CN113403334B - Plasmid kit for Saccharomyces cerevisiae multi-copy integration - Google Patents

Plasmid kit for Saccharomyces cerevisiae multi-copy integration Download PDF

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CN113403334B
CN113403334B CN202110659681.2A CN202110659681A CN113403334B CN 113403334 B CN113403334 B CN 113403334B CN 202110659681 A CN202110659681 A CN 202110659681A CN 113403334 B CN113403334 B CN 113403334B
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周景文
高松
曾伟主
陈坚
沐万孟
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Abstract

The invention discloses a plasmid kit for Saccharomyces cerevisiae multicopy integration, belonging to the field of gene and metabolic engineering. The multi-copy integrated plasmid kit constructed and verified by the invention can realize multi-copy, stable and integrated expression of a plurality of exogenous genes on a saccharomyces cerevisiae genome through one-time transformation. And can realize the stable and integrated expression of exogenous genes 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 plasmid kit for Saccharomyces cerevisiae multicopy integration, belonging to the field of gene and metabolic engineering.
Background
Saccharomyces cerevisiae is used as a model strain of eukaryotes, often to introduce heterologous pathways to synthesize high value-added products. These approaches typically involve several to tens of genes, and co-expression of multiple genes in s.cerevisiae requires integration multiple times, and are inefficient, and the integration site and the screening tag cannot meet the subsequent integration operations. Therefore, there is a need for efficient, high copy and stable insertion sites into which genes to be overexpressed are integrated, enabling efficient expression of heterologous genes.
There are a variety of long terminal repeats in the Saccharomyces cerevisiae genome (Long terminal repeats, LTRs), one type of which is a transposon, the transposon in yeast being designated Ty transposon (Ty retrotransposons). The degree of matching of the sequences of Ty transposons can be divided into five types, ty1Cons, ty2Cons, ty3Cons, ty4Cons and Ty5Cons, and the degree of matching of sequences between transposons is also low, and the copy numbers of the five Ty transposons on the genome are about 20 to 50, and are uniformly distributed on the genome, homologous recombination rarely occurs, and the structure is stable, so that these Ty sites are ideal foreign gene insertion sites (Maury, J.; germann, S.M.; baall Jacobsen, S.A.; et al, easy cloning Multi: A Set of Vectors for Simultaneous and Multiple Genomic Integrations in Saccharomyces cerevisiae [ J ]. PloS One 2016,11 (3), e 0150394.). However, at present, the integration expression based on Ty locus has various defects such as insufficient screening tag and low copy number of obtained transformant; the integrated expression frame uses the same promoter, the same terminator and other expression elements, so that the homologous recombination probability is increased; the ability to coordinate integration between different Ty sites is unknown, etc., limiting the use of such integration sites.
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 selection tag categories including 5 auxotroph tags were first expanded: uracil-deficient tag (KlURA 3), leucine-deficient tag (KlLEU 2), histidine-deficient tag (SpHIS 5), tryptophan-deficient tag (scrip 1) and methionine-deficient tag (ScMET 15); 5 antibiotic selection tags: nociceptin resistance tag (Nourseothricin resistance, natMX), hygromycin resistance tag (Hygromycin resistance, hphmX), bialaphos resistance tag (Bialaphos resistance, patMX) (Goldstein A L, mcCusker J H.Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae [ J ]. Yeast (Chichester, england) 1999,15 (14), 1541-53), geneticin resistance tag (Geneticin resistance, kanMX) (Wach A, brachat A, pohlmann R, et al, new heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae [ J ]. Yeast (Chichester, england) 1994,10 (13), 1793-808), and bleMx resistance tag (Phleomycin resistance, bleMx) (Gueldener U, heinischer J, koehler G J, et al, A second set of loxP marker cassettes for Cre-mediated multiple gene knockouts in budding Yeast [ J ]. Nucleic Acids Res 2002,30 (6), e23.). Meanwhile, when constructing a screening gene expression cassette, a weak promoter is used for expressing a relevant screening gene, and a degradation tag (Degradation signal; deg) is added at the C-terminal of the screening gene (Gilon T, chomsky O, kulka R G.degradation signals for ubiquitin system proteolysis in Saccharomyces cerevisiae [ 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 enhanced to improve the integrated copy number of the exogenous gene. Finally, all the elements such as promoters and terminators are different in order to reduce the probability of homologous recombination of the foreign gene. And (3) connecting the constructed expression package with a reporter gene green fluorescent protein EGFP, respectively converting the expression package into a saccharomyces cerevisiae strain, and determining the distribution condition of integration difficulty and integration copy number of corresponding labels and sites by detecting the number of obtained transformants and the intensity distribution of the transformant green fluorescent protein.
After a high-copy integration tool was obtained and its integration performance was verified, the huperzine synthesis pathway genes were integrated at different Ty sites on the saccharomyces cerevisiae genome using different screening tags for verification of the performance of multicopy integration.
Disclosure of Invention
Saccharomyces cerevisiae includes model strain S288c and its derivatives. The plasmid can realize multicopy integration in Saccharomyces cerevisiae genome after one-time transformation, and the integration sites are mutually independent, so that the plasmid has good stability and can be used for over-expression of genes.
The invention provides a Saccharomyces cerevisiae multicopy integrated gene expression frame, which consists of a weak promoter sequence, a screening gene sequence with a degradation tag deg anda terminator; the weak promoter comprises P ADE6 、P LEU2 、P URA3 、P PMA1 、P ZWF1 、P ARO7 、P PYC1 、P ADE3 、P YEF3 、P ERG1 The screening genes comprise ScTRP1, klLEU2, klURA3, scMET15, spHIS5 and natMX, hphMX, kanMX, patMX, bleMX.
