CN114181964B - Expression cassette combination, recombinant vector, recombinant saccharomyces cerevisiae and application of recombinant saccharomyces cerevisiae - Google Patents

Abstract

The invention provides an expression cassette combination, a recombinant vector, a recombinant saccharomyces cerevisiae and application thereof, belonging to the technical field of bioengineering; the recombinant saccharomyces cerevisiae constructed based on the expression cassette combination can synthesize santalene and santalene, and the product of the engineering strain of the saccharomyces cerevisiae for producing santalene contains 1 alpha-santalene, 2 alpha-bergamotene, 3 epi-beta-santalene and 4 beta-santalene; the engineering strain products of the saccharomyces cerevisiae for producing the santalol comprise 1 alpha-santalol, 2 alpha-bergamotene, 3 epi-beta-santalol, 4 beta-santalol, 5 (Z) -alpha-santalol, 6 (Z) -alpha-bergamotol and 7 (Z) -beta-santalol.

Description

Expression cassette combination, recombinant vector, recombinant saccharomyces cerevisiae and application of recombinant saccharomyces cerevisiae

Technical Field

The invention relates to the technical field of bioengineering, in particular to an expression cassette combination, a recombinant vector, a recombinant saccharomyces cerevisiae and application thereof.

Background

Sesquiterpene santalene and santalol derived from Santalum album are main components of sandalwood essential oil, and have important application value. However, due to the severe growth conditions, long growth period, low essential oil content and vigorous market demand of the sandalwood, the sandalwood is excessively harvested, the yield of the sandalwood essential oil is low, and the price is increased. If the microbial cell factory is adopted to carry out heterologous biosynthesis on the santalene and the santalol, the efficiency can be improved, the cost can be reduced, the natural condition limitation can be broken, and the contradiction between supply and demand of the santalol essential oil can be effectively relieved.

The biosynthesis route of santalene and santalol is as follows: firstly, alpha-/beta-santalene in the album is subjected to MVA (mechanical vapor deposition) to obtain FPP, then the FPP is cyclized by SaSS enzyme and subjected to multiple reactions to synthesize alpha-, beta-and epi-beta-santalene, and finally, C12 is hydroxylated to form santalol under the cooperation of CYP450 and NADPH dependent Cytochrome P450 Reductase (CPR). Therefore, FPP is used as a general substrate for synthesizing sesquiterpenes such as santalene and other terpenes (C is more than or equal to 15), the yield of the FPP directly influences the yield of target terpenes, and due to the fact that the prior art lacks sufficient enzyme kinetic information for various enzymes in the MVA pathway of a host, and the factors influencing the flux of metabolic substances of the host are complex and various, a high-efficiency and general system for producing santalene and santalene is not constructed yet.

Disclosure of Invention

In view of the above, the present invention aims to provide an expression cassette combination, a recombinant vector, a recombinant saccharomyces cerevisiae and applications thereof.

The invention provides an expression cassette combination, which is sequentially connected with the following components from 5 'to 3': mva + EGFP expression cassette and SaSS-delta expression cassette; the nucleotide sequence of the mva + EGFP expression cassette is shown in SEQ ID NO:2 is shown in the figure; the nucleotide sequence of the SaSS-delta expression cassette is shown as SEQ ID NO: 3.

Preferably, an erg20 expression cassette is also linked between the mva + EGFP expression cassette and the SaSS-delta expression cassette; the nucleotide sequence of the erg20 expression cassette is shown in SEQ ID NO: 4.

Preferably, after the SaSS-delta expression cassette, a P76F39V 1-delta expression cassette is also ligated; the nucleotide sequence of the P76F39V 1-delta expression cassette is shown in SEQ ID NO: shown at 5.

Preferably, a screening gene expression cassette is also linked before the mva + EGFP expression cassette; the screening gene expression cassette comprises a delta first half-URA 3 expression cassette; the nucleotide sequence of the delta first half-URA 3 expression cassette is shown in SEQ ID NO: 1.

The invention also provides a recombinant vector which comprises the expression cassette combination according to the scheme.

