CN114907997A

CN114907997A - Construction and application of diosgenin synthesis strain

Info

Publication number: CN114907997A
Application number: CN202110176223.3A
Authority: CN
Inventors: 张学礼; 许丽萍; 樊飞宇; 戴住波; 陈晶; 刘萍萍; 李清艳
Original assignee: Tianjin Institute of Industrial Biotechnology of CAS
Current assignee: Tianjin Institute of Industrial Biotechnology of CAS
Priority date: 2021-02-07
Filing date: 2021-02-07
Publication date: 2022-08-16
Anticipated expiration: 2041-02-07
Also published as: CN114907997B

Abstract

The invention discloses construction and application of a diosgenin synthesis strain. The invention provides a recombinant bacterium, which is obtained by carrying out the following 1) transformation in a chassis yeast containing a relevant gene of a diosgenin synthesis path: 1) allowing the saccharomyces cerevisiae with the chassis to express a steroid 22-hydroxylase gene and improving the expression of an SvvCPR gene in the genome of the saccharomyces cerevisiae with the chassis; experiments prove that the invention perfects the heterogenous synthetic route of the diosgenin and obviously improves the heterogenous production of the diosgenin by comparing the activities of key enzymes from different sources. In addition, the bacterial strain is modified by a metabolic engineering method to obtain the diosgenin artificial cell factory. Finally, the yield of the diosgenin in a 5L tank reaches 1.3g/L, which is the highest yield reported at present, and provides a good reference case for the heterologous synthesis of other steroid hormone medicaments.

Description

Construction and application of diosgenin synthesis strain

Technical Field

The invention belongs to the technical field of biology, and relates to construction and application of a diosgenin synthesis strain.

Background

Steroids, which are widely distributed in nature, are an important component of cell membranes, such as cholesterol, which is common in animals, campesterol, sitosterol and stigmasterol in plants, and ergosterol in lower eukaryotes, such as saccharomyces cerevisiae, which play a vital role in the survival of animals and plants. In addition, the steroid compounds can further synthesize other steroid hormones in organisms, such as progesterone, vitamin D3 and the like. Steroid hormones, also called steroid hormones, are endogenous medicines of human bodies and have irreplaceable effects on maintaining the health of the human bodies. Steroid drugs are interesting molecules, and have various pharmacological activities due to the structural diversity, so that the steroid drugs are mainly applied to the aspects of medicine and health care, have strong pharmacological effects of resisting infection, allergy, virus and shock, and can improve protein metabolism, recover and enhance physical strength, promote urination and reduce blood pressure. According to statistics, the sales of steroid hormone medicines is higher than 280 hundred million dollars in 2011, which accounts for about 6 percent of the total sales of medicines in the world; steroid hormone drugs have been sold for over $ 1000 billion in 2016, and are second only to antibiotics in the second main group.

Diosgenin (Diosgenin), also called Diosgenin, has a structure shown in fig. 1. Since the discovery of diosgenin (Diosgenia) in the rhizome of the genus dioscorea as a source of steroid hormone drugs in 1940, a great deal of attention has been paid by experts in botany and pharmacology to the study of this genus of plants. Diosgenin has a greater similarity with steroid hormone medicines in structure, is a basic raw material of steroid hormone medicines, has the efficacies of antianaphylaxis, antiviral property, antishock property and the like, and is called as medicinal gold. The water-soluble steroid saponin and the derivatives thereof have various pharmacological activities such as gastric mucosa protection, anti-inflammation, anti-cancer and the like. With the ever-expanding market demand of steroid hormones, the biosynthesis of diosgenin, a precursor thereof, is receiving wide attention, and the current synthesis process of diosgenin is mainly a traditional acidolysis method. The diosgenin is finally obtained by the steps of crushing, acidolysis, rinsing, extraction, crystallization and the like of the plant turmeric. Due to the factors of uncertainty and long period (more than 2 years) of the planting state of the turmeric, serious pollution of the extraction process and the like, the supply and price of the dioscin have large fluctuation, an efficient, clean and stable steroid compound production mode is expected, and if the conversion from plant extraction to biosynthesis can be realized, the dioscin production in China can be greatly promoted, and the development of steroid hormone industry in China is promoted.

Disclosure of Invention

An object of the present invention is to provide a recombinant bacterium.

The recombinant bacterium provided by the invention is obtained by carrying out the following 1) transformation in the base yeast containing the relevant gene of the dioscin synthetic path:

1) allowing the saccharomyces cerevisiae with the chassis to express a steroid 22 hydroxylase gene;

or allowing the saccharomyces cerevisiae chassis to express a steroid 22-hydroxylase gene and increasing the expression of a nicotinamide adenine dinucleotide phosphate-cytochrome P450 reductase SvvCPR gene in the genome of the saccharomyces cerevisiae chassis;

the dioscin synthetic path related gene is 3-hydroxy-3-methylglutaryl coenzyme A reductase 1 gene tHMG1, alpha-aminooxalate reductase gene Lys2, mevalonate kinase gene ERG12, isopentenyl pyrophosphate isomerase IDI1, mevalonate decarboxylase ERG19, (derived from Ruje) 3-hydroxy-3-methylglutaryl coenzyme A reductase HMGR, 3-hydroxy-3-methylglutaryl coenzyme A ERG13, mevalonate kinase ERG8, acetyl coenzyme A acyltransferase ERG10, squalene synthase AtSQS derived from Arabidopsis thaliana, farnesyl pyrophosphate synthase SmFPS derived from Salvia miltiorrhiza, squalene epoxidase ERG1, liraginose derived sterol 26-position hydroxylase VcCYP94N, grape-derived nicotinamide adenine dinucleotide phosphate-cytochrome P450 reductase SvCPR CPR, dioscorea origin sterol 16, 22 dihydroxy oxidase DGCYP90G, potato-derived sterol 7-site reductase StDWF5 and/or raw chicken-derived sterol 24-site reductase GgDHCR 24.

In the recombinant bacteria, the steroid 22-hydroxylase is derived from rhizoma et radix Veratri, Arabidopsis thaliana, tomato or dioscorea zingiberensis.

In the recombinant bacterium, the steroid 22-hydroxylase is any one of the following 1) -3):

1) a protein encoded by the nucleotide shown in the 482-1937 position of the sequence 19 in the sequence table;

2) protein derived from the protein with the same function as 1) obtained by substituting and/or deleting and/or adding 1) one or more amino acid residues;

3) a protein having 95% or more identity to 1) and having the same function as 1).

In the recombinant bacteria, the recombinant bacteria are obtained by modifying the 1) and the following 2) in the base yeast containing the relevant genes of the diosgenin synthesis path:

2) improving the expression of sterol 26-hydroxylase VcCYP94N gene (a chenopodium album derived enzyme) gene, sterol 16, 22-dioxygenase gene DGCYP90G dioscorea zingiberensis derived and the steroid 22-hydroxylase in the genome of the saccharomyces cerevisiae with the chassis.

In the recombinant bacteria, the expression of sterol 26-hydroxylase gene VcCYP94N (a chenopodium quinoa-derived enzyme) gene, sterol 16, 22-dioxygenase gene DGCYP90G dioscorea zingiberensis-derived gene and the steroid 22-hydroxylase in the genome of the saccharomyces cerevisiae with the improved chassis is obtained by integrating sterol 26-reductase VcCYP94N gene, sterol 16, 22-dioxygenase gene DGCYP90G and the steroid 22-hydroxylase gene expression cassette into the genome of the saccharomyces cerevisiae containing tHMG1 and Lys2 genes;

the VcCYP94N gene comprises a TEF1 promoter, a VcCYP94N gene derived from veratrum nigrum and a CYC1 terminator;

the DGCYP90G expression cassette comprises a TDH3 promoter, a dioscorea zingiberensis-derived DGCYP90G gene and a TPI1 terminator;

the expression cassette of the steroid 22-position hydroxylase gene expression cassette comprises a PGK1 promoter, a yangtao uniflower-derived VcCYP90B27 gene and an ADH1 terminator.

In the recombinant bacteria, the recombinant bacteria are obtained by carrying out the transformation of 1), 2) and 3) in the chassis yeast containing relevant genes of the diosgenin synthesis path:

3) the DGCYP90G gene is expressed in the saccharomyces cerevisiae with high copy in a plasmid form, and specifically pRS426-URA3-pTEF1-DGCYP90G-tCYC1 plasmid is transferred.

In the recombinant strain, the saccharomyces cerevisiae on the chassis is obtained by modifying according to at least one of the following A) -D):

A) increasing the expression of tmg 1, mevalonate kinase ERG12, isopentenyl pyrophosphate isomerase IDI1, mevalonate decarboxylase ERG19, 3-hydroxy-3-methylglutaryl coa reductase hmg, 3-hydroxy-3-methylglutaryl coa ERG13, mevalonate kinase ERG8 and acetyl coa acyl transferase ERG10 in the s.cerevisiae genome containing the 3-hydroxy-3-methylglutaryl coa reductase 1 gene tmg 1 and the α -aminooxalate reductase gene Lys 2;

B) improving the expression of squalene synthase AtSQS, farnesyl pyrophosphate synthase SmFPS and squalene epoxidase ERG1 in the saccharomyces cerevisiae genome containing 3-hydroxy-3-methylglutaryl coenzyme A reductase 1 gene tHMG1 and alpha-aminooxalate reductase gene Lys 2;

C) allowing the saccharomyces cerevisiae genome containing the 3-hydroxy-3-methylglutaryl coenzyme A reductase 1 gene tHMG1 and the alpha-aminooxalate reductase gene Lys2 to express sterol 26 hydroxylase VcCYP94N, nicotinamide adenine dinucleotide phosphate-cytochrome P450 reductase SvvCPR, sterol 16, 22 dihydroxy oxidase DGCYP90G, sterol 7 reductase StDWF5 and/or sterol 24 reductase GgDHCR 24;

D) knocking out alcohol acetylase ATF2 gene in the saccharomyces cerevisiae genome containing tHMG1 and Lys2 genes.

In the recombinant bacterium B, the Saccharomyces cerevisiae genome containing tHMG1 and Lys2 genes is expressed by VcCYP94N, SvvCPR, DGCYP90G, StDWF5 and/or GgDHCR24, and the expression cassettes of VcCYP94N, SvvCPR, DGCYP90G, StDWF5 and/or GgDHCR24 are integrated into the Saccharomyces cerevisiae genome containing the tHMG1 and Lys2 genes;

the VcCYP94N expression cassette comprises a PGK1 promoter, a VcCYP94N gene derived from veratrum nigrum and an ADH1 terminator;

the SvvCPR expression cassette comprises a TDH3 promoter, a grape-derived SvvCPR gene and a TPI1 terminator;

the DGCYP90G expression cassette comprises a TEF1 promoter, a dioscorea zingiberensis-derived DGCYP90G gene and a CYC1 terminator;

the StDWF5 expression cassette comprises a PGK1 promoter, a potato-derived StDWF5 gene and an ADH1 terminator;

the GgDHCR24 expression cassette comprises a TEF1 promoter, a chick-derived GgDHCR24 gene and a CYC1 terminator.

It is another object of the invention to provide a method.

The method for preparing the first target recombinant bacterium provided by the invention is prepared according to a transformation method in the recombinant bacterium.

The recombinant bacterium obtained by the method is also within the protection scope of the invention.

The application of the recombinant bacterium or the recombinant bacterium obtained by the method in producing diosgenin or improving the yield of diosgenin is also within the protection scope of the invention;

the invention also provides a method for producing diosgenin, which comprises the following steps: fermenting and culturing the recombinant bacteria to obtain the diosgenin.

Experiments prove that the invention perfects the heterogenous synthesis path of the diosgenin and obviously improves the heterogenous production of the diosgenin by comparing the activities of key enzymes from different sources. In addition, the bacterial strain is modified by a metabolic engineering method to obtain the diosgenin artificial cell factory. Finally, the yield of the diosgenin in a 5L tank reaches 1.3g/L, which is the highest yield reported at present, and provides a good reference case for the heterologous synthesis of other steroid hormone medicaments.

Drawings

Fig. 1 is the structural formula of diosgenin.

FIG. 2 shows GC-MS detection analysis of fermentation products of strain LP-074.

FIG. 3 shows the yield of diosgenin in the LP-085 series of strains.

FIG. 4 shows the yield of diosgenin.

FIG. 5 is a graph of LP-BC fed-batch fermentation.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The Saccharomyces cerevisiae BY-T1 strain (described in Chinese patent 201210453416.X) in the following examples, which contains tHMG1 (3-hydroxy-3-methylglutaryl coenzyme A reductase 1 derived from a part of Saccharomyces cerevisiae) and Lys2 (alpha-aminooxalate reductase), was obtained from Tianjin Industrial Biotechnology research institute and can synthesize squalene with higher yield.

Example 1 getting through the pathway for the synthesis of Saccharomyces cerevisiae dioscin

Firstly, constructing a strain BY-T5 and increasing MVA path flux

1. Construction of gRNA plasmid of endogenous Leu2 gene of Saccharomyces cerevisiae

PCR amplification was first performed using the primers 43803-up and 43803-Leu2gRNA-down1 (see Table 1) using the plasmid p426-SNR52p-gRNA. CAN. Y-SUP4t (#43803, purchased from Addgene, containing cas9 binding region) as template.

The amplification system is TAKARA

10. mu.l of HS DNA polymerase, 10. mu.l of Dntp mix, 4. mu.l of each primer (see Table 1), 0.5. mu.l of template, 0.5. mu.l of Primerstar HS polymerase (2.5U/. mu.L), and distilled water to a total volume of 50. mu.l.

Amplification conditions were 98 ℃ pre-denaturation for 2min (1 cycle); denaturation at 98 ℃ for 10 seconds, annealing at 56 ℃ for 15 seconds, and extension at 72 ℃ for 5 minutes (30 cycles); extension at 72 ℃ for 8 min (1 cycle).

And (3) carrying out Dpn1 digestion treatment on the PCR product obtained by amplification, wherein the Dpn1 treatment system is as follows: mu.L of 10 XDpn 1 Buffer (Thermo Co.), 5. mu.L Dpn1(Therom Co., 400,000 chemical end units/ml), 100. mu.L of PCR amplification product, digestion treatment for 4 hours, and gel recovery treatment of the treated product for use.

The digest obtained after gel recovery was transferred to Trans1-T1 competent cells in an ice bath for 30 min, heat-shocked at 42 ℃ for 30 sec, and immediately placed on ice for 2 min. Adding 800 mul LB culture medium, incubating at 250rpm and 37 ℃ for 1 hour, coating the bacterial liquid on LB plate containing ampicillin, directly selecting two monoclonal plasmids to carry out sequencing verification after overnight culture, and the sequencing result shows that the correct plasmids

Named pLeu2 gRNA.

Table 1 shows gRNA plasmid primers for constructing Leu2, NDT80, ATF2 and Gal80 sites

The gRNA sequences of N20 corresponding to each gene are bolded in the table above. .

2. Construction of homologous recombination fragments

Yeast endogenous genes tHMG1 (3-hydroxy-3-methylglutaryl coenzyme A reductase 1 from a part of Saccharomyces cerevisiae), ERG12 (mevalonate kinase of Saccharomyces cerevisiae), IDI1 (isopentenyl pyrophosphate isomerase of Saccharomyces cerevisiae), ERG19 (mevalonate decarboxylase of Saccharomyces cerevisiae), ERG13 (3-hydroxy-3-methylglutaryl coenzyme A of Saccharomyces cerevisiae), ERG8 (phosphomevalonate kinase of Saccharomyces cerevisiae), ERG10 (acetyl coenzyme A acyltransferase of Saccharomyces cerevisiae) were amplified from the genome of Saccharomyces cerevisiae BY4742(Saccharomyces cerevisiae BY4742, described in Carrier baker brachhmann et al, 1998, Yeast,14: 115-fold 132, publicly available from the institute of Biotechnology in Tianjin) strain BY an upstream primer with a SexA I cleavage site and a downstream primer with an Asc I cleavage site (amplification primers are shown in Table 2).

The amplification system is TAKARA

10. mu.l of HS DNA polymerase, 10. mu.l of Dntp mix, 4. mu.l of each primer (see Table 2), 0.5. mu.l of template, 0.5. mu.l of Primerstar HS polymerase (2.5U/. mu.L), and distilled water to a total volume of 50. mu.l.

Amplification conditions were 98 ℃ pre-denaturation for 2min (1 cycle); denaturation at 98 ℃ for 10 seconds, annealing at 56 ℃ for 15 seconds, and extension at 72 ℃ for 2 minutes (30 cycles); extension at 72 ℃ for 8 min (1 cycle). And (5) recovering and treating the glue for later use.

TABLE 2 Saccharomyces cerevisiae MVA pathway gene amplification primers

Primer name	Sequence (5 '-3')
		SexAI-tHMG1-F	ACCTGGTAAAACAATGGCTGCAGACCAATTG
AscI-tHMG1-R	GGCGCGCCTTAGGATTTAATGCAGGTGACGGA
		SexAI-ERG12-F	ACCTGGTAAAACAATGTCATTACCGTTCTTAACTTCTG
AscI-ERG12-R	GGCGCGCCTTAGGATTTATGAAGTCCATGGTAAATTCGTGTTT
		SexAI-IDI1-F	ACCTGGTAAAACAATGACTGCCGACAACAATAGTA
AscI-IDI1-R	GGCGCGCCTTAGGATTTATAGCATTCTATGAATTTGCCTGTCAT
		SexAI-ERG19-F	ACCTGGTAAAACAATGACCGTTTACACAGCATC
AscI-ERG19-R	GGCGCGCCTTAGGATTTATTCCTTTGGTAGACCAGTCTTTGC
		SexAI-ERG13-F	ACCTGGTAAAACAATGAAACTCTCAACTAAACTTTGTTGG
AscI-ERG13-R	GGCGCGCCTTATTTTTTAACATCGTAAGATCTTCTAAATTTGT
		SexAI-ERG8-F	ACCTGGTAAAACAATGTCAGAGTTGAGAGCCTT
AscI-ERG8-R	GGCGCGCCTTATTTATCAAGATAAGTTTCCGGATCTTTTTCT
		SexAI-ERG10-F	ACCTGGTAAAACAATGTCTCAGAACGTTTACATTGTAT
AscI-ERG10-R	TGCTTCCTCTATTGTCATTGAAAAGATATGAGGCGCGCC

The sequence of the exogenous gene HMGR-N (derived from 3-hydroxy-3-methylglutaryl coenzyme A reductase of the genus Rujie) is optimized according to the codon preference of the saccharomyces cerevisiae and is synthesized by the Kinsley company. When synthesizing the gene, a SexA I enzyme cutting site is introduced at the 5 'end, and an Asc I enzyme cutting site is introduced at the 3' end. The obtained total gene synthetic product and the 7 PCR amplified fragments are subjected to double enzyme digestion by using SexA I and Asc I of Thermo company, and simultaneously, the enzyme digestion products of plasmids M2, M9, M16, M5, M7, M8, M4 and M3 (described in Chinese patent 201210453416.X) are recovered for later use.

Plasmids M2-tHMG1, M9-ERG12, M16-IDI1, M5-ERG19, M7-HMGR-N, M8-ERG13, M4-ERG8 and M3-ERG10 are obtained by ligation. A connection system: fragments and scaffold were 50ng each, 5. mu.L of 2 XQuick ligation Buffer (NEB), 0.5. mu.L of Quick ligation (NEB, 400,000 covalent end units/ml), supplemented with distilled water to 10. mu.L, reacted at room temperature for 10min to give a ligation product, transferred to Trans1-T1 competent cells, incubated in ice for 30 min, heat-shocked at 42 ℃ for 30 sec, and immediately placed on ice for 2 min. Adding 800 mu.l LB culture medium, incubating at 250rpm and 37 ℃ for 1 hour, smearing the bacterial liquid on LB plate containing ampicillin, after overnight culture, PCR screening 5 positive single colonies, and verifying the correct single colony sequencing to obtain plasmids M2-tHMG1, M9-ERG12, M16-IDI1, M5-ERG19, M7-HMGR-N, M8-ERG13, M4-ERG8 and M3-ERG 10.

PCR amplification was carried out using Leu2-50-pPGK1-F and S-7G-1-M-tADH1-pPDC1-R (see Table 3) using the constructed plasmid M2-tHMG1 as a template (the same procedure as in step 1) to obtain Leu2-50-pPGK1-tHMG1-tADH1-pPDC1-50 fragment (SEQ ID NO: 1) comprising the PGK1 promoter (position 51-801 of SEQ ID NO: 1), the Saccharomyces cerevisiae endogenous truncated tHMG1 (position 802-2386 of SEQ ID NO: 1) and the ADH1 terminator (position 2387-2545 of SEQ ID NO: 1).

PCR amplification was carried out using the constructed plasmid M9-ERG12 as a template and using primers S-7G-1-M-tADH1-pPDC1-F and 3G-1-M-tADH2-pENO2-R (see Table 3) (the same procedure as in step 1) to obtain a fragment of tADH1-50-pPDC1-ERG12-tADH2-pENO2-50 (SEQ ID NO: 2) comprising a PDC1 promoter (position 51-851 of SEQ ID NO: 2), an endogenous ERG12 gene of Saccharomyces cerevisiae (position 852-2184 of SEQ ID NO: 2) and an ADH2 terminator (position 2185-2585 of SEQ ID NO: 2).

PCR amplification was carried out using the constructed plasmid M16-IDI1 as a template and using primers 3G-2-M-tADH2-pENO2-F and 3G-2-M-tPDC1-pPYK1-R (see Table 3) (the same procedure as in step 1) to obtain a tADH2-50-pENO2-IDI1-tPDC1-pPYK1-50 fragment (SEQ ID NO: 3) comprising the ENO2 promoter (position 51-1051 of SEQ ID NO: 3), the Saccharomyces cerevisiae endogenous IDI1 gene (position 1052-1949 of SEQ ID NO: 3) and the PDC1 terminator (position 1950-2350 of SEQ ID NO: 3).

PCR amplification was performed using the constructed plasmid M5-ERG19 as a template and using primers 3G-3-M-tPDC1-pPYK1-F and S-8G-1-M-tPGGI 1-pTEF2-R (see Table 3) (the method is the same as step 1), and a tPDC1-50-pPYK1-ERG 19-tPGGI 1-pTEF2-50 fragment (SEQ ID NO: 4) was obtained, which fragment contains the PYK1 promoter (position 51-1051 of SEQ ID NO: 4), the endogenous Saccharomyces cerevisiae ERG19 gene (position 1052-2243 of SEQ ID NO: 4) and the PGI1 terminator (position 2244-2643 of SEQ ID NO: 4).

PCR amplification was performed using primers S-8G-1-M-tPGI1-pTEF2-F and S-8G-1-M-tENO2-pFBA1-R (see Table 3) using the constructed plasmid M7-HMGR-N as a template (the method is the same as step 1), to obtain tPGI1-50-pTEF2-HMGR-N-tENO2-pFBA1-50 fragment (SEQ ID NO: 5) containing TEF2 promoter (SEQ ID NO: 51-613), HMGR-N gene (SEQ ID NO: 5: 614-1916) and ENO2 terminator (SEQ ID NO: 5: 1917-2317).

PCR amplification was performed using the constructed plasmid M8-ERG13 as a template and primers S-8G-1-M-tENO2-pFBA1-F and S-4G-4-M-tTDH2-pTDH3-R (see Table 3) (the method is the same as step 1), and a tENO 2-50-pFBBA 1-ERG13-tTDH2-pTDH3-50 fragment (SEQ ID NO: 6) was obtained, which comprises the FBA1 promoter (position 51-873 of SEQ ID NO: 6), the Saccharomyces cerevisiae endogenous ERG13 gene (position 874-2299 of SEQ ID NO: 6) and the TDH2 terminator (position 2300-2701 of SEQ ID NO: 6).

PCR amplification was performed using the constructed plasmid M4-ERG8 as a template and primers S-4G-3-M-tTDH2-pTDH3-F and 3G-3-M-tTPI1-pTEF1-R (see Table 3) (the same procedure as in step 1) to obtain a tTDH2-50-pTDH3-ERG8-tTPI1-pTEF1-50 fragment (SEQ ID NO: 7) comprising the TDH3 promoter (position 51-851 of SEQ ID NO: 7), the Saccharomyces cerevisiae endogenous ERG8 gene (position 852-2208 of SEQ ID NO: 7) and TPI1A terminator (at positions 2209-2609 of SEQ ID NO: 7).

PCR amplification was performed using the constructed plasmid M3-ERG10 as a template and primers 3G-2-M-tTPI1-pTEF1-F and Leu2-50-tCYC1-R (see Table 3) (the same procedure as step 1) to obtain a tTPI1-50-pTEF1-ERG10-tCYC1-Leu2-50 fragment (SEQ ID NO: 8) comprising a TEF1 promoter (position 51-454 of SEQ ID NO: 8), an endogenous ERG10 gene of Saccharomyces cerevisiae (position 455-1652 of SEQ ID NO: 8) and a CYC1 terminator (position 1653-1960 of SEQ ID NO: 8).