In one embodiment, the terminator is T TDH3 The 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 ERG1 The nucleotide is shown as SEQ ID NO. 1-10; the screening gene is provided with a degradation tag, the degradation tag is deg, and the nucleotide sequence is shown as SEQ ID NO. 46; the nucleotide sequences formed by the screening genes and the degradation labels are respectively shown in SEQ ID NO. 11-20.
In one embodiment, the weak Promoter is disclosed in the document Promoter-library-based pathway optimization for efficient (2S) -naringenin production from p-coumaric acid in Saccharomyces cerevisiae (disclosed in 2020).
The invention provides a Saccharomyces cerevisiae multicopy integration plasmid, which consists of the gene expression frame and a pre-integration expression frame; the pre-integrated expression frame consists of an upstream and downstream homologous arm sequence of the Ty transposon, a terminator sequence and a green fluorescent protein expression frame.
In one embodiment, the Ty transposon sequences comprise Ty1 conn, ty2 conn, ty3 conn, ty4 conn, and Ty5 conn; the terminator includes 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 ADH1 And T CYC1 。
In one embodiment, the Saccharomyces cerevisiae multicopy integration plasmid is linked 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 respectively shown as SEQ ID NO.23 and SEQ ID NO. 24; the upstream and downstream homology arms of Ty2 are respectively shown as SEQ ID NO.25 and SEQ ID NO. 26; the upstream and downstream homology arms of Ty3 are respectively shown as SEQ ID NO.27 and SEQ ID NO. 28; the upstream and downstream homology arms of Ty4 are respectively shown as SEQ ID NO.29 and SEQ ID NO. 30; the upstream and downstream sequences of all Ty elements have been disclosed in literature Maury, j; germann, s.m.; baall Jacobsen, S.A., et al, easy CloneMulti A Set of Vectors for Simultaneous and Multiple Genomic Integrations in Saccharomyces cerevisiae [ J ]. PloS One 2016,11 (3), e0150394 (disclosed in 2016)
In one embodiment, the terminator 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 ADH1 And T CYC1 The sequences of (2) are respectively shown as SEQ ID NO. 31-40; such terminator sequences are disclosed in the literature Promoter-library-based pathway optimization for efficient (2S) -naringenin production from p-coumaric acid in Saccharomyces cerevisiae (published in 2020).
In one embodiment, the green fluorescent protein expression cassette comprises a promoter P GAL7 Promoter P GAL7 Upstream of (a) comprises a terminator sequence T GAL10 With terminator T GAL10 Promoter P of (C) GAL7 The nucleotide sequence of the fluorescent protein is SEQ ID NO.41, and the nucleotide sequence of the green fluorescent protein is SEQ ID NO.42.
In one embodiment, the screening gene is preferably KlLEU2, klURA3, spHIS5, hphMX, natMX.
The invention provides a strain for expressing a target protein, which expresses the target protein through the 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 the taxifolin, which expresses related genes for producing the taxifolin through the saccharomyces cerevisiae multicopy integration plasmid, wherein the genes are expressed through the following three modules:
module one: overexpression of genes involved in the synthesis of p-coumaric acid from tyrosine or phenylalanine:
from tyrosine to p-coumaric acid genes including ARO4 fbr 、ARO7 fbr 、FjTAL、EcaroL；
Genes ranging from phenylalanine to p-coumaric acid include ARO4 fbr 、ARO7 fbr 、FjTAL、EcaroL、SmPAL、SmC4H；
And a second module: overexpression of genes related to production of p-coumaric acid to naringin, including Pc4CL, phCHS and MsCHI;
and a third module: naringenin to huperzine synthesis related genes including SmF3' H, smCPR and SmF3H.
In one embodiment, the strain further expresses the malonyl-coa pathway related gene ScACC1 S659A ,S1157A 、SeACS2 S641P And SpHIS5.
In one embodiment, C800 is used as the starting strain, which is disclosed in the patent document with publication number CN111424020 a.
The invention provides a method for producing huperzine, which is to take the strain for producing the huperzine and glucose or ethanol as a substrate to synthesize the huperzine from the beginning.
In one embodiment, the strain is cultured to obtain an OD 600 Inoculating 20-30 seed solution to YPD culture medium at 1-5 mL/100mL, culturing at 30-35 deg.C and 200-250 rpm, fermenting for 72 hr, and adding 12 hr, 24 hr, 36 hr and 48 hr95% ethanol or glucose.
In one embodiment, the 95% ethanol is added in an amount of 0.5% (0.5 mL/100 mL), the glucose concentration is 500g/L, and the addition amount is 0.5% (0.5 mL/100 mL).
In one embodiment, the temperature is controlled to 30.+ -. 0.1 ℃.
In one embodiment, the strain is cultured to obtain an OD 600 Inoculating 20-30 seed solution into YPD culture medium according to the proportion of 1-5 mL/100mL, culturing at 30-35 ℃, 500-600 rpm and aeration rate of 1-3 vvn, supplementing fed-batch culture medium at the flow rate of 5.0mL/L when the glucose concentration in the reaction system is reduced to 0g/L, controlling the pH value to be 5.5+/-0.1, and fermenting for 72h.
The invention provides an application of the saccharomyces cerevisiae multicopy integrated gene expression frame or the saccharomyces cerevisiae multicopy integrated plasmid in production of target proteins.
The invention provides application of the strain in production of picea.
