcatcgcct acgccggccc ctggaaggcc 240

cgcaacgcct acgactggac cgtcgagtcg acggtgtacg tctcccaccg gcaccagcgg 300

ctcggactgg gctccaccct ctacacccac ctgctgaagt ccatggaggc ccagggcttc 360

aagagcgtgg tcgccgtcat cggactgccc aacgacccga gcgtgcgcct gcacgaggcg 420

ctcggataca ccgcgcgcgg gacgctgcgg gcagccggct acaagcacgg gggctggcac 480

gacgtggggt tctggcagcg cgacttcgag ctgccggccc cgccccgccc cgtccggccc 540

gtcacacaga tcgcttgtaa aaattggttc tcttctttgt ctcatttcgt tattcatttg 600

taa 603

<210> 20

<211> 429

<212> DNA

<213> Artificial sequence

<400> 20

atggccgacc aagcgacgcc caacctgcca tcacgagatt tcgatcccac cgccgccttc 60

tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc 120

ggggatctca agctggagtt cttcgcccac cccgggctcg atcccctcgc gagttggttc 180

agctgctgcc tgaggctgga cgacctcgcg gagttctacc ggcagtgcaa atccgtcggc 240

atccaggaaa ccagcagcgg ctatccgcgc atccatgccc ccgaactgca ggagtgggga 300

ggcacgatgg ccgctttggt cgacccggac gggacgctcc tgcgcctgat acagaacgaa 360

ttgcttgcag gcatctcagc ttgtaaaaat tggttctctt ctttgtctca tttcgttatt 420

catttgtaa 429

<210> 21

<211> 211

<212> DNA

<213> Artificial sequence

<400> 21

tccgcgctga gggtttaatg gcgcgccgcg gccgcccgcg gtgttggaat aaaaatccac 60

tatcgtctat caactaatag ttatattatc aatatattat catatacggt gttaagatga 120

tgacataagt tatgagaagc tgtcatcgaa gttagaggaa gctgaagtgc aaggattgat 180

aatgtaatag gatcaatgaa tataaacata t 211

<210> 22

<211> 213

<212> DNA

<213> Artificial sequence

<400> 22

aaaacggaat gaggaataat cgtaatatta gtatgtagaa atatagattc cattttgagg 60

attcctatat cctcgaggag aacttctagt gtatattctg tatacctaat attatagcct 120

ttatcaacaa tggaatccca acaattatct aattacccac aaatttctca agatctgcgg 180

ccgcactcag acctgaagtg aagttcctat act 213

<210> 23

<211> 208

<212> DNA

<213> Artificial sequence

<400> 23

ccgcgctgag ggtttaatgg cgcgccgcgg ccgcccgcgg tgttggaata aaaatcaact 60

atcatctact aactagtatt tacgttacta gtatattatc atatacggtg ttagaagatg 120

acgcaaatga tgagaaatag tcatctaaat tagtggaagc tgaaacgcaa ggattgataa 180

tgtaatagga tcaatgaata ttaacata 208

<210> 24

<211> 211

<212> DNA

<213> Artificial sequence

<400> 24

taaaacggaa tgatgaataa tatttataga attgtgtaga attgcagatt cccttttatg 60

gattcctaaa tcctcgagga gaacttctag tatattctgt atacctaata ttatagcctt 120

tatcaacaat ggaatcccaa caattatctc aaaattcaca tatttctcaa gatctgcggc 180

cgcactcaga cctgaagtga agttcctata c 211

<210> 25

<211> 209

<212> DNA

<213> Artificial sequence

<400> 25

gtgtccgcgc tgagggttta atggcgcgcc gcggccgccc gcggtgttgg aataaaaatc 60

aactatcatc tactaactag tatttacgtt actagtatat tatcatatac ggtgttagaa 120

gatgacgcaa atgatgagaa atagtcatct aaattagtgg aagctgaaac gcaaggattg 180

ataatgtaat aggatcaatg aatattaac 209

<210> 26

<211> 205

<212> DNA

<213> Artificial sequence

<400> 26

atataaaatg atgataataa tatttataga attgtgtaga attgcagatt cccttttatg 60

gattcctaaa tcctgaggag aacttctagt atattctaca tacctaatat tattgcctta 120

ttaaaaatgg aatcccaaca attacatcaa aatccacatt ctcagatctg cggccgcact 180

cagacctgaa gtgaagttcc tatac 205

<210> 27

<211> 212

<212> DNA

<213> Artificial sequence

<400> 27

gtccgcgctg agggtttaat ggcgcgccgc ggccgcccgc ggtgttgtat ctcaaaatga 60

gatatgtcag tatgacaata cgtcatcctg aacgttcata aaacacatat gaaacaacct 120

tataacaaaa cgaacaacat gagacaaaac ccgtccttcc ctagctgaac tacccaaaag 180

tataaatgcc tgaacaatta gtttagatcc ga 212

<210> 28

<211> 213

<212> DNA

<213> Artificial sequence

<400> 28

gattccgcgc ttccaccact tagtatgatt catattttat ataatatata agataagtaa 60

cattccgtga attaatctga taaactgttt tgacaactgg ttacttccct aagactgttt 120

atattaggat tgtcaagaca ctccggtatt actcgagccc gtaatacaac aagatctgcg 180

gccgcactca gacctgaagt gaagttccta tac 213

<210> 29

<211> 298

<212> DNA

<213> Artificial sequence

<400> 29

gaacttctga agtggggatt taaatgcggc cgcgctgagg gtttaatggc gcgccgcggc 60

cgcccgcggt gttggaacga gagtaattaa tagtgacatg agttgctatg gtaacaatct 120

aatgcttaca tcgtatatta atgtacaact cgtatacgtt taagtgtgat tgcgcctatt 180

gcagaaggaa tgttaaacga gaagctcaga caatactgaa gctgtgttaa agacctatta 240

gttgaacatg ttatgctagc attaagtcct cagcgagctc gcatggaatg cgtgcgat 298

<210> 30

<211> 401

<212> DNA

<213> Artificial sequence

<400> 30

taggtgatat cagatccact agtggcctat gcacccaatt cgccctatag tgagtcgtat 60

tacgcgcgct cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc 120

cctacaggac tagtgctgag gcattaattg atcaggtagg tacatatatg aggaatatga 180

gtcgtcacat caatgtatag taactaccgg aatcactatt atattggtca tgattaatat 240

gaccaatcgg cgtgtgtttt atatacctct cttatttagt ataagaagat cagtactcac 300

ttcttcatta atactaattt ttaacctcta attatcaaca agatctgcgg ccgcggccgc 360

aaatttaaat aaaatgaagt gaagttccta tactttctag a 401

<210> 31

<211> 343

<212> DNA

<213> Artificial sequence

<400> 31

caagtgtttt tgacgaaaga ttatcacttg gaaacaaagc aatattccat ttggaagggt 60

tcatagcaaa agttatgtgc tgtctagatt aatgtaagat atgtcataaa tactgtataa 120

gtcacacaaa aagctgatat ttaacgcatc ttagtcttta ttttctttgt tatttatttt 180

catttaaaac aaactttact gttttttttt tgtttattat ttttagtata caactatata 240

gataatttac atttattctt cgtcattaaa tttaggagcc aagaaaaact gtaggaaccc 300

actcttcaaa tcaaattgga ataaagcagg agcttcgctg gag 343

<210> 32

<211> 352

<212> DNA

<213> Artificial sequence

<400> 32

agagtatcaa gaatttaaaa tgaaacatct caaaagaaaa aagaatgcaa atgagtctat 60

cgacgaattt gaagggaaat aaacgcataa tgtacagtaa cgtataacaa ttaaagattt 120

gtggaagttt tcaaaaactt ttcaactttt ttctttgttt tttttttgca acttcttata 180

ttaatattgt catagatatt tcttatacaa aaacaagcga acaaaaataa tcgacgtata 240