TABLE 3 primers for homologous recombination fragments for amplifying Leu sites

3. Transformation of Cas9 plasmid

The strain BY-T1 (described in Chinese patent 201210453416.X) was used, SD-Ura-His-Leu-Trp (Beijing Pankeno technology Co., Ltd.), 2% glucose, 0.005% His., 0.01% Leu, 0.01% Ura, 0.01% Trp (each percentage indicates g/100 mL). 1mL (OD: about 0.6-1.0) was taken and dispensed into 1.5mL EP tubes, centrifuged at 10000g for 1min at 4 ℃ and the supernatant was discarded, the precipitate was washed with sterile water (4 ℃), centrifuged under the same conditions and the supernatant discarded. The cells were incubated at 25 ℃ for 20min with 1mL of a treatment solution (10mM LiAc; 10mM DTT; 0.6M sorbitol; 10mM Tris-HCl (pH7.5) added thereto, and the treatment solution was used. After centrifugation, the supernatant was discarded, 1mL of 1M sorbitol (0.22 μ M aqueous membrane filtration sterilization) was added to the cells for resuspension, and the cells were centrifuged, and the supernatant was discarded (resuspended twice with 1M sorbitol) to a final volume of about 80. mu.L. Cas9 plasmid p414-TEF1p-Cas9-CYC1t (#43802 purchased from Addgene)1 uL is added, the mixture is transferred to an electric transfer cup after being mixed evenly, electric shock is carried out for 5.6ms at 2.7kv, 1mL of 1M sorbitol is added, the mixture is revived at 30 ℃ for 1h, and the mixture is coated on a screening medium plate (formula: 0.8% yeast selection medium SD-Ura-His-Leu-Trp, 2% glucose, 0.005% His, 0.01% Leu, 0.01% Ura, 1.5% agar, and each percentage number represents g/100 mL). The conditions of the screening culture are as follows: culturing at 30 deg.C for 36 hr or more.

Optionally selecting a strain named as strain BY-T1(Cas9), wherein the strain is a recombinant strain obtained BY transferring plasmid p414-TEF1p-Cas9-CYC1T into Saccharomyces cerevisiae BY-T1.

4. Co-transformation of gRNA plasmids and gene homologous recombination fragments

BY-T1(Cas9) was cultured overnight in screening medium. The screening medium consisted of: SD-Trp (beijing pan kino (functional genome) science and technology ltd), 2% glucose, 0.005% his, 0.01% leu, 0.01% Ura (each percentage number indicates g/100 mL). Preparing saccharomyces cerevisiae competence (the method is the same as the step 3), adding pLeu2gRNA plasmid and 2 muL of each of eight homologous recombination fragments into prepared By-T1(Cas9) competent cells, uniformly mixing, transferring the mixture into an electric transfer cup, shocking BY 2.7kv for 5.6ms, adding 1mL of 1M sorbitol, recovering at 30 ℃ for 1h, and coating the mixture on a screening medium plate (the formula: 0.8% of yeast selection medium SD-Ura-His-Leu-Trp, 2% of glucose, 0.005% of His., 0.01% of Leu., 1.5% of agar, wherein each percentage number represents g/100 mL). The conditions of the screening culture are as follows: culturing at 30 deg.C for 36 hr or more. PCR identified the correct positive clone, designated strain BY-T5(Cas 9).

The strain BY-T5 is a recombinant strain which is obtained BY replacing a Leu2 Gene (Gene ID:850342, updated on 14-Jan-2021) in a BY-T1(Cas9) genome with a pPGK1-tHMG1-tADH1-pPDC1-ERG12-tADH2-pENO2-IDI 1-tPMDC 1-pPYK1-ERG19-tPGI1-pTE F2-HMGR-N-tENO2-pFBA1-ERG13-tTDH2-pTDH3-ERG8-tTPI1-pTEF1-ERG10-tCYC1 (nucleotide sequences are composed of sequence 51-2545, sequence 51-2585, sequence 2 51-2585, sequence 3 51-2600, sequence 4 51-2643, sequence 5, sequence 51-1967, sequence 51-2317, sequence 2706-2317, sequence 2311-51-2318 and sequence 51-2358 of the number of the bacterium, and a recombinant strain.

5. gRNA plasmid elimination

BYT5 was streaked onto three zones on screening media plates. The screening medium consisted of: 5-FOA-Trp (Beijing Pankeno (functional genome) science and technology Co., Ltd.), 2% glucose 0.005% 5-FOA, 0.005% His, 0.01% Leu, 0.01% Ura.1.5% agar (each percentage indicates g/100 mL). After single colonies grew out on the plates, they were screened back on SD-Trp (Beijing Pankeno (functional genome) technologies, Ltd.), 2% glucose, 0.005% His, 0.01% Leu, 0.01% Ura (each percentage indicates g/100mL) and SD-Trp-URA (formulation: 0.8% yeast selection medium SD-Ura-His-Leu-Trp, 2% glucose, 0.005% His, 0.01% Leu, 1.5% agar; each percentage indicates g/100mL), and strains which could not grow on SD-Trp-URA plates and could grow on SD-Trp were selected for use.

Secondly, constructing a strain BY-T30 to increase the yield of lanosterol

1. Construction of gRNA plasmid of endogenous NDT80 gene of saccharomyces cerevisiae

PCR amplification was first performed using the primers 43803-up and 43803-NDT80gRNA-down1 (see Table 1) using the plasmid p426-SNR52p-gRNA. CAN. Y-SUP4t (#43803, purchased from Addgene, containing cas9 binding region) as template.

The amplification system is TAKARA

And (3) carrying out Dpn1 digestion treatment on the PCR product obtained by amplification, wherein the Dpn1 treatment system is as follows: 10. mu.L of 10 XDpn 1 Buffer (Thermo Co.), 5. mu.L of Dpn1(Therom Co., 400,000 covalent end units/ml), 100. mu.L of the PCR amplification product, digestion treatment for 4 hours, and subsequent gel recovery treatment of the treated product for use.

The digest obtained after the gel recovery was transferred to Trans1-T1 competent cells in an ice bath for 30 minutes, heat-shocked at 42 ℃ for 30 seconds and immediately placed on ice for 2 minutes. Adding 800 mul LB culture medium, incubating at 250rpm and 37 ℃ for 1 hour, coating the bacterial liquid on LB plate containing ampicillin, directly selecting two monoclonal plasmids for sequencing verification after overnight culture, and the sequencing result shows that the plasmid is correct

The plasmid of (3) was designated pNDT80 gRNA.

2. Construction of homologous recombination fragments

The yeast endogenous gene ERG1 (squalene epoxidase from Saccharomyces cerevisiae) was amplified from the genome of Saccharomyces cerevisiae BY4742 strain BY an upstream primer with SexA I cleavage site and a downstream primer with Asc I cleavage site (shown in Table 4).

The amplification system is TAKARA

10. mu.l of HS DNA polymerase, 10. mu.l of Dntp mix, 4. mu.l of each primer (see Table 4), 0.5. mu.l of template, 0.5. mu.l of PrimerSTAR HS polymerase (2.5U/. mu.L), and distilled water were added to a total volume of 50. mu.l.

TABLE 4 Saccharomyces cerevisiae ERG1 gene amplification primers

Name of primer	Sequence (5 '-3')
		SexAI-ERG1-F	ACCTGGTAAAACAATGTCTGCTGTTAACGTTGCA
AscI-ERG1-R	GGCGCGCCTTAACCAATCAACTCACCAAACAAAAATG

The sequences of the foreign genes AtSQS (squalene synthase from Arabidopsis thaliana) and SmFPS (farnesyl pyrophosphate synthase from Salvia miltiorrhiza) were optimized according to the codon preference of Saccharomyces cerevisiae, and were synthesized by Kinseri. When synthesizing the gene, a SexA I enzyme cutting site is introduced at the 5 'end, and an Asc I enzyme cutting site is introduced at the 3' end. The obtained total gene synthetic product and the PCR amplified fragment are subjected to double enzyme digestion by SexA I and Asc I of Thermo company, and simultaneously, the enzyme digestion products of plasmids M2, M3 and M4 (described in Chinese patent 201210453416.X) are recovered for later use.

The plasmids M2-AtSQS, M3-SmFPS and M4-ERG1 are obtained by ligation. A connection system: the fragments and the backbone were each 50ng, 5. mu.L of 2 Xquick ligation Buffer (NEB), 0.5. mu.L of Quick ligation (NEB, 400,000 synergistic end units/ml), supplemented with distilled water to 10. mu.L, reacted at room temperature for 10min to give a ligation product, transferred into Trans1-T1 competent cells, incubated in ice for 30 min, heat-shocked at 42 ℃ for 30 sec, and immediately placed on ice for 2 min. Adding 800. mu.l LB culture medium, incubating at 250rpm and 37 ℃ for 1 hour, coating the bacterial liquid on LB plate containing ampicillin, culturing overnight, PCR screening 5 positive single colonies, verifying the correct single colony sequencing to obtain plasmids M2-AtSQS, M3-SmFPS and M4-ERG 1.

PCR amplification was performed using the constructed plasmid M2-AtSQS as a template and primers NDT80-50-pPGK1-F and 3G-2M-pTDH3-tADH1-R (see Table 5) (the same procedure as in step 1) to obtain an NDT80-50-pPGK1-AtSQS-tADH1-pTDH3-50 fragment (SEQ ID NO: 9) comprising a PGK1 promoter (position 51-801 of SEQ ID NO: 9), an Arabidopsis derived AtSQS (position 802-22035 of SEQ ID NO: 9) and an ADH1 terminator (position 2036-2194 of SEQ ID NO: 9).

PCR amplification was performed using the constructed plasmid M4-ERG1 as a template and using primers 3G-2M-tADH1-pTDH3-F and 3G-2M-pTEF1-tTPI1-R (see Table 5) (the same procedure as step 1) to obtain a tADH1-50-pTDH3-ERG1-tTPI1-pTEF1-50 fragment (sequence 10) comprising a TDH3 promoter (position 51-851 of sequence 10), a Saccharomyces cerevisiae-derived ERG1 gene (position 852-2343 of sequence 10) and a TPI1 terminator (position 2344-2746 of sequence 10). And performing gel recovery treatment on the target fragment obtained by amplification for later use.

PCR amplification was performed using the constructed plasmid M3-SmFPS as a template and using primers 3G-2M-tTPI1-pTEF1-F and NDT80-50-tCYC1-R (see Table 5) (the same procedure as in step 1) to obtain a tTPI1-50-pTEF 1-pTFPS-tCYC 1-NDT80-50 fragment (SEQ ID NO: 11) comprising a TEF1 promoter (position 51-481 of SEQ ID NO: 11), a SmFPS gene derived from Salvia miltiorrhiza (position 482-1532 of SEQ ID NO: 11), and a CYC1 terminator (position 1533-1840 of SEQ ID NO: 11).

Table 5 shows primers for amplifying homologous recombination fragments at NDT80, ATF2 and Gal80 sites

3. Co-transformation of gRNA plasmids and gene homologous recombination fragments

BY-T5 was cultured overnight in selection medium. The screening medium consisted of: SD-Trp (beijing pan keno (functional genome) science and technology ltd), 2% glucose, 0.005% his, 0.01% leu, 0.01% Ura (each percentile represents g/100 mL). Preparing saccharomyces cerevisiae competence (same step 3 of the method), adding pNDT80gRNA plasmid and three homologous recombination fragments (sequence 9 to sequence 11) into prepared BY-T5 competent cells, uniformly mixing, transferring to an electric rotating cup, shocking BY 2.7kv for 5.6ms, adding 1mL of 1M sorbitol, recovering at 30 ℃ for 1h, and coating on a screening medium plate (formula: 0.8% yeast selection medium SD-Ura-His-Leu-Trp, 2% glucose, 0.005% His, 0.01% Leu, 1.5% agar, wherein each percentage represents g/100 mL). The conditions of the screening culture are as follows: culturing at 30 deg.C for 36 hr or more. The correct positive clone was identified BY PCR and was designated strain BY-T30.

The strain BY-T30 is a recombinant bacterium obtained BY replacing NDT80 Gene (Gene ID:856524, updated on 6-Oct-2020) in BY-T5 genome with pPGK1-AtSQS-tADH1-pTDH3-ERG1-tTPI1-pTEF1-SmFPS-tCYC 1(the nucleotide sequence consists of 51 st to 2194 of a sequence 9, 51 st to 2746 of the sequence 10 and 51 st to 1840 of the sequence 11).

4. gRNA plasmid elimination

BYT30 was streaked onto three zones on screening media plates. The screening medium consisted of: 5-FOA-Trp (Beijing Pankeno (functional genome) science and technology Co., Ltd.), 2% glucose 0.005% 5-FOA, 0.005% His., 0.01% Leu., 0.01% Ura.1.5% agar (each percentage indicates g/100 mL). After single colonies grew out on the plates, they were back-screened on SD-Trp (Beijing Pankeno (functional genome) science and technology Co., Ltd.), 2% glucose, 0.005% His, 0.01% Leu, 0.01% Ura (each percentage number indicates g/100mL) and SD-Trp-URA (formulation: 0.8% yeast selection medium SD-Ura-His-Leu-Trp, 2% glucose, 0.005% His, 0.01% Leu, 1.5% agar; each percentage number indicates g/100mL), and strains that could not grow on SD-Trp-URA plates and could grow on SD-Trp were selected for use.

Thirdly, construction of saccharomyces cerevisiae chassis strain LP-034

The alcohol acetylase (ATF2) gene of the saccharomyces cerevisiae can perform acetylation reaction on the C3 position of the steroid compound to form an ester precipitate, so that subsequent reaction cannot be performed. Therefore, in order to avoid the influence of acetylation reaction of steroid C3-hydroxyl on the experiment, a saccharomyces cerevisiae chassis strain LP-034 is constructed by a Crispr-Cas9 method at an ATF2 site by a knocking-up operation:

1. construction of Saccharomyces cerevisiae endogenous ATF2 gene gRNA plasmid

PCR amplification was first performed using the primers 43803-up and 43803-ATF2gRNA-down1 (see Table 1) using the plasmid p426-SNR52p-gRNA. CAN. Y-SUP4t (#43803, purchased from Addgene, containing cas9 binding region) as template.

The amplification system is TAKARA

The digest obtained after gel recovery was transferred to Trans1-T1 competent cells in an ice bath for 30 min, heat-shocked at 42 ℃ for 30 sec, and immediately placed on ice for 2 min. Adding 800 μ l LB medium, incubating at 250rpm and 37 deg.C for 1 hr, spreading the culture solution on LB plate containing ampicillin, culturing overnight, and randomly selecting two monoclonal substancesThe particles are subjected to sequencing verification, and the sequencing result shows that the correct plasmid is obtained

Was designated pATF2 gRNA.

The pATF2gRNA plasmid contains the gRNA sequence of N20 corresponding to the ATF2 gene.

2. Construction of homologous recombination fragments

Exogenous genes VcCYP94N (a 26-position hydroxylase derived from rhizoma et radix Veratri), DGCYP90G (a 16, 22-dihydroxyoxidase derived from rhizoma Dioscoreae Zingiberensis), and SvvCPR (a phosphate-cytochrome P450 reductase derived from glucose adenine dinucleotide) are related genes for synthesizing diosgenin, and sequences are optimized according to the codon preference of Saccharomyces cerevisiae and synthesized by Kinry corporation. When synthesizing the gene, a SexA I enzyme cutting site is introduced at the 5 'end, and an Asc I enzyme cutting site is introduced at the 3' end.

The obtained whole gene synthetic product is subjected to double enzyme digestion by SexA I and Asc I of Thermo company, and simultaneously SexA I and Asc I are used for enzyme digestion of plasmids M2, M3 and M4, and enzyme digestion product glue is recovered for later use.

50ng of enzyme digestion plasmids M2, M3 and M4 are respectively added into a connection system with 50ng of each of the obtained VcCYP94N, DGCYP90G and SvvCPR gene fragments: mu.L of 2 XQuick ligation Buffer (NEB), 0.5. mu.L of Quick ligation Buffer (NEB, 400,000 covalent end units/ml), distilled water was added to 10. mu.L, and the mixture was reacted at room temperature for 10min to obtain a ligation product, which was transferred to Trans1-T1 competent cells for 30 min in ice bath, heat-shocked at 42 ℃ for 30 sec, and immediately placed on ice for 2 min. Adding 800 mul LB culture medium, incubating at 250rpm and 37 ℃ for 1 hour, smearing bacterial liquid on LB plate containing ampicillin, after overnight culture, PCR screening 5 positive single colonies, verifying the correct single colony sequencing to obtain plasmids M2-VcCYP94N, M3-DGCYP90G and M4-SvvCPR.

PCR amplification was performed using the constructed plasmid M2-VcCYP94N as a template and primers ATF2-50-pPGK1-F and 3G-2M-pTDH3-tADH1-R (see Table 5) (the method is the same as step 1), and an ATF2-50-pPGK1-VcCYP94N-ADH1t-pTDH3-50 fragment (sequence 12) was obtained, which comprises a PGK1 promoter (position 51-801 of sequence 12), a Veratrum nigrum-derived VcCYP94N gene (position 802-2257 of sequence 12) and an ADH1 terminator (position 2258-2416 of sequence 12).

PCR amplification was carried out using the constructed plasmid M3-DGCYP90G as a template and using primers 3G-2M-tTPI1-pTEF1-F and ATF2-50-tCYC1-R (see Table 5) (the same procedure as in step 1) to obtain a pTEF1-DGCYP90G-tCYC1 fragment (SEQ ID NO: 13) comprising a TEF1 promoter (position 51-481 of SEQ ID NO: 13), a Dioscorea zingiberensis-derived DGCYP90G gene (position 482-1949 of SEQ ID NO: 13) and a CYC1 terminator (position 1950-2257 of SEQ ID NO: 13).

PCR was performed using the constructed plasmid M4-SvvCPR as a template and primers 3G-2M-tADH1-pTDH3-F and 3G-2M-pTEF1-tTPI1-R (see Table 5) (the same procedure as in step 1), to obtain a pTDH3-SvvCPR-tTPI1 fragment (SEQ ID NO: 14) comprising the TDH3 promoter (position 51-851 of SEQ ID NO: 14), the grape-derived SvvCPR gene (position 852-2964 of SEQ ID NO: 14) and the TPI1 terminator (position 2965-3367 of SEQ ID NO: 14). And performing gel recovery treatment on the target fragment obtained by amplification for later use.

BY-T30 was grown overnight in selection medium. The screening medium consisted of: SD-Trp (beijing pan kino (functional genome) science and technology ltd), 2% glucose, 0.005% his, 0.01% leu, 0.01% Ura (each percentage number indicates g/100 mL). Preparation of Saccharomyces cerevisiae competence (method same as step 3) to the prepared BY-T30 competent cells, pATF2gRNA plasmid (containing Cas9 binding region) and three homologous recombination fragments (sequence 12 to sequence 14) each 2. mu.L were added, mixed well, transferred to an electric cuvette, shocked at 2.7kv for 5.6ms, added with 1mL of sorbitol, revived at 30 ℃ for 1h, and spread on a screening medium plate (formulation: 0.8% yeast selection medium SD-Ura-His-Leu-Trp, 2% glucose, 0.005% His, 0.01% Leu, 1.5% agar, each percentage number indicates g/100 mL). The conditions of the screening culture are as follows: culturing at 30 deg.C for 36 hr or more. The correct positive clone was identified by PCR and was designated strain LP-034.

The strain LP-034 is a recombinant bacterium obtained by replacing an ATF2 Gene (Gene ID:853088, updated on 21-Mar-2020) in a BYT30 genome with pPGK1-VcCYP94N-tADH1-pTDH3-SvvCPR-tTPI1-TEF1-DGCYP90G-tCYC1 fragment (a nucleotide sequence sequentially consists of 51 th to 2416 th nucleotides of a sequence 12, 51 th to 2964 th nucleotides of a sequence 14 and 51 th to 2257 th nucleotides of the sequence 13).

Secondly, construction of saccharomyces cerevisiae chassis strain LP-074

1. Introducing cholesterol synthesis gene to break through the synthetic path of dioscin

Exogenous genes StDWF5 (potato-derived sterol 7-site reductase) and GgDHCR24 (chicken-derived sterol 24-site reductase) are enzymes required by cholesterol precursors in a diosgenin synthesis path, sequences are optimized according to the codon preference of saccharomyces cerevisiae, and are synthesized by Kinserin. When synthesizing the gene, a SexA I enzyme cutting site is introduced at the 5 'end, and an Asc I enzyme cutting site is introduced at the 3' end. The obtained whole gene synthetic product is subjected to double enzyme digestion by SexA I and Asc I of Thermo company, and simultaneously, the SexA I and Asc I are used for enzyme digestion of plasmid M2 and enzyme digestion product glue of M3 are recovered for later use. Plasmids M2-StDWF5, M3-GgDHCR24 were obtained (step 1 of the same procedure).

PCR was performed using the constructed plasmid M2-StDWF5 as a template and primers 1M-pEASY-pPGK1-F and 1M-tADH1-pTEF1-R (see Table 6) (step 1 of the same method), to obtain pPGK1-StDWF5-tADH1t fragment (SEQ ID NO: 15) comprising PGK1 promoter (positions 63-813 of SEQ ID NO: 15), potato-derived StDWF5 gene (positions 814-2119 of SEQ ID NO: 15) and ADH1 terminator (position 2120-2278 of SEQ ID NO: 15).

PCR amplification was performed using the constructed plasmid M3-GgDHCR24 as a template using primers 2M-tADH1-pTEF1-F and 2M-tCYC1-pEASY-R (see Table 6) (step 1 of the same method), to obtain a pTEF1-GgDHCR24-tCYC1 fragment (SEQ ID NO: 16) comprising the TEF1 promoter (position 51-481 of SEQ ID NO: 16), the original chicken-derived GgDHCR24 gene (position 482-2033 of SEQ ID NO: 16), and the CYC1 terminator (position 2034-2341 of SEQ ID NO: 16).

Saccharomyces cerevisiae LP-034 competence was prepared (same method as step 3), pPGK1-StDWF5-tADH1 fragment, pTEF1-GgDHCR24-tCYC1 and laboratory-established homologous arm marker fragment TRP-His3-up, TRP-down (described in Chinese patent ZL201210453416. X) were added to the prepared competent cells, each 2. mu.L was mixed, transferred to an electric cuvette, shocked with 2.7kv for 5.7ms, added with 1mL of 1M sorbitol, revived at 30 ℃ for 1h, and spread on a solid screening medium (formulation: yeast selection medium SD-Ura-His-Leu-Trp, 2% glucose, 0.01% Leu., 0.01% Ura, 1.5% agar, each percentage representing g/100 mL). The conditions of the screening culture are as follows: culturing at 30 deg.C for 36 hr or more. The correct positive clone was identified by PCR and was designated strain LP-074.

Through sequencing, the strain LP-074 is a recombinant bacterium obtained by replacing TRP1 Gene (Gene ID:851570, updated on 10-Oct-2020) of saccharomyces cerevisiae LP-034 with His-pPGK1-StDWF5-tADH1-TEF1-GgDHCR24-tCYC1 (nucleotide sequence is composed of His sequence (Gene ID:854377, updated on 6-Oct-2020), nucleotide 63-2278 of sequence 15 and nucleotide 51-2341 of sequence 16), namely StDWF5 and GgDHCR24 Gene segments are integrated at TRP1 site of LP-034.

Table 6 shows StDWF5 and GgDHCR24 gene integration amplification primers

2. Identification of Saccharomyces cerevisiae LP-074 strain product

And (3) shaking flask fermentation: activating a saccharomyces cerevisiae LP-074 strain in a corresponding solid selection medium (formula: solid yeast screening medium SD-Ura-His-Leu-Trp, 2% glucose, 1.5% agar; each percentage number represents g/100 mL); then inoculating the strain in a corresponding liquid selection culture medium (formula: liquid yeast screening culture medium SD-Ura-His-Leu-Trp, 2% glucose; each percentage number represents g/100mL) to prepare a seed solution (30 ℃, 250rpm, 16 h); the original OD 0.1 inoculum sizes were inoculated into 3 bottles of 100mL flasks containing the corresponding liquid selection medium (formulation: Yeast selection Medium SD-Ura-His-Leu-Trp, 2% glucose; each percentage number indicates g/100mL) respectively, and shaking cultured at 30 ℃ and 250rpm for 5 days to obtain fermentation broth.

The product extraction method comprises the following steps: taking 5mL of fermentation liquor in a 15mL centrifuge tube, centrifuging at 11000r/min for 2min, removing the culture medium, washing with sterile water for 2 times, adding 1mL of 3M HCL, boiling for 10min, washing with sterile water for 1 time, resuspending the precipitate with 1mL of 1.5M NaOH-methanol solution, and incubating at 60 ℃ for 6 h. Add 1ml of n-hexane and vortex for 10 min. Taking the upper layer to pass through a 0.22 mu m organic nylon filter membrane for detection and standby.

Gas chromatography detection: an agilent technologies 5975C gas chromatograph was used, equipped with a chromatography column: HP-5ms (30m 0.25mm 0.5 μm). The injection port temperature is 300 ℃, the injection volume is 1 mu L, the flow is not divided, and the solvent is delayed for 5 min; chromatographic conditions are as follows: maintaining at 240 deg.C for 5min, heating at 10 deg.C/min to 300 deg.C, and maintaining at 300 deg.C for 25min, for 36 min.