In one embodiment of the invention, the microbial cell is Saccharomyces cerevisiae strain C800 (CEN.PK2-1D, MAT. Alpha.; ura3-52; leu2-3,112; trp1-289; his3Δ1; MAL 2-8) C The method comprises the steps of carrying out a first treatment on the surface of the SUC2; gal 80:KanMX) strain C800 is disclosed in the patent document with publication number CN 111424020A. .
The invention provides a recombinant bacterium for producing yellow fir and a production method thereof, wherein the method comprises the steps of selecting the multicopy vector, integrating a plurality of copies of yellow fir synthetic genes at 3 different Ty sites, and fermenting and producing yellow fir by utilizing the recombinant bacterium.
The invention has the beneficial effects that: the multi-copy integrated plasmid kit constructed and verified by the invention can realize multi-copy, stable and integrated expression of a plurality of exogenous genes on a saccharomyces cerevisiae genome through one-time transformation. And can realize the expression of exogenous genes in multiple batches, multiple genes, stability and integration by selecting different screening labels at different Ty sites, thus realizing the efficient expression of exogenous genes.
Drawings
FIG. 1 is a schematic genotype of a multicopy plasmid kit (A: 10 selection gene expression cassette combination schematic for multicopy integration; B:5 pre-integration expression cassettes schematic; C-G:50 plasmid kits for multicopy integration of Saccharomyces cerevisiae; all element positions and sequences in this figure are positions and sequences in the actual plasmid map).
FIG. 2 shows the distribution of the number of transformants that can be obtained after one transformation (screening tag on the abscissa and number of transformants obtained after one transformation on the ordinate. Different shapes represent different Ty integration sites).
FIG. 3 shows the fluorescence intensity distribution of multicopy recombinant strains (50 combined expression cassettes in the expression cassette are integrated on the Saccharomyces cerevisiae genome to obtain transformants, 10-30 transformants are randomly selected, the fluorescence intensity distribution is detected, and a box diagram is drawn, and the fluorescence intensity distribution of each transformant is simultaneously drawn beside the box diagram, panel A. Blank is the fluorescence intensity distribution of strain C800 in YNB medium, and only one EGFP copy is present on the genome of C887. Panel B-Panel H. The selection tags are ScTRP1deg, klURA3deg, scMET15deg, hphMXdeg, kanMXdeg, patMXdeg and bleneg in this order, the distribution of the fluorescence intensities of the transformants is shown in Ty1Cons1, ty1Cons2, ty2Cons, ty3Cons and Ty4Cons in this order, the selection tags are KlLUE2deg at Ty1Cons1 locus, the selection tags are shown in FIG. 5deg, the selection tags are shown at Ty1Cons1 locus, the selection tags are shown in this order, and the other transformants are not shown at Ty1Cons2 locus X. The other positions are not shown, and the distribution of the fluorescence intensity of the transformants is shown in this order, and the distribution of Ty1Cons is shown in this diagram.
FIG. 4 is a schematic diagram of the pathway for the synthesis of huperzine in Saccharomyces cerevisiae (huperzine synthesis pathway is divided into three modules, module one: p-coumaric acid synthesis pathway (ARO 4/ARO7/TAL or ARO4/ARO7/PAL/C4H is required to be overexpressed), module two: naringenin synthesis pathway (4 CL/CHS/CHI is required to be overexpressed), and module three: huperzine synthesis pathway (F3' H/CPR/F3H is required to be overexpressed).
FIG. 5 shows the effect of expression in the single Ty site and double Ty site integration pathway genes.
FIG. 6 is a graph showing the effect of expression of the gene of the three Ty site integration pathway; the numbers in brackets on the abscissa of panel a are fluorescence intensities.
FIG. 7 shows copy numbers of integrated genes at different sites.
FIG. 8 is a graph showing the de novo production of huperzine from a 5-L fermenter.
Detailed Description
YNB medium: 0.72g/L yeast nitrogen source basal medium and 20g/L glucose.
YPD medium: 10g/L yeast powder, 20g/L peptone and 20g/L glucose.
A final concentration of 50mg/L leucine, 50mg/L tryptophan, 50mg/L histidine or 50mg/L uracil was added to YNB medium as required.
Various antibiotics are added into a non-scalding culture medium in a mother solution form, wherein the concentration of the nociceptin mother solution is 100mg/L, and the working concentration is 100 mug/L; the concentration of hygromycin mother liquor is 300mg/L, and the working concentration is 300 mug/L; the concentration of the glufosinate-ammonium mother solution is 800mg/L, the working concentration is 800 mug/L (if bialaphos is used, the concentration of the mother solution is 200mg/L, and the working concentration is 200 mug/L); the concentration of the geneticin mother solution is 200mg/L, and the working concentration is 200 mug/L; the concentration of the bleomycin mother solution is 100mg/L, and the working concentration is 100 mug/L. Transformants using amino acid-deficient selection tags and glufosinate antibiotic selection tags were plated on YNB plates and transformants using selection tags of the other four antibiotics were plated on YPD plates.
20g/L of agar powder is added into the solid culture medium.