The invention also provides a recombinant saccharomyces cerevisiae, wherein the genome of the saccharomyces cerevisiae is used as a vector, and the expression cassette combination in the scheme is inserted.

Preferably, the genome of the recombinant saccharomyces cerevisiae is also inserted with a fusion gene comprising CYP736A167 and CPR genes; the nucleotide sequence of CYP736A167 is shown in SEQ ID NO:6 is shown in the figure; the CPR gene comprises Sa-CPR2 or 46tATR1; the nucleotide sequence of the Sa-CPR2 is shown as SEQ ID NO: shown in figure 7; the nucleotide sequence of the 46tATR1 is shown as SEQ ID NO: shown at 8.

Preferably, the 5' end of the ERG9 gene of the recombinant Saccharomyces cerevisiae is also inserted with P _HXT1 Promoters or P _ERG1 A promoter; the P is _HXT1 The nucleotide sequence of the promoter is shown in SEQ ID NO: shown as 9; the P is _ERG1 The nucleotide sequence of the promoter is shown in SEQ ID NO: shown at 10.

The invention also provides an expression cassette combination, the recombinant vector or the application of the recombinant saccharomyces cerevisiae in preparation of santalene and/or santalene.

The invention provides an expression cassette combination, which is sequentially connected with the following components from 5 'to 3': mva + EGFP expression cassette and SaSS-delta expression cassette. In the invention, the mva + EGFP expression cassette is obtained by sequentially connecting an ADH1 promoter, mva + EGFP fusion gene and NOS terminator; the SaSS-delta expression cassette is obtained by sequentially connecting a GAP promoter, a SaSS gene, a CYC1 terminator and a delta latter half sequence. In the invention, the delta sequence is derived from saccharomyces cerevisiae, is divided into two halves and is respectively connected to two ends of a constructed metabolic pathway, and is recombined at a saccharomyces cerevisiae delta site as a homologous arm; the mva Gene is a hydroxymethylglutaryl-CoA reductase (3-hydroxy-3-methylglutaryl-CoA reductase mva, NCBI Gene No CP 009361) Gene, and is derived from staphylococcus aureus (Staphylococcus aureus ATCC 25923), the mva Gene can increase the supply of substrate FPP, and promote the improvement of the yield of santalene, and the EGFP Gene is a green fluorescent protein Gene, so that the screening of high-expression recombinant strains is facilitated; the SaSS gene in the SaSS-delta expression cassette is a santalene synthase gene, is derived from S.album and is subjected to yeast codon optimization; the promoter and the terminator used in the expression cassette are both derived from a saccharomyces cerevisiae constitutive promoter, and can stably and efficiently promote gene expression in saccharomyces cerevisiae. The recombinant saccharomyces cerevisiae constructed based on the expression cassette combination can synthesize santalene and santalene, and the product of the engineering strain of the saccharomyces cerevisiae for producing santalene contains 1 alpha-santalene, 2 alpha-bergamotene, 3 epi-beta-santalene and 4 beta-santalene; the engineering strain products of the saccharomyces cerevisiae for producing the santalol comprise 1 alpha-santalol, 2 alpha-bergamotene, 3 epi-beta-santalol, 4 beta-santalol, 5 (Z) -alpha-santalol, 6 (Z) -alpha-bergamotol and 7 (Z) -beta-santalol.

Drawings

FIG. 1 is a schematic diagram of a delta-URA 3 expression cassette;

FIG. 2 is a schematic diagram of a mva + EGFP expression cassette structure;

FIG. 3 is a schematic diagram of the structure of an erg20 box expression cassette;

FIG. 4 is a schematic diagram of the structure of a SaSS-delta box expression cassette;