Gas chromatography-mass spectrometry (GC-MS) detection: a gas chromatograph tandem mass spectrometry (GC-MS) agilent technologies 5975C gas chromatograph and a three-axis insert xl MSD detector equipped with a chromatographic column: HP-5ms (30m 0.25mm 0.5 μm). GC-MS measurement conditions: the injection port temperature is 300 ℃, the injection volume is 1 mu L, the flow is not divided, and the solvent is delayed for 5 min; chromatographic conditions are as follows: maintaining at 240 deg.C for 5min, heating at 10 deg.C/min to 300 deg.C, and maintaining at 300 deg.C for 25min, and totally 36 min. MS conditions: SIM: 69,139,282, and 414.

Diosgenin standards were purchased from alatin for qualitative and quantitative analysis.

The product was detected using an agilent technologies 5975C gas chromatograph.

The yield of diosgenin is shown in figure 2, and trace amount of diosgenin can be produced.

Example 2 identification of key enzyme for heterologous synthesis of diosgenin by Saccharomyces cerevisiae and screening of sterol 22-hydroxylase activity of different sources

Identification of key enzyme for heterogeneously synthesizing diosgenin by saccharomyces cerevisiae

Constructing a saccharomyces cerevisiae chassis strain LP-085-DG by a method of Crispr-Cas 9:

1. construction of gRNA plasmid of endogenous Gal80 gene of Saccharomyces cerevisiae

First, using a plasmid p426-SNR52p-gRNA.CAN.Y-SUP4t as a template, and using primers 43803-up and 43803-Gal80gRNA-down1 (see Table 1), PCR amplification was performed (the same procedure as in step 1 of example 1), and the obtained plasmid was named pGal80 gRNA.

The pGal80gRNA plasmid contains the gRNA sequence of N20 corresponding to the Gal80 gene.

2. Construction of homologous recombination fragments

Exogenous genes SvvCPR (glucose-derived nicotinamide adenine dinucleotide phosphate-cytochrome P450 reductase) and DGCYP059 (dioscorea zingiberensis-derived steroid 22-hydroxylase gene) were optimized in sequence according to the codon preference of Saccharomyces cerevisiae, and synthesized by Kinsley. When synthesizing the gene, a SexA I enzyme cutting site is introduced at the 5 'end, and an Asc I enzyme cutting site is introduced at the 3' end. The obtained whole gene synthetic product is subjected to double enzyme digestion by SexA I and Asc I of Thermo company, and simultaneously, the SexA I and Asc I are used for enzyme digestion of plasmid M2 and enzyme digestion product glue of M3 are recovered for later use. The plasmid M2-SvvCPR, M3-DGCYP059 (procedure same as step 2 of example 1).

PCR was performed using the constructed plasmid M2-SvvCPR as a template and using primers Gal80-50-pPGK1-F and 1M-tADH1-pTEF1-R (see Table 5) (step 1 of example 1), to obtain a pPGK1-SvvCPR-ADH1t fragment (SEQ ID NO: 17) comprising a PGK1 promoter (position 51-801 of SEQ ID NO: 17), a grape-derived SvvCPR gene (position 802-2914 of SEQ ID NO: 17) and an ADH1 terminator (position 2915-3073 of SEQ ID NO: 17).

PCR amplification was performed using the constructed plasmid M3-DGCYP059 as a template using primers 2M-tADH1-pTEF1-F and Gal80-50-tCYC1-R (see Table 5) (the same procedure as in step 1 of example 1), to obtain a pTEF1-DGCYP059-tCYC1 fragment (SEQ ID NO: 18) comprising the TEF1 promoter (position 51-481 of SEQ ID NO: 18), the dioscorea zingiberensis-derived DGCYP059 gene (position 482-1934 of SEQ ID NO: 18), and the CYC1 terminator (position 1935-2242 of SEQ ID NO: 18).

Saccharomyces cerevisiae LP-074 strain competence was prepared (same procedure as example 1, step 3), pGal80gRNA plasmid and two homologous recombination fragments (pPGK1-SvvCPR-ADH1t fragment and pTEF1-DGCYP059-tCYC1 fragment) were added at 2. mu.L each, mixed well and transferred to an electric cuvette, shocked at 2.7kv for 5.6ms, added with 1mL of 1M sorbitol, thawed at 30 ℃ for 1h, spread on screening medium plates (formulation: 0.8% yeast selection medium SD-Ura-His-Leu-Trp, 2% glucose, 0.01% Leu, 1.5% agar, each percentage number indicating g/100 mL). The conditions of the screening culture are as follows: culturing at 30 deg.C for 36 hr or more.

A correct positive clone was identified by PCR and named strain LP-085-DG.

LP-085-DG is a recombinant bacterium obtained by replacing the Gal80 Gene (Gene ID:854954, updated on 14-Jan-2021) in the genome of LP-074 bacterium with pPGK1-SvvCPR-tADH1-pTEF1-DGCYP059-tCYC 1(the nucleotide sequence consists of 51 st to 3073 nd nucleotides in sequence 17 and 51 st to 2242 nd nucleotides in sequence 18).

3. Detection of saccharomyces cerevisiae LP-085-DG fermentation product

The yield of the recombinant strain LP-085-DG diosgenin was determined according to 2 of example 1, and the result is shown in FIG. 3, wherein the yield of the recombinant strain LP-085-DG diosgenin was 3.75mg/l of fermentation broth supernatant; therefore, the externally introduced steroid 22-hydroxylase improves the synthetic pathway of the diosgenin and improves the capability of the saccharomyces cerevisiae for synthesizing the diosgenin.

Screening of steroid 22-hydroxylase activity from different sources

In order to screen suitable steroid 22 hydroxylase, steroid 22 hydroxylase of different sources is introduced by a method of Crispr-Cas9, and saccharomyces cerevisiae chassis strains LP-085-Vc, LP-085-At and LP-085-Sl are constructed:

The same procedure as described in step 1 above gave pGal80gRNA plasmid.

2. Construction of homologous recombination fragments

Sequences of exogenous genes VcCYP90B27, AtDWF4 and SlCYP90B3 are optimized according to the codon preference of saccharomyces cerevisiae and are synthesized by Kinsley. When synthesizing the gene, a SexA I enzyme cutting site is introduced at the 5 'end, and an Asc I enzyme cutting site is introduced at the 3' end. The obtained whole gene synthetic product is subjected to double enzyme digestion by SexA I and Asc I of Thermo company, and meanwhile, the enzyme digestion product glue of SexA I and Asc I enzyme digestion plasmid M3 is recovered for later use. Plasmids M3-VcCYP90B27, M3-AtDWF4 and M3-SlCYP90B3 were obtained (same procedure as in example 1, step 2). PCR amplification is carried out by taking the constructed plasmids M3-VcCYP90B 27M 3-AtDWF4 and M3-SlCYP90B3 as templates and using primers 2M-tADH1-pTEF1-F and Gal80-50-tCYC1t-R (see tables 5 and 6) (the method is the same as the step 1 of the example 1) to obtain a pTEF1-VcCYP90B27-tCYC1 fragment (sequence 19) which comprises a TEF1 promoter (position 51-481 of the sequence 19), a Vera derived VcCYP90B27 gene (position 482-1937 of the sequence 19) and a CYC1 terminator (position 1938-2245 of the sequence 19), a pTEF1-AtDWF4-tCYC1 fragment (sequence 20) which comprises a TEF 5 promoter (position 51-1 of the sequence 20), an AtDWF 4-ATF 4 gene (position 2025) and an Arabidopsis thaliana derived sequence 2022-2025-position 20224-AMT CYC 465 of the sequence). pTEF1-Sl-CYP90B3-tCYC1 fragment (SEQ ID NO: 21), which comprises a TEF1 promoter (position 51-481 of SEQ ID NO: 21), a Veratrum-derived SlCYP90B3 gene (position 482-1955 of SEQ ID NO: 21) and a CYC1 terminator (position 1956-2263 of SEQ ID NO: 21).

Saccharomyces cerevisiae LP-074 strain competence was prepared (same method as example 1, step 3), pGal80gRNA plasmid and 2. mu.L of each of the two homologous recombination fragments were added, mixed well and transferred to an electric cuvette, shocked at 2.7kv for 5.6ms, added with 1mL of 1M sorbitol, revived at 30 ℃ for 1h, and spread on a screening medium plate (formulation: 0.8% yeast selection medium SD-Ura-His-Leu-Trp, 2% glucose, 0.01% Leu., 1.5% agar; each percentage indicates g/100 mL). The conditions of the screening culture are as follows: culturing at 30 deg.C for 36 hr or more. The correct positive clones were identified by PCR and named strains LP-085-Vc (the corresponding 2 homologous fragments were pPGK1-SvvCPR-ADH1t fragment and pTEF1-VcCYP90B27-tCYC1 fragment), LP-085-At (the corresponding 2 homologous fragments were pPGK1-SvvCPR-ADH1t fragment and pTEF 1-DWF 4-tCYC1 fragment) and LP-085-Sl (the corresponding 2 homologous fragments were pPGK 1-SvvCPR-1 t fragment and pTEF1-Sl-CYP90B3-tCYC1 fragment).

LP-085-Vc is a recombinant bacterium obtained by replacing Gal80 Gene (Gene ID:854954, updated on 14-Jan-2021) in the genome of LP-074 bacterium with pPGK1-SvvCPR-tADH1-pTEF1-VcCYP90B27-tCYC 1(the nucleotide sequence consists of 51-3073 th nucleotides in a sequence 17 and 51-2245 th nucleotides in a sequence 19).

LP085-At is a recombinant bacterium obtained by replacing the Gal80 Gene (Gene ID:854954, updated on 14-Jan-2021) in the genome of LP-074 bacterium with pPGK1-SvvCPR-tADH1-pTEF1-AtDWF4-tCYC 1(the nucleotide sequence is composed of 51-3073 th nucleotides of sequence 17 and 51-2332 th nucleotides of sequence 20).

LP-085-Sl is a recombinant bacterium obtained by replacing Gal80 Gene (Gene ID:854954, updated on 14-Jan-2021) in the genome of LP-074 bacterium with pPGK1-SvvCPR-tADH1-pTEF1-SlCYP90B3-tCYC 1(the nucleotide sequence consists of 51-3073 nucleotides in a sequence 17 and 51-2263 nucleotides in a sequence 21).

3. Detection of fermentation products of Saccharomyces cerevisiae LP-085-Vc, LP-085-At and LP-085-Sl

The recombinant strain was tested according to 2 of example 1, and the yield of diosgenin is shown in fig. 3, and it can be seen that the yield of diosgenin of the recombinant strain introduced with VcCYP90B27 is the highest.

Example 3 construction of a high Dioscorea saponin-producing Strain LP-104

Due to the large accumulation of precursor cholesterol, diosgenin production is increased by increasing the diosgenin synthesis module copy number.

Plasmids M2-VcCYP90B27, M3-VcCYP94N and M4-DGCYP90G were constructed (the same procedure as in step 2 of example 1).

PCR amplification was performed using the constructed plasmid M2-VcCYP90B27 as a template and using primers 1M-pEASY-pPGK1-F and 3G-2M-tADH1-pTDH3-R (see tables 5 and 6) (the method is the same as step 1 of example 1) to obtain pPGK1-VcCYP90B27-ADH1t fragment (SEQ ID NO: 22) comprising PGK1 promoter (position 1-750 of SEQ ID NO: 22), Veratrum-derived VcCYP90B27 gene (position 751-236 of SEQ ID NO: 22) and ADH1 terminator (position 2207-2365 of SEQ ID NO: 22).

PCR amplification was performed using the constructed plasmid M3-VcCYP94N as a template and using primers 3G-2M-tTPI1-pTEF1-F and 2M-TCYC1-pEASY-R (see tables 2 and 3) (the method is the same as step 1 of example 1), to obtain a pTEF1-VcCYP94N-tCYC1 fragment (SEQ ID NO: 23) comprising a TEF1 promoter (position 51-481 of SEQ ID NO: 23), a Veratrum album-derived VcCYP94N gene (position 482-2027 of SEQ ID NO: 23) and a CYC1 terminator (position 2028-2335 of SEQ ID NO: 23). PCR amplification was carried out using the constructed plasmid M4-DGCYP90G as a template and using primers 3G-2M-tADH1-pTDH3-F and 3G-2M-pTEF1-tTPI1-R (see Table 5) (the same procedure as in step 1 of example 1) to obtain a pTDH3-DGCYP90G-tCYC1 fragment (SEQ ID NO: 24) comprising the TDH3 promoter (position 51-851 of SEQ ID NO: 24), the dioscorea zingiberensis-derived DGCYP90G gene (position 852-2319 of SEQ ID NO: 24) and the TPI1 terminator (position 2320-2722 of SEQ ID NO: 24). Preparing Saccharomyces cerevisiae LP-085 competence, adding pPGK1-VcCYP90B27-ADH1t fragment, pTDH3-DGCYO90G-tTPI1 fragment, pTEF1-VcCYP94N-tCYC1 fragment, laboratory-existing homologous arm marker fragment rDNA-Leu2-up (sequence 25; the homologous arm fragment comprises 400bp homologous region upstream of rDNA site, Leu2 marker Gene (Gene ID:850342, updated on 14-Jan-2021), PGK promoter 400bp homologous region, rDNA-Leu2-down (sequence 26; the homologous arm fragment comprises ADH1 terminator 200bp homologous region, and rDNA site downstream 300bp homologous region), mixing, transferring into electroporation cuvette, 2.7kv 5.7ms, adding 1mL 1M sorbitol, applying to the cell culture medium with temperature of 30 ℃, inoculating the solid culture medium, and resuscitating, screening and culturing on the medium (Tr-Ura selection Gene), 2% glucose, 1.5% agar; each percentage number represents g/100 mL). The conditions of the screening culture are as follows: culturing at 30 deg.C for 36 hr or more. PCR identified the correct positive clone, designated strain LP-104.

The strain LP-104 is a recombinant bacterium obtained by replacing rDNA (Chinese patent 201210453416.X) in an LP-085-Vc genome with Leu2-pPGK1-VcCYP90B27-tADH1-pTDH3-DGCYP90G-tTPI1-TEF1-VcCYP94N-tCYC 1(the nucleotide sequence consists of 687 th and 1746 th nucleotides of the sequence 25, 1 st to 2365 th nucleotides of the sequence 22, 51 st to 2335 th nucleotides of the sequence 23 and 51 st to 2722 th nucleotides of the sequence 27) to realize that gene segments of the VcCYP90B27, the DGCYP90G and the VcCYP94N are integrated at the rDNA site of the saccharomyces cerevisiae LP-085.

Example 4 overexpression of DGCYP90G using a high copy number plasmid to obtain strain LP-BC

By utilizing the efficient homologous recombination capability of saccharomyces cerevisiae, a high-copy plasmid pRS426-URA3-pTEF1-DGCYP90G-tCYC1 is constructed

1. Obtaining homologous recombination fragments

First, using pRS425 plasmid as a template, PCR amplification was performed using primers 425-F and 425-50-tCYC1-R (see Table 7) (method identity 1) to obtain fragment 425 (SEQ ID NO: 27), which was then recovered and processed for further use.

PCR amplification was performed using Saccharomyces cerevisiae BY4742 genome as template and primers 425-50-URA3-R and pTEF1-50-URA3-F (see Table 7) (1 of the same protocol) to obtain fragment 425-50-URA3-pTEF1-50 (SEQ ID NO: 28), and gel was recovered and processed for further use.

M3-DGCYP90G plasmid is used as a template, primers pTEF1-up-F and tCYC1-down-R (shown in Table 7) are used for PCR amplification (1 of the same method), a fragment pTEF1-DGCYP90G-tCYC1 (sequence 29) is obtained, and the gel is recovered for standby.

2. In vivo homologous recombination of Saccharomyces cerevisiae

Saccharomyces cerevisiae LP-104 strain competence was prepared (same method as example 1, step 3), 2. mu.L of each of the three fragments was added, mixed well, transferred to an electric cuvette, shocked at 2.7kv for 5.6ms, added with 1mL of 1M sorbitol, revived at 30 ℃ for 1h, and spread on a screening medium plate (formulation: 0.8% yeast selection medium SD-Ura-His-Leu-Trp, 2% glucose, 0.01% Leu., 1.5% agar; each percentage number indicates g/100 mL). The conditions of the screening culture are as follows: culturing at 30 deg.C for 36 hr or more. 5 single clones were randomly selected and verified to be 3274bp in correct size with primers 425-50-URA3-R and tCYC 1-down-R. Was named LP-BC.

The recombinant strain LP-BC is obtained by introducing high-copy plasmid pRS426-URA3-pTEF1-DGCYP90G-tCYC1 into Saccharomyces cerevisiae LP-104.

TABLE 7 primers for high copy plasmid construction

3. Detection of fermentation products of Saccharomyces cerevisiae LP-034, LP-074, LP-085-Vc, LP-104, LP-BC (genotypes are shown in Table 8)

The method is the same as step 2 of example 1, and the yield of diosgenin is shown in FIG. 4.

The data show that the yield of the diosgenin of the strain LP-074 is extremely low, and the yield of the diosgenin of the strain LP-085 expressing the VcCYP90B27 derived from the chenopodium quinoa is obviously improved on the basis of the strain LP-074. Further integrating a diosgenin synthesis module at rDNA multicopy sites, screening a LP-104 strain capable of producing 24.6mg/L diosgenin, further integrating high-copy plasmids, and over-expressing DGCYP90G to increase the yield of diosgenin to 32.3 mg/L.

4. Fed-batch fermentation

Specific methods and procedures reference the references [ Paddon C J, Westfall P J, Pitera D J, et al, high-Level Semi-Synthetic Production of the post anti metabolic ingredient [ J ] Nature,2013,496(7446), 528-32 ], or Westfall P J, Pitera D J, Lenihan J R, et al, Production of Amorphodiene in Yeast, and Its Conversion to hydrolytic ingredient, Precursor the anti enzymatic ingredient [ J ]. Proc Natl Acad Sci USA,2012,109(3): E111-8 ], for the final tank of 5 liter fermentation of LP-BC strain diosgenin yield 1.3g/L, final OD600 (final 401: OD 401).

The discovery result shows that the VcCYP90B27 from the chenopodium quinoa has a great help effect on the heterologous production of diosgenin by saccharomyces cerevisiae. In addition, the yield of diosgenin of the LP-BC strain is the highest reported yield, and a solid foundation is laid for biosynthesis of steroid hormones in a one-step method.