The components of the fed-batch culture medium: glucose 400g/L, KH 2 PO 4 18g/L，MgSO 4 ·7H 2 O 10.24g/L，K 2 SO 4 7g/L，Na 2 SO 4 0.56g/L, metal salt mother liquor 20 mL.L -1 Vitamin mother liquor 24 mL.L -1 Various amino acids were added at 1g/L as required. Metal salt mother liquor: znSO (ZnSO) 4 ·7H 2 O 5.75g/L，MnCl 2 ·4H 2 O 0.32g/L，CoCl 2 ·6H 2 O 0.47g/L，NaMoO 4 ·2H 2 O 0.48g/L，CaCl 2 ·2H 2 O 2.9g/L，FeSO 4 ·7H 2 O2.8 g/L,80mL of 0.5M EDTA (pH 8.0). Vitamin mother liquor: raw materials0.05g/L of Biotin (Biotin), 1g/L of calcium pantothenate (Calcium pantothenate), 1g/L of Nicotinic acid (Nicotinic acid), 25g/L of myo-Inositol (myo-Inositol), 1g/L of Thiamine hydrochloride (Thiamine HCl), 1g/L of Pyridoxal hydrochloride (Pyridoxal HCl), 0.02g/L of p-aminobenzoic acid (p-Aminobenzoic acid)
Saccharomyces cerevisiae CEN.PK2-1D (MAT. Alpha.; ura3-52; leu2-3,112; trp1-289; his3Δ1; MALD2-8) C The method comprises the steps of carrying out a first treatment on the surface of the SUC 2) is used for expression of genes.
Coli JM109 was used for molecular cloning.
Plasmids pcfB2989, pcfB2988, pcfB2797, pcfB2990, pcfB2796 and pcfB2803 (Addgene plasmid #63636, #63638, #63639, #63645, #63641, # 63646) were offered by Irina Borodina & Jerome Maury (Maury J, germann S M, baallal Jacobsen S A, et al.easy Clonemulti: A set of vectors for simultaneous and multiple genomic integrations in Saccharomyces cerevisiae [ J ]. PloS One 2016,11 (3), e 0150394.).
Plasmids pMDT-SmPAL and pMDT-SmC4H are Silybum-derived phenylalanine ammonia lyase (Phenylalanine ammonia lyase, PAL) and cinnamic acid hydroxylase (Cinnamic acid hydroxylase, C4H), obtained from Silybum marianum transcriptome identification and reverse transcription by tBLASTN, corresponding to Contig 5930 and Contig265 (Lv Y, gao S, xu S, et al spatial organization of silybin biosynthesis in milk thistle [ Silybum marianum (L.) Gaertn ] [ J ]. Plant J2017,92 (6), 995-1004.), respectively, the corresponding gene DNA sequences are shown in Table 1, with the nucleotide sequences SEQ ID NO.44 and SEQ ID NO.45, respectively.
P-coumaric acid, naringenin, eriodictyol, and huperzine markers were purchased from Sigma-Aldrich (st.louis, MO).
North Silk fungus (Nourseothricin sulfate, CAS: 96736-11-7) element was purchased from Solarbio (N9210).
Hygromycin (Hygromycin B Solution, CAS: 3128-04-9) is purchased from biological engineering (Shanghai) Inc. (B540725).
Glufosinate (Ammonium glufosinate, CAS: 77182-82-2) was purchased from Ark Pharm (AGZ 938).
Geneticin (G418 Sulfate, CAS: 108321-42-2) was purchased from Biotechnology (Shanghai) Inc. (A100859).
Bleomycin (Bleomycin, CAS: 11006-33-0) is available from biological engineering (Shanghai) Inc. (A620212).
The detection method comprises the following steps: mixing 100 μl of fermentation broth with 900 μl of methanol, vortex shaking, mixing for 30s, centrifuging at 13500rpm for 5min, and filtering supernatant. And (3) using Shimadzu high performance liquid phase detection. Chromatographic column: ZORBAX SB-C18 (4.6 mm. Times.150 mm, shimadzu). Mobile phase a:100% acetonitrile, mobile phase B:100% of water, 1 per mill (V/V) of trifluoroacetic acid is added into all mobile phases, and then the solution is filtered and removed from bubbles by ultrasonic waves for later use. Mobile phase flow rate: 1mL/min, column temperature: 30 ℃, sample injection amount: 10 μl, detection wavelength: 290nm. Liquid phase procedure: 0-10 min, 10-40% mobile phase A; 10-15 min, 40-60% of mobile phase A; 15-18 min, 60-10% of mobile phase A, and distinguishing various substances according to different peak time of different substances.
Example 1: construction of multicopy plasmid expression kits
In the present invention, gibson assembly technique (Gibson assembly) is used as assembly means, and all sequences are assembled in seamless cloning. The corresponding nucleotide sequences are amplified by PCR, homologous arms of about 20-30bp are included between the sequences, and all plasmid expression kits can be obtained through Gibbsen assembly.
(1) Construction of multiple copy integrated screening gene expression cassette combinations
10 combinations of selectable gene expression cassettes (Marker genes, shown as pT 0-pT 9 in Table 2 and FIG. 1) for multicopy integration were selected, comprising 10 weak promoter sequences, 10 selectable gene sequences with degradation tag deg, and terminator T TDH3 The nucleotide sequence is SEQ ID NO.43.
10 weak promoters P ADE6 、P LEU2 、P URA3 、P PMA1 、P ZWF1 、P ARO7 、P PYC1 、P ADE3 、P YEF3 、P ERG1 The corresponding nucleotide sequences are 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 in this order (Gao, S.; zhou, H.; zhou, J.; et al, promot.)er-library-based pathway optimization for efficient(2S)-naringenin production from p-coumaric acid in Saccharomyces cerevisiae[J].J Agric Food Chem 2020,68(25),6884-6891.)。
The 10 screening genes with degradation tag deg are ScTRP1-deg, klLEU2-deg, klURA3-deg, scMET15-deg, spHIS5-deg, natMX-deg, hphMX-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 cassettes
According to the integration site of Saccharomyces cerevisiae, 5 pre-integration expression frames are constructed, including 5 groups of upstream and downstream homologous arm sequences of Ty sequences, 8 bidirectional terminator sequences, 2 unidirectional terminator sequences and one green fluorescent protein expression frame.