tacaatagat atataagact gtttttcttc aatagaacag gcgaaattat tctaccggcc 300

gaaggtacat cttcccgcta tgtaataaat agaggtattt aagttataac aa 352

<210> 33

<211> 356

<212> DNA

<213> Artificial sequence

<400> 33

agcttctctt tacgttccaa tgactggtaa agttaccatt gcaatgttgt tgagaaacat 60

gttacgttta gtaaggaacg tagaactgtc taaagaaaaa tagaacattt gtggctgttc 120

aaataagcat aggtaaaaca gaatacataa aaataaggga aaaaaagaaa gatcatttgt 180

aatgtactta ctacatacat atagaattct cctctcttag attatcatat aatatacaaa 240

gtttatgggt cttatttctt ctgtctttta gcagagggct caatatcagt ggcactgacg 300

aactcttctt cgtattcttg tccatcatcc gaatacgacg cttcatcttc gtagtc 356

<210> 34

<211> 358

<212> DNA

<213> Artificial sequence

<400> 34

aaagattctc tttttttatg atatttgtac ataaacttta taaatgaaat tcataataga 60

aacgacacga aattacaaaa tggaatatgt tcatagggta gacgaaacta tatacgcaat 120

ctacatacat ttatcaagaa ggagaaaaag gaggatgtaa aggaatacag gtaagcaaat 180

tgatactaat ggctcaacgt gataaggaaa aagaattgca ctttaacatt aatattgaca 240

aggaggaggg caccacacaa aaagttaggt gtaacagaaa atcatgaaac tatgattcct 300

aatttatata ttggaggatt ttctctaaaa aaaaaaaaat acaacaaata aaaaacac 358

<210> 35

<211> 439

<212> DNA

<213> Artificial sequence

<400> 35

ttttaaaggt gaactgatct acgcgccctc gatagtaatg actaaatatc ttgggtagag 60

tatatataat gtcgtatttt tgtatattgt tttatttaga caaatagtaa cgtgttatgt 120

tccttcaatc gcatctttca tgatctttaa tcgatcgtca aatggatcca tttagagttt 180

ctcatcacca tccccatatc atttcactcc accccgcttt acgtaaaaaa aaaaaaaaaa 240

attgaataaa tgactaagaa ttagacacaa ttttgtctta atgaatgctt tttacttatg 300

acacatgcca gtttgtacat atgttgatct tcatagctcc gataatcttc ataaattcgt 360

gacaaattaa aattacacat tattatgtaa actataatat acaatgttgc ctatcaagac 420

aaacatatgc actctatga 439

<210> 36

<211> 400

<212> DNA

<213> Artificial sequence

<400> 36

cactcatacg ccatccttaa agacctggtc tacgatcaaa tgattttttt agtttacaat 60

ctatttttgt ttctaagcaa gtttatcacg caaatacata agtatatttt tactttctat 120

tcttcctagt ttatatttat ttcattgtaa ctttcttaga agctcggtcc tctcgctata 180

tagtaggatc tgcaacatat ttggatgtgg gtgggcgttc tccttctttt ttagatgtaa 240

ggtccaacac gtataacagg tgatacacat agaaagacac gtggaaataa cagtcattta 300

cgaatattta aaacctgagc aactccgtca aatttgatct taatcttttc tggggcccca 360

tctaattccc agaaagccct tcgaattaga aaccggatgc 400

<210> 37

<211> 331

<212> DNA

<213> Artificial sequence

<400> 37

ggtaacaaga ttggcaaaca taataagaaa ggtccaaagt tcaaatctag aaaaaaatta 60

tagaagattg aaactgagca atatggctaa ttacacacct ggagaaaaaa tcagatatgt 120

atatataaga atattataat actgtatatt aaaaatgatt aaaataaaga aaaaaatgaa 180

tcgggcgttt aattgcttat tatcttgaag aagcgaaagt acactatata gtaataatgt 240

gaggttaatt aaatatggat gagataatga cgaaagaaaa tgcagaaatg tcgttttaaa 300

agtaaccccc ataatctagt gaggttcgac g 331

<210> 38

<211> 301

<212> DNA

<213> Artificial sequence

<400> 38

gttaacatta cgttaataaa taggtatata tgaatattta taccaacaca tctattataa 60

taggcgaacc tctgtatgta attaagtaaa aaaaaaacga tgtgacagga tagttaaggt 120

gcctcgtaca taaataaaaa cggaaatagt taattctttc aaaaatatgg caatagccaa 180

actcattcag aaggtacagg aaacactctg tttctgtgcg tttatataac catgcttata 240

aaagaaagat tgaacaaaat atacatgaat ttatgaacgg taatcaccgt taattgttac 300

a 301

<210> 39

<211> 205

<212> DNA

<213> Artificial sequence

<400> 39

cattccgttg gtagatacgt tgttgacact tctaaataag cgaatttctt atgatttatg 60

atttttatta ttaaataagt tataaaaaaa ataagtgtat acaaatttta aagtgactct 120

taggttttaa aacgaaaatt cttattcttg agtaactctt tcctgtaggt caggttgctt 180

tctcaggtat agcatgaggt cgctc 205

<210> 40

<211> 190

<212> DNA

<213> Artificial sequence

<400> 40

atccgctcta accgaaaagg aaggagttag acaacctgaa gtctaggtcc ctatttattt 60

ttttatagtt atgttagtat taagaacgtt atttatattt caaatttttc ttttttttct 120

gtacagacgc gtgtacgcat gtaacattat actgaaaacc ttgcttgaga aggttttggg 180

acgctcgaag 190

<210> 41

<211> 725

<212> DNA

<213> Artificial sequence

<400> 41

tttgccagct tactatcctt cttgaaaata tgcactctat atcttttagt tcttaattgc 60

aacacataga tttgctgtat aacgaatttt atgctatttt ttaaatttgg agttcagtga 120

taaaagtgtc acagcgaatt tcctcacatg tagggaccga attgtttaca agttctctgt 180

accaccatgg agacatcaaa aattgaaaat ctatggaaag atatggacgg tagcaacaag 240

aatatagcac gagccgcgga gttcatttcg ttacttttga tatcactcac aactattgcg 300

aagcgcttca gtgaaaaaat cataaggaaa agttgtaaat attattggta gtattcgttt 360

ggtaaagtag agggggtaat ttttcccctt tattttgttc atacattctt aaattgcttt 420

gcctctcctt ttggaaagct atacttcgga gcactgttga gcgaaggctc attagatata 480

ttttctgtca ttttccttaa cccaaaaata agggaaaggg tccaaaaagc gctcggacaa 540

ctgttgaccg tgatccgaag gactggctat acagtgttca caaaatagcc aagctgaaaa 600

taatgtgtag ctatgttcag ttagtttggc tagcaaagat ataaaagcag gtcggaaata 660

tttatgggca ttattatgca gagcatcaac atgataaaaa aaaacagttg aatattccct 720

caaaa 725

<210> 42

<211> 717

<212> DNA

<213> Artificial sequence

<400> 42

atgggtaagg gagaagaact tttcactgga gttgtcccaa ttcttgttga attagatggt 60

gatgttaatg ggcacaaatt ttctgtcagt ggagagggtg aaggtgatgc aacatacgga 120

aaacttaccc ttaaatttat ttgcactact ggaaagcttc ctgttccttg gccaacactt 180

gtcactactc ttacttatgg tgttcaatgc ttttcaagat acccagatca tatgaagcgg 240

cacgacttct tcaagagcgc catgcctgag ggatacgtgc aggagaggac catcttcttc 300

aaggacgacg ggaactacaa gacacgtgct gaagtcaagt ttgagggaga caccctcgtc 360

aacagaatcg agcttaaggg aatcgatttc aaggaggacg gaaacatcct cggccacaag 420

ttggaataca actacaactc ccacaacgta tacatcatgg cagacaaaca aaagaatgga 480