Table 8 shows the genotypes of the strains according to the invention

SEQUENCE LISTING

<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences

Construction and application of <120> dioscin synthetic strain

<160> 29

<170> PatentIn version 3.5

<210> 1

<211> 2592

<212> DNA

<213> Artificial sequence

<400> 1

gccaggtgac cacgttggtc aagaaatcac agccgaagcc attaaggttc acgcacagat 60

attataacat ctgcacaata ggcatttgca agaattactc gtgagtaagg aaagagtgag 120

gaactatcgc atacctgcat ttaaagatgc cgatttgggc gcgaatcctt tattttggct 180

tcaccctcat actattatca gggccagaaa aaggaagtgt ttccctcctt cttgaattga 240

tgttaccctc ataaagcacg tggcctctta tcgagaaaga aattaccgtc gctcgtgatt 300

tgtttgcaaa aagaacaaaa ctgaaaaaac ccagacacgc tcgacttcct gtcttcctat 360

tgattgcagc ttccaatttc gtcacacaac aaggtcctag cgacggctca caggttttgt 420

aacaagcaat cgaaggttct ggaatggcgg gaaagggttt agtaccacat gctatgatgc 480

ccactgtgat ctccagagca aagttcgttc gatcgtactg ttactctctc tctttcaaac 540

agaattgtcc gaatcgtgtg acaacaacag cctgttctca cacactcttt tcttctaacc 600

aagggggtgg tttagtttag tagaacctcg tgaaacttac atttacatat atataaactt 660

gcataaattg gtcaatgcaa gaaatacata tttggtcttt tctaattcgt agtttttcaa 720

gttcttagat gctttctttt tctctttttt acagatcatc aaggaagtaa ttatctactt 780

tttacaacaa atataaaaca atggctgcag accaattggt gaaaactgaa gtcaccaaga 840

agtcttttac tgctcctgta caaaaggctt ctacaccagt tttaaccaat aaaacagtca 900

tttctggatc gaaagtcaaa agtttatcat ctgcgcaatc gagctcatca ggaccttcat 960

catctagtga ggaagatgat tcccgcgata ttgaaagctt ggataagaaa atacgtcctt 1020

tagaagaatt agaagcatta ttaagtagtg gaaatacaaa acaattgaag aacaaagagg 1080

tcgctgcctt ggttattcac ggtaagttac ctttgtacgc tttggagaaa aaattaggtg 1140

atactacgag agcggttgcg gtacgtagga aggctctttc aattttggca gaagctcctg 1200

tattagcatc tgatcgttta ccatataaaa attatgacta cgaccgcgta tttggcgctt 1260

gttgtgaaaa tgttataggt tacatgcctt tgcccgttgg tgttataggc cccttggtta 1320

tcgatggtac atcttatcat ataccaatgg caactacaga gggttgtttg gtagcttctg 1380

ccatgcgtgg ctgtaaggca atcaatgctg gcggtggtgc aacaactgtt ttaactaagg 1440

atggtatgac aagaggccca gtagtccgtt tcccaacttt gaaaagatct ggtgcctgta 1500

agatatggtt agactcagaa gagggacaaa acgcaattaa aaaagctttt aactctacat 1560

caagatttgc acgtctgcaa catattcaaa cttgtctagc aggagattta ctcttcatga 1620

gatttagaac aactactggt gacgcaatgg gtatgaatat gatttctaaa ggtgtcgaat 1680

actcattaaa gcaaatggta gaagagtatg gctgggaaga tatggaggtt gtctccgttt 1740

ctggtaacta ctgtaccgac aaaaaaccag ctgccatcaa ctggatcgaa ggtcgtggta 1800

agagtgtcgt cgcagaagct actattcctg gtgatgttgt cagaaaagtg ttaaaaagtg 1860

atgtttccgc attggttgag ttgaacattg ctaagaattt ggttggatct gcaatggctg 1920

ggtctgttgg tggatttaac gcacatgcag ctaatttagt gacagctgtt ttcttggcat 1980

taggacaaga tcctgcacaa aatgttgaaa gttccaactg tataacattg atgaaagaag 2040

tggacggtga tttgagaatt tccgtatcca tgccatccat cgaagtaggt accatcggtg 2100

gtggtactgt tctagaacca caaggtgcca tgttggactt attaggtgta agaggcccgc 2160

atgctaccgc tcctggtacc aacgcacgtc aattagcaag aatagttgcc tgtgccgtct 2220

tggcaggtga attatcctta tgtgctgccc tagcagccgg ccatttggtt caaagtcata 2280

tgacccacaa caggaaacct gctgaaccaa caaaacctaa caatttggac gccactgata 2340

taaatcgttt gaaagatggg tccgtcacct gcattaaatc ctaaagttat aaaaaaaata 2400

agtgtataca aattttaaag tgactcttag gttttaaaac gaaaattctt attcttgagt 2460

aactctttcc tgtaggtcag gttgctttct caggtatagc atgaggtcgc tcttattgac 2520

cacacctcta ccggcatgcc gacatgcgac tgggtgagca tatgttccgc tgatgtgatg 2580

tgcaagataa ac 2592

<210> 2

<211> 2632

<212> DNA

<213> Artificial sequence

<400> 2

ggtatagcat gaggtcgctc ttattgacca cacctctacc ggcatgccga catgcgactg 60

ggtgagcata tgttccgctg atgtgatgtg caagataaac aagcaaggca gaaactaact 120

tcttcttcat gtaataaaca caccccgcgt ttatttacct atctctaaac ttcaacacct 180

tatatcataa ctaatatttc ttgagataag cacactgcac ccataccttc cttaaaaacg 240

tagcttccag tttttggtgg ttccggcttc cttcccgatt ccgcccgcta aacgcatatt 300

tttgttgcct ggtggcattt gcaaaatgca taacctatgc atttaaaaga ttatgtatgc 360

tcttctgact tttcgtgtga tgaggctcgt ggaaaaaatg aataatttat gaatttgaga 420

acaattttgt gttgttacgg tattttacta tggaataatc aatcaattga ggattttatg 480

caaatatcgt ttgaatattt ttccgaccct ttgagtactt ttcttcataa ttgcataata 540

ttgtccgctg cccctttttc tgttagacgg tgtcttgatc tacttgctat cgttcaacac 600

caccttattt tctaactatt ttttttttag ctcatttgaa tcagcttatg gtgatggcac 660

atttttgcat aaacctagct gtcctcgttg aacataggaa aaaaaaatat ataaacaagg 720

ctctttcact ctccttgcaa tcagatttgg gtttgttccc tttattttca tatttcttgt 780

catattcctt tctcaattat tattttctac tcataacctc acgcaaaata acacagtcaa 840

atcaatcaaa atgtcattac cgttcttaac ttctgcaccg ggaaaggtta ttatttttgg 900

tgaacactct gctgtgtaca acaagcctgc cgtcgctgct agtgtgtctg cgttgagaac 960

ctacctgcta ataagcgagt catctgcacc agatactatt gaattggact tcccggacat 1020

tagctttaat cataagtggt ccatcaatga tttcaatgcc atcaccgagg atcaagtaaa 1080

ctcccaaaaa ttggccaagg ctcaacaagc caccgatggc ttgtctcagg aactcgttag 1140

tcttttggat ccgttgttag ctcaactatc cgaatccttc cactaccatg cagcgttttg 1200

tttcctgtat atgtttgttt gcctatgccc ccatgccaag aatattaagt tttctttaaa 1260

gtctacttta cccatcggtg ctgggttggg ctcaagcgcc tctatttctg tatcactggc 1320

cttagctatg gcctacttgg gggggttaat aggatctaat gacttggaaa agctgtcaga 1380

aaacgataag catatagtga atcaatgggc cttcataggt gaaaagtgta ttcacggtac 1440

cccttcagga atagataacg ctgtggccac ttatggtaat gccctgctat ttgaaaaaga 1500

ctcacataat ggaacaataa acacaaacaa ttttaagttc ttagatgatt tcccagccat 1560

tccaatgatc ctaacctata ctagaattcc aaggtctaca aaagatcttg ttgctcgcgt 1620

tcgtgtgttg gtcaccgaga aatttcctga agttatgaag ccaattctag atgccatggg 1680

tgaatgtgcc ctacaaggct tagagatcat gactaagtta agtaaatgta aaggcaccga 1740

tgacgaggct gtagaaacta ataatgaact gtatgaacaa ctattggaat tgataagaat 1800

aaatcatgga ctgcttgtct caatcggtgt ttctcatcct ggattagaac ttattaaaaa 1860

tctgagcgat gatttgagaa ttggctccac aaaacttacc ggtgctggtg gcggcggttg 1920

ctctttgact ttgttacgaa gagacattac tcaagagcaa attgacagct tcaaaaagaa 1980

attgcaagat gattttagtt acgagacatt tgaaacagac ttgggtggga ctggctgctg 2040

tttgttaagc gcaaaaaatt tgaataaaga tcttaaaatc aaatccctag tattccaatt 2100

atttgaaaat aaaactacca caaagcaaca aattgacgat ctattattgc caggaaacac 2160

gaatttacca tggacttcat aagcggatct cttatgtctt tacgatttat agttttcatt 2220

atcaagtatg cctatattag tatatagcat ctttagatga cagtgttcga agtttcacga 2280

ataaaagata atattctact ttttgctccc accgcgtttg ctagcacgag tgaacaccat 2340

ccctcgcctg tgagttgtac ccattcctct aaactgtaga catggtagct tcagcagtgt 2400

tcgttatgta cggcatcctc caacaaacag tcggttatag tttgtcctgc tcctctgaat 2460

cgtctccctc gatatttctc attttccttc gcatgccagc attgaaatga tcgaagttca 2520

atgatgaaac ggtaattctt ctgtcattta ctcatctcat ctcatcaagt tatataattc 2580

taaatcctac tcttgccgtt gccatccaaa atgagctaga aggtggatta ac 2632

<210> 3

<211> 2367

<212> DNA

<213> Artificial sequence

<400> 3

taattcttct gtcatttact catctcatct catcaagtta tataattcta aatcctactc 60

ttgccgttgc catccaaaat gagctagaag gtggattaac aaatataatg acaaatcgtt 120

gcttgtctga cttgattcca ctacagttac aaatatttga cattgtatat aagttttgca 180

agttcatcaa atctatgaga gcaaaattat gtcaactgga ccccgtacta tatgagaaac 240

acaaaagcgg gatgatgaaa acactaaacg aaggctatcg tacaaacaat ggcggtcagg 300

aagatgttgg ttaccaagaa gatgccgccc tggaattaat tcagaagctg attgaataca 360

ttagcaacgc gtccagcatt tttcggaagt gtctcataaa ctttactcaa gagttaagta 420

ctgaaaaatt cgacttttat gatagttcaa gtgtcgacgc tgcgggtata gaaagggttc 480

tttactctat agtacctcct cgctcagcat ctgcttcttc ccaaagatga acgcggcgtt 540

atgtcactaa cgacgtgcac caacttgcgg aaagtggaat cccgttccaa aactggcatc 600

cactaattga tacatctaca caccgcacgc cttttttctg aagcccactt tcgtggactt 660

tgccatatgc aaaattcatg aagtgtgata ccaagtcagc atacacctca ctagggtagt 720

ttctttggtt gtattgatca tttggttcat cgtggttcat taattttttt tctccattgc 780

tttctggctt tgatcttact atcatttgga tttttgtcga aggttgtaga attgtatgtg 840

acaagtggca ccaagcatat ataaaaaaaa aaagcattat cttcctacca gagttgattg 900

ttaaaaacgt atttatagca aacgcaattg taattaattc ttattttgta tcttttcttc 960

ccttgtctca atcttttatt tttattttat ttttcttttc ttagtttctt tcataacacc 1020

aagcaactaa tactataaca tacaataata atgactgccg acaacaatag tatgccccat 1080

ggtgcagtat ctagttacgc caaattagtg caaaaccaaa cacctgaaga cattttggaa 1140

gagtttcctg aaattattcc attacaacaa agacctaata cccgatctag tgagacgtca 1200

aatgacgaaa gcggagaaac atgtttttct ggtcatgatg aggagcaaat taagttaatg 1260

aatgaaaatt gtattgtttt ggattgggac gataatgcta ttggtgccgg taccaagaaa 1320

gtttgtcatt taatggaaaa tattgaaaag ggtttactac atcgtgcatt ctccgtcttt 1380

attttcaatg aacaaggtga attactttta caacaaagag ccactgaaaa aataactttc 1440

cctgatcttt ggactaacac atgctgctct catccactat gtattgatga cgaattaggt 1500

ttgaagggta agctagacga taagattaag ggcgctatta ctgcggcggt gagaaaacta 1560

gatcatgaat taggtattcc agaagatgaa actaagacaa ggggtaagtt tcacttttta 1620

aacagaatcc attacatggc accaagcaat gaaccatggg gtgaacatga aattgattac 1680

atcctatttt ataagatcaa cgctaaagaa aacttgactg tcaacccaaa cgtcaatgaa 1740

gttagagact tcaaatgggt ttcaccaaat gatttgaaaa ctatgtttgc tgacccaagt 1800

tacaagttta cgccttggtt taagattatt tgcgagaatt acttattcaa ctggtgggag 1860

caattagatg acctttctga agtggaaaat gacaggcaaa ttcatagaat gctataagcg 1920

atttaatctc taattattag ttaaagtttt ataagcattt ttatgtaacg aaaaataaat 1980

tggttcatat tattactgca ctgtcactta ccatggaaag accagacaag aagttgccga 2040

cagtctgttg aattggcctg gttaggctta agtctgggtc cgcttcttta caaatttgga 2100

gaatttctct taaacgatat gtatattctt ttcgttggaa aagatgtctt ccaaaaaaaa 2160

aaccgatgaa ttagtggaac caaggaaaaa aaaagaggta tccttgatta aggaacactg 2220

tttaaacagt gtggtttcca aaaccctgaa actgcattag tgtaatagaa gactagacac 2280

ctcgatacaa ataatggtta ctcaattcaa aactgccaat gctactattt tggagattaa 2340

tctcagtaca aaacaatatt aaaaaga 2367

<210> 4

<211> 2691

<212> DNA

<213> Artificial sequence

<400> 4

gaagactaga cacctcgata caaataatgg ttactcaatt caaaactgcc aatgctacta 60

ttttggagat taatctcagt acaaaacaat attaaaaaga ggtgaattat ttttcccccc 120

ttattttttt tttgttaaaa ttgatccaaa tgtaaataaa caatcacaag gaaaaaaaaa 180

aaaaaaaaaa aaatagccgc catgaccccg gatcgtcggt tgtgatacgg tcagggtagc 240

gccctggtca aacttcagaa ctaaaaaaat aataaggaag aaaaaaatag ctaatttttc 300

cggcagaaag attttcgcta cccgaaagtt tttccggcaa gctaaatgga aaaaggaaag 360

attattgaaa gagaaagaaa gaaaaaaaaa aaatgtacac ccagacatcg ggcttccaca 420

atttcggctc tattgttttc catctctcgc aacggcggga ttcctctatg gcgtgtgatg 480

tctgtatctg ttacttaatc cagaaactgg cacttgaccc aactctgcca cgtgggtcgt 540

tttgccatcg acagattggg agattttcat agtagaattc agcatgatag ctacgtaaat 600

gtgttccgca ccgtcacaaa gtgttttcta ctgttctttc ttctttcgtt cattcagttg 660

agttgagtga gtgctttgtt caatggatct tagctaaaat gcatattttt tctcttggta 720

aatgaatgct tgtgatgtct tccaagtgat ttcctttcct tcccatatga tgctaggtac 780

ctttagtgtc ttcctaaaaa aaaaaaaagg ctcgccatca aaacgatatt cgttggcttt 840

tttttctgaa ttataaatac tctttggtaa cttttcattt ccaagaacct cttttttcca 900

gttatatcat ggtccccttt caaagttatt ctctactctt tttcatattc attctttttc 960

atcctttggt tttttattct taacttgttt attattctct cttgtttcta tttacaagac 1020

accaatcaaa acaaataaaa catcatcaca atgaccgttt acacagcatc cgttaccgca 1080

cccgtcaaca tcgcaaccct taagtattgg gggaaaaggg acacgaagtt gaatctgccc 1140

accaattcgt ccatatcagt gactttatcg caagatgacc tcagaacgtt gacctctgcg 1200

gctactgcac ctgagtttga acgcgacact ttgtggttaa atggagaacc acacagcatc 1260

gacaatgaaa gaactcaaaa ttgtctgcgc gacctacgcc aattaagaaa ggaaatggaa 1320

tcgaaggacg cctcattgcc cacattatct caatggaaac tccacattgt ctccgaaaat 1380

aactttccta cagcagctgg tttagcttcc tccgctgctg gctttgctgc attggtctct 1440

gcaattgcta agttatacca attaccacag tcaacttcag aaatatctag aatagcaaga 1500

aaggggtctg gttcagcttg tagatcgttg tttggcggat acgtggcctg ggaaatggga 1560

aaagctgaag atggtcatga ttccatggca gtacaaatcg cagacagctc tgactggcct 1620

cagatgaaag cttgtgtcct agttgtcagc gatattaaaa aggatgtgag ttccactcag 1680

ggtatgcaat tgaccgtggc aacctccgaa ctatttaaag aaagaattga acatgtcgta 1740

ccaaagagat ttgaagtcat gcgtaaagcc attgttgaaa aagatttcgc cacctttgca 1800

aaggaaacaa tgatggattc caactctttc catgccacat gtttggactc tttccctcca 1860

atattctaca tgaatgacac ttccaagcgt atcatcagtt ggtgccacac cattaatcag 1920

ttttacggag aaacaatcgt tgcatacacg tttgatgcag gtccaaatgc tgtgttgtac 1980

tacttagctg aaaatgagtc gaaactcttt gcatttatct ataaattgtt tggctctgtt 2040

cctggatggg acaagaaatt tactactgag cagcttgagg ctttcaacca tcaatttgaa 2100

tcatctaact ttactgcacg tgaattggat cttgagttgc aaaaggatgt tgccagagtg 2160

attttaactc aagtcggttc aggcccacaa gaaacaaacg aatctttgat tgacgcaaag 2220

actggtctac caaaggaata aacaaatcgc tcttaaatat atacctaaag aacattaaag 2280

ctatattata agcaaagata cgtaaatttt gcttatatta ttatacacat atcatatttc 2340

tatattttta agatttggtt atataatgta cgtaatgcaa aggaaataaa ttttatacat 2400

tattgaacag cgtccaagta actacattat gtgcactaat agtttagcgt cgtgaagact 2460

ttattgtgtc gcgaaaagta aaaattttaa aaattagagc accttgaact tgcgaaaaag 2520

gttctcatca actgtttaaa aggaggatat caggtcctat ttctgacaaa caatatacaa 2580

atttagtttc aaagatgaat cagtgcgcga aggacataac tcatgaagcc tccagtatac 2640

catggggccg tatacttaca tatagtagat gtcaagcgta ggcgcttccc c 2691

<210> 5

<211> 2364

<212> DNA

<213> Artificial sequence

<400> 5

aagatgaatc agtgcgcgaa ggacataact catgaagcct ccagtatacc atggggccgt 60

atacttacat atagtagatg tcaagcgtag gcgcttcccc tgccggctgt gagggcgcca 120

taaccaaggt atctatagac cgccaatcag caaactacct ccgtacattc atgttgcacc 180

cacacattta tacacccaga ccgcgacaaa ttacccataa ggttgtttgt gacggcgtcg 240

tacaagagaa cgtgggaact ttttaggctc accaaaaaag aaagaaaaaa tacgagttgc 300

tgacagaagc ctcaagaaaa aaaaaattct tcttcgacta tgctggaggc agagatgatc 360

gagccggtag ttaactatat atagctaaat tggttccatc accttctttt ctggtgtcgc 420

tccttctagt gctatttctg gcttttccta tttttttttt tccatttttc tttctctctt 480

tctaatatat aaattctctt gcattttcta tttttctctc tatctattct acttgtttat 540

tcccttcaag gttttttttt aaggagtact tgtttttaga atatacggtc aacgaactat 600

aattaactaa acatgacagg caagacgggt cacatcgatg gtttgaactc gcgcattgaa 660

aagatgcgag atctcgaccc cgcacaacgg ctggtgcgcg ttgccgaggc ggcgggcctc 720

gagcccgagg cgatcagcgc gctggcgggt aacggcgccc tgcccctctc gctggccaac 780

gggatgatcg agaacgtcat cggcaaattc gaactgccgc tgggcgtggc cacgaatttc 840

actgtgaacg gccgcgacta tctgatcccg atggcggtcg aagagccctc ggtggtggcg 900

gccgcgtcct atatggcgcg tatcgcgcgc gagaatggcg gattcaccgc gcatggcacc 960

gcgcccttga tgcgcgccca gatccaggtg gtcgggttgg gtgatcccga gggcgcccgg 1020

cagcgtctcc tcgcccacaa ggccgcgttc atggaggcgg cggacgctgt cgatccggtg 1080

cttgtcgggc tgggtggcgg ctgccgcgat atcgaggttc acgtgttccg ggatacgccg 1140

gtgggcgcga tggtcgtcct gcacctgatc gtcgatgtgc gcgacgcgat gggggccaat 1200

acggtcaaca cgatggccga acggctggcc cccgaggtcg agcggattgc cggtggcacc 1260

gtgcggctgc gcatcctgtc gaacctcgcc gacctgcgat tggtccgggc gcgggtggaa 1320

ctggccccgg aaacactgac aacgcagggc tatgacggcg ccgacgtggc gcggggcatg 1380

gtcgaggcct gcgcgcttgc catcgtcgac ccctatcgcg cggcgaccca taacaagggg 1440

atcatgaacg gcatcgaccc ggtcgtcgtc gccaccggca atgactggcg cgcgatcgag 1500

gcgggtgccc atgcctatgc cgcccgcacg ggtcattata cctcgctgac ccgctgggaa 1560

ctggcgaatg acgggcggct tgtgggcacg atcgaactgc ccctggcgct tggccttgtc 1620

ggcggcgcga ccaagacgca cccgaccgca cgggcggcgc tggccctgat gcaggtagag 1680

actgcaaccg aactggccca ggtcaccgcc gccgtgggtc tggcgcagaa catggccgcc 1740

atccgcgcgc tggcgaccga aggcatccag cgcggtcaca tgacccttca tgcgcgcaac 1800

atcgcgatca tggccggcgc aacaggcgcc gatatcgacc gcgtcacccg ggtcattgtc 1860

gaagcgggcg acgtcagcgt ggcccgtgca aaacaggtgc tggaaaacac ctgaagtgct 1920

tttaactaag aattattagt cttttctgct tattttttca tcatagttta gaacacttta 1980

tattaacgaa tagtttatga atctatttag gtttaaaaat tgatacagtt ttataagtta 2040

ctttttcaaa gactcgtgct gtctattgca taatgcactg gaaggggaaa aaaaaggtgc 2100

acacgcgtgg ctttttcttg aatttgcagt ttgaaaaata actacatgga tgataagaaa 2160

acatggagta cagtcacttt gagaaccttc aatcagctgg taacgtcttc gttaattgga 2220

tactcaaaaa agatggatag catgaatcac aagatggaag gaaatgcggg ccacgaccac 2280

agtgatatgc atatgggaga tggagatgat acctgatcca actggcaccg ctggcttgaa 2340

caacaatacc agccttccaa cttc 2364

<210> 6

<211> 2799

<212> DNA

<213> Artificial sequence

<400> 6

tgcgggccac gaccacagtg atatgcatat gggagatgga gatgatacct gatccaactg 60

gcaccgctgg cttgaacaac aataccagcc ttccaacttc tgtaaataac ggcggtacgc 120

cagtgccacc agtaccgtta cctttcggta tacctccttt ccccatgttt ccaatgccct 180

tcatgcctcc aacggctact atcacaaatc ctcatcaagc tgacgcaagc cctaagaaat 240

gaataacaat actgacagta ctaaataatt gcctacttgg cttcacatac gttgcatacg 300

tcgatataga taataatgat aatgacagca ggattatcgt aatacgtaat agttgaaaat 360

ctcaaaaatg tgtgggtcat tacgtaaata atgataggaa tgggattctt ctatttttcc 420

tttttccatt ctagcagccg tcgggaaaac gtggcatcct ctctttcggg ctcaattgga 480

gtcacgctgc cgtgagcatc ctctctttcc atatctaaca actgagcacg taaccaatgg 540

aaaagcatga gcttagcgtt gctccaaaaa agtattggat ggttaatacc atttgtctgt 600

tctcttctga ctttgactcc tcaaaaaaaa aaaatctaca atcaacagat cgcttcaatt 660

acgccctcac aaaaactttt ttccttcttc ttcgcccacg ttaaatttta tccctcatgt 720

tgtctaacgg atttctgcac ttgatttatt ataaaaagac aaagacataa tacttctcta 780

tcaatttcag ttattgttct tccttgcgtt attcttctgt tcttcttttt cttttgtcat 840

atataaccat aaccaagtaa tacatattca aaatgaaact ctcaactaaa ctttgttggt 900

gtggtattaa aggaagactt aggccgcaaa agcaacaaca attacacaat acaaacttgc 960

aaatgactga actaaaaaaa caaaagaccg ctgaacaaaa aaccagacct caaaatgtcg 1020

gtattaaagg tatccaaatt tacatcccaa ctcaatgtgt caaccaatct gagctagaga 1080

aatttgatgg cgtttctcaa ggtaaataca caattggtct gggccaaacc aacatgtctt 1140

ttgtcaatga cagagaagat atctactcga tgtccctaac tgttttgtct aagttgatca 1200

agagttacaa catcgacacc aacaaaattg gtagattaga agtcggtact gaaactctga 1260

ttgacaagtc caagtctgtc aagtctgtct tgatgcaatt gtttggtgaa aacactgacg 1320

tcgaaggtat tgacacgctt aatgcctgtt acggtggtac caacgcgttg ttcaactctt 1380

tgaactggat tgaatctaac gcatgggatg gtagagacgc cattgtagtt tgcggtgata 1440

ttgccatcta cgataagggt gccgcaagac caaccggtgg tgccggtact gttgctatgt 1500

ggatcggtcc tgatgctcca attgtatttg actctgtaag agcttcttac atggaacacg 1560

cctacgattt ttacaagcca gatttcacca gcgaatatcc ttacgtcgat ggtcattttt 1620

cattaacttg ttacgtcaag gctcttgatc aagtttacaa gagttattcc aagaaggcta 1680

tttctaaagg gttggttagc gatcccgctg gttcggatgc tttgaacgtt ttgaaatatt 1740

tcgactacaa cgttttccat gttccaacct gtaaattggt cacaaaatca tacggtagat 1800

tactatataa cgatttcaga gccaatcctc aattgttccc agaagttgac gccgaattag 1860

ctactcgcga ttatgacgaa tctttaaccg ataagaacat tgaaaaaact tttgttaatg 1920

ttgctaagcc attccacaaa gagagagttg cccaatcttt gattgttcca acaaacacag 1980

gtaacatgta caccgcatct gtttatgccg cctttgcatc tctattaaac tatgttggat 2040

ctgacgactt acaaggcaag cgtgttggtt tattttctta cggttccggt ttagctgcat 2100

ctctatattc ttgcaaaatt gttggtgacg tccaacatat tatcaaggaa ttagatatta 2160

ctaacaaatt agccaagaga atcaccgaaa ctccaaagga ttacgaagct gccatcgaat 2220

tgagagaaaa tgcccatttg aagaagaact tcaaacctca aggttccatt gagcatttgc 2280

aaagtggtgt ttactacttg accaacatcg atgacaaatt tagaagatct tacgatgtta 2340

aaaaataaat ttaactcctt aagttacttt aatgatttag tttttattat taataattca 2400