The 5 groups of Ty sequences are sequentially Ty1Cons1, ty1Cons2, ty3 and Ty4, the Ty1Con1 upstream/downstream homology arm, ty1Con2 upstream/downstream homology arm, ty3 upstream/downstream homology arm and Ty4 upstream/downstream homology arm, and the nucleotide sequences corresponding to the 5 groups of upstream/downstream homology arms are sequentially 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.
The 8 bidirectional terminators are sequentially 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 The 2 unidirectional terminators are T in sequence ADH1 And T CYC1 The nucleotide sequences corresponding to the bidirectional terminator are SEQ ID NO.31, SEQ ID NO.32, SEQ ID NO.33, SEQ ID NO.34, SEQ ID NO.35, SEQ ID NO.36, SEQ ID NO.37 and SEQ ID NO.38 in sequence, and the sequences corresponding to the unidirectional terminator are SEQ ID NO.39 and SEQ ID NO.40 in sequence.
Expression of green fluorescent proteinThe box includes a promoter P GAL7 Promoter P GAL7 Upstream of (a) comprises a terminator sequence T GAL10 The green fluorescent protein is EGFP with terminator T GAL10 Promoter P of (C) GAL7 The nucleotide sequence of the EGFP is SEQ ID NO.41, and the nucleotide sequence of the green fluorescent protein EGFP is SEQ ID NO.42. A terminator sequence T is also used in the present invention TDH3 The nucleotide sequence is SEQ ID NO.43. In the invention, two genes SmPAL and SmC4H derived from silybum marianum are used, and the nucleotide sequences are SEQ ID NO.44 and SEQ ID NO.45 in sequence.
50 plasmid kits for Saccharomyces cerevisiae multicopy integration were constructed: the 10 screening gene expression frames (Marker genes) are respectively inserted into the middle of two double terminators (Gibson assembly technology is used, no redundant base insertion is carried out, and no trace insertion is carried out), namely, a group of saccharomyces cerevisiae multicopy integration tool kit containing 50 plasmids is obtained. Wherein the Ty1Cons1 pre-integration expression frame is combined with 10 screening genes to sequentially obtain 10 plasmids in total of pcT111, pcT112, pcT113, pcT114, pcT115, pcT116, pcT117, pcT118, pcT119 and pcT. Wherein the Ty1Cons2 pre-integration expression frame is combined with 10 screening genes to sequentially obtain 10 plasmids in total of pcT121, pcT122, pcT123, pcT124, pcT125, pcT126, pcT127, pcT128, pcT129 and pcT. The Ty2 pre-integration expression cassette was combined with 10 selection genes, sequentially obtaining pcT, pcT22, pcT, pcT24, pcT25, pcT, pcT, pcT, 28, pcT, 29, and pcT20 total 10 plasmids. The Ty3 pre-integration expression cassette was combined with 10 selection genes, sequentially obtaining pcT, pcT, pcT, pcT, 34, pcT, pcT, pcT, pcT, pcT, 39 and pcT30 total 10 plasmids. Wherein the Ty4 pre-integration expression frame is combined with 10 screening genes to sequentially obtain 10 plasmids of pcT, pcT42, pcT43, pcT44, pcT45, pcT46, pcT47, pcT48, pcT49 and pcT 40.
The actual positions of all the genes, promoters, terminators, etc. in 10 combinations of selectable gene expression cassettes for multicopy integration, 5 pre-integrated expression cassettes, and 50 plasmid kits for multicopy integration of Saccharomyces cerevisiae are shown in FIG. 1.
Gene ScACC1 S659A,S1157A 、SeACS S641P 、SmF3′H D284N 、SmCPR I453V 、ARO4 fbr 、ARO7 fbr FjTAL, ecaroL, smF3H, the nucleotide sequences corresponding to the sequences are SEQ ID NO. 47-SEQ ID NO.55. Other promoters P mentioned GAL1 And P GAL10 The nucleotide sequences corresponding to the sequence are SEQ ID NO.56 and SEQ ID NO.57.
All nucleotide sequences in 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.
All nucleotide sequences of Table 1
TABLE 2 genotypes of plasmids and strains
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TABLE 3 Critical primer sequences
Example 2: multi-copy integration plasmid tool integration capability verification
Plasmid pcT to pcT119 was amplified with primer Ty 11-inter-up/down, plasmids pcT to pcT129 were amplified with primer Ty 12-inter-up/down, plasmids 3828 to pcT29 were amplified with primer pellet Ty 2-inter-up/down, plasmids pcT to pcT39 were amplified with primer Ty 3-inter-up/down, and plasmids pcT40 to pcT were amplified with primer Ty 4-inter-up/down, respectively, to obtain integrated expression cassette portions of the vector. The PCR products were recovered and purified and integrated into the Saccharomyces cerevisiae strain by efficient transformation methods (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 selection tags were TRP1, LEU2, URA3, HIS5, natMX, hphMX, patMX, bleMX, transformation into strain C800 was performed. When the screening tag is MET15, strain C815 is selected.
When the screening tag was KanMX, strain C850 was selected. The transformed cells were plated on the corresponding screening plates (YNB-TRP when the screening gene was TRP 1) - Plate, and when screening gene LEU2, coating on YNB-LEU - Plate, when screening gene URA3, is coated on YNB-URA - Flat plate, when screening gene is HIS5, coating on YNB-HIS - Plate, screening gene is MET15 and coated on YNB-MET - A plate, wherein the screening gene is a natMX YPD plate coated with norrissin-containing YPD plate, the screening gene is a hpMX YPD plate coated with hygromycin-containing YPD plate, the screening gene is a KanMX YPD plate coated with geneticin-containing YPD plate, the screening gene is a patMX YPD plate coated with glufosinate-containing YPD plate, and the screening gene is a bleMX plate coated with bleomycin-containing YPD plateYPD plate), 30 ℃, 3-5d until single colonies were obtained. The obtained single colonies were counted and inoculated with single colonies for cultivation, and the fluorescence intensity was detected. The integration efficiency of each combination in the kit was determined from the obtained number of transformants and the distribution of fluorescence intensity of the transformants. After one transformation, the distribution of the number of transformants obtainable for each combination is shown in FIG. 2. The range of fluorescence intensity distribution of the multicopy recombinant strain obtained for each combination is shown in FIG. 3.