atcaaagtta acttcaaaat tagacacaac attgaagatg gaagcgttca actagcagac 540

cattatcaac aaaatactcc aattggcgat ggccctgtcc ttttaccaga caaccattac 600

ctgtccacac aatctgccct ttcgaaagat cccaacgaaa agagagacca catggtcctt 660

cttgagtttg taacagctgc tgggattaca catggcatgg atgaactata caaataa 717

<210> 43

<211> 580

<212> DNA

<213> Artificial sequence

<400> 43

gtgaatttac tttaaatctt gcatttaaat aaattttctt tttatagctt tatgacttag 60

tttcaattta tatactattt taatgacatt ttcgattcat tgattgaaag ctttgtgttt 120

tttcttgatg cgctattgca ttgttcttgt ctttttcgcc acatgtaata tctgtagtag 180

atacctgata cattgtggat gctgagtgaa attttagtta ataatggagg cgctcttaat 240

aattttgggg atattggctt ttttttttaa agtttacaaa tgaatttttt ccgccaggat 300

aacgattctg aagttactct tagcgttcct atcggtacag ccatcaaatc atgcctataa 360

atcatgccta tatttgcgtg cagtcagtat catctacatg aaaaaaactc ccgcaatttc 420

ttatagaata cgttgaaaat taaatgtacg cgccaagata agataacata tatctagatg 480

cagtaatata cacagattcc cgcggacgtg ggaaggaaaa aattagataa caaaatctga 540

gtgatatgga aattccgctg tatagctcat atctttccct 580

<210> 44

<211> 2157

<212> DNA

<213> Artificial sequence

<400> 44

atggatcaat ataccaatgg acattccact agtaagatga atcatgatga tcatagtagt 60

ttatgccaga ctactacgac ggacccattg aattggggtg tggctgcggc ggcgttaagc 120

ggcagccacc tcaacgaggt gaagaagatg gtggaggagt atcggaatcc ggtggtgagg 180

ttgggtggcg aaacgctgac tattggtcag gtagcagcgg tcgccaccac tagggacgtt 240

caggttgagt tatcggaatc ttctcgtgcc ggagttacag ccagcagcga ttgggtgatg 300

gatagcatga aaagaggagg agacacctat ggtgtcacca ccggtttcgg tgccacctct 360

caccggagaa caaaggaagg tggtgctctt caacaagagc tcattagatt cttgaacgcc 420

ggaatcttcg gtaccggaac ggaatccgac catacgctgc cgcaatccac cacaagagcc 480

gccatgttgg tcagaatcaa caccctcctc caaggctact ccggcatccg attcgaaatc 540

ttagaagcca tcactaaatt tctcaaccac aacatcaccc catgcctacc cttacgtggc 600

actatcacag cctccggtga cctggtccca ctctcctaca ttgccggaat cttgaccggt 660

cgtcacaatt ccaaggccgt cggccccacc ggagaattac tcgatgccgc caaatctttc 720

gatcgtgccg gtatcgatac tggatttttc gagctacaac cgaaagaagg acttgcgtta 780

gtgaacggga ccgccgtggg gtccggtatg gcttcggtgg ttctttttga agctaatatc 840

ctagcggttt tatcggaggt tttatcggcc atttttgcgg aagttatgca aggaaaacct 900

gagtttacgg atcatctgac ccataaactc aagcatcacc ccggccagat tgaggccgcg 960

gcgatcatgg agcacattct tgatggtagc tcctacgtaa aagaagcaca aaagatgcac 1020

gaaatggatc cgcttcaaaa accaaagcaa gatcgttacg ctcttcgtac gtcaccgcaa 1080

tggctcggtc cgcttatcga ggtgatacgt acatcgacga aatcgatcga gagagagatt 1140

aattcggtta atgataaccc tttaattgat gtttctcgaa ataaggcact tcatggaggg 1200

aattttcagg gtaccccaat aggagtctcg atggataata cccgattagc cgttgcatcc 1260

atcgggaagc ttttgtttgc gcagttttcc gagcttgtga atgattttta taacaatggg 1320

cttccatcca atctttctgg aagccgtaat ccaagtttgg attacggctt caaaggagca 1380

gagattgcga tggcatccta ctgctcggaa cttcagttcc tcgcaaaccc tgtaacgagt 1440

cacgttcaaa gcgcagagca acataaccag gatgtgaact cattagggtt gatttcttca 1500

aggaaaacgg ccgaagcagt tgagatttta aaactcatgt catccactta cttagtggca 1560

ctatgccaag cagtcgactt gaggcactta gaagaaaacc tcaagtcgac tgtcaagaac 1620

gttgttagcc aggtggccaa gaaagtcttg accaccggcc acaatggcca gcttcaccca 1680

tctcgctttt gcgaaaagga tttgctcaaa gtggtcgacc gtgagcacat cttttcgtac 1740

atcgatgacc cgtgtagtgc aacttaccct ctaatgcaaa aattacgaca agttatagtc 1800

gatcacgcgc tcgcaaacgg tgaggccgaa atggactcga gtacgtcaat cttccaaaag 1860

atcggggctt tcgagcaaga actcgaaacc cttttgccaa aagaagtcga aagcactcgg 1920

gtcgatcacg aaggtggaaa gttagcgatt tttaacagaa tcgaagaatg tcgatcatac 1980

ccgttgtata agtttgtaag gatggaactc ggaaccggct atttgaccgg cgagaagacg 2040

gtttcaccgg gagaggagtt tgagaaggtg ttctcggcga tatgtgccgg aaaattgatg 2100

gatccattgt tggattgttt gaagatgtgg gacggaaagc cacttccaat ttcataa 2157

<210> 45

<211> 1518

<212> DNA

<213> Artificial sequence

<400> 45

atggatcttc tccttttgga gaaagctctt gtagggctct tcgtagccat tttaggagcg 60

atcttcatat ctaagttacg tggaaagcgt ttcaagctcc cgccgggacc aattccggta 120

ccgattttcg gaaactggct tcaagtcggc gatgatctca accaccggaa cttaacagat 180

ctagccaaga agttcggcca gatcttcctt ctccgtatgg gccaacggaa cctcgtcgtc 240

gtatcatcgc cggatctcgc caaggaagtc ctccacacac aaggcgtgga gttcggatct 300

cgaactagaa acgtcgtgtt cgacattttc acagggaaag gacaagatat ggtgtttacg 360

gtttacggcg agcactggcg gaagatgcgg aggatcatga cggttccgtt tttcaccaat 420

aaagttgttc agcagtacag gttcggatgg gaggcggagg cggcggcggt ggtggaggat 480

gtgaagaaga atccggcagc agcgacggaa gggattgtga tcaggagacg gttacagctg 540

atgatgtata acaatatgtt cagaattatg tttgatagaa ggttcgagag tgaggacgat 600

cctttgttct tgaagctcaa ggcgttgaat ggggagagga gtcgattggc acagagcttc 660

gattacaact atggcgattt catcccaatt ttgaggccgt ttttgaaagg ttatttgaag 720

atgtgcaaag aagtcaaaga gaagaggttg cagctattca aggattactt cgttgatgaa 780

aggaagaaga tgggaagcat aaaaaccatg gacaacaacc aaatcaaatg tgcaattgat 840

catatacttg aagctcagga caaaggagag atcaacgagg acaatgtcct ttacatcgtt 900

gagaatatca atgttgccgc aatcgaaacc accctctggt ccatcgaatg gggaatcgcg 960

gaactcgtga accaccctga aatccaatcg aaactgagac acgaactcga caccaaactc 1020

ggacccggag tccaagtcac cgaaccagac atccaaaagc ttccatacct ccaagccgtg 1080

gttaaggaga ctctccgcct tcggatggct atcccgctcc tggtcccaca catgaacctc 1140

cacgacgcca agcttaacgg ctacgacatt ccagccgaaa gcaagatctt ggtcaacgcc 1200

tggtggctag ccaacaaccc cgaacaatgg aagaaacccg atgaattccg acccgaaaga 1260

ttcttcgaag aagaaagcca cgtggaggct aacggaaatg atttccgtta cttgccgttt 1320

ggagtcggga gaaggagttg tcccgggatt atccttgcgt taccgatctt ggggataacg 1380