tgctcatgac atctcatata cacgtttata aaacttaaat agattgaaaa tgtattaaag 2460

attcctcagg gattcgattt ttttggaagt ttttgttttt ttttccttga gatgctgtag 2520

tatttgggaa caattataca atcgaaagat atatgcttac attcgaccgt tttagccgtg 2580

atcattatcc tatagtaaca taacctgaag cataactgac actactatca tcaatacttg 2640

tcacatgaga actctgtgaa taattaggcc actgaaattt gatgcctgaa ggaccggcat 2700

cacggatttt cgataaagca cttagtatca cactaattgg cttttcgcca tactagcgtt 2760

gaatgttagc gtcaacaaca agaagtttaa tgacgcgga 2799

<210> 7

<211> 2658

<212> DNA

<213> Artificial sequence

<400> 7

tcacggattt tcgataaagc acttagtatc acactaattg gcttttcgcc atactagcgt 60

tgaatgttag cgtcaacaac aagaagttta atgacgcgga ggccaaggca aaaagattcc 120

ttgattacgt aagggagtta gaatcatttt gaataaaaaa cacgcttttt cagttcgagt 180

ttatcattat caatactgcc atttcaaaga atacgtaaat aattaatagt agtgattttc 240

ctaactttat ttagtcaaaa aattagcctt ttaattctgc tgtaacccgt acatgcccaa 300

aatagggggc gggttacaca gaatatataa catcgtaggt gtctgggtga acagtttatt 360

cctggcatcc actaaatata atggagcccg ctttttaagc tggcatccag aaaaaaaaag 420

aatcccagca ccaaaatatt gttttcttca ccaaccatca gttcataggt ccattctctt 480

agcgcaacta cagagaacag gggcacaaac aggcaaaaaa cgggcacaac ctcaatggag 540

tgatgcaacc tgcctggagt aaatgatgac acaaggcaat tgacccacgc atgtatctat 600

ctcattttct tacaccttct attaccttct gctctctctg atttggaaaa agctgaaaaa 660

aaaggttgaa accagttccc tgaaattatt cccctacttg actaataagt atataaagac 720

ggtaggtatt gattgtaatt ctgtaaatct atttcttaaa cttcttaaat tctactttta 780

tagttagtct tttttttagt tttaaaacac caagaactta gtttcgaata aacacacata 840

aacaaacaaa atgtcagagt tgagagcctt cagtgcccca gggaaagcgt tactagctgg 900

tggatattta gttttagata caaaatatga agcatttgta gtcggattat cggcaagaat 960

gcatgctgta gcccatcctt acggttcatt gcaagggtct gataagtttg aagtgcgtgt 1020

gaaaagtaaa caatttaaag atggggagtg gctgtaccat ataagtccta aaagtggctt 1080

cattcctgtt tcgataggcg gatctaagaa ccctttcatt gaaaaagtta tcgctaacgt 1140

atttagctac tttaaaccta acatggacga ctactgcaat agaaacttgt tcgttattga 1200

tattttctct gatgatgcct accattctca ggaggatagc gttaccgaac atcgtggcaa 1260

cagaagattg agttttcatt cgcacagaat tgaagaagtt cccaaaacag ggctgggctc 1320

ctcggcaggt ttagtcacag ttttaactac agctttggcc tccttttttg tatcggacct 1380

ggaaaataat gtagacaaat atagagaagt tattcataat ttagcacaag ttgctcattg 1440

tcaagctcag ggtaaaattg gaagcgggtt tgatgtagcg gcggcagcat atggatctat 1500

cagatataga agattcccac ccgcattaat ctctaatttg ccagatattg gaagtgctac 1560

ttacggcagt aaactggcgc atttggttga tgaagaagac tggaatatta cgattaaaag 1620

taaccattta ccttcgggat taactttatg gatgggcgat attaagaatg gttcagaaac 1680

agtaaaactg gtccagaagg taaaaaattg gtatgattcg catatgccag aaagcttgaa 1740

aatatataca gaactcgatc atgcaaattc tagatttatg gatggactat ctaaactaga 1800

tcgcttacac gagactcatg acgattacag cgatcagata tttgagtctc ttgagaggaa 1860

tgactgtacc tgtcaaaagt atcctgaaat cacagaagtt agagatgcag ttgccacaat 1920

tagacgttcc tttagaaaaa taactaaaga atctggtgcc gatatcgaac ctcccgtaca 1980

aactagctta ttggatgatt gccagacctt aaaaggagtt cttacttgct taatacctgg 2040

tgctggtggt tatgacgcca ttgcagtgat tactaagcaa gatgttgatc ttagggctca 2100

aaccgctaat gacaaaagat tttctaaggt tcaatggctg gatgtaactc aggctgactg 2160

gggtgttagg aaagaaaaag atccggaaac ttatcttgat aaataagatt aatataatta 2220

tataaaaata ttatcttctt ttctttatat ctagtgttat gtaaaataaa ttgatgacta 2280

cggaaagctt ttttatattg tttctttttc attctgagcc acttaaattt cgtgaatgtt 2340

cttgtaaggg acggtagatt tacaagtgat acaacaaaaa gcaaggcgct ttttctaata 2400

aaaagaagaa aagcatttaa caattgaaca cctctatatc aacgaagaat attactttgt 2460

ctctaaatcc ttgtaaaatg tgtacgatct ctatatgggt tactcataag tgtaccgaag 2520

actgcattga aagtttatgt tttttcactg gaggcgtcat tttcgcgttg agaagatgtt 2580

cttatccaaa tttcaactgt tatatagaag tgatccccca cacaccatag cttcaaaatg 2640

tttctactcc ttttttac 2658

<210> 8

<211> 2034

<212> DNA

<213> Artificial sequence

<400> 8

attttcgcgt tgagaagatg ttcttatcca aatttcaact gttatataga agtgatcccc 60

cacacaccat agcttcaaaa tgtttctact ccttttttac tcttccagat tttctcggac 120

tccgcgcatc gccgtaccac ttcaaaacac ccaagcacag catactaaat ttcccctctt 180

tcttcctcta gggtgtcgtt aattacccgt actaaaggtt tggaaaagaa aaaagagacc 240

gcctcgtttc tttttcttcg tcgaaaaagg caataaaaat ttttatcacg tttctttttc 300

ttgaaaattt ttttttttga tttttttctc tttcgatgac ctcccattga tatttaagtt 360

aataaacggt cttcaatttc tcaagtttca gtttcatttt tcttgttcta ttacaacttt 420

ttttacttct tgctcattag aaagaaagca tagcaatcta atctaagttt taattacaaa 480

atgtctcaga acgtttacat tgtatcgact gccagaaccc caattggttc attccagggt 540

tctctatcct ccaagacagc agtggaattg ggtgctgttg ctttaaaagg cgccttggct 600

aaggttccag aattggatgc atccaaggat tttgacgaaa ttatttttgg taacgttctt 660

tctgccaatt tgggccaagc tccggccaga caagttgctt tggctgccgg tttgagtaat 720

catatcgttg caagcacagt taacaaggtc tgtgcatccg ctatgaaggc aatcattttg 780

ggtgctcaat ccatcaaatg tggtaatgct gatgttgtcg tagctggtgg ttgtgaatct 840

atgactaacg caccatacta catgccagca gcccgtgcgg gtgccaaatt tggccaaact 900

gttcttgttg atggtgtcga aagagatggg ttgaacgatg cgtacgatgg tctagccatg 960

ggtgtacacg cagaaaagtg tgcccgtgat tgggatatta ctagagaaca acaagacaat 1020

tttgccatcg aatcctacca aaaatctcaa aaatctcaaa aggaaggtaa attcgacaat 1080

gaaattgtac ctgttaccat taagggattt agaggtaagc ctgatactca agtcacgaag 1140

gacgaggaac ctgctagatt acacgttgaa aaattgagat ctgcaaggac tgttttccaa 1200

aaagaaaacg gtactgttac tgccgctaac gcttctccaa tcaacgatgg tgctgcagcc 1260

gtcatcttgg tttccgaaaa agttttgaag gaaaagaatt tgaagccttt ggctattatc 1320

aaaggttggg gtgaggccgc tcatcaacca gctgatttta catgggctcc atctcttgca 1380

gttccaaagg ctttgaaaca tgctggcatc gaagacatca attctgttga ttactttgaa 1440

ttcaatgaag ccttttcggt tgtcggtttg gtgaacacta agattttgaa gctagaccca 1500

tctaaggtta atgtatatgg tggtgctgtt gctctaggtc acccattggg ttgttctggt 1560

gctagagtgg ttgttacact gctatccatc ttacagcaag aaggaggtaa gatcggtgtt 1620

gccgccattt gtaatggtgg tggtggtgct tcctctattg tcattgaaaa gatatgaccg 1680

ctgatcctag agggccgcat catgtaatta gttatgtcac gcttacattc acgccctccc 1740

cccacatccg ctctaaccga aaaggaagga gttagacaac ctgaagtcta ggtccctatt 1800

tattttttta tagttatgtt agtattaaga acgttattta tatttcaaat ttttcttttt 1860

tttctgtaca gacgcgtgta cgcatgtaac attatactga aaaccttgct tgagaaggtt 1920

ttgggacgct cgaaggcttt aatttgcaag ctgcggccct gcattaatga atcggccaac 1980

gcgcggatgc aggtatcaga actggtgatt taggtggttc caacagtacc accg 2034

<210> 9

<211> 2241

<212> DNA

<213> Artificial sequence

<400> 9

tactaacctt tcattaaaga gaaataacaa tattataaaa agcgcttaaa acgcacagat 60

attataacat ctgcacaata ggcatttgca agaattactc gtgagtaagg aaagagtgag 120

gaactatcgc atacctgcat ttaaagatgc cgatttgggc gcgaatcctt tattttggct 180

tcaccctcat actattatca gggccagaaa aaggaagtgt ttccctcctt cttgaattga 240

tgttaccctc ataaagcacg tggcctctta tcgagaaaga aattaccgtc gctcgtgatt 300

tgtttgcaaa aagaacaaaa ctgaaaaaac ccagacacgc tcgacttcct gtcttcctat 360

tgattgcagc ttccaatttc gtcacacaac aaggtcctag cgacggctca caggttttgt 420

aacaagcaat cgaaggttct ggaatggcgg gaaagggttt agtaccacat gctatgatgc 480

ccactgtgat ctccagagca aagttcgttc gatcgtactg ttactctctc tctttcaaac 540

agaattgtcc gaatcgtgtg acaacaacag cctgttctca cacactcttt tcttctaacc 600

aagggggtgg tttagtttag tagaacctcg tgaaacttac atttacatat atataaactt 660

gcataaattg gtcaatgcaa gaaatacata tttggtcttt tctaattcgt agtttttcaa 720

gttcttagat gctttctttt tctctttttt acagatcatc aaggaagtaa ttatctactt 780

tttacaacaa atataaaaca atggggagct tggggacgat gctgagatat ccggatgaca 840

tatatccgct cctgaagatg aaacgagcga ttgagaaagc ggagaagcag atccctcctg 900

agccacactg gggtttctgc tattcgatgc tccacaaggt ttctcgaagc ttttctctcg 960

ttattcagca actcaacacc gagctccgta acgccgtgtg tgtgttctac ttggttctcc 1020

gagctcttga tactgttgag gatgatacta gcataccaac tgatgaaaag gttcccatcc 1080

tgatagcttt tcaccggcac atatacgata ctgattggca ttattcatgt ggtacgaagg 1140

agtacaagat tctaatggac caatttcacc atgtttctgc agcttttttg gaacttgaaa 1200

aagggtatca agaggctatc gaggaaatta ctagaagaat gggtgcaggg atggccaagt 1260

ttatctgcca agaggtagaa actgttgatg actacgatga atactgccac tatgttgctg 1320

ggcttgttgg tttaggtttg tcgaaactct tcctcgctgc aggatcagag gttttgacac 1380

cagattggga ggcgatttcc aattcaatgg gtttatttct gcagaaaaca aacattatca 1440

gagattatct tgaggacatt aatgagatac caaaatcccg catgttttgg cctcgcgaga 1500

tttggggcaa atatgctgac aagcttgagg atttaaaata cgaggagaac acaaacaaat 1560

ccgtacagtg cttaaatgaa atggttacca atgcgttgat gcatattgaa gattgcctga 1620

aatacatggt ttccttgcgt gatccttcca tatttcggtt ctgtgccatc cctcagatca 1680

tggcgattgg aacacttgca ttatgctata acaatgaaca agtattcaga ggcgttgtga 1740

aactgaggcg aggtcttact gctaaagtca ttgatcgtac aaagacaatg gctgatgtct 1800

atggtgcttt ctatgatttt tcctgcatgc tgaagacaaa ggttgacaag aacgatccaa 1860

atgccagtaa gacactaaac cgacttgaag ccgttcagaa actctgcaga gacgctggag 1920

ttcttcaaaa cagaaaatct tatgttaatg acaaaggaca accaaacagt gtctttatta 1980

taatggttgt gattctactg gccatagtct ttgcatatct cagagcaaac tgaagttata 2040

aaaaaaataa gtgtatacaa attttaaagt gactcttagg ttttaaaacg aaaattctta 2100

ttcttgagta actctttcct gtaggtcagg ttgctttctc aggtatagca tgaggtcgct 2160

cttattgacc acacctctac cggcatgccg aatactagcg ttgaatgtta gcgtcaacaa 2220

caagaagttt aatgacgcgg a 2241

<210> 10

<211> 2793

<212> DNA

<213> Artificial sequence

<400> 10

ggtatagcat gaggtcgctc ttattgacca cacctctacc ggcatgccga atactagcgt 60

tgaatgttag cgtcaacaac aagaagttta atgacgcgga ggccaaggca aaaagattcc 120

ttgattacgt aagggagtta gaatcatttt gaataaaaaa cacgcttttt cagttcgagt 180

ttatcattat caatactgcc atttcaaaga atacgtaaat aattaatagt agtgattttc 240

ctaactttat ttagtcaaaa aattagcctt ttaattctgc tgtaacccgt acatgcccaa 300

aatagggggc gggttacaca gaatatataa catcgtaggt gtctgggtga acagtttatt 360

cctggcatcc actaaatata atggagcccg ctttttaagc tggcatccag aaaaaaaaag 420

aatcccagca ccaaaatatt gttttcttca ccaaccatca gttcataggt ccattctctt 480

agcgcaacta cagagaacag gggcacaaac aggcaaaaaa cgggcacaac ctcaatggag 540

tgatgcaacc tgcctggagt aaatgatgac acaaggcaat tgacccacgc atgtatctat 600

ctcattttct tacaccttct attaccttct gctctctctg atttggaaaa agctgaaaaa 660

aaaggttgaa accagttccc tgaaattatt cccctacttg actaataagt atataaagac 720

ggtaggtatt gattgtaatt ctgtaaatct atttcttaaa cttcttaaat tctactttta 780

tagttagtct tttttttagt tttaaaacac caagaactta gtttcgaata aacacacata 840

aacaaacaaa atgtctgctg ttaacgttgc acctgaattg attaatgccg acaacacaat 900

tacctacgat gcgattgtca tcggtgctgg tgttatcggt ccatgtgttg ctactggtct 960

agcaagaaag ggtaagaaag ttcttatcgt agaacgtgac tgggctatgc ctgatagaat 1020

tgttggtgaa ttgatgcaac caggtggtgt tagagcattg agaagtctgg gtatgattca 1080

atctatcaac aacatcgaag catatcctgt taccggttat accgtctttt tcaacggcga 1140

acaagttgat attccatacc cttacaaggc cgatatccct aaagttgaaa aattgaagga 1200

cttggtcaaa gatggtaatg acaaggtctt ggaagacagc actattcaca tcaaggatta 1260

cgaagatgat gaaagagaaa ggggtgttgc ttttgttcat ggtagattct tgaacaactt 1320

gagaaacatt actgctcaag agccaaatgt tactagagtg caaggtaact gtattgagat 1380

attgaaggat gaaaagaatg aggttgttgg tgccaaggtt gacattgatg gccgtggcaa 1440

ggtggaattc aaagcccact tgacatttat ctgtgacggt atcttttcac gtttcagaaa 1500

ggaattgcac ccagaccatg ttccaactgt cggttcttcg tttgtcggta tgtctttgtt 1560

caatgctaag aatcctgctc ctatgcacgg tcacgttatt cttggtagtg atcatatgcc 1620

aatcttggtt taccaaatca gtccagaaga aacaagaatc ctttgtgctt acaactctcc 1680

aaaggtccca gctgatatca agagttggat gattaaggat gtccaacctt tcattccaaa 1740

gagtctacgt ccttcatttg atgaagccgt cagccaaggt aaatttagag ctatgccaaa 1800

ctcctacttg ccagctagac aaaacgacgt cactggtatg tgtgttatcg gtgacgctct 1860

aaatatgaga catccattga ctggtggtgg tatgactgtc ggtttgcatg atgttgtctt 1920

gttgattaag aaaataggtg acctagactt cagcgaccgt gaaaaggttt tggatgaatt 1980

actagactac catttcgaaa gaaagagtta cgattccgtt attaacgttt tgtcagtggc 2040

tttgtattct ttgttcgctg ctgacagcga taacttgaag gcattacaaa aaggttgttt 2100

caaatatttc caaagaggtg gcgattgtgt caacaaaccc gttgaatttc tgtctggtgt 2160

cttgccaaag cctttgcaat tgaccagggt tttcttcgct gtcgcttttt acaccattta 2220

cttgaacatg gaagaacgtg gtttcttggg attaccaatg gctttattgg aaggtattat 2280

gattttgatc acagctatta gagtattcac cccatttttg tttggtgagt tgattggtta 2340

agattaatat aattatataa aaatattatc ttcttttctt tatatctagt gttatgtaaa 2400

ataaattgat gactacggaa agctttttta tattgtttct ttttcattct gagccactta 2460

aatttcgtga atgttcttgt aagggacggt agatttacaa gtgatacaac aaaaagcaag 2520

gcgctttttc taataaaaag aagaaaagca tttaacaatt gaacacctct atatcaacga 2580

agaatattac tttgtctcta aatccttgta aaatgtgtac gatctctata tgggttactc 2640

ataagtgtac cgaagactgc attgaaagtt tatgtttttt cactggaggc gtcattttcg 2700

cgttgagaag atgttcttat ccaaatttca actgttatat agaagtgatc ccccacacac 2760

catagcttca aaatgtttct actccttttt tac 2793

<210> 11

<211> 1880

<212> DNA

<213> Artificial sequence

<400> 11

attttcgcgt tgagaagatg ttcttatcca aatttcaact gttatataga agtgatcccc 60

cacacaccat agcttcaaaa tgtttctact ccttttttac tcttccagat tttctcggac 120

tccgcgcatc gccgtaccac ttcaaaacac ccaagcacag catactaaat ttcccctctt 180

tcttcctcta gggtgtcgtt aattacccgt actaaaggtt tggaaaagaa aaaagagacc 240

gcctcgtttc tttttcttcg tcgaaaaagg caataaaaat ttttatcacg tttctttttc 300

ttgaaaattt ttttttttga tttttttctc tttcgatgac ctcccattga tatttaagtt 360

aataaacggt cttcaatttc tcaagtttca gtttcatttt tcttgttcta ttacaacttt 420

ttttacttct tgctcattag aaagaaagca tagcaatcta atctaagttt taattacaaa 480

atggctaatt tgaatggtga atctgctgat ttgagagcaa catttttggg tgtttactct 540

gttttgaagt cagaattgtt gaatgatcca gcatttgaat ggacagatgg ttcaagacaa 600

tgggttgaaa gaatgttgga ttacaacgtt ccaggtggta aattgaacag aggtttgtct 660

gttattgatt catacaaatt gttgaagggt ggtaaagatt tgactgatga tgaagttttc 720

ttggcttctg cattaggttg gtgtgttgaa tggttacaag catacttttt ggttttggat 780

gatatcatgg ataactcaca tacaagaaga ggtcaaccat gttggtttag agttccaaaa 840

gttggtatga tcgcaattaa tgatggtatc atcttgagaa atcatattcc aagaattttg 900

aagaaacatt ttagaactaa accatactac gttgatttgt tggatttgtt taatgaagtt 960

gaattccaaa cagcttctgg tcaaatgatc gatttgatca ctacaatcga aggtgaaaag 1020

gatttgtcta agtactcatt gccattgcat agaagaatcg ttcaatacaa gactgcttat 1080

tactcatttt acttgccagt tgcttgtgca ttgttaatgg caggtgaaga tttggaaaaa 1140

catccaacag ttaaggatgt tttgattaat atgggtatct atttccaagt tcaagatgat 1200

tacttagatt gttttggtga accagaaaag attggtaaaa tcggtactga tatcgaagat 1260

ttcaagtgtt cttggttggt tgttaaagca ttggaattgt gtaacgaaga acaaaagaaa 1320

actttatttg aacattatgg taaagaagat ccagctgatg ttgcaaagat taaagttttg 1380

tacaacgaaa ttaatttgca aggtgttttc gcagaattcg aatctaagtc atacgaaaaa 1440

ttgaattctt caattgaagc tcatccatct aagtcagttc aagcagtttt gaaatcattt 1500

ttgggtaaaa tctataaaag acaaaaatga ccgctgatcc tagagggccg catcatgtaa 1560

ttagttatgt cacgcttaca ttcacgccct ccccccacat ccgctctaac cgaaaaggaa 1620

ggagttagac aacctgaagt ctaggtccct atttattttt ttatagttat gttagtatta 1680

agaacgttat ttatatttca aatttttctt ttttttctgt acagacgcgt gtacgcatgt 1740

aacattatac tgaaaacctt gcttgagaag gttttgggac gctcgaaggc tttaatttgc 1800

aagctgcggc cctgcattaa tgaatcggcc aacgcgcata aactaatgat tttaaatcgt 1860

taaaaaaata tgcgaattct 1880

<210> 12

<211> 2553

<212> DNA

<213> Artificial sequence

<400> 12

gaattcaacg agtttcctat tttggtattc tatatttttt catacaccta acgcacagat 60

attataacat ctgcacaata ggcatttgca agaattactc gtgagtaagg aaagagtgag 120

gaactatcgc atacctgcat ttaaagatgc cgatttgggc gcgaatcctt tattttggct 180

tcaccctcat actattatca gggccagaaa aaggaagtgt ttccctcctt cttgaattga 240

tgttaccctc ataaagcacg tggcctctta tcgagaaaga aattaccgtc gctcgtgatt 300

tgtttgcaaa aagaacaaaa ctgaaaaaac ccagacacgc tcgacttcct gtcttcctat 360

tgattgcagc ttccaatttc gtcacacaac aaggtcctag cgacggctca caggttttgt 420

aacaagcaat cgaaggttct ggaatggcgg gaaagggttt agtaccacat gctatgatgc 480

ccactgtgat ctccagagca aagttcgttc gatcgtactg ttactctctc tctttcaaac 540

agaattgtcc gaatcgtgtg acaacaacag cctgttctca cacactcttt tcttctaacc 600

aagggggtgg tttagtttag tagaacctcg tgaaacttac atttacatat atataaactt 660

gcataaattg gtcaatgcaa gaaatacata tttggtcttt tctaattcgt agtttttcaa 720

gttcttagat gctttctttt tctctttttt acagatcatc aaggaagtaa ttatctactt 780

tttacaacaa atataaaaca atggatttgc catctgcttc agctgctgtt gctgctgcta 840

ctgctgcagt tattttcttg ttgactatct acctgctgcc aaagaaaaaa tctccagctt 900

ctactggtaa gaacggttct acttctttgg aatcttaccc agttattggt aacttgccac 960

acttcgttaa gaacagaaac agattcttgg attgggttgc cgaaatcatt tctcaatctc 1020

caactggtac tgttattgct gctccattgg tttttacttc taacccagaa aacgttgaac 1080

ataccgctaa gtctagattt gatgcttatg ctagaggtcc agctgctaca gctgttttac 1140

atgatttttt aggttccggc atcttgaacg ttgatggtga tagttggagg gctcaaagaa 1200

agactgcttc ttctgaattc actaccagat ctttgagagc cttcattttg gatgctgttg 1260

acggtgaagc tgctggtaga ttattgccat tattgtctag agctgctgct tctggtgaag 1320

tttttgactt gcaagatgtc ttggaaagat tcgccttcga taacatttgc tccattattt 1380

tcgatgccga tccaaactgt ttgaacgata ctcatgatgg tgttggtgaa agattctacc 1440

atgcttttca tgatgctacc ttgttgtcta ctggcagata ttactatcca ttccattggg 1500

tttggaggtt gttgagatgg ttgaatttgg gtactgaaaa gcgtttgaga gatgccgttt 1560

ctgatgttca taaggccatt gatgaattgg tcggttctag aaaaactgaa gttggtacta 1620

ctgttagaag gcaaggtggt ggttctgatt tgttgtcaag atttgctgaa ggtggtgatt 1680

actccgatga tgttttaagg gatgtcttga tcaacttcgt tttggctggt agagatacaa 1740

ctccatcagc tttgacttgg tttttcttta tgatctcctc cagaccagat gttgttgatc 1800

aaattttgga cgagatcagg tccatcagag atcatcaaga tagatctaat ccaaacggtg 1860

gtggtggcgg ttttactttg gaagaattga gagaaatgaa ctacttgcat gctgccatta 1920

ccgaatcctt gagattgaat ccaccagttc cattgatgcc aaagatgtgt atggaagatg 1980

atgtattgcc agatggtact gtagttagaa gaggttggac tgttatgtac tctgcttttg 2040

ctatgggtag aaaggctgaa atttggggtg aagattgcat ggaattcaaa ccagaaagat 2100

ggttggatga tggtggttgt tttaaatctg cttccgctta tagattgcca gcatttcatg 2160

ctggtcctag aatttgtttg ggtaaagata tggcctacat tcagatgaag gctgttgctt 2220

catctttgtt ggaaaggttc gaagttgaag tcgttgaaaa aagaggtaag ccagaattgt 2280

ccatcaccat gagaatggac agaggtttgc cagttagagt gaaagaaaga aagcgtggtt 2340

gttaaagtta taaaaaaaat aagtgtatac aaattttaaa gtgactctta ggttttaaaa 2400

cgaaaattct tattcttgag taactctttc ctgtaggtca ggttgctttc tcaggtatag 2460

catgaggtcg ctcttattga ccacacctct accggcatgc cgaatactag cgttgaatgt 2520

tagcgtcaac aacaagaagt ttaatgacgc gga 2553

<210> 13

<211> 2304

<212> DNA

<213> Artificial sequence

<400> 13

attttcgcgt tgagaagatg ttcttatcca aatttcaact gttatataga agtgatcccc 60

cacacaccat agcttcaaaa tgtttctact ccttttttac tcttccagat tttctcggac 120

tccgcgcatc gccgtaccac ttcaaaacac ccaagcacag catactaaat ttcccctctt 180

tcttcctcta gggtgtcgtt aattacccgt actaaaggtt tggaaaagaa aaaagagacc 240

gcctcgtttc tttttcttcg tcgaaaaagg caataaaaat ttttatcacg tttctttttc 300

ttgaaaattt ttttttttga tttttttctc tttcgatgac ctcccattga tatttaagtt 360

aataaacggt cttcaatttc tcaagtttca gtttcatttt tcttgttcta ttacaacttt 420

ttttacttct tgctcattag aaagaaagca tagcaatcta atctaagttt taattacaaa 480

atgttcccat tggccattat cgttttgttg ttcccaactc tgctgttgtt gtttattggt 540

gttgctttgg gtttgagatc tggtgctaat gaatcttgga aaaagagggg tttgaatatc 600