As can be seen from FIG. 2, the majority of plasmids in the Ty expression cassette can obtain about 100-500 transformants after one transformation, but pcT122, pcT22, pcT32, pcT42; pcT25, pcT, 35, pcT45 and pcT, pcT, 26, pcT were transformed multiple times to obtain no transformants, whereas pcT, pcT, 124, pcT, pcT, 34 and pcT with ScMET15deg as the screening tag gave more than 1 ten thousand transformants, however the transformants were almost non-fluorescent (fig. 3), and it was seen that ScMET15deg was not suitable for high copy integration here.
As can be seen from FIG. 3, the fluorescence intensity values of the blank strain and the strain C887 having only one EGFP copy were about 8000 and 25000 (FIG. 3A). When ScMET15deg, kanMXdeg, patMXdeg, bleMXdeg was used as a screening tag, the obtained transformants were weak in fluorescence intensity, most of which were only one copy (fig. 3D, 3F, 3G, 3H), and when ScTRR1deg was used as a screening tag, most of the transformants were weak in fluorescence intensity (fig. 3B). And the transformant (FIG. 3C, FIG. 3E and FIG. 3I) which almost has high probability of obtaining strong fluorescence signals by using KlURA3deg, klLEU2deg and SpHIS5deg, natMXdeg, hphMXdeg, the average value of the fluorescence intensity is 7.9-16.5 times of that of the control, and the strongest fluorescent intensity of one strain reaches 48.2 times of that of the control group. Thus, these multicopy integrative plasmids can be used to boost the copy number in Saccharomyces cerevisiae.
Example 3: application of multi-copy integrated plasmid kit
The synthesis of huperzine involved the introduction and overexpression of multiple genes and was therefore used to verify the performance of the multicopy integrated plasmid kit. The synthesis route of the huperzine is shown in figure 4, wherein the first module is glucose to p-coumaric acid, the second module is p-coumaric acid to naringenin, and the third module is naringenin to huperzine. The multi-copy integrated plasmid kit with significantly increased copy number in example 2 was selected for use in the production of huperzine or related intermediates.
1. Construction of single Ty site integrated recombinant bacterium
The primers Ty 4-inter-up/Ty 4-inter-down are used for respectively amplifying pcfB4-47LL and pcfB4-P05m4, PCR products are efficiently transformed by saccharomyces cerevisiae and are respectively integrated into the Ty4 site of a saccharomyces cerevisiae strain C800 to respectively obtain strains C8011 and C805. The strain C8011 integrates the related genes of the synthesis of the p-coumaric acid in the first module, the p-coumaric acid can be synthesized from the head, the strain C805 integrates the related genes of the synthesis of the naringenin to the huperzine in the third module, and the huperzine can be synthesized from the naringenin.
2. Construction of double Ty site integrated recombinant bacterium
The plasmid pcfB3-P03 is amplified by using a primer Ty 3-inter-up/Ty 3-inter-down, and the obtained PCR product is purified and refined and is integrated into a Ty3 locus of a genome of a strain C8011 by efficient transformation of saccharomyces cerevisiae, so as to obtain a strain C824. The plasmid pcfB4-P05m4 is amplified by using a primer Ty 4-inter-up/Ty 4-inter-down, and the obtained PCR product is purified and refined and is integrated into a Ty4 locus of a genome of a strain C803 by efficient transformation of saccharomyces cerevisiae, so as to obtain the strain C857. Strain C824 integrates genes related to the synthesis of naringenin from glucose in the first and second modules, naringenin can be synthesized from naringenin in the first and third modules, and strain C857 integrates genes related to the synthesis of p-coumaric acid to huperzine in the second and third modules, and huperzine can be synthesized from p-coumaric acid.
pT820 was amplified using primers Pf-gal80/Pf-gal80D, and after recovery of the PCR products, the products were transformed into strain C803 and strain C857, respectively, by the Saccharomyces cerevisiae efficient transformation method, to obtain strain C823 and strain C877, respectively. Strain C823 and strain C877 potentiate the malonyl-CoA pathway based on the original strains C803 and C857, respectively.
3. Construction of recombinant bacteria integrated at site of three Ty
Integrating the genes related to the first module on the basis of the strain C877, and respectively obtaining a strain C900 and a strain C901 according to the use of tyrosine or phenylalanine as a precursor: pcT21-LL and pcT-LHL are amplified by using primers Ty 2-inter-up/Ty 2-inter-down, and PCR products are recovered and transformed to Ty2 locus in strain C877 by a Saccharomyces cerevisiae efficient transformation method to obtain strains C900 and C901 respectively. Both strain C900 and strain C901 can synthesize huperzine from the head, wherein strain C900 uses tyrosine as precursor, p-coumaric acid is obtained via TAL approach to synthesize huperzine, and strain C901 uses phenylalanine as precursor, p-coumaric acid is obtained via PAL/C4H/CPR approach to obtain huperzine.