atcgggcgat tggtgcagaa tttcgagcta ttgccgccac cggggatgtc gaagatcgat 1440

gtgaaggaga aaggtggaca gtttagtttg catattttga atcattccac cgttgttgct 1500

aaaccaagat cattgtga 1518

<210> 46

<211> 48

<212> DNA

<213> Artificial sequence

<400> 46

gcttgtaaaa attggttctc ttctttgtct catttcgtta ttcatttg 48

<210> 47

<211> 6702

<212> DNA

<213> Artificial sequence

<400> 47

atgagcgaag aaagcttatt cgagtcttct ccacagaaga tggagtacga aattacaaac 60

tactcagaaa gacatacaga acttccaggt catttcattg gcctcaatac agtagataaa 120

ctagaggagt ccccgttaag ggactttgtt aagagtcacg gtggtcacac ggtcatatcc 180

aagatcctga tagcaaataa tggtattgcc gccgtgaaag aaattagatc cgtcagaaaa 240

tgggcatacg agacgttcgg cgatgacaga accgtccaat tcgtcgccat ggccacccca 300

gaagatctgg aggccaacgc agaatatatc cgtatggccg atcaatacat tgaagtgcca 360

ggtggtacta ataataacaa ctacgctaac gtagacttga tcgtagacat cgccgaaaga 420

gcagacgtag acgccgtatg ggctggctgg ggtcacgcct ccgagaatcc actattgcct 480

gaaaaattgt cccagtctaa gaggaaagtc atctttattg ggcctccagg taacgccatg 540

aggtctttag gtgataaaat ctcctctacc attgtcgctc aaagtgctaa agtcccatgt 600

attccatggt ctggtaccgg tgttgacacc gttcacgtgg acgagaaaac cggtctggtc 660

tctgtcgacg atgacatcta tcaaaagggt tgttgtacct ctcctgaaga tggtttacaa 720

aaggccaagc gtattggttt tcctgtcatg attaaggcat ccgaaggtgg tggtggtaaa 780

ggtatcagac aagttgaacg tgaagaagat ttcatcgctt tataccacca ggcagccaac 840

gaaattccag gctcccccat tttcatcatg aagttggccg gtagagcgcg tcacttggaa 900

gttcaactgc tagcagatca gtacggtaca aatatttcct tgttcggtag agactgttcc 960

gttcagagac gtcatcaaaa aattatcgaa gaagcaccag ttacaattgc caaggctgaa 1020

acatttcacg agatggaaaa ggctgccgtc agactgggga aactagtcgg ttatgtctct 1080

gccggtaccg tggagtatct atattctcat gatgatggaa aattctactt tttagaattg 1140

aacccaagat tacaagtcga gcatccaaca acggaaatgg tctccggtgt taacttacct 1200

gcagctcaat tacaaatcgc tatgggtatc cctatgcata gaataagtga cattagaact 1260

ttatatggta tgaatcctca ttctgcctca gaaatcgatt tcgaattcaa aactcaagat 1320

gccaccaaga aacaaagaag acctattcca aagggtcatt gtaccgcttg tcgtatcaca 1380

tcagaagatc caaacgatgg attcaagcca tcgggtggta ctttgcatga actaaacttc 1440

cgttcttcct ctaatgtttg gggttacttc tccgtgggta acaatggtaa tattcactcc 1500

ttttcggact ctcagttcgg ccatattttt gcttttggtg aaaatagaca agcttccagg 1560

aaacacatgg ttgttgccct gaaggaattg tccattaggg gtgatttcag aactactgtg 1620

gaatacttga tcaaactttt ggaaactgaa gatttcgagg ataacactat taccaccggt 1680

tggttggacg atttgattac tcataaaatg accgctgaaa agcctgatcc aactcttgcc 1740

gtcatttgcg gtgccgctac aaaggctttc ttagcatctg aagaagcccg ccacaagtat 1800

atcgaatcct tacaaaaggg acaagttcta tctaaagacc tactgcaaac tatgttccct 1860

gtagatttta tccatgaggg taaaagatac aagttcaccg tagctaaatc cggtaatgac 1920

cgttacacat tatttatcaa tggttctaaa tgtgatatca tactgcgtca actagctgat 1980

ggtggtcttt tgattgccat aggcggtaaa tcgcatacca tctattggaa agaagaagtt 2040

gctgctacaa gattatccgt tgactctatg actactttgt tggaagttga aaacgatcca 2100

acccagttgc gtactccatc ccctggtaaa ttggttaaat tcttggtgga aaatggtgaa 2160

cacattatca agggccaacc atatgcagaa attgaagtta tgaaaatgca aatgcctttg 2220

gtttctcaag aaaatggtat cgtccagtta ttaaagcaac ctggttctac cattgttgca 2280

ggtgatatca tggctattat gactcttgac gatccatcca aggtcaagca cgctctacca 2340

tttgaaggta tgctgccaga ttttggttct ccagttatcg aaggaaccaa acctgcctat 2400

aaattcaagt cattagtgtc tactttggaa aacattttga agggttatga caaccaagtt 2460

attatgaacg cttccttgca acaattgata gaggttttga gaaatccaaa actgccttac 2520

tcagaatgga aactacacat ctctgcttta cattcaagat tgcctgctaa gctagatgaa 2580

caaatggaag agttagttgc acgttctttg agacgtggtg ctgttttccc agctagacaa 2640

ttaagtaaat tgattgatat ggccgtgaag aatcctgaat acaaccccga caaattgctg 2700

ggcgccgtcg tggaaccatt ggcggatatt gctcataagt actctaacgg gttagaagcc 2760

catgaacatt ctatatttgt ccatttcttg gaagaatatt acgaagttga aaagttattc 2820

aatggtccaa atgttcgtga ggaaaatatc attctgaaat tgcgtgatga aaaccctaaa 2880

gatctagata aagttgcgct aactgttttg tctcattcga aagtttcagc gaagaataac 2940

ctgatcctag ctatcttgaa acattatcaa ccattgtgca agttatcttc taaagtttct 3000

gccattttct ctactcctct acaacatatt gttgaactag aatctaaggc taccgctaag 3060

gtcgctctac aagcaagaga aattttgatt caaggcgctt taccttcggt caaggaaaga 3120

actgaacaaa ttgaacatat cttaaaatcc tctgttgtga aggttgccta tggctcatcc 3180

aatccaaagc gctctgaacc agatttgaat atcttgaagg acttgatcga ttctaattac 3240

gttgtgttcg atgttttact tcaattccta acccatcaag acccagttgt gactgctgca 3300

gctgctcaag tctatattcg tcgtgcttat cgtgcttaca ccataggaga tattagagtt 3360

cacgaaggtg tcacagttcc aattgttgaa tggaaattcc aactaccttc agctgcgttc 3420

tccacctttc caactgttaa atctaaaatg ggtatgaaca gggctgttgc tgtttcagat 3480

ttgtcatatg ttgcaaacag tcagtcatct ccgttaagag aaggtatttt gatggctgtg 3540

gatcatttag atgatgttga tgaaattttg tcacaaagtt tggaagttat tcctcgtcac 3600

caatcttctt ctaacggacc tgctcctgat cgttctggta gctccgcatc gttgagtaat 3660

gttgctaatg tttgtgttgc ttctacagaa ggtttcgaat ctgaagagga aattttggta 3720

aggttgagag aaattttgga tttgaataag caggaattaa tcaatgcttc tatccgtcgt 3780

atcacattta tgttcggttt taaagatggg tcttatccaa agtattatac ttttaacggt 3840

ccaaattata acgaaaatga aacaattcgt cacattgagc cggctttggc cttccaactg 3900

gaattaggaa gattgtccaa cttcaacatt aaaccaattt tcactgataa tagaaacatc 3960

catgtctacg aagctgttag taagacttct ccattggata agagattctt tacaagaggt 4020

attattagaa cgggtcatat ccgtgatgac atttctattc aagaatatct gacttctgaa 4080

gctaacagat tgatgagtga tatattggat aatttagaag tcaccgacac ttcaaattct 4140

gatttgaatc atatcttcat caacttcatt gcggtgtttg atatctctcc agaagatgtc 4200