FIG. 5 shows the biosynthetic pathways of santalene and santalol; wherein AACT (ERG 10) is acetoacetyl-CoA thiolase; HMGS (ERG 13) 3-hydroxy-3-methylglutaryl-CoA synthase e; HMGR 3-hydroxy-3-methylglutaryl-CoA reductase; MK (ERG 12) mevalonate kinase; MPK (ERG 8) mevalonate-5-phosphate kinase; MVD (ERG 19) mevalonate diphosphate decarboxylase; IDI is isopentenyl diphosphate isomerase; GPPS, geranyl diphosphate synthase; FPPS (ERG 20) farnesyl diphosphate synthase; saSS, santalene synthase; CYP76/CPR2 S.album cytochrome P450/S.album cytochrome P450 reductase; HMG-CoA 3-hydroxy-3-methylglutaryl-CoA; MVA, mevalonic acid; MVP, mevalonate phosphate; MVPP mevalonate-5-phosphate; IPP is isopentenyl diphosphate; DMAPP, dimethyl allyl diphosphate; GPP geranyl diphosphate; FPP, farnesyl diphosphate;

FIG. 6 is a schematic diagram of the synthesis of santalene and santalol of example 1;

FIG. 7 shows 3 synthetic pathways for santalene production;

FIG. 8 is 7P 450/CPR combinations of transformed santalene engineered strains;

FIG. 9 is a graph of squalene down-regulation patterns;

FIG. 10 is a mass spectrum of α -santalene;

FIG. 11 is a mass spectrum of α -bergamotene;

FIG. 12 is a mass spectrum of epi- β -santalene;

FIG. 13 is a mass spectrum of β -santalene;

FIG. 14 is a mass spectrum of (Z) - α -santalol;

FIG. 15 is a mass spectrum of (Z) - α -bergamotol;

FIG. 16 is a mass spectrum of (Z) - β -santalol.

Detailed Description

The invention provides an expression cassette combination, which is sequentially connected with the following components from 5 'to 3': mva + EGFP expression cassette and SaSS-delta expression cassette; the nucleotide sequence of the mva + EGFP expression cassette is shown in SEQ ID NO: 2.

In the invention, the mva + EGFP expression cassette is obtained by sequentially connecting an ADH1 promoter, mva, EGFP and NOS terminator box, and the total length is 2956bp; the ADH1 promoter, mva, EGFP and NOS terminator box are 705bp, 1281bp, 720bp and 253bp in sequence. The construction method of the mva +EGFP expression cassette is not particularly limited, and conventional methods in the art can be adopted. In the present invention, the mva Gene is a hydroxymethylglutaryl-CoA reductase (3-hydroxy-3-methylglutaryl-CoA reductase mva, NCBI Gene No CP 009361) Gene derived from Staphylococcus aureus (Staphylococcus aureus ATCC 25923); the mva gene can increase the supply of substrate FPP and promote the increase of the yield of santalene.

In the invention, the nucleotide sequence of the SaSS-delta expression cassette is shown as SEQ ID NO: 3. In the invention, the SaSS-delta expression cassette is obtained by sequentially connecting a GAP promoter, a SaSS, a CYC1 terminator and a delta latter half, and the total length is 2850bp; the sequence lengths of the GAP promoter, the SaSS, the CYC1 terminator and the delta backup are respectively as follows: 644bp, 1710bp, 248bp and 248bp. The construction method of the SaSS-delta expression cassette is not particularly limited, and conventional methods in the art can be adopted. In the invention, the SaSS gene in the SaSS-delta expression cassette is a santalene synthase gene, is derived from S.album and is subjected to Saccharomyces cerevisiae type codon optimization. In the invention is particularly practicalIn the application process, the SaSS gene is synthesized by Nanjing Jinsri biotechnology Co., ltd,

GenBank: HQ 343276), the synthesized gene was ligated to vectors pUC57 and pPIC9K, and amplified again when used.

In the invention, an erg20 expression cassette is also connected between the mva + EGFP expression cassette and the SaSS-delta expression cassette; the nucleotide sequence of the erg20 expression cassette is shown in SEQ ID NO: 4. In the invention, the erg20 expression cassette is preferably obtained by sequentially connecting a TEF promoter, erg20 and a TEF terminator, and the total length of the erg20 expression cassette is 1601bp; the sequence lengths of the TEF promoter, erg20 and TEF terminator box are 344bp, 1059bp and 198bp respectively. In the invention, the erg20 gene (2E, 6E) -farnesyl diphosphate synthase/dimethylallyl transferase is amplified from S.cerevisiae BY4742 genome as a template; the erg20 gene functions to further