cctccaggtt ctatgggttg gcctttgttg ggtgaaacta ttgcttttag aaagttgcat 660

ccatgcacat ctttgggtga gtatatggaa gatagattgc agagatacgg taagatctac 720

aggtctaatt tgtttggtgc tccaactgtt gtttctgctg atgctgaatt gaacagattc 780

gttttgatga acgatggcaa gttgtttgaa ccatcttggc caaaatctgt tgccgatatt 840

ttgggtaaga cctccatgtt ggttttgact ggtgaaatgc acaggtacat gaagtctttg 900

tctgttaact tcatgggtat cgccagattg agaaatcatt tcttgggtga ttccgagagg 960

tacattttgg aaaatttggc tacttggaaa gagggtgttc catttccagc taaagaagaa 1020

gcttgtaaga tcacctttaa cctgatggtc aagaacatct tgtctatgaa tccaggtgaa 1080

ccagaaaccg aaagattgag gatcttgtac atgtctttca tgaagggtgt tattgccatg 1140

ccattgaatt ttccaggtac tgcttacaga aaggccattc aatctagagc cactatcttg 1200

aaaaccatcg aacacttgat ggaagatcgt ttggaaaaaa agaaggccgg tactgataat 1260

attggtgaag ctgatttgtt gggcttcatc ttggaacaat ctaacttgga tgctgaacaa 1320

ttcggtgact tgttgttggg tttgttattt ggtggtcacg aaacatcttc taccgctatt 1380

actttggcta tctacttctt ggaaggttgt ccaaaagctg tccaagaatt gagagaagaa 1440

catttgaact tggtcaggat gaagaaacag agaggtgaat ctaaagcttt gacgtgggaa 1500

gattacaagt ctatggattt cgctcaatgc gttgtctctg aaactttgag attgggtaac 1560

atcatcaagt tcgttcacag aaaagctaac accgatgtcc aattcaaggg ttacgatatt 1620

ccatctggtt ggtctgttat tccagttttt gctgctgctc atttggatcc tactgtttac 1680

gataatccac aaaagttcga tccttggaga tggcaaacta tctcttcatc tactgccaga 1740

atcgataact acatgccatt tggtcaaggt ttgagaaatt gtgctggttt ggaattggct 1800

aagatggaaa ttgctgtttt cttgcaccac ttggtcttga atttcgattg ggaattagct 1860

gaaccagatc atccattggc ttatgctttt ccagaattcg aaaaaggctt gccaatcaag 1920

gtcagaaagt tgtctatttt ggagtaaccg ctgatcctag agggccgcat catgtaatta 1980

gttatgtcac gcttacattc acgccctccc cccacatccg ctctaaccga aaaggaagga 2040

gttagacaac ctgaagtcta ggtccctatt tattttttta tagttatgtt agtattaaga 2100

acgttattta tatttcaaat ttttcttttt tttctgtaca gacgcgtgta cgcatgtaac 2160

attatactga aaaccttgct tgagaaggtt ttgggacgct cgaaggcttt aatttgcaag 2220

ctgcggccct gcattaatga atcggccaac gcgctgcgat actgccgtag cgggccttcg 2280

tatagctcgg ccgagctcgt acaa 2304

<210> 14

<211> 3414

<212> DNA

<213> Artificial sequence

<400> 14

ggtatagcat gaggtcgctc ttattgacca cacctctacc ggcatgccga atactagcgt 60

tgaatgttag cgtcaacaac aagaagttta atgacgcgga ggccaaggca aaaagattcc 120

ttgattacgt aagggagtta gaatcatttt gaataaaaaa cacgcttttt cagttcgagt 180

ttatcattat caatactgcc atttcaaaga atacgtaaat aattaatagt agtgattttc 240

ctaactttat ttagtcaaaa aattagcctt ttaattctgc tgtaacccgt acatgcccaa 300

aatagggggc gggttacaca gaatatataa catcgtaggt gtctgggtga acagtttatt 360

cctggcatcc actaaatata atggagcccg ctttttaagc tggcatccag aaaaaaaaag 420

aatcccagca ccaaaatatt gttttcttca ccaaccatca gttcataggt ccattctctt 480

agcgcaacta cagagaacag gggcacaaac aggcaaaaaa cgggcacaac ctcaatggag 540

tgatgcaacc tgcctggagt aaatgatgac acaaggcaat tgacccacgc atgtatctat 600

ctcattttct tacaccttct attaccttct gctctctctg atttggaaaa agctgaaaaa 660

aaaggttgaa accagttccc tgaaattatt cccctacttg actaataagt atataaagac 720

ggtaggtatt gattgtaatt ctgtaaatct atttcttaaa cttcttaaat tctactttta 780

tagttagtct tttttttagt tttaaaacac caagaactta gtttcgaata aacacacata 840

aacaaacaaa atgcaatcat cctccgtaaa ggtatcccca ttcgacttaa tgtcagcaat 900

catcaagggt tctatggacc aatcaaacgt atcatcagaa tcaggtggtg ctgcagccat 960

ggttttggaa aacagagaat tcattatgat cttgactaca tccattgctg ttttgatcgg 1020

ttgtgttgtc gtattgatat ggagaagatc aggtcaaaaa caatccaaga ctccagaacc 1080

acctaaacct ttgattgtta aggatttgga agtagaagtt gatgacggta aacaaaaggt 1140

tacaatattt ttcggtacac aaaccggtac tgctgaaggt ttcgcaaaag ccttggctga 1200

agaagcaaag gccagatacg aaaaggcaat ttttaaggtt gtcgatttgg atgactatgc 1260

cggtgacgac gatgaatacg aagaaaaatt gaaaaaggaa actttggcct ttttcttttt 1320

ggctacatat ggtgacggtg aaccaaccga caatgctgca agattctaca aatggtttgc 1380

tgagggtaaa gaacgtggtg aatggttgca aaacttaaag tatggtgttt tcggtttggg 1440

taacagacaa tacgaacatt tcaacaaagt tgcaaaggta gttgacgata taatcacaga 1500

acaaggtggt aaaagaatcg tcccagtagg tttgggtgac gatgaccaat gtattgaaga 1560

tgacttcgcc gcttggagag aattattatg gcctgaatta gatcaattgt taagagacga 1620

agatgacgct accactgtat ctacaccata taccgcagcc gttttggaat acagagtcgt 1680

atttcatgat cctgaaggtg catcattaca agacaagtca tggggttccg ccaatggtca 1740

tactgttcac gatgctcaac acccatgtag agccaacgtt gctgtcagaa aagaattgca 1800

tactcctgct agtgatagat cttgcacaca cttggaattc gacatttctg gtactggttt 1860

aacatatgaa accggtgacc atgtaggtgt ttactgtgaa aatttgccag aaacagtcga 1920

agaagcagaa agattgttag gtttctcacc tgatgtatat ttttccatac acaccgaaag 1980

agaagacggt actccattaa gtggttcttc attgtctcca ccttttccac cttgcacttt 2040

gagaacagca ttaaccagat acgccgatgt tttgtccagt cctaaaaagt ctgcattggt 2100

cgccttagct gcacatgcat cagatccatc cgaagccgac agattgaaat atttggctag 2160

tccttctggt aaagatgaat acgctcaatg ggttgtcgca agtcaaagat ctttgttaga 2220

aattatggcc gaatttccat ctgctaagcc acctttgggt gtcttctttg ccgctgtagc 2280

tccaagattg caacctagat actacagtat ctcttcatcc ccaaagatgg ttccttctag 2340

aatacatgtt acctgtgcat tggtctgcga taaaatgcca actggtagaa tccacaaggg 2400

tatttgttca acatggatga aatatgccgt tccattagaa gaatcacaag attgctcctg 2460

ggcacctatc ttcgttagac aatcaaactt caaattgcca gctgatacct ccgtccctat 2520

cattatgatt ggtccaggta caggtttagc tcctttcaga ggtttcttgc aagaaagatt 2580

tgcattgaag gaagctggtg cagaattggg tagttctatc ttgttctttg gttgtagaaa 2640

cagaaagatg gattacatct acgaagacga attgaacggt ttcgtagaaa gtggtgcttt 2700

gtctgaattg atcgttgcat tttcaagaga aggtccaact aaggaatacg ttcaacataa 2760

gatgatggaa aaggctagtg atatctggaa cgtcatctct caaggtggtt atatatacgt 2820

atgcggtgac gctaagggta tggcaagaga cgttcataga actttgcaca caatcttaca 2880

agaacaaggt tctttagatt catccaaggc tgaatcaatg gtaaagaact tacaaatgac 2940

tggtagatac ttgagagatg tcgattaata taattatata aaaatattat cttcttttct 3000

ttatatctag tgttatgtaa aataaattga tgactacgga aagctttttt atattgtttc 3060

tttttcattc tgagccactt aaatttcgtg aatgttcttg taagggacgg tagatttaca 3120

agtgatacaa caaaaagcaa ggcgcttttt ctaataaaaa gaagaaaagc atttaacaat 3180

tgaacacctc tatatcaacg aagaatatta ctttgtctct aaatccttgt aaaatgtgta 3240

cgatctctat atgggttact cataagtgta ccgaagactg cattgaaagt ttatgttttt 3300

tcactggagg cgtcattttc gcgttgagaa gatgttctta tccaaatttc aactgttata 3360

tagaagtgat cccccacaca ccatagcttc aaaatgtttc tactcctttt ttac 3414

<210> 15

<211> 2325

<212> DNA

<213> Artificial sequence

<400> 15

ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccttaatt 60

aaacgcacag atattataac atctgcacaa taggcatttg caagaattac tcgtgagtaa 120

ggaaagagtg aggaactatc gcatacctgc atttaaagat gccgatttgg gcgcgaatcc 180

tttattttgg cttcaccctc atactattat cagggccaga aaaaggaagt gtttccctcc 240

ttcttgaatt gatgttaccc tcataaagca cgtggcctct tatcgagaaa gaaattaccg 300

tcgctcgtga tttgtttgca aaaagaacaa aactgaaaaa acccagacac gctcgacttc 360

ctgtcttcct attgattgca gcttccaatt tcgtcacaca acaaggtcct agcgacggct 420

cacaggtttt gtaacaagca atcgaaggtt ctggaatggc gggaaagggt ttagtaccac 480

atgctatgat gcccactgtg atctccagag caaagttcgt tcgatcgtac tgttactctc 540

tctctttcaa acagaattgt ccgaatcgtg tgacaacaac agcctgttct cacacactct 600

tttcttctaa ccaagggggt ggtttagttt agtagaacct cgtgaaactt acatttacat 660

atatataaac ttgcataaat tggtcaatgc aagaaataca tatttggtct tttctaattc 720

gtagtttttc aagttcttag atgctttctt tttctctttt ttacagatca tcaaggaagt 780

aattatctac tttttacaac aaatataaaa caatggccga atctcaattg gttcatccac 840

ctttgttcac ctacatttct atgttggctt tgttgacttt ggttccacca ttcgttattt 900

tgatgtggta cactaacgtt catgctgatg gttctgttct gcaaactttc aactacctga 960

aagaaaatgg cttgcaaggt ttgattgata tctggccaag accaactgct attgctggta 1020

agattattat ctgctacgct ttgtttgaag ccaccttgca attgctattg ccaggtaaaa 1080

gagttcaagg tccaatttct ccaactggtc atagacctgt ttacaaggct aatggtatgg 1140

ctgcttatac cgttactttg attacctact tgtctttgtg gtggttcggt attttcaacc 1200

caactgttgt ttacgatcac ttgggtgaaa ttctgtccac tttgaatttc ggctcactga 1260

ttttctgcct gttcttgtat attaagggtc atgttgctcc atcctctact gatcatggtt 1320

cttcaggtaa catcatcgtt gattattact ggggcatgga actgtatcca agaattggta 1380

aacacttcga catcaaggtt ttcaccaact gtagattcgg tatgatttct tggggtttgt 1440

tgccaattac ttactgcatc aagcagtacg aagaatacgg ttctttgtct gactctatgt 1500

tgatccatac catcatcacc ttggtttacg ttactaagtt tttctggtgg gaagctggtt 1560

attggaacac tatggatatt gctcatgata gagccggttt ctatatttgt tggggttgtt 1620

tggttttcct gccatgtatg tatacttctc caggtatgta cttggttaag cacccagtta 1680

atttgggtcc acaattggcc atttcaattt tggttgctgg tatcttgtgc gtctacatta 1740

actacgattg cgatagacag agacaagaat tcagaagaac taacggtaaa gctttggttt 1800

ggggtaaagc tccttctaaa atcgttgctt cttacactac tactaccggt gaaactaagt 1860

cctctttgtt gttaacttct ggttggtggg gtttgagcag acattttcat tatgttccag 1920

aaatcctggc ctcattcttt tggtctgttc cagctttgtt taaccacatc atgccatact 1980

tctacgtcat ctatttgacc ggtttgttgt tggatagagc taaaagggat gacgaaagat 2040

gcaaatccaa gtacggtaaa tactggaaga agtactgcga aaaggttcca tacagagtta 2100

ttccaggcat ctactgaagt tataaaaaaa ataagtgtat acaaatttta aagtgactct 2160

taggttttaa aacgaaaatt cttattcttg agtaactctt tcctgtaggt caggttgctt 2220

tctcaggtat agcatgaggt cgctcttatt gaccacacct ctaccggcat gccgaagtga 2280

tcccccacac accatagctt caaaatgttt ctactccttt tttac 2325

<210> 16

<211> 2416

<212> DNA

<213> Artificial sequence

<400> 16

ggtatagcat gaggtcgctc ttattgacca cacctctacc ggcatgccga agtgatcccc 60

cacacaccat agcttcaaaa tgtttctact ccttttttac tcttccagat tttctcggac 120

tccgcgcatc gccgtaccac ttcaaaacac ccaagcacag catactaaat ttcccctctt 180

tcttcctcta gggtgtcgtt aattacccgt actaaaggtt tggaaaagaa aaaagagacc 240

gcctcgtttc tttttcttcg tcgaaaaagg caataaaaat ttttatcacg tttctttttc 300

ttgaaaattt ttttttttga tttttttctc tttcgatgac ctcccattga tatttaagtt 360

aataaacggt cttcaatttc tcaagtttca gtttcatttt tcttgttcta ttacaacttt 420

ttttacttct tgctcattag aaagaaagca tagcaatcta atctaagttt taattacaaa 480

atgtcagctg tttggtcttt aggtgcaggt ttgttgttgt tgttgttgtg ggttagacat 540

agaggtttag aagctgtttt ggttcatcat agatggatct tcgtttgttt ctttttgatg 600

ccattgtcta tcttgttcga tgtttactac caattaagag cttgggcagt tagaagaatg 660

cattcagcac caagattgca tggtcaaaga gttagacata tccaagaaca agttagagaa 720

tggaaagaag aaggtggtag aagatatatg tgtactggta gaccaggatg gttaacagtt 780

tctttgagag ttggtaaata caagaaaact cataagaaca tcatgatcaa tttgatggat 840

gttttggaag ttgattcaga aagacaagtt gttagagttg aaccattggt tactatgggt 900

caattaacag cttatttgaa tccaatgggt tggactattc cagttgttcc agaattagat 960

gatttgactg ttggtggttt aattatgggt acaggtatcg aatcttcatc tcatatctat 1020

ggtttgttcc aacatacatg tatggcttac gaattggttt tagcagatgg ttcattagtt 1080

agatgttctc caactgaaaa ctcagatttg ttttatgctg ttccatggtc ttgtggtaca 1140

ttaggtttct tggttgctgc agaaattaaa atgatcccag ctaagaaata cattagatta 1200

cattacgaac cagttagagg tttgagatct atctgtgaaa agtttactga agaatctaaa 1260

aataaggaaa attcatttgt tgaaggttta gtttactctt tggaagaagc tgttattatg 1320

actggtgttt taacagatga agcagaacca tcaaagatta atagaatcgg taactactac 1380

aaaccatggt ttttcaagca tgttgaaaag tatttgaagg ctaataagac tggtatcgaa 1440

tacatcccat ctagacatta ctaccataga catacaagat caattttctg ggaattgcaa 1500

gatatcatcc cattcggtaa caatccagtt tttagatatt tgtttggttg gatggttcca 1560

ccaaagatct ctttgttgaa gttgactcaa ggtgaagcaa tcagaaaatt gtacgaacaa 1620

catcatgttg ttcaagatat gttggttcca atgaagtcat tggaaaaatc tatccaaaca 1680

ttccatgttg atttgaacgt ttacccattg tggttgtgtc catttttgtt gccaaacaac 1740

cctggtatgg ttcatccaaa aggtgacgaa actgaattgt atgttgatat tggtgcttac 1800

ggtgaaccaa aaacaaaaca atttgaagct agagcatcta tgagacaaat ggaaaagttc 1860

gttagatcag ttcatggttt ccaaatgttg tacgctgatt gttacatgac tagagaagaa 1920

ttctgggata tgttcgatgg ttcattgtac cattctttga gagaacaaat gaactgtaag 1980

gatgcattcc cagaagttta cgataagatc tgtaaggctg caagacatta accgctgatc 2040

ctagagggcc gcatcatgta attagttatg tcacgcttac attcacgccc tccccccaca 2100

tccgctctaa ccgaaaagga aggagttaga caacctgaag tctaggtccc tatttatttt 2160

tttatagtta tgttagtatt aagaacgtta tttatatttc aaatttttct tttttttctg 2220

tacagacgcg tgtacgcatg taacattata ctgaaaacct tgcttgagaa ggttttggga 2280

cgctcgaagg ctttaatttg caagctgcgg ccctgcatta atgaatcggc caacgcgccg 2340

tattacaatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgcgttg 2400

gccgattcat taatgc 2416

<210> 17

<211> 3120

<212> DNA

<213> Artificial sequence

<400> 17

ccagcgtata caatctcgat agttggtttc ccgttctttc cactcccgtc acgcacagat 60

attataacat ctgcacaata ggcatttgca agaattactc gtgagtaagg aaagagtgag 120

gaactatcgc atacctgcat ttaaagatgc cgatttgggc gcgaatcctt tattttggct 180

tcaccctcat actattatca gggccagaaa aaggaagtgt ttccctcctt cttgaattga 240

tgttaccctc ataaagcacg tggcctctta tcgagaaaga aattaccgtc gctcgtgatt 300

tgtttgcaaa aagaacaaaa ctgaaaaaac ccagacacgc tcgacttcct gtcttcctat 360

tgattgcagc ttccaatttc gtcacacaac aaggtcctag cgacggctca caggttttgt 420

aacaagcaat cgaaggttct ggaatggcgg gaaagggttt agtaccacat gctatgatgc 480

ccactgtgat ctccagagca aagttcgttc gatcgtactg ttactctctc tctttcaaac 540

agaattgtcc gaatcgtgtg acaacaacag cctgttctca cacactcttt tcttctaacc 600

aagggggtgg tttagtttag tagaacctcg tgaaacttac atttacatat atataaactt 660

gcataaattg gtcaatgcaa gaaatacata tttggtcttt tctaattcgt agtttttcaa 720

gttcttagat gctttctttt tctctttttt acagatcatc aaggaagtaa ttatctactt 780

tttacaacaa atataaaaca atgcaatcat cctccgtaaa ggtatcccca ttcgacttaa 840

tgtcagcaat catcaagggt tctatggacc aatcaaacgt atcatcagaa tcaggtggtg 900

ctgcagccat ggttttggaa aacagagaat tcattatgat cttgactaca tccattgctg 960

ttttgatcgg ttgtgttgtc gtattgatat ggagaagatc aggtcaaaaa caatccaaga 1020

ctccagaacc acctaaacct ttgattgtta aggatttgga agtagaagtt gatgacggta 1080

aacaaaaggt tacaatattt ttcggtacac aaaccggtac tgctgaaggt ttcgcaaaag 1140

ccttggctga agaagcaaag gccagatacg aaaaggcaat ttttaaggtt gtcgatttgg 1200

atgactatgc cggtgacgac gatgaatacg aagaaaaatt gaaaaaggaa actttggcct 1260

ttttcttttt ggctacatat ggtgacggtg aaccaaccga caatgctgca agattctaca 1320

aatggtttgc tgagggtaaa gaacgtggtg aatggttgca aaacttaaag tatggtgttt 1380

tcggtttggg taacagacaa tacgaacatt tcaacaaagt tgcaaaggta gttgacgata 1440

taatcacaga acaaggtggt aaaagaatcg tcccagtagg tttgggtgac gatgaccaat 1500

gtattgaaga tgacttcgcc gcttggagag aattattatg gcctgaatta gatcaattgt 1560

taagagacga agatgacgct accactgtat ctacaccata taccgcagcc gttttggaat 1620

acagagtcgt atttcatgat cctgaaggtg catcattaca agacaagtca tggggttccg 1680

ccaatggtca tactgttcac gatgctcaac acccatgtag agccaacgtt gctgtcagaa 1740

aagaattgca tactcctgct agtgatagat cttgcacaca cttggaattc gacatttctg 1800

gtactggttt aacatatgaa accggtgacc atgtaggtgt ttactgtgaa aatttgccag 1860

aaacagtcga agaagcagaa agattgttag gtttctcacc tgatgtatat ttttccatac 1920

acaccgaaag agaagacggt actccattaa gtggttcttc attgtctcca ccttttccac 1980

cttgcacttt gagaacagca ttaaccagat acgccgatgt tttgtccagt cctaaaaagt 2040

ctgcattggt cgccttagct gcacatgcat cagatccatc cgaagccgac agattgaaat 2100

atttggctag tccttctggt aaagatgaat acgctcaatg ggttgtcgca agtcaaagat 2160

ctttgttaga aattatggcc gaatttccat ctgctaagcc acctttgggt gtcttctttg 2220

ccgctgtagc tccaagattg caacctagat actacagtat ctcttcatcc ccaaagatgg 2280

ttccttctag aatacatgtt acctgtgcat tggtctgcga taaaatgcca actggtagaa 2340

tccacaaggg tatttgttca acatggatga aatatgccgt tccattagaa gaatcacaag 2400

attgctcctg ggcacctatc ttcgttagac aatcaaactt caaattgcca gctgatacct 2460

ccgtccctat cattatgatt ggtccaggta caggtttagc tcctttcaga ggtttcttgc 2520

aagaaagatt tgcattgaag gaagctggtg cagaattggg tagttctatc ttgttctttg 2580

gttgtagaaa cagaaagatg gattacatct acgaagacga attgaacggt ttcgtagaaa 2640

gtggtgcttt gtctgaattg atcgttgcat tttcaagaga aggtccaact aaggaatacg 2700

ttcaacataa gatgatggaa aaggctagtg atatctggaa cgtcatctct caaggtggtt 2760

atatatacgt atgcggtgac gctaagggta tggcaagaga cgttcataga actttgcaca 2820

caatcttaca agaacaaggt tctttagatt catccaaggc tgaatcaatg gtaaagaact 2880

tacaaatgac tggtagatac ttgagagatg tcagttataa aaaaaataag tgtatacaaa 2940

ttttaaagtg actcttaggt tttaaaacga aaattcttat tcttgagtaa ctctttcctg 3000

taggtcaggt tgctttctca ggtatagcat gaggtcgctc ttattgacca cacctctacc 3060

ggcatgccga agtgatcccc cacacaccat agcttcaaaa tgtttctact ccttttttac 3120

<210> 18

<211> 1988

<212> DNA

<213> Artificial sequence

<400> 18

ggtatagcat gaggtcgctc ttattgacca cacctctacc ggcatgccga agtgatcccc 60

cacacaccat agcttcaaaa tgtttctact ccttttttac tcttccagat tttctcggac 120

tccgcgcatc gccgtaccac ttcaaaacac ccaagcacag catactaaat ttcccctctt 180

tcttcctcta gggtgtcgtt aattacccgt actaaaggtt tggaaaagaa aaaagagacc 240

gcctcgtttc tttttcttcg tcgaaaaagg caataaaaat ttttatcacg tttctttttc 300

ttgaaaattt ttttttttga tttttttctc tttcgatgac ctcccattga tatttaagtt 360

aataaacggt cttcaatttc tcaagtttca gtttcatttt tcttgttcta ttacaacttt 420

ttttacttct tgctcattag aaagaaagca tagcaatcta atctaagttt taattacaaa 480

atggctccaa tggaattgct gttgatagtt tctccattgg ttttggccct gatcatcttt 540

tttagcttca ggggtacttc taaaggtggt gataaggctg aaaaaattcc tccaggtact 600

atgggttggc cattgattgg tcatacaatt ccttttatgc aaccccattc ttctgcttca 660

ttgggtttgt ttgttgacca gaatattgct aagcacggta gaatcttcag gatgaatttg 720

ttgggtaagc caactatcgt ttctgctgat gctgatttca acagattcat cttgcaatcc 780

gaaggtagga tgttcgaaaa ttcttgtcca acctccattg ccgaaattat gggtagatgg 840

tctatgttgg ctttggctgg tgatgttcat agagaaatga gatccattgc tgtcaacttc 900

atgtccaacg ttaagttgag aacttacttc ttgccagacg ttgaacaaca agccattaag 960

attttgtctg cttggagaca tggttctact ttctctgctc aagaagaagg taagaagttc 1020

gcttttaact tgatggtcaa gcacttgatg tctatggatc caggtatgcc agaaactgaa 1080

caattgagaa aagagtacat cacgttcatg aagggtatgg cttctattcc attgaatttg 1140

ccaggtactg cttacagaaa ggcattgcaa tctagatcca tcatcttgaa gatcatgggt 1200

caaaagttgg acgaaagagt tgaaaaagtt aagagaggtt gcgaaggttt ggaagaagat 1260

gatttgttag cttctgttgc tgcccaatct aacattacca gagatcaaat tctggacctg 1320

atcctgtcta tgttatttgc tggtcacgaa acttcatctg ctgctatttg tttggctatc 1380

tacttcttgg aatcttctcc aaaagccttg caacagttga gagaagaaca tatcaacata 1440

gccaagatga aggaagaaaa aggcgaaact ggtttgactt gggatgatta caaacagatg 1500

gaattcaccc attgcgttat caacgaaact ttgagattgg gtaacatcgt caagttcttg 1560

catagaaagg ccattaagga tgttcagtac aagggttacg atattccatg tggttgggaa 1620

gttgttccaa ttatttcttc cgctcatttg gacccatcta tctatgatga tccacaatct 1680

tacaatcctt ggagatggca aactatttct actgctactt ccaagaacaa caacatcatg 1740

tctttttcag gtggtccaag agtttgtcca ggtgctgaat tggctaaaat ggaaatggct 1800

gttttcttgc atcacttggt ccaaaagttc aactgggaat tagctgaaca tgactaccca 1860

gtttcttttc cattcttggg tttcccaaaa cacttgccaa tcaaggttca tgccattgat 1920

cataaggctt ccgcttaaaa gcatcttgcc ctgtgcttgg cccccagtgc agcgaacgtt 1980

ataaaaac 1988

<210> 19

<211> 2292

<212> DNA

<213> Artificial sequence

<400> 19

ggtatagcat gaggtcgctc ttattgacca cacctctacc ggcatgccga agtgatcccc 60

cacacaccat agcttcaaaa tgtttctact ccttttttac tcttccagat tttctcggac 120

tccgcgcatc gccgtaccac ttcaaaacac ccaagcacag catactaaat ttcccctctt 180

tcttcctcta gggtgtcgtt aattacccgt actaaaggtt tggaaaagaa aaaagagacc 240