gaagccgcct tcggtggttt cttagaaaga tttggtaaga gattgttgag attgcgtgtt 4260

tcttctgccg aaattagaat catcatcaaa gatcctcaaa caggtgcccc agtaccattg 4320

cgtgccttga tcaataacgt ttctggttat gttatcaaaa cagaaatgta caccgaagtc 4380

aagaacgcaa aaggtgaatg ggtatttaag tctttgggta aacctggatc catgcattta 4440

agacctattg ctactcctta ccctgttaag gaatggttgc aaccaaaacg ttataaggca 4500

cacttgatgg gtaccacata tgtctatgac ttcccagaat tattccgcca agcatcgtca 4560

tcccaatgga aaaatttctc tgcagatgtt aagttaacag atgatttctt tatttccaac 4620

gagttgattg aagatgaaaa cggcgaatta actgaggtgg aaagagaacc tggtgccaac 4680

gctattggta tggttgcctt taagattact gtaaagactc ctgaatatcc aagaggccgt 4740

caatttgttg ttgttgctaa cgatatcaca ttcaagatcg gttcctttgg tccacaagaa 4800

gacgaattct tcaataaggt tactgaatat gctagaaagc gtggtatccc aagaatttac 4860

ttggctgcaa actcaggtgc cagaattggt atggctgaag agattgttcc actatttcaa 4920

gttgcatgga atgatgctgc caatccggac aagggcttcc aatacttata cttaacaagt 4980

gaaggtatgg aaactttaaa gaaatttgac aaagaaaatt ctgttctcac tgaacgtact 5040

gttataaacg gtgaagaaag atttgtcatc aagacaatta ttggttctga agatgggtta 5100

ggtgtcgaat gtctacgtgg atctggttta attgctggtg caacgtcaag ggcttaccac 5160

gatatcttca ctatcacctt agtcacttgt agatccgtcg gtatcggtgc ttatttggtt 5220

cgtttgggtc aaagagctat tcaggtcgaa ggccagccaa ttattttaac tggtgctcct 5280

gcaatcaaca aaatgctggg tagagaagtt tatacttcta acttacaatt gggtggtact 5340

caaatcatgt ataacaacgg tgtttcacat ttgactgctg ttgacgattt agctggtgta 5400

gagaagattg ttgaatggat gtcttatgtt ccagccaagc gtaatatgcc agttcctatc 5460

ttggaaacta aagacacatg ggatagacca gttgatttca ctccaactaa tgatgaaact 5520

tacgatgtaa gatggatgat tgaaggtcgt gagactgaaa gtggatttga atatggtttg 5580

tttgataaag ggtctttctt tgaaactttg tcaggatggg ccaaaggtgt tgtcgttggt 5640

agagcccgtc ttggtggtat tccactgggt gttattggtg ttgaaacaag aactgtcgag 5700

aacttgattc ctgctgatcc agctaatcca aatagtgctg aaacattaat tcaagaacct 5760

ggtcaagttt ggcatccaaa ctccgccttc aagactgctc aagctatcaa tgactttaac 5820

aacggtgaac aattgccaat gatgattttg gccaactgga gaggtttctc tggtggtcaa 5880

cgtgatatgt tcaacgaagt cttgaagtat ggttcgttta ttgttgacgc attggtggat 5940

tacaaacaac caattattat ctatatccca cctaccggtg aactaagagg tggttcatgg 6000

gttgttgtcg atccaactat caacgctgac caaatggaaa tgtatgccga cgtcaacgct 6060

agagctggtg ttttggaacc acaaggtatg gttggtatca agttccgtag agaaaaattg 6120

ctggacacca tgaacagatt ggatgacaag tacagagaat tgagatctca attatccaac 6180

aagagtttgg ctccagaagt acatcagcaa atatccaagc aattagctga tcgtgagaga 6240

gaactattgc caatttacgg acaaatcagt cttcaatttg ctgatttgca cgataggtct 6300

tcacgtatgg tggccaaggg tgttatttct aaggaactgg aatggaccga ggcacgtcgt 6360

ttcttcttct ggagattgag aagaagattg aacgaagaat atttgattaa aaggttgagc 6420

catcaggtag gcgaagcatc aagattagaa aagatcgcaa gaattagatc gtggtaccct 6480

gcttcagtgg accatgaaga tgataggcaa gtcgcaacat ggattgaaga aaactacaaa 6540

actttggacg ataaactaaa gggtttgaaa ttagagtcat tcgctcaaga cttagctaaa 6600

aagatcagaa gcgaccatga caatgctatt gatggattat ctgaagttat caagatgtta 6660

tctaccgatg ataaagaaaa attgttgaag actttgaaat aa 6702

<210> 48

<211> 1959

<212> DNA

<213> Artificial sequence

<400> 48

atgtcacaaa ctcataaaca tgctattcca gcaaacatcg ctgatagatg tttgattaat 60

ccagaacaat acgaaactaa gtacaagcaa tctattaatg atccagatac attttggggt 120

gaacaaggta aaattttgga ttggatcact ccataccaaa aggttaaaaa tacatcattt 180

gctcctggta atgtttctat taaatggtac gaagatggta ctttgaattt ggctgcaaac 240

tgtttggata gacatttgca agaaaatggt gacagaacag caattatttg ggaaggtgac 300

gatgcttcac aatctaagca tatctcttac agagaattac atagagatgc atgtagattc 360

gctaacactt tgttagattt gggtattaag aaaggtgacg ttgttgctat ctatatgcca 420

atggttccag aagctgcagt tgcaatgtta gcttgtgcaa gaattggtgc tgttcattca 480

gttatttttg gtggtttttc tccagaagct gttgcaggta gaatcatcga ttcttcatct 540

agattggtta ttacagcaga tgaaggtgtt agagctggta gatcaatccc attgaagaaa 600

aatgttgatg atgctttgaa aaatccaaac gttacttcag ttgaacatgt tatcgttttg 660

aaaagaacag gttctgatat tgattggcaa gaagatagag atttgtggtg gagagatttg 720

attgaaaaag cttctccaga acatcaacca gaagcaatga acgctgaaga tccattgttt 780

attttgtaca cttcaggttc tacaggtaaa ccaaaaggtg ttttacatac tacaggtggt 840

tatttggttt acgctgcaac tacttttaaa tacgttttcg attaccatcc aggtgacatc 900

tattggtgta ctgctgatgt tggttgggtt acaggtcatt catatttgtt atacggtcca 960

ttagcatgtg gtgctactac attgatgttt gaaggtgttc caaattggcc aactccagct 1020

agaatgtgtc aagttgttga taagcatcaa gttaacatct tgtacactgc accaacagct 1080

attagagcat tgatggctga aggtgacaaa gcaattgaag gtacagatag atcatctttg 1140

agaattttag gttctgttgg tgaaccaatt aatccagaag cttgggaatg gtactggaag 1200

aaaattggta aagaaaagtg tccagttgtt gatacttggt ggcaaactga aacaggtggt 1260

tttatgatta caccattgcc aggtgctatt gaattaaaag caggttcagc tactagacca 1320

tttttcggtg ttcaaccagc attagttgat aatgaaggtc atccacaaga aggtgctact 1380

gagggtaatt tggttattac agattcttgg ccaggtcaag caagaacatt gtttggtgac 1440

catgaaagat ttgaacaaac ttacttctca acttttaaaa acatgtactt ttctggtgac 1500

ggtgctagaa gagatgaaga tggttattac tggatcactg gtagagttga tgatgttttg 1560

aacgtttcag gtcatagatt gggtacagca gaaattgaat ctgcattggt tgctcatcca 1620

aaaattgcag aagctgcagt tgttggtatt ccacatgcta ttaaaggtca agcaatctat 1680

gcttacgtta ctttaaatca tggtgaagaa ccatcaccag aattgtatgc agaagttaga 1740

aactgggtta gaaaggaaat tggtccattg gctacaccag atgttttaca ttggactgat 1800

tcattgccaa agacaagatc aggtaaaatc atgagaagaa tcttgagaaa gattgctgca 1860

ggtgacactt caaatttggg tgacacttct acattggctg atccaggtgt tgttgaaaaa 1920