4. RNA expression level of gene related to picrin synthesis in recombinant strain (detection of integrated copy number of different Ty sites and stable orientation verification)
(1) Copy number detection
Strain culture: the single colony of the strain C900 and the strain C901 obtained by the construction is selected and inoculated in a 250mL shaking flask containing 5mL YNB culture medium, and the culture is carried out at 30 ℃ and 220rpm for 24 hours, thus obtaining a seed culture medium. Seed cultures were grown to the initial OD 600 =0.1 transfer to 250mL shake flasks of 50mL YNB broth. Culturing at 30 deg.C and 220rpm for 6-8 hr to logarithmic early phase. The fermentation broth was centrifuged at 13500rpm for 3min. The supernatant was removed and yeast cells were collected.
RT-PCR procedure: total RNA from yeast was extracted using the RNAprep pure Plant Kit kit (TIANGEN, beijing, china). The total RNA was used as a template, and the DNA was removed using PrimeScript RT kit, followed by reverse transcription to obtain cDNA. RT-PCR was performed using a SYBR Premix Ex Taq (Tli RNAseH Plus) kit and cDNA as a template. The reference gene ACT1 for homogenization was subjected to RT-PCR by the primer pair ACT1-F/ACT 1-R. RT-PCR was performed on a LightCycler 480II instrument. All the results were normalized to the reference gene ACT 1. The expression intensity of the gene was according to 2 -ΔΔCt Method (Livak K J, schmittgen 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 numbers of genes for modules one, two and three were quantified using primers qEGFP-F/R, qCHS-F/R and qF H-F/R, respectively.
Through RT-PCR verification, the genes of the first module, the second module and the third module in the strain C900 are respectively integrated at a Ty2 site, a Ty3 site and a Ty4 site, the copy numbers are sequentially 4, 5 and 7, and the copy numbers of the genes in the strain C901 at the Ty2 site, the Ty3 site and the Ty4 site are sequentially 8, 5 and 7 (figure 7).
(2) Stability verification
Strains C900 and C901 were inoculated into 250mL shake flasks containing 20mL of liquid YPD medium, cultured at 30℃at 220rpm, inoculated into 250mL shake flasks containing fresh 20mL of liquid YPD medium every 12 hours at 1% (mL/100 mL), passaged as such, after a total of 60 passages over 30 days, the two strains obtained after the passage and their corresponding starting strains were inoculated into 250mL shake flasks containing 20mL of liquid YPD medium, respectively, cultured at 30℃at 220rpm for 14-16 hours, inoculated into 250mL shake flasks containing 20mL of YPD medium at 1mL/100mL of inoculation ratio, cultured at 30℃at 220rpm, fermented for 72 hours, and added with 0.5% (0.5 mL/100 mL) of 95% ethanol at 12 hours, 24 hours, 36 hours and 48 hours, respectively, and the production of taxifolin was determined after the end. The results show that the yields of the yellow fir of the original strains C900 and C901 are 50.93mg/L and 33.83mg/L respectively, and the yields of the yellow fir produced by the strains after passage of the strains C900 and C901 are 52.34mg/L and 33.48mg/L respectively, and obviously the capability of producing yellow fir of the strains after passage is not significantly changed, thereby proving that the stability of the three-locus integration strain based on the Ty transposon is good.
5. Recombinant bacteria fermentation production of huperzine
(1) Using the constructed strain C8011, strain C803, strain C805 and strain C824, strain C857 and strain C877: single colonies are respectively picked and inoculated into 250mL shake flasks of 20mL YPD culture medium, and the shake flasks are controlled at 30 ℃ and 220rpm to culture for 14-16h until the strain OD 600 Seed solution was obtained to about 25, at which time the strain was in mid-log phase. A250 mL shake flask containing 20mL YPD medium was inoculated at a ratio of 1mL/100mL, cultured at 30℃and 220rpm, and 0.5% (0.5 mL/100 mL) of a 500g/L glucose solution or 0.5% (0.5 mL/100 mL) of 95% ethanol was added at 12h, 24h, 36h and 48h, respectively, as required. Sampling after fermentation is finished, and detecting the content of the final product and the intermediate product.
Strain C8011 integrates the relevant genes of module one and can be fermented in 250mL shake flasks for 72h to synthesize 544.01mg/L p-coumaric acid starting from glucose (FIG. 5A). Strain C803 integrates the relevant genes of module two and can synthesize 623.60mg/L naringenin from 1000mg/L p-coumaric acid in 250mL shake flasks for 72h (FIG. 5B). Strain C805 integrated the relevant genes of module three and was able to synthesize 603.90mg/L of huperzine from 1000mg/L of naringenin in 250mL for 72h while accumulating 392.30mg/L of eriodictyol with the remaining 83.80mg/L of naringenin (FIG. 5A). Single site integration based on Ty transposon can result in constitutive strains with stable gene phenotype.
Strain C824 is a related gene integrated with a second module based on strain C8011, and naringenin can be synthesized from glucose as a substrate, and 264.05mg/L naringenin can be synthesized by 72h fermentation in a 250mL shake flask (FIG. 5A). Strain C857 is a related gene integrated with the second module on the basis of strain C803, can synthesize the yellow fir element by taking p-coumaric acid as a substrate, can synthesize 346.43mg/L yellow fir element from 1000mg/L p-coumaric acid by 72h fermentation in a 250mL shake flask, and meanwhile, the accumulation amounts of intermediate naringenin and eriodictyol are 66.32mg/L and 142.66mg/L, and the substrate p-coumaric acid still remains 76.52mg/L (FIG. 5C). Double site integration based on the Ty transposon still allows to obtain a constitutive strain with good stability.