gcctcgtttc tttttcttcg tcgaaaaagg caataaaaat ttttatcacg tttctttttc 300

ttgaaaattt ttttttttga tttttttctc tttcgatgac ctcccattga tatttaagtt 360

aataaacggt cttcaatttc tcaagtttca gtttcatttt tcttgttcta ttacaacttt 420

ttttacttct tgctcattag aaagaaagca tagcaatcta atctaagttt taattacaaa 480

atggctatgg aattgttgtt gttgatccca gcttttattg ttgcaatcat cattttcttt 540

tcttttaaat caactaacgg tacttctaca aagccattga agttgcctcc aggtcaaatg 600

ggttggcctt ttattggtca tacaatccct tttatgcaac cacattcttc agcttctttg 660

ggtccataca tcgatttgaa cactgcaaga tacggtacaa tttttagaat gaatttgttg 720

gctaagccaa ctatcgtttc agcagatcca gaattcaata gatacatctt gcaaaacgaa 780

ggtagattgt tcgaaaactc ttgtccaact tcaattgctg aaattatggg tagatggtct 840

atgttggcat taacaggtga cgttcataga gaaatgagat caatcgctgt ttcttttatg 900

tcaaacgtta agttgagaac atacttcatc ggtgacatcg aacaacaagc aattaaagtt 960

ttggcttctt gggcaggtag agatgctcca ttttcagcac aagatgaagg taaaaagttc 1020

gcttttaatt tgatggttaa acatttgatg tctatggaac caggaatgaa ggaaactgaa 1080

caattgagat ctgaatacca tgcttttatg aagggtatgg catcaattcc aattaatttg 1140

ccaggtacag cttacagaaa agcattgcaa tctagatcaa tcatcttgaa gattatgggt 1200

gaaaaattag atgaaagaat taaacaagtt aaagaaggtt gtgaaggttt ggaacaagat 1260

gatttgttag cttctgtttc aaagcatcca aatttggcaa aggaacaaat cttggatttg 1320

atcttgtcta tgttgtttgc tggtcatgaa acttcttcag ctgcaatcgc tttggcaata 1380

tactttttgg aatcatgtcc aaaagctgtt gaacaattga gagaagaaca taaggaaatc 1440

gcaagacaaa agaaagaaag aggtgaaaca ggtttgaact gggatgatta caagaaaatg 1500

gaattcactc attgtgttat taatgaaaca ttgagaatgg gtaacatcgt taagttctta 1560

catagaagag ctattaaaga tgttcaattc aaaggttacg atatcccatg tggttgggaa 1620

gttgttccaa ttatttctgc tgcacatttg gattcttcaa tctatgatga tccacaaaga 1680

tacgatccat ggagatggca agctatttta gctggtaaca ctaaaaataa caacgttaca 1740

tcaattatgt ctttttcagg tggtccaaga ttgtgtccag gtgctgaatt ggcaaagttg 1800

gaaatcgctg ttttcttgca tcatttggtt caaaagtacc aatgggaaat ggcagaacat 1860

gattacccag tttctttccc atttttaggt ttcccaaaga gattgccaat taaagttaga 1920

ccattgggtg actaaccgct gatcctagag ggccgcatca tgtaattagt tatgtcacgc 1980

ttacattcac gccctccccc cacatccgct ctaaccgaaa aggaaggagt tagacaacct 2040

gaagtctagg tccctattta tttttttata gttatgttag tattaagaac gttatttata 2100

tttcaaattt ttcttttttt tctgtacaga cgcgtgtacg catgtaacat tatactgaaa 2160

accttgcttg agaaggtttt gggacgctcg aaggctttaa tttgcaagct gcggccctgc 2220

attaatgaat cggccaacgc gcaagcatct tgccctgtgc ttggccccca gtgcagcgaa 2280

cgttataaaa ac 2292

<210> 20

<211> 2379

<212> DNA

<213> Artificial sequence

<400> 20

ggtatagcat gaggtcgctc ttattgacca cacctctacc ggcatgccga agtgatcccc 60

cacacaccat agcttcaaaa tgtttctact ccttttttac tcttccagat tttctcggac 120

tccgcgcatc gccgtaccac ttcaaaacac ccaagcacag catactaaat ttcccctctt 180

tcttcctcta gggtgtcgtt aattacccgt actaaaggtt tggaaaagaa aaaagagacc 240

gcctcgtttc tttttcttcg tcgaaaaagg caataaaaat ttttatcacg tttctttttc 300

ttgaaaattt ttttttttga tttttttctc tttcgatgac ctcccattga tatttaagtt 360

aataaacggt cttcaatttc tcaagtttca gtttcatttt tcttgttcta ttacaacttt 420

ttttacttct tgctcattag aaagaaagca tagcaatcta atctaagttt taattacaaa 480

atgttcgaaa ccgaacatca taccttgttg cctttgttgt tgttgccatc tttgctgtcc 540

ttgctgttgt tcttgattct gttgaagaga agaaaccgta agaccagatt caatttgcct 600

ccaggtaaat ctggttggcc atttttgggt gaaactatcg gttatttgaa gccatacact 660

gctactactt tgggtgattt catgcaacaa cacgtttcta agtacggtaa gatctacagg 720

tctaatttgt tcggtgaacc tactatcgtt tctgctgatg ctggtttgaa tagattcatc 780

ttgcaaaacg aaggcaggtt gttcgaatgt tcttacccaa gatctatcgg tggtatttta 840

ggtaagtggt ccatgttggt tttggttggt gatatgcata gagatatgag gtccatctcc 900

ttgaatttct tgtctcatgc tagattgagg accatcttgt tgaaggatgt tgaaagacac 960

accttgttcg ttttggattc ttggcaacaa aactccattt tctccgctca agatgaagct 1020

aagaagttca cttttaactt gatggccaag cacatcatgt ctatggatcc aggtgaagaa 1080

gaaactgagc aactgaagaa agaatacgtc actttcatga agggtgttgt ttctgctcca 1140

ttgaatttgc caggtactgc ttatcataag gccttgcaat ctagagctac catcttgaag 1200

ttcatcgaac gtaagatgga agagagaaag ttggacatca aagaagagga ccaagaggaa 1260

gaagaggtta agactgaaga tgaggctgaa atgtctaagt ccgatcatgt tagaaagcaa 1320

agaaccgatg atgacttgtt aggttgggtt ttgaagcact ctaacttgtc cactgaacaa 1380

atcttggacc tgatcttgtc tttgttattt gctggtcacg aaacctcatc tgttgctatt 1440

gctttggcta tattcttctt gcaagcttgt ccaaaagccg tcgaagaatt gagagaagaa 1500

catttggaaa ttgccagggc caaaaaagaa ttgggtgaat ctgaattgaa ctgggacgat 1560

tacaagaaga tggatttcac tcaatgcgtc atcaacgaaa ctttgagatt gggtaacgtt 1620

gtcagattct tgcatagaaa ggccttgaaa gatgtcagat acaagggtta cgatattcca 1680

tcaggttgga aagttttgcc agttatttct gccgttcact tggataactc tagatacgat 1740

caacccaatt tgttcaatcc ttggagatgg cagcagcaaa acaatggtgc ttcttcttct 1800

ggttctggtt cattttctac ttggggtaac aattacatgc catttggtgg tggtcctaga 1860

ttgtgtgctg gttcagaatt ggctaaattg gaaatggccg ttttcatcca tcatctggtg 1920

ttgaagttta actgggaatt agccgaagat gataagccat ttgctttccc atttgttgat 1980

ttcccaaacg gtttgccaat cagagtttcc agaattttgt gaccgctgat cctagagggc 2040

cgcatcatgt aattagttat gtcacgctta cattcacgcc ctccccccac atccgctcta 2100

accgaaaagg aaggagttag acaacctgaa gtctaggtcc ctatttattt ttttatagtt 2160

atgttagtat taagaacgtt atttatattt caaatttttc ttttttttct gtacagacgc 2220

gtgtacgcat gtaacattat actgaaaacc ttgcttgaga aggttttggg acgctcgaag 2280

gctttaattt gcaagctgcg gccctgcatt aatgaatcgg ccaacgcgca agcatcttgc 2340

cctgtgcttg gcccccagtg cagcgaacgt tataaaaac 2379

<210> 21

<211> 2313

<212> DNA

<213> Artificial sequence

<400> 21

ggtatagcat gaggtcgctc ttattgacca cacctctacc ggcatgccga agtgatcccc 60

cacacaccat agcttcaaaa tgtttctact ccttttttac tcttccagat tttctcggac 120

tccgcgcatc gccgtaccac ttcaaaacac ccaagcacag catactaaat ttcccctctt 180

tcttcctcta gggtgtcgtt aattacccgt actaaaggtt tggaaaagaa aaaagagacc 240

gcctcgtttc tttttcttcg tcgaaaaagg caataaaaat ttttatcacg tttctttttc 300

ttgaaaattt ttttttttga tttttttctc tttcgatgac ctcccattga tatttaagtt 360

aataaacggt cttcaatttc tcaagtttca gtttcatttt tcttgttcta ttacaacttt 420

ttttacttct tgctcattag aaagaaagca tagcaatcta atctaagttt taattacaaa 480

atgtctgatt tggaattttt cttgtttttg attccaccaa tcttagcagt tttgatcatc 540

ttgaatttgt ttaaaagaaa acataatttt caaaatttgc caccaggaga tatgggttgg 600

ccatttttag gtgaaactat cggttatttg agaccatact ctgctactac aattggtgac 660

ttcatgcaag atcatatctc tagatacggt aaaattttta agtcaaattt gtttggtgaa 720

ccaacaattg tttcagctga tgcaggtttg aacagataca tcttgcaaaa cgaaggtaga 780

ttgttcgaat gtaactaccc aagatctatt ggtggtattt tgggtaaatg gtcaatgttg 840

gttcaagttg gtcaaatgca tagagatatg agaatgatcc cattgaattt cttgtctaac 900

gctagattga gaaatcaatt gttatctgaa gttgaaaagc atactttgtt ggttttgggt 960

tcttggaaac aagattcagt tgtttgtgct caagatgaag ctaagaaatt gacttttaat 1020

ttcatggcag aacatattat gtctttacaa ccaggaaatc cagaaactga aaagttgaag 1080

aaagaataca tcacttttat gaaaggtgtt gtttcagctc cattaaattt tccaggtact 1140

gcttacagaa aggcattgca atctagatca acaatcttgg gttttattga aagaaagatg 1200

gaagaaagat tgaaagaaat gaatagaaac gaaaacgatt tgttgggttg ggttttgaaa 1260

aattctaatt tgtcaaagga acaaatctta gatttgttat tgtctttatt gtttgcaggt 1320

catgaaactt cttcagttgc tattgcattg tctattttct tgttggaatc atgtccagct 1380

gcagttcaac aattgactga agaacatttg gaaatctcta gagctaagaa acaatcaggt 1440

gaaacagaat tgaactggga tgattacaag aaaatggaat tcactcaatg tgttattaat 1500

gaaacattga gattgggtaa cgttgttaga tttttacata gaaaagcagt taaggatgtt 1560

agatacaagg gttacgatat cccatgtggt tggaaagttt tgccagttat ttctgctgca 1620

catttggatc catcattgtt tgatagacca catgattttg atccatggag atggcaaaat 1680

gctgaagaat ctccatctgg taaaggtggt tctactggta catcttcaac tacaaagtct 1740

tcaaacaact tcatgccatt tggtggtggt ccaagattgt gtgctggttc agaattggca 1800

aagttggaaa tggctatttt cattcattac ttagttttga acttccattg gaagttggct 1860

gcaacagatc aagcttttgc atatccatac gttgatttcc caaacgcttt gccaattaat 1920

atccaacata gatctttaaa taagttgcat gattaaccgc tgatcctaga gggccgcatc 1980

atgtaattag ttatgtcacg cttacattca cgccctcccc ccacatccgc tctaaccgaa 2040

aaggaaggag ttagacaacc tgaagtctag gtccctattt atttttttat agttatgtta 2100

gtattaagaa cgttatttat atttcaaatt tttctttttt ttctgtacag acgcgtgtac 2160

gcatgtaaca ttatactgaa aaccttgctt gagaaggttt tgggacgctc gaaggcttta 2220

atttgcaagc tgcggccctg cattaatgaa tcggccaacg cgcaagcatc ttgccctgtg 2280

cttggccccc agtgcagcga acgttataaa aac 2313

<210> 22

<211> 2413

<212> DNA

<213> Artificial sequence

<400> 22

acgcacagat attataacat ctgcacaata ggcatttgca agaattactc gtgagtaagg 60

aaagagtgag gaactatcgc atacctgcat ttaaagatgc cgatttgggc gcgaatcctt 120

tattttggct tcaccctcat actattatca gggccagaaa aaggaagtgt ttccctcctt 180

cttgaattga tgttaccctc ataaagcacg tggcctctta tcgagaaaga aattaccgtc 240

gctcgtgatt tgtttgcaaa aagaacaaaa ctgaaaaaac ccagacacgc tcgacttcct 300

gtcttcctat tgattgcagc ttccaatttc gtcacacaac aaggtcctag cgacggctca 360

caggttttgt aacaagcaat cgaaggttct ggaatggcgg gaaagggttt agtaccacat 420

gctatgatgc ccactgtgat ctccagagca aagttcgttc gatcgtactg ttactctctc 480

tctttcaaac agaattgtcc gaatcgtgtg acaacaacag cctgttctca cacactcttt 540

tcttctaacc aagggggtgg tttagtttag tagaacctcg tgaaacttac atttacatat 600

atataaactt gcataaattg gtcaatgcaa gaaatacata tttggtcttt tctaattcgt 660

agtttttcaa gttcttagat gctttctttt tctctttttt acagatcatc aaggaagtaa 720

ttatctactt tttacaacaa atataaaaca atggctatgg aattgttgtt gttgatccca 780

gcttttattg ttgcaatcat cattttcttt tcttttaaat caactaacgg tacttctaca 840

aagccattga agttgcctcc aggtcaaatg ggttggcctt ttattggtca tacaatccct 900

tttatgcaac cacattcttc agcttctttg ggtccataca tcgatttgaa cactgcaaga 960

tacggtacaa tttttagaat gaatttgttg gctaagccaa ctatcgtttc agcagatcca 1020

gaattcaata gatacatctt gcaaaacgaa ggtagattgt tcgaaaactc ttgtccaact 1080

tcaattgctg aaattatggg tagatggtct atgttggcat taacaggtga cgttcataga 1140

gaaatgagat caatcgctgt ttcttttatg tcaaacgtta agttgagaac atacttcatc 1200

ggtgacatcg aacaacaagc aattaaagtt ttggcttctt gggcaggtag agatgctcca 1260

ttttcagcac aagatgaagg taaaaagttc gcttttaatt tgatggttaa acatttgatg 1320

tctatggaac caggaatgaa ggaaactgaa caattgagat ctgaatacca tgcttttatg 1380

aagggtatgg catcaattcc aattaatttg ccaggtacag cttacagaaa agcattgcaa 1440

tctagatcaa tcatcttgaa gattatgggt gaaaaattag atgaaagaat taaacaagtt 1500

aaagaaggtt gtgaaggttt ggaacaagat gatttgttag cttctgtttc aaagcatcca 1560

aatttggcaa aggaacaaat cttggatttg atcttgtcta tgttgtttgc tggtcatgaa 1620

acttcttcag ctgcaatcgc tttggcaata tactttttgg aatcatgtcc aaaagctgtt 1680

gaacaattga gagaagaaca taaggaaatc gcaagacaaa agaaagaaag aggtgaaaca 1740

ggtttgaact gggatgatta caagaaaatg gaattcactc attgtgttat taatgaaaca 1800

ttgagaatgg gtaacatcgt taagttctta catagaagag ctattaaaga tgttcaattc 1860

aaaggttacg atatcccatg tggttgggaa gttgttccaa ttatttctgc tgcacatttg 1920

gattcttcaa tctatgatga tccacaaaga tacgatccat ggagatggca agctatttta 1980

gctggtaaca ctaaaaataa caacgttaca tcaattatgt ctttttcagg tggtccaaga 2040

ttgtgtccag gtgctgaatt ggcaaagttg gaaatcgctg ttttcttgca tcatttggtt 2100

caaaagtacc aatgggaaat ggcagaacat gattacccag tttctttccc atttttaggt 2160

ttcccaaaga gattgccaat taaagttaga ccattgggtg actaaagtta taaaaaaaat 2220

aagtgtatac aaattttaaa gtgactctta ggttttaaaa cgaaaattct tattcttgag 2280

taactctttc ctgtaggtca ggttgctttc tcaggtatag catgaggtcg ctcttattga 2340

ccacacctct accggcatgc cgaatactag cgttgaatgt tagcgtcaac aacaagaagt 2400

ttaatgacgc gga 2413

<210> 23

<211> 2382

<212> DNA

<213> Artificial sequence

<400> 23

attttcgcgt tgagaagatg ttcttatcca aatttcaact gttatataga agtgatcccc 60

cacacaccat agcttcaaaa tgtttctact ccttttttac tcttccagat tttctcggac 120

tccgcgcatc gccgtaccac ttcaaaacac ccaagcacag catactaaat ttcccctctt 180

tcttcctcta gggtgtcgtt aattacccgt actaaaggtt tggaaaagaa aaaagagacc 240

gcctcgtttc tttttcttcg tcgaaaaagg caataaaaat ttttatcacg tttctttttc 300

ttgaaaattt ttttttttga tttttttctc tttcgatgac ctcccattga tatttaagtt 360

aataaacggt cttcaatttc tcaagtttca gtttcatttt tcttgttcta ttacaacttt 420

ttttacttct tgctcattag aaagaaagca tagcaatcta atctaagttt taattacaaa 480

atggatttgc catctgcttc agctgctgtt gctgctgcta ctgctgcagt tattttcttg 540

ttgactatct acctgctgcc aaagaaaaaa tctccagctt ctactggtaa gaacggttct 600

acttctttgg aatcttaccc agttattggt aacttgccac acttcgttaa gaacagaaac 660

agattcttgg attgggttgc cgaaatcatt tctcaatctc caactggtac tgttattgct 720

gctccattgg tttttacttc taacccagaa aacgttgaac ataccgctaa gtctagattt 780

gatgcttatg ctagaggtcc agctgctaca gctgttttac atgatttttt aggttccggc 840

atcttgaacg ttgatggtga tagttggagg gctcaaagaa agactgcttc ttctgaattc 900

actaccagat ctttgagagc cttcattttg gatgctgttg acggtgaagc tgctggtaga 960

ttattgccat tattgtctag agctgctgct tctggtgaag tttttgactt gcaagatgtc 1020

ttggaaagat tcgccttcga taacatttgc tccattattt tcgatgccga tccaaactgt 1080

ttgaacgata ctcatgatgg tgttggtgaa agattctacc atgcttttca tgatgctacc 1140

ttgttgtcta ctggcagata ttactatcca ttccattggg tttggaggtt gttgagatgg 1200

ttgaatttgg gtactgaaaa gcgtttgaga gatgccgttt ctgatgttca taaggccatt 1260

gatgaattgg tcggttctag aaaaactgaa gttggtacta ctgttagaag gcaaggtggt 1320

ggttctgatt tgttgtcaag atttgctgaa ggtggtgatt actccgatga tgttttaagg 1380

gatgtcttga tcaacttcgt tttggctggt agagatacaa ctccatcagc tttgacttgg 1440

tttttcttta tgatctcctc cagaccagat gttgttgatc aaattttgga cgagatcagg 1500

tccatcagag atcatcaaga tagatctaat ccaaacggtg gtggtggcgg ttttactttg 1560

gaagaattga gagaaatgaa ctacttgcat gctgccatta ccgaatcctt gagattgaat 1620

ccaccagttc cattgatgcc aaagatgtgt atggaagatg atgtattgcc agatggtact 1680

gtagttagaa gaggttggac tgttatgtac tctgcttttg ctatgggtag aaaggctgaa 1740

atttggggtg aagattgcat ggaattcaaa ccagaaagat ggttggatga tggtggttgt 1800

tttaaatctg cttccgctta tagattgcca gcatttcatg ctggtcctag aatttgtttg 1860

ggtaaagata tggcctacat tcagatgaag gctgttgctt catctttgtt ggaaaggttc 1920

gaagttgaag tcgttgaaaa aagaggtaag ccagaattgt ccatcaccat gagaatggac 1980

agaggtttgc cagttagagt gaaagaaaga aagcgtggtt gttaaccgct gatcctagag 2040

ggccgcatca tgtaattagt tatgtcacgc ttacattcac gccctccccc cacatccgct 2100

ctaaccgaaa aggaaggagt tagacaacct gaagtctagg tccctattta tttttttata 2160

gttatgttag tattaagaac gttatttata tttcaaattt ttcttttttt tctgtacaga 2220

cgcgtgtacg catgtaacat tatactgaaa accttgcttg agaaggtttt gggacgctcg 2280

aaggctttaa tttgcaagct gcggccctgc attaatgaat cggccaacgc gcaagcatct 2340

tgccctgtgc ttggccccca gtgcagcgaa cgttataaaa ac 2382

<210> 24

<211> 2769

<212> DNA

<213> Artificial sequence

<400> 24

ggtatagcat gaggtcgctc ttattgacca cacctctacc ggcatgccga atactagcgt 60

tgaatgttag cgtcaacaac aagaagttta atgacgcgga ggccaaggca aaaagattcc 120

ttgattacgt aagggagtta gaatcatttt gaataaaaaa cacgcttttt cagttcgagt 180

ttatcattat caatactgcc atttcaaaga atacgtaaat aattaatagt agtgattttc 240

ctaactttat ttagtcaaaa aattagcctt ttaattctgc tgtaacccgt acatgcccaa 300

aatagggggc gggttacaca gaatatataa catcgtaggt gtctgggtga acagtttatt 360

cctggcatcc actaaatata atggagcccg ctttttaagc tggcatccag aaaaaaaaag 420

aatcccagca ccaaaatatt gttttcttca ccaaccatca gttcataggt ccattctctt 480

agcgcaacta cagagaacag gggcacaaac aggcaaaaaa cgggcacaac ctcaatggag 540

tgatgcaacc tgcctggagt aaatgatgac acaaggcaat tgacccacgc atgtatctat 600

ctcattttct tacaccttct attaccttct gctctctctg atttggaaaa agctgaaaaa 660

aaaggttgaa accagttccc tgaaattatt cccctacttg actaataagt atataaagac 720

ggtaggtatt gattgtaatt ctgtaaatct atttcttaaa cttcttaaat tctactttta 780

tagttagtct tttttttagt tttaaaacac caagaactta gtttcgaata aacacacata 840

aacaaacaaa atgttcccat tggccattat cgttttgttg ttcccaactc tgctgttgtt 900

gtttattggt gttgctttgg gtttgagatc tggtgctaat gaatcttgga aaaagagggg 960

tttgaatatc cctccaggtt ctatgggttg gcctttgttg ggtgaaacta ttgcttttag 1020

aaagttgcat ccatgcacat ctttgggtga gtatatggaa gatagattgc agagatacgg 1080

taagatctac aggtctaatt tgtttggtgc tccaactgtt gtttctgctg atgctgaatt 1140

gaacagattc gttttgatga acgatggcaa gttgtttgaa ccatcttggc caaaatctgt 1200

tgccgatatt ttgggtaaga cctccatgtt ggttttgact ggtgaaatgc acaggtacat 1260

gaagtctttg tctgttaact tcatgggtat cgccagattg agaaatcatt tcttgggtga 1320

ttccgagagg tacattttgg aaaatttggc tacttggaaa gagggtgttc catttccagc 1380

taaagaagaa gcttgtaaga tcacctttaa cctgatggtc aagaacatct tgtctatgaa 1440

tccaggtgaa ccagaaaccg aaagattgag gatcttgtac atgtctttca tgaagggtgt 1500

tattgccatg ccattgaatt ttccaggtac tgcttacaga aaggccattc aatctagagc 1560

cactatcttg aaaaccatcg aacacttgat ggaagatcgt ttggaaaaaa agaaggccgg 1620

tactgataat attggtgaag ctgatttgtt gggcttcatc ttggaacaat ctaacttgga 1680

tgctgaacaa ttcggtgact tgttgttggg tttgttattt ggtggtcacg aaacatcttc 1740

taccgctatt actttggcta tctacttctt ggaaggttgt ccaaaagctg tccaagaatt 1800

gagagaagaa catttgaact tggtcaggat gaagaaacag agaggtgaat ctaaagcttt 1860

gacgtgggaa gattacaagt ctatggattt cgctcaatgc gttgtctctg aaactttgag 1920

attgggtaac atcatcaagt tcgttcacag aaaagctaac accgatgtcc aattcaaggg 1980

ttacgatatt ccatctggtt ggtctgttat tccagttttt gctgctgctc atttggatcc 2040

tactgtttac gataatccac aaaagttcga tccttggaga tggcaaacta tctcttcatc 2100

tactgccaga atcgataact acatgccatt tggtcaaggt ttgagaaatt gtgctggttt 2160

ggaattggct aagatggaaa ttgctgtttt cttgcaccac ttggtcttga atttcgattg 2220

ggaattagct gaaccagatc atccattggc ttatgctttt ccagaattcg aaaaaggctt 2280

gccaatcaag gtcagaaagt tgtctatttt ggagtaagat taatataatt atataaaaat 2340

attatcttct tttctttata tctagtgtta tgtaaaataa attgatgact acggaaagct 2400

tttttatatt gtttcttttt cattctgagc cacttaaatt tcgtgaatgt tcttgtaagg 2460

gacggtagat ttacaagtga tacaacaaaa agcaaggcgc tttttctaat aaaaagaaga 2520

aaagcattta acaattgaac acctctatat caacgaagaa tattactttg tctctaaatc 2580

cttgtaaaat gtgtacgatc tctatatggg ttactcataa gtgtaccgaa gactgcattg 2640

aaagtttatg ttttttcact ggaggcgtca ttttcgcgtt gagaagatgt tcttatccaa 2700

atttcaactg ttatatagaa gtgatccccc acacaccata gcttcaaaat gtttctactc 2760

cttttttac 2769

<210> 25

<211> 2081

<212> DNA

<213> Artificial sequence

<400> 25

tcctctaatc aggttccacc aaacagatac cccggtgttt cacggaatgg tacgtttgat 60

atcgctgatt tgagaggagg ttacacttga agaatcacag tcttgcgacc ggctattcaa 120

caaggcattc ccccaagttt gaattctttg aaatagattg ctattagcta gtaatccacc 180

aaatccttcg ctgctcacca atggaatcgc aagatgccca cgatgagact gttcaggtta 240

aacgcaaaag aaacacactc tgggaatttc ttcccaaatt gtatctctca atacgcatca 300

acccatgtca attaaacacg ctgtatagag actaggcaga tctgacgatc acctagcgac 360

tctctccacc gtttgacgag gccatttaca aaaacataac gaacgacaag cctactcgaa 420

ttcgtttcca aactcttttc gaacttgtct tcaactgctt tcgcatgaag tacctcccaa 480

ctacttttcc tcacacttgt actccatgac taaacccccc ctcccattac aaactaaaat 540

cttactttta ttttcttttg ccctctctgt cgctctgcct taactacgta tttctcgccg 600

agaaaaactt caatttaagc tattctccaa aaatcttagc gtatattttt tttccaaagt 660

gacaggtgcc ccgggtaacc cagttcatgt ctgcccctaa gaagatcgtc gttttgccag 720

gtgaccacgt tggtcaagaa atcacagccg aagccattaa ggttcttaaa gctatttctg 780

atgttcgttc caatgtcaag ttcgatttcg aaaatcattt aattggtggt gctgctatcg 840

atgctacagg tgttccactt ccagatgagg cgctggaagc ctccaagaag gctgatgccg 900

ttttgttagg tgctgtgggt ggtcctaaat ggggtaccgg tagtgttaga cctgaacaag 960