ccattggaag aaaaacaagc tattgcaatg ccatcttaa 1959

<210> 49

<211> 1557

<212> DNA

<213> Artificial sequence

<400> 49

atgactatcc tacccctgct actctacgcc tccataactg gtttactaat ctatgtattg 60

cttaacctac gcaccacccc tcgttctaac cacctcccac tcccacccgg cccaacccca 120

tggccaatca tcggaaactt acctcatctt ggaagaatac cgcaccatgc gctggcggcc 180

atggctacaa agtacggccc gttgatgcat ctccggctcg gcgtcgttga cgtggtggtg 240

gcggcgtctg cgtcggtggc ggcacagttt ttgaaggttc atgacgccaa tttcgcgagt 300

aggccgccga actccggcgc gaaacacatc gcgtataatt atcaggatct ggtgtttgca 360

ccttatggtc agaaatggcg gatgcttagg aagatttgct ccgtgcatct gttctctaac 420

aaagcactcg atgatttccg tcacgttcgt caggaggagg tggcgattct ggtgcgcgct 480

ttggccggag ccggtcgatc tacggcggcg gcgttaggtc aactacttaa cgtttgcacc 540

acaaacgcgt tggcacgagt gatgttaggt cggagagtgt tcgtggacgg aagtgaaggc 600

aatcgagacg cggatgaatt caaggatatg gtggttgaag tgatggtatt ggccggagaa 660

ttcaacatcg gcgacttcat tccggcgctt gattggctgg atctgcaaag cgtgacgaag 720

aagatgaaga aactccatct ccgattcgat tcgtttctta acaaaatcct ggaagaccat 780

agaaatggag gtgacgtcac ttcgggtaac gtggatttgc tgagcacgtt gatttcgctc 840

aaggatgacg ccaatgggga gggcgggaag ctttcagata tcgaaatcaa agctttgctt 900

ctgaatttat tcactgcggg aacagacaca tcatctagta cggtggaatg ggcaatggct 960

gaactcattc gccatccgca attattgaag caagcccaag aagaattgga cactgttgtt 1020

ggtaaagacc ggcttgtatc cgaattggac ctgagtagac taacattcct cgaagccatt 1080

gtgaaggaaa ccttcaggct ccacccatcg accccactct ctttgccacg gattgcatca 1140

gagagctgtg aagtcgatgg gtattacatt cctaagggaa ccacacttct tgttaacgtg 1200

tgggccattg cccgagaccc aaaaatgtgg accgacccgc ttgaattccg acccacccgg 1260

ttcttgccgg gaggtgaaaa gccgaatgct aatgtaaagg gaaatgattt tgaaataata 1320

ccgtttgggg ctggtcgaag gatttgtgcg ggtatgagcc tagggttacg gatggttcag 1380

ttgctcactg cgactctggt tcatgccttt gattggaaat tggctaacgg gttagaccca 1440

gagaagctca atatggaaga agcttatggg ttgacccttc aaagggctgc acccttgatg 1500

gtgcacccaa ccccacggtt agctccccat ttgtatgaaa gcagtcaagg tttataa 1557

<210> 50

<211> 2133

<212> DNA

<213> Artificial sequence

<400> 50

atgcaatcgg actcgtctct ggaaacgtcg tcgtttgatt tgattaccgc agctcttaag 60

gagaaagtta ttgatacagc aaacgcatct gatagcggag attcaacgat gcctccggct 120

ttggcgatga ttttggaaaa ccgtgagctg tttatgatgc tgactacaac agtggctctt 180

ttgcttggat ttattgtcgt ttcgttctgg aagagatctt ctgagaagaa gtcggctaag 240

gatttggagc taccgaagat cgttgtgcct aagagacagc aggaacagga ggttgatgac 300

ggtaagaaga aggttacgat tctttttgga acgcagaccg gaacggcgga aggtttcgct 360

aaggcactgt tggaagaagc taaagcgcga tatgaaaagg cgacctttaa agtagtcgat 420

ttggatgatt atgctgttga tgatgatgag tacgaagaga aactaaagaa ggagtcattt 480

gctttcttct tcttggctac atatggagat ggtgagccaa ctgataatgc tgccagattt 540

tataaatggt ttacagaggg aggtgagaaa ggagtttggc ttgaaaagct tcaatatgga 600

gtatttggcc ttggcaatag acaatacgag catttcaaca agattgcaaa agaggttgac 660

gatggtctcg cagagcaggg tgcaaagcgc cttgttccag ttggccttgg agatgatgat 720

caatccattg aagatgattt tactgcatgg aaagagttag tgtggcctga gttggatgaa 780

ttgcttcgtg acgaggatga caaaggcgtt gctactccct acacagctgc tattccagaa 840

taccgagttg tgtttcatga gaaacatgat acatctgctg aagatcaaat tcagacaaat 900

ggtcatgctg ttcatgatgc tcaacatcca tgcagatcca atgtggctgt taaaaaggag 960

ctccataccc ctgaatctga tcgctcttgc acgcatctgg aatttgacat ctcacacact 1020

ggactatcat acgaaactgg ggaccatgtt ggtgtctact gtgagaactt aagtgaagtt 1080

gtggaggagg ctgagaggtt aataggttta ccatcggata cttatttctc agttcacacg 1140

gataacgaag atggaacacc acttggtgga gcttccttac tacctccttt ccctccatgc 1200

actttaagaa aagcattggc taattacgca gatgtattga cttctcccaa aaagtcggcc 1260

ttgattgctc tagctgctca tgcttctgat cctactgaag ctgaacgact aaaatttctt 1320

gcatctcctg ctgggaagga tgaatattct caatgggtta ttgcaagcca aagaagcctg 1380

cttgaggtca tggaagcttt cccatcggct aagcctccac ttggggtttt ctttgcagct 1440

attgctccac gcttacagcc tcgatactac tctatttctt cctccccgaa gatggcacct 1500

agcaggattc atgttacttg tgcattagtt tatgagaaaa cacctgcagg ccgtctccat 1560

aaaggaatct gttcaacctg gatgaagaat gctgtgccta tgacggaaag tcaggattgc 1620

agctgggcac ctattttcgt tagaacgtct aacttcagac ttcccactga tccaaaagtt 1680

cctgttatca tgattggccc tggaaccgga ttggctccgt tcagaggttt tcttcaagaa 1740

agattagctc tgaaggaagc cggaactgaa ctgggatcat ccattttatt cttcggatgt 1800

agaaatcgca aagtggattt catatatgag aatgaactga aagactttgt tgagaatggt 1860

gctgtttccg agcttattgt tgccttctcc cgtgaaggcc ccaataagga atatgtgcaa 1920

cataaaatga gcgatagggc ttcggatcta tggaacttgc tttcggaggg agcatattta 1980

tacgtttgtg gtgatgccaa aggcatggct aaagatgtac accggaccct tcacacaatt 2040

gtgcaagaac agggatctct agactcgtca aaggcagagc tgtatgtgaa gaatctacaa 2100

atgtcaggaa gatacctccg tgatgtttgg tag 2133

<210> 51

<211> 1113

<212> DNA

<213> Artificial sequence

<400> 51

atgagtgaat ctccaatgtt cgctgccaac ggcatgccaa aggtaaatca aggtgctgaa 60

gaagatgtca gaattttagg ttacgaccca ttagcttctc cagctctcct tcaagtgcaa 120

atcccagcca caccaacttc tttggaaact gccaagagag gtagaagaga agctatagat 180

attattaccg gtaaagacga cagagttctt gtcattgtcg gtccttgttc catccatgat 240

ctagaagccg ctcaagaata cgctttgaga ttaaagaaat tgtcagatga attaaaaggt 300

gatttatcca tcattatgag agcatacttg gagaagccaa gaacaaccgt cggctggaaa 360

ggtctaatta atgaccctga tgttaacaac actttcaaca tcaacaaggg tttgcaatcc 420

gctagacaat tgtttgtcaa cttgacaaat atcggtttgc caattggttc tgaaatgctt 480

gataccattt ctcctcaata cttggctgat ttggtctcct tcggtgccat tggtgccaga 540

accaccgaat ctcaactgca cagagaattg gcctccggtt tgtctttccc agttggtttc 600