(2) Strains C900 and C901 can both synthesize the huperzine from glucose (C900 takes tyrosine as a precursor, C901 takes phenylalanine as a precursor), 10 strains with different fluorescence intensities are selected from a transformant library of the strains C900 and C901 respectively in order to verify the relation between the synthesis capacity of the huperzine and the copy number of one gene of the module, and fermentation verification is carried out by using a 48 deep-hole plate, so that the higher the fluorescence intensity is, the higher the accumulation amount of the huperzine and other flavone related compounds is (the numbers in the horizontal brackets are the fluorescence intensities in FIG. 6A). Wherein the highest accumulation of 20.66mg/L of the yellow fir element was obtained for the strain C901 which uses phenylalanine as a precursor, and the accumulation of 56.67 mg/L, 31.24 mg/L and 19.01mg/L of the coumaric acid, naringenin and eriodictyol, respectively. While the highest accumulation of yellow fir element obtained by the strain C900 using tyrosine as the precursor was 19.77mg/L, while the accumulation of coumaric acid, naringenin and eriodictyol was 31.42, 23.67 and 15.56mg/L, respectively.
Single colonies of C900 and C901 were picked separately and inoculated into 250mL shake flasks of 20mL YPD medium, and controlledShaking table is prepared at 30 ℃ and 220rpm, and cultured for 14-16 hours until the strain OD 600 Seed solution was obtained to about 25, at which time the strain was in mid-log phase. The seed solution was inoculated into a 250mL shake flask containing 20mL of YPD medium at an inoculation ratio of 1mL/100mL, cultured at 30℃and 220rpm, fermented for 72 hours, and added with 0.5% (0.5 mL/100 mL) of 95% ethanol at 12h, 24h, 36h and 48h, respectively. Transformants with the highest accumulation of the obtained huperzine were designated as C900 and C901. Then, re-screening was performed at the shake flask level, and at 12h, 24h, 36h and 48h, 500g/L of glucose solution was added in a proportion of 0.5% (0.5 mL/100 mL) or 95% ethanol was added in a proportion of 0.5% (0.5 mL/100 mL), respectively, to examine the optimal fermentation carbon source. At 72h, strain C900 had accumulated, fermentatively, amounts of 11.88/8.78, 13.35/10.85, 22.72/13.40 and 50.93/33.83mg/L for p-coumaric acid, naringenin, eriodictyol and taxifolin, respectively, using glucose/ethanol as a carbon source; the amounts of p-coumaric acid, naringenin, eriodictyol, and taxifolin accumulated by fermentation 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, for strain C901 (FIG. 6B).
(3) Strain C901 was fermented in a 5L fermenter: seed medium was transferred to a 5L fermenter containing 2.5L of YPD medium at 1% as above. The temperature was controlled to 30.+ -. 0.1 ℃ and the rotational speed was 600rpm, aeration rate 3vvn. Starting a feed pump when the glucose concentration is reduced to 0g/L, setting the feeding speed of the culture medium to 5.0mL/L, controlling the pH value to 5.5+/-0.1, and measuring the concentration of various substances in the fermentation liquid every 12h in the fermentation process until the fermentation is finished. As a result, as shown in FIG. 8, it was possible to synthesize 135.83mg/L of taxifolin from the head within 72 hours while accumulating 40.37, 10.32 and 41.10mg/L of p-coumaric acid, naringenin and eriodictyol.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and 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 Jiangnan
<120> a set of plasmid kits for Saccharomyces cerevisiae multicopy integration
<130> BAA210539A
<160> 57
<170> PatentIn version 3.3
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<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 (3)

1. The saccharomyces cerevisiae multicopy integration plasmid is characterized by consisting of a gene expression frame and a pre-integration expression frame; the pre-integrated expression frame consists of an upstream and downstream homologous arm sequence of the Ty transposon, a terminator sequence and a green fluorescent protein expression frame;
the expression frame consists of a weak promoter sequence and a screening gene sequence with a degradation tag deg;
the gene expression frame is any one of the following (a) - (d):
(a) From promoter P URA3 And screening genesKlURA3Composition;
(b) From promoter P ZWF1 And screening genesSpHIS5Composition;
(c) From promoter P ARO7 And screening genesnatMXComposition;
(d) From promoter P LEU2 And screening genesKlLEU2Composition;
(e) From promoter P PYC1 And screening geneshphMXComposition;
the gene expression cassette (a) is used for integration of Ty1 cos 1, ty2, ty3 or Ty4 sites;
the gene expression cassette (b) is used for integration of Ty1 cos 1 or Ty1 cos 2 sites;
the gene expression cassette (c) is used for integration of Ty1 cos 2 or Ty3 sites;
the gene expression cassette (d) is for integration of the Ty1 cos 1 site;
the gene expression cassette (e) is used for integration of Ty1 cos 1, ty1 cos 2, ty3 or Ty4 sites;
the weak promoter P LEU2 、P URA3 、P ZWF1 、P ARO7 、P PYC1 The nucleotide of (2) is shown as SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7 respectively; the screening gene is provided with a degradation tag, and the nucleotide sequences formed by the screening gene and the degradation tag are respectively shown as SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.16 and SEQ ID NO. 17;
the upstream homology arm of the Ty transposon, the green fluorescent protein expression cassette, the terminator sequence, the gene expression cassette and the downstream homology arm of the Ty transposon are connected in this order.
2. The s.cerevisiae multicopy integration plasmid of claim 1 wherein 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 ADH1 And T CYC1 。
3. The saccharomyces cerevisiae multicopy integration plasmid of claim 1 wherein the green fluorescent protein expression cassette comprises a promoter P GAL7 Promoter P GAL7 Upstream of (a) comprises a terminator sequence T GAL10 。
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