gtttactaaa aatccgtaaa gaacttcaat tgtacgccaa cttaagacca tgtaactttg 1020

catccgactc tcttttagac ttatctccaa tcaagccaca atttgctaaa ggtactgact 1080

tcgttgttgt cagagaatta gtgggaggta tttactttgg taagagaaag gaagacgatg 1140

gtgatggtgt cgcttgggat agtgaacaat acaccgttcc agaagtgcaa agaatcacaa 1200

gaatggccgc tttcatggcc ctacaacatg agccaccatt gcctatttgg tccttggata 1260

aagctaatgt tttggcctct tcaagattat ggagaaaaac tgtggaggaa accatcaaga 1320

acgaattccc tacattgaag gttcaacatc aattgattga ttctgccgcc atgatcctag 1380

ttaagaaccc aacccaccta aatggtatta taatcaccag caacatgttt ggtgatatca 1440

tctccgatga agcctccgtt atcccaggtt ccttgggttt gttgccatct gcgtccttgg 1500

cctctttgcc agacaagaac accgcatttg gtttgtacga accatgccac ggttctgctc 1560

cagatttgcc aaagaataag gtcaacccta tcgccactat cttgtctgct gcaatgatgt 1620

tgaaattgtc attgaacttg cctgaagaag gtaaggccat tgaagatgca gttaaaaagg 1680

ttttggatgc aggtatcaga actggtgatt taggtggttc caacagtacc accgaagtcg 1740

gtgatgctgt cgccgaagaa gttaagaaaa tccttgctta aatactagcg ttgaatgtta 1800

gcgtcaacaa caagaagttt aatgacgcgg aggccaaggc aaaaagattc cttgattacg 1860

taagggagtt agaatcattt tgaataaaaa acacgctttt tcagttcgag tttatcatta 1920

tcaatactgc catttcaaag aatacgtaaa taattaatag tagtgatttt cctaacttta 1980

tttagtcaaa aaattagcct tttaattctg ctgtaacccg tacatgccca aaataggggg 2040

cgggttacac agaatatata acatcgtagg tgtctgggtg a 2081

<210> 26

<211> 705

<212> DNA

<213> Artificial sequence

<400> 26

agtctaggtc cctatttatt tttttatagt tatgttagta ttaagaacgt tatttatatt 60

tcaaattttt cttttttttc tgtacagacg cgtgtacgca tgtaacatta tactgaaaac 120

cttgcttgag aaggttttgg gacgctcgaa ggctttaatt tgcaagctgc ggccctgcat 180

taatgaatcg gccaacgcgc ctcactattt tttactgcgg aagcggaagc ggaaaatacg 240

gaaacgcgcg ggaacataca aaacatacaa aatatacctt tctcacacaa gaaatatatg 300

ctacttgcaa aatatcatac caaaaaactt ttcacaaccg aaaccaaaac caacggatat 360

catacattac actaccacca ttcaaacttt actactatcc tcccttcagt ttcccttttt 420

ctgccttttt cggtgacgga aatacgcttc agagacccta aagggaaatc catgccataa 480

caggaaagta acatcccaat gcggactata ccaccccacc acactcctac caataacggt 540

aactattcta tgttttctta ctcctatgtc tattcatctt tcatctgact acctaatact 600

atgcaaaaat gtaaaatcat cacacaaaac ataaacaatc aaaatcagcc atttccgcac 660

cttttcctct gtccactttc aaccgtccct ccaaatgtaa aatgg 705

<210> 27

<211> 5337

<212> DNA

<213> Artificial sequence

<400> 27

aactgcggtc aagatatttc ttgaatcagg cgccttagac cgctcggcca aacaaccaat 60

tacttgttga gaaatagagt ataattatcc tataaatata acgtttttga acacacatga 120

acaaggaagt acaggacaat tgattttgaa gagaatgtgg attttgatgt aattgttggg 180

attccatttt taataaggca ataatattag gtatgtggat atactagaag ttctcctcga 240

ccgtcgatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcagga 300

aattgtaaac gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt 360

ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat 420

agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa 480

cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta 540

atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc 600

ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc 660

gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac 720

acccgccgcg cttaatgcgc cgctacaggg cgcgtcgcgc cattcgccat tcaggctgcg 780

caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg 840

gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg 900

taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgggtaccgg 960

gccccccctc gaggtcgacg gtatcgataa gcttgatatc gaattcctgc agcccggggg 1020

atccactagt tctagagcgg ccgccaccgc ggtggagctc cagcttttgt tccctttagt 1080

gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt 1140

atccgctcac aattccacac aacataggag ccggaagcat aaagtgtaaa gcctggggtg 1200

cctaatgagt gaggtaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg 1260

gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc 1320

gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc 1380

ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata 1440

acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg 1500

cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct 1560

caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa 1620

gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc 1680

tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt 1740

aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg 1800

ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg 1860

cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct 1920

tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc 1980

tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg 2040

ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc 2100

aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt 2160

aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa 2220

aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat 2280

gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct 2340

gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg 2400

caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag 2460

ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta 2520

attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg 2580

ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg 2640

gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct 2700

ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta 2760

tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg 2820

gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc 2880

cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg 2940

gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga 3000

tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg 3060

ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat 3120

gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc 3180

tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca 3240

catttccccg aaaagtgcca cctgaacgaa gcatctgtgc ttcattttgt agaacaaaaa 3300

tgcaacgcga gagcgctaat ttttcaaaca aagaatctga gctgcatttt tacagaacag 3360

aaatgcaacg cgaaagcgct attttaccaa cgaagaatct gtgcttcatt tttgtaaaac 3420

aaaaatgcaa cgcgagagcg ctaatttttc aaacaaagaa tctgagctgc atttttacag 3480

aacagaaatg caacgcgaga gcgctatttt accaacaaag aatctatact tcttttttgt 3540

tctacaaaaa tgcatcccga gagcgctatt tttctaacaa agcatcttag attacttttt 3600

ttctcctttg tgcgctctat aatgcagtct cttgataact ttttgcactg taggtccgtt 3660

aaggttagaa gaaggctact ttggtgtcta ttttctcttc cataaaaaaa gcctgactcc 3720

acttcccgcg tttactgatt actagcgaag ctgcgggtgc attttttcaa gataaaggca 3780

tccccgatta tattctatac cgatgtggat tgcgcatact ttgtgaacag aaagtgatag 3840

cgttgatgat tcttcattgg tcagaaaatt atgaacggtt tcttctattt tgtctctata 3900

tactacgtat aggaaatgtt tacattttcg tattgttttc gattcactct atgaatagtt 3960

cttactacaa tttttttgtc taaagagtaa tactagagat aaacataaaa aatgtagagg 4020

tcgagtttag atgcaagttc aaggagcgaa aggtggatgg gtaggttata tagggatata 4080

gcacagagat atatagcaaa gagatacttt tgagcaatgt ttgtggaagc ggtattcgca 4140

atattttagt agctcgttac agtccggtgc gtttttggtt ttttgaaagt gcgtcttcag 4200

agcgcttttg gttttcaaaa gcgctctgaa gttcctatac tttctagaga ataggaactt 4260

cggaatagga acttcaaagc gtttccgaaa acgagcgctt ccgaaaatgc aacgcgagct 4320

gcgcacatac agctcactgt tcacgtcgca cctatatctg cgtgttgcct gtatatatat 4380

atacatgaga agaacggcat agtgcgtgtt tatgcttaaa tgcgtactta tatgcgtcta 4440

tttatgtagg atgaaaggta gtctagtacc tcctgtgata ttatcccatt ccatgcgggg 4500

tatcgtatgc ttccttcagc actacccttt agctgttcta tatgctgcca ctcctcaatt 4560

ggattagtct catccttcaa tgctatcatt tcctttgata ttggatcata ctaagaaacc 4620

attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg 4680

cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct 4740

tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc 4800

gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat 4860

atcgactacg tcgtaaggcc gtttctgaca gagtaaaatt cttgagggaa ctttcaccat 4920

tatgggaaat gcttcaagaa ggtattgact taaactccat caaatggtca ggtcattgag 4980

tgttttttat ttgttgtatt tttttttttt tagagaaaat cctccaatat caaattagga 5040

atcgtagttt catgattttc tgttacacct aactttttgt gtggtgccct cctccttgtc 5100

aatattaatg ttaaagtgca attctttttc cttatcacgt tgagccatta gtatcaattt 5160

gcttacctgt attcctttac tatcctcctt tttctccttc ttgataaatg tatgtagatt 5220

gcgtatatag tttcgtctac cctatgaaca tattccattt tgtaatttcg tgtcgtttct 5280

attatgaatt tcatttataa agtttatgta caaatatcat aaaaaaagag aatcttt 5337

<210> 28

<211> 1120

<212> DNA

<213> Artificial sequence

<400> 28

atttcattta taaagtttat gtacaaatat cataaaaaaa gagaatcttt ttagttttgc 60

tggccgcatc ttctcaaata tgcttcccag cctgcttttc tgtaacgttc accctctacc 120

ttagcatccc ttccctttgc aaatagtcct cttccaacaa taataatgtc agatcctgta 180

gagaccacat catccacggt tctatactgt tgacccaatg cgtctccctt gtcatctaaa 240

cccacaccgg gtgtcataat caaccaatcg taaccttcat ctcttccacc catgtctctt 300

tgagcaataa agccgataac aaaatctttg tcgctcttcg caatgtcaac agtaccctta 360

gtatattctc cagtagatag ggagcccttg catgacaatt ctgctaacat caaaaggcct 420

ctaggttcct ttgttacttc ttctgccgcc tgcttcaaac cgctaacaat acctgggccc 480

accacaccgt gtgcattcgt aatgtctgcc cattctgcta ttctgtatac acccgcagag 540

tactgcaatt tgactgtatt accaatgtca gcaaattttc tgtcttcgaa gagtaaaaaa 600

ttgtacttgg cggataatgc ctttagcggc ttaactgtgc cctccatgga aaaatcagtc 660

aagatatcca catgtgtttt tagtaaacaa attttgggac ctaatgcttc aactaactcc 720

agtaattcct tggtggtacg aacatccaat gaagcacaca agtttgtttg cttttcgtgc 780

atgatattaa atagcttggc agcaacagga ctaggatgag tagcagcacg ttccttatat 840

gtagctttcg acatgattta tcttcgtttc ctgcaggttt ttgttctgtg cagttgggtt 900

aagaatactg ggcaatttca tgtttcttca acactacata tgcgtatata taccaatcta 960

agtctgtgct ccttccttcg ttcttccttc tgttcggaga ttaccgaatc aaaaaaattt 1020

caaagaaacc gaaatcaaaa aaaagaataa aaaaaaaatg atgaattgaa agtgatcccc 1080

cacacaccat agcttcaaaa tgtttctact ccttttttac 1120

<210> 29

<211> 2204

<212> DNA

<213> Artificial sequence

<400> 29

agtgatcccc cacacaccat agcttcaaaa tgtttctact ccttttttac tcttccagat 60

tttctcggac tccgcgcatc gccgtaccac ttcaaaacac ccaagcacag catactaaat 120

ttcccctctt tcttcctcta gggtgtcgtt aattacccgt actaaaggtt tggaaaagaa 180

aaaagagacc gcctcgtttc tttttcttcg tcgaaaaagg caataaaaat ttttatcacg 240

tttctttttc ttgaaaattt ttttttttga tttttttctc tttcgatgac ctcccattga 300

tatttaagtt aataaacggt cttcaatttc tcaagtttca gtttcatttt tcttgttcta 360

ttacaacttt ttttacttct tgctcattag aaagaaagca tagcaatcta atctaagttt 420

taattacaaa atgttcccat tggccattat cgttttgttg ttcccaactc tgctgttgtt 480

gtttattggt gttgctttgg gtttgagatc tggtgctaat gaatcttgga aaaagagggg 540

tttgaatatc cctccaggtt ctatgggttg gcctttgttg ggtgaaacta ttgcttttag 600

aaagttgcat ccatgcacat ctttgggtga gtatatggaa gatagattgc agagatacgg 660

taagatctac aggtctaatt tgtttggtgc tccaactgtt gtttctgctg atgctgaatt 720

gaacagattc gttttgatga acgatggcaa gttgtttgaa ccatcttggc caaaatctgt 780

tgccgatatt ttgggtaaga cctccatgtt ggttttgact ggtgaaatgc acaggtacat 840

gaagtctttg tctgttaact tcatgggtat cgccagattg agaaatcatt tcttgggtga 900

ttccgagagg tacattttgg aaaatttggc tacttggaaa gagggtgttc catttccagc 960

taaagaagaa gcttgtaaga tcacctttaa cctgatggtc aagaacatct tgtctatgaa 1020

tccaggtgaa ccagaaaccg aaagattgag gatcttgtac atgtctttca tgaagggtgt 1080

tattgccatg ccattgaatt ttccaggtac tgcttacaga aaggccattc aatctagagc 1140

cactatcttg aaaaccatcg aacacttgat ggaagatcgt ttggaaaaaa agaaggccgg 1200

tactgataat attggtgaag ctgatttgtt gggcttcatc ttggaacaat ctaacttgga 1260

tgctgaacaa ttcggtgact tgttgttggg tttgttattt ggtggtcacg aaacatcttc 1320

taccgctatt actttggcta tctacttctt ggaaggttgt ccaaaagctg tccaagaatt 1380

gagagaagaa catttgaact tggtcaggat gaagaaacag agaggtgaat ctaaagcttt 1440

gacgtgggaa gattacaagt ctatggattt cgctcaatgc gttgtctctg aaactttgag 1500

attgggtaac atcatcaagt tcgttcacag aaaagctaac accgatgtcc aattcaaggg 1560

ttacgatatt ccatctggtt ggtctgttat tccagttttt gctgctgctc atttggatcc 1620

tactgtttac gataatccac aaaagttcga tccttggaga tggcaaacta tctcttcatc 1680

tactgccaga atcgataact acatgccatt tggtcaaggt ttgagaaatt gtgctggttt 1740

ggaattggct aagatggaaa ttgctgtttt cttgcaccac ttggtcttga atttcgattg 1800

ggaattagct gaaccagatc atccattggc ttatgctttt ccagaattcg aaaaaggctt 1860

gccaatcaag gtcagaaagt tgtctatttt ggagtaaccg ctgatcctag agggccgcat 1920

catgtaatta gttatgtcac gcttacattc acgccctccc cccacatccg ctctaaccga 1980

aaaggaagga gttagacaac ctgaagtcta ggtccctatt tattttttta tagttatgtt 2040

agtattaaga acgttattta tatttcaaat ttttcttttt tttctgtaca gacgcgtgta 2100

cgcatgtaac attatactga aaaccttgct tgagaaggtt ttgggacgct cgaaggcttt 2160

aatttgcaag ctgcggccct gcattaatga atcggccaac gcgc 2204

Claims

1. A recombinant bacterium is obtained by carrying out the following 1) transformation on a base yeast containing a relevant gene of a diosgenin synthesis path:

the genes related to the dioscin synthetic pathway are 3-hydroxy-3-methylglutaryl coenzyme A reductase 1 gene tHMG1, alpha-aminooxalate reductase gene Lys2, mevalonate kinase gene ERG12, isopentenyl pyrophosphate isomerase gene IDI1, mevalonate decarboxylase gene ERG19, 3-hydroxy-3-methylglutaryl coenzyme A reductase gene HMGR, 3-hydroxy-3-methylglutaryl coenzyme A gene ERG13, mevalonate kinase gene ERG8, acetyl coenzyme A acyl transferase gene ERG10, squalene synthase gene AtSQS, farnesyl pyrophosphate synthase gene SmFPS, squalene epoxide gene ERG1, sterol 26-position nicotinamide hydroxylase gene CYP94N, adenine dinucleotide phosphate-cytochrome P450 reductase gene SvvvvvvvvvVsQS, sterol 16, 22 dihydroxy oxidase gene DG 90G, and alpha-aminoglutaryl reductase gene Lys2, Sterol 7-position reductase gene StDWF5 and/or sterol 24-position reductase gene GgDHCR 24.

2. The recombinant bacterium according to claim 1, wherein: the steroid 22-hydroxylase gene is derived from rhizoma et radix Veratri, Arabidopsis thaliana, tomato or dioscorea zingiberensis.

3. The recombinant bacterium according to claim 1 or 2, wherein:

the steroid 22-hydroxylase gene is any one of the following 1) -3):

4. The recombinant bacterium according to any one of claims 1 to 3, wherein:

the recombinant bacteria are obtained by carrying out the transformation of the step 1) and the step 2) in the Chassis yeast containing the relevant genes of the diosgenin synthesis path:

2) and improving the expression of sterol 26 hydroxylase genes VcCYP94N, sterol 16, 22 dioxygenase genes DGCYP90G and steroid 22 hydroxylase genes in the genome of the saccharomyces cerevisiae with the chassis.

5. The recombinant bacterium according to claim 4, wherein:

the sterol 26-site hydroxylase gene VcCYP94N, sterol 16, 22-site dioxygenase gene DGCYP90G and the expression of the steroid 22-site hydroxylase gene in the genome of the saccharomyces cerevisiae with the improved chassis are obtained by integrating an expression cassette of the sterol 26-site reductase gene VcCYP94N, an expression cassette of the sterol 16, 22-site dioxygenase gene DGCYP90G and an expression cassette of the steroid 22-site hydroxylase gene into the genome of the saccharomyces cerevisiae containing the tHMG1 and Lys2 genes;

the expression cassette of the VcCYP94N comprises a TEF1 promoter, a VcCYP94N gene derived from veratrum mongolicum and a CYC1 terminator;

the expression cassette of the steroid 22-position hydroxylase gene comprises a PGK1 promoter, a rhizoma et radix Veratri-derived VcCYP90B27 gene and an ADH1 terminator.

6. The recombinant bacterium according to claim 4 or 5, wherein:

the recombinant bacteria are obtained by carrying out the transformation of 1), 2) and 3) in the base yeast containing the relevant genes of the diosgenin synthesis path:

3) expressing the sterol 16, 22 dioxygenase gene DGCYP90G in a plasmid form in saccharomyces cerevisiae of the chassis in high copy.

7. The recombinant bacterium according to any one of claims 1 to 6, wherein:

the saccharomyces cerevisiae on the chassis is obtained by modifying according to at least one of the following A) to D):

A) increasing the expression of 3-hydroxy-3-methylglutaryl-coa reductase 1 gene tmg 1, mevalonate kinase gene ERG12, isopentenyl pyrophosphate isomerase gene IDI1, mevalonate decarboxylase gene ERG19, 3-hydroxy-3-methylglutaryl-coa reductase gene HMGR, 3-hydroxy-3-methylglutaryl-coa gene ERG13, mevalonate kinase gene ERG8 and acetyl-coa acyl transferase gene ERG10 in the genome of said s.cerevisiae containing 3-hydroxy-3-methylglutaryl-coa reductase 1 gene tmg 1 and said α -aminooxalate reductase gene Lys 2;

B) increasing the expression of squalene synthase gene AtSQS, farnesyl pyrophosphate synthase gene SmFPS and squalene epoxidase gene ERG1 in the Saccharomyces cerevisiae genome containing 3-hydroxy-3-methylglutaryl coenzyme A reductase 1 gene tHMG1 and the alpha-aminooxalate reductase gene Lys 2;

C) expressing sterol 26 hydroxylase gene VcCYP94N, nicotinamide adenine dinucleotide phosphate-cytochrome P450 reductase gene SvvCPR, sterol 16, 22 dihydroxyoxidase gene DGCYP90G, sterol 7 reductase gene StDWF5 and/or sterol 24 reductase gene GgDHCR24 in the Saccharomyces cerevisiae genome containing 3-hydroxy-3-methylglutaryl coenzyme A reductase 1 gene tHMG1 and the alpha-aminooxalate reductase gene Lys 2;

D) knocking out alcohol acetylase gene ATF2 in the saccharomyces cerevisiae genome containing 3-hydroxy-3-methylglutaryl coenzyme A reductase 1 gene tHMG1 and the alpha-aminooxalate reductase gene Lys 2.

8. The recombinant bacterium according to claim 7, wherein:

in the B, the improvement is obtained by integrating a VcCYP94N expression cassette, a SvvCPR expression cassette, a DGCYP90G expression cassette, a StDWF5 expression cassette and/or a GgDHCR24 expression cassette into the saccharomyces cerevisiae genome containing the tHMG1 and Lys2 genes;

the SvvCPR expression cassette comprises a TDH3 promoter, a SvvCPR gene derived from grapes and a TPI1 terminator;

the DGCYP90G expression cassette comprises a TEF1 promoter, a DGCYP90G gene derived from dioscorea zingiberensis and a CYC1 terminator;

the StDWF5 expression cassette comprises a PGK1 promoter, a StDWF5 gene derived from potatoes and an ADH1 terminator;

the GgDHCR24 expression cassette comprises a TEF1 promoter, a GgDHCR24 gene derived from a chicken and a CYC1 terminator.

9. A method for preparing the recombinant bacterium of any one of claims 1 to 8, which is prepared by the method for modifying the recombinant bacterium of any one of claims 1 to 8.

10. A recombinant bacterium obtained by the method according to any one of claims 4 to 9;

or, the recombinant bacterium or the recombinant bacterium obtained by the method of claim 9 is applied to producing diosgenin or improving the yield of diosgenin;

or, a method for producing diosgenin, comprising the following steps: fermenting and culturing the recombinant bacteria to obtain the diosgenin.