aagaacggta ccgatggtac cttaaatgtt gctgtggatg cttgtcaagc cgctgctcat 660

tctcaccatt tcatgggtgt tactttgcat ggtgttgctg ctatcaccac tactaagggt 720

aacgaacact gcttcgttat tctaagaggt ggtaaaaagg gtaccaacta cgacgctaag 780

tccgttgcag aagctaaggc tcaattgcct gccggttcca acggtctaat gattgactac 840

tctcacggta actccaataa ggatttcaga aaccaaccaa aggtcaatga cgttgtttgt 900

gagcaaatcg ctaacggtga aaacgccatt accggtgtca tgattgaatc aaacatcaac 960

gaaggtaacc aaggcatccc agccgaaggt aaagccggct tgaaatatgg tgtttccatc 1020

actgatgctt gtataggttg ggaaactact gaagacgtct tgaggaaatt ggctgctgct 1080

gtcagacaaa gaagagaagt taacaagaaa tag 1113

<210> 52

<211> 771

<212> DNA

<213> Artificial sequence

<400> 52

atggatttca caaaaccaga aactgtttta aatctacaaa atattagaga tgaattagtt 60

agaatggagg attcgatcat cttcaaattt attgagaggt cgcatttcgc cacatgtcct 120

tcagtttatg aggcaaacca tccaggttta gaaattccga attttaaagg atctttcttg 180

gattgggctc tttcaaatct tgaaattgcg cattctcgca tcagaagatt cgaatcacct 240

gatgaaactc ccttctttcc tgacaagatt cagaaatcat tcttaccgag cattaactac 300

ccacaaattt tggcgcctta tgccccagaa gttaattaca atgataaaat aaaaaaagtt 360

tatattgaaa agattatacc attaatttcg aaaagagatg gtgatgataa gaataacttc 420

agttctgttg ccactagaga tatagaatgt ttgcaaagct tgagtaggag aatccacttt 480

ggcaagtttg ttgctgaagc caagttccaa tcggatatcc cgctatacac aaagctgatc 540

aaaagtaaag atgtcgaggg gataatgaag aatatcacca attctgccgt tgaagaaaag 600

attctagaaa gattaactaa gaaggctgaa gtctatggtg tggaccctac caacgagtca 660

ggtgaaagaa ggattactcc agaatatttg gtaaaaattt ataaggaaat tgttatacct 720

atcactaagg aagttgaggt ggaatacttg ctaagaaggt tggaagagta a 771

<210> 53

<211> 1521

<212> DNA

<213> Artificial sequence

<400> 53

atgaacacca ttaatgaata cttgagttta gaagaattcg aagcaataat cttcggtaac 60

caaaaagtaa ctatctctga tgttgtcgta aacagagtta acgaaagttt taacttctta 120

aaggaatttt ctggtaataa ggttatatat ggtgtaaaca ctggtttcgg tccaatggct 180

caatacagaa tcaaggaatc tgatcaaatc caattgcaat acaatttgat aagaagtcat 240

tcttcaggta ctggtaaacc attatctcct gtttgtgcta aggctgcaat cttggcaaga 300

ttgaacacat tgtctttagg caactcaggt gttcacccat ctgttattaa tttgatgtct 360

gaattgataa acaaagacat cactcctttg atattcgaac atggtggtgt tggtgcatct 420

ggtgacttgg tccaattgtc ccacttggcc ttagtattga taggtgaagg tgaagttttc 480

tataaaggtg aaagaagacc aacacctgaa gtcttcgaaa tcgaaggttt aaagcctata 540

caagtagaaa tcagagaagg tttagctttg attaatggta cttctgtcat gacaggtata 600

ggtgttgtca acgtatacca tgctaagaaa ttgttggatt ggtcattgaa gtccagttgt 660

gccattaatg aattggttca agcatatgat gaccatttct ctgcagaatt gaaccaaacc 720

aagagacaca agggtcaaca agaaatcgca ttgaagatga gacaaaattt gtccgatagt 780

acattgatca gaaagagaga agaccactta tactcaggtg aaaacaccga agaaattttc 840

aaagaaaagg ttcaagaata ctactccttg agatgcgtcc cacaaatctt gggtcctgta 900

ttggaaacta ttaataacgt tgcctcaatc ttggaagatg aattcaattc cgctaacgat 960

aacccaatca tcgacgttaa aaatcaacat gtttatcacg gtggtaactt ccatggtgac 1020

tacatttctt tagaaatgga caaattgaag atagttatca caaaattgac catgttggct 1080

gaaagacaat tgaactactt gttgaactca aagattaacg aattgttgcc acctttcgtt 1140

aatttgggta cattgggttt taacttcggt atgcaaggtg ttcaattcac cgccacttca 1200

actacagctg aatcccaaat gttgagtaac ccaatgtacg ttcattccat ccctaacaac 1260

aacgataacc aagacatcgt ctctatgggt accaactcag ccgtcattac ttccaaagta 1320

atagaaaacg cattcgaagt tttggccatc gaaatgatca caattgtcca agctatcgat 1380

tacttgggtc aaaaggacaa gatctcttct gtttctaaga aatggtacga tgaaataaga 1440

aacataatcc caacctttaa ggaagaccaa gttatgtacc ctttcgtaca aaaggttaag 1500

gatcatttga ttaacaatta a 1521

<210> 54

<211> 525

<212> DNA

<213> Artificial sequence

<400> 54

atgacacaac ctctttttct gatcgggcct cggggctgtg gtaaaacaac ggtcggaatg 60

gcccttgccg attcgcttaa ccgtcggttt gtcgataccg atcagtggtt gcaatcacag 120

ctcaatatga cggtcgcgga gatcgtcgaa agggaagagt gggcgggatt tcgcgccaga 180

gaaacggcgg cgctggaagc ggtaactgcg ccatccaccg ttatcgctac aggcggcggc 240

attattctga cggaatttaa tcgtcacttc atgcaaaata acgggatcgt ggtttatttg 300

tgtgcgccag tatcagtcct ggttaaccga ctgcaagctg caccggaaga agatttacgg 360

ccaaccttaa cgggaaaacc gctgagcgaa gaagttcagg aagtgctgga agaacgcgat 420

gcgctatatc gcgaagttgc gcatattatc atcgacgcaa caaacgaacc cagccaggtg 480

atttctgaaa ttcgcagcgc cctggcacag acgatcaatt gttga 525

<210> 55

<211> 1035

<212> DNA

<213> Artificial sequence

<400> 55

atgcttgaaa acaggttcgt tcgcgatgaa gacgagcgtc caaaagtggc gtacaataat 60

tttagcaacg agattccggt gatctcactt gaaggtatcg acgatactag tagtagggcg 120

gagatttgcg agaagatcgt taaggcttgt gaagattggg gggtttttca ggtggtggat 180

cacgggatcg ataatagatt gttgacggag atgacgaggc tcgccacgga gttcttcatg 240

atgccgccgg aggagaaact ccgatttgat atgagtggcg ggaaaaaagg cggtttcatt 300

gtttccagcc atcttcaagg agaaacggtg caagattgga gggagattgt aaccttcttc 360

tcgtacccaa caaaagcaag agactactct aggtggcccg ataagcccaa agagtggagg 420

gcagttactg aggaatatag caaggtgtta atgggcctgg cctgcaagct actagaggta 480

ttgtctgagg caatgggcct tgagaaagag gccttgacca aagcttgtgt agatatggac 540

caaaaggtgg tggtcaatta ctatccaaaa tgccctcatc ccgacctcac gttgggcctg 600

aaacgacata cggatccggg aacaatcacg ttgttgcttc aggaccaagt tggtgggctt 660

caggcgactc gtgatggtgg tcaaagttgg atcacagttc agccgattga aggtgctttt 720

gtggttaatc ttggtgatca tggacattat ttgagcaacg ggaggttcaa gaacgcagac 780

caccaagccg tggtgaactc aaacacgagc cgactctcca tagctacgtt tcaaaaccct 840

gcaccggatg cgattgtata cccgctgaaa gtgaatgagg gagataaatc gataatggaa 900

gaagctataa ctttcatgga gatgtacaag aagaagatgg gtcgagacct tgagttggct 960

cggcttaaga agctagccaa ggacaagcaa caagatttgg agaaagagaa gccaatcgag 1020

aatatatttg cttag 1035

<210> 56

<211> 668

<212> DNA

<213> Artificial sequence

<400> 56

ttatattgaa ttttcaaaaa ttcttacttt ttttttggat ggacgcaaag aagtttaata 60

atcatattac atggcattac caccatatac atatccatat ctaatcttac ttatatgttg 120

tggaaatgta aagagcccca ttatcttagc ctaaaaaaac cttctctttg gaactttcag 180

taatacgctt aactgctcat tgctatattg aagtacggat tagaagccgc cgagcgggcg 240

acagccctcc gacggaagac tctcctccgt gcgtcctcgt cttcaccggt cgcgttcctg 300

aaacgcagat gtgcctcgcg ccgcactgct ccgaacaata aagattctac aatactagct 360

tttatggtta tgaagaggaa aaattggcag taacctggcc ccacaaacct tcaaattaac 420

gaatcaaatt aacaaccata ggatgataat gcgattagtt ttttagcctt atttctgggg 480

taattaatca gcgaagcgat gatttttgat ctattaacag atatataaat ggaaaagctg 540

cataaccact ttaactaata ctttcaacat tttcagtttg tattacttct tattcaaatg 600

tcataaaagt atcaacaaaa aattgttaat atacctctat actttaacgt caaggagaaa 660

aaactata 668

<210> 57

<211> 668

<212> DNA

<213> Artificial sequence

<400> 57

tatagttttt tctccttgac gttaaagtat agaggtatat taacaatttt ttgttgatac 60

ttttatgaca tttgaataag aagtaataca aactgaaaat gttgaaagta ttagttaaag 120

tggttatgca gcttttccat ttatatatct gttaatagat caaaaatcat cgcttcgctg 180

attaattacc ccagaaataa ggctaaaaaa ctaatcgcat tatcatccta tggttgttaa 240

tttgattcgt taatttgaag gtttgtgggg ccaggttact gccaattttt cctcttcata 300

accataaaag ctagtattgt agaatcttta ttgttcggag cagtgcggcg cgaggcacat 360

ctgcgtttca ggaacgcgac cggtgaagac gaggacgcac ggaggagagt cttccgtcgg 420

agggctgtcg cccgctcggc ggcttctaat ccgtacttca atatagcaat gagcagttaa 480

gcgtattact gaaagttcca aagagaaggt ttttttaggc taagataatg gggctcttta 540

catttccaca acatataagt aagattagat atggatatgt atatggtggt aatgccatgt 600

aatatgatta ttaaacttct ttgcgtccat ccaaaaaaaa agtaagaatt tttgaaaatt 660

caatataa 668

Claims (10)

1. The saccharomyces cerevisiae multicopy integration gene expression frame is characterized in that the expression frame consists of a weak promoter sequence and a screening gene sequence with a degradation label deg; the weak promoter comprises P_ADE6、P_LEU2、P_URA3、P_PMA1、P_ZWF1、P_ARO7、P_PYC1、P_ADE3、P_YEF3、P_ERG1The screening gene comprises ScTRP1, KlLEU2, KlURA3, ScMET15, SpHIS5, natMX, hpHMX, kanMX, patMX and bleMX.

2. The gene expression cassette of claim 1, wherein the weak promoter P is a promoter for expression of a gene_ADE6、P_LEU2、P_URA3、P_PMA1、P_ZWF1、P_ARO7、P_PYC1、P_ADE3、P_YEF3、P_ERG1The nucleotide is shown in SEQ ID NO. 1-10 respectively; the screening gene is provided with a degradation label, and a nucleotide sequence formed by the screening gene and the degradation label is shown in SEQ ID NO. 11-20.

3. A saccharomyces cerevisiae multicopy integration plasmid consisting of a gene expression cassette and a pre-integration expression cassette according to claim 1 or 2; the pre-integration expression frame consists of an upstream and downstream homologous arm sequence of a Ty transposon, a terminator sequence and a green fluorescent protein expression frame.

4. The Saccharomyces cerevisiae multicopy integration plasmid of claim 3, wherein the plasmid is a plasmid that integrates multiple copies of Saccharomyces cerevisiae gene into a single geneTy transposon sequences include Ty1Cons, Ty2Cons, Ty3Cons, Ty4Cons and Ty5 Cons; the terminator comprises T_RFC5-T_POL30、T_SEC13-T_PNP1、T_MTD1-T_RPF2、T_LEU2-T_NFS1、T_DSF1-T_HXT13、T_TIM21-T_GSC2、T_RRP12-T_TAF3、T_RNA14-T_BUB2、T_ADH1And T_CYC1。

5. The Saccharomyces cerevisiae multicopy integration plasmid of claim 3, wherein the upstream homology arm, the green fluorescent protein expression cassette, the terminator sequence, the gene expression cassette, the downstream homology arm are ligated in that order.

6. The Saccharomyces cerevisiae multicopy integration plasmid of claim 3, wherein the green fluorescent protein expression cassette comprises a promoter P_GAL7Promoter P_GAL7Upstream of (2) comprises a terminator sequence T_GAL10。

7. A strain for producing taxifolin, which expresses relevant genes for producing taxifolin by the saccharomyces cerevisiae multicopy integration plasmid of any one of claims 3-6, and the genes are expressed by the following three modules:

a first module: overexpression of the relevant genes from tyrosine or phenylalanine to p-coumaric acid synthesis:

from tyrosine to p-coumaric acid genes include ARO4^fbr、ARO7^fbr、FjTAL、EcaroL；

The genes from phenylalanine to p-coumaric acid include ARO4^fbr、ARO7^fbr、FjTAL、EcaroL、SmPAL、SmC4H；

And a second module: overexpresses genes related to p-coumaric acid to naringenin production, including Pc4CL, PhCHS, and MsCHI;

and a third module: naringenin-to-taxifolin synthesis related genes including SmF 3' H, SmCPR and SmF 3H.

8. The strain of claim 7, wherein the strain is a starting strain of Saccharomyces cerevisiae C800.

9. A method for synthesizing taxifolin, which is characterized in that the strain of claim 7 or 8 is used for de novo synthesis of taxifolin by using glucose or ethanol as a substrate.

10. Use of the saccharomyces cerevisiae multicopy integration gene expression cassette of claim 1 or 2, or the saccharomyces cerevisiae multicopy integration plasmid of any one of claims 3 to 6, or the strain of claim 7 or 8 for the production of a protein of interest or taxifolin and derivatives thereof.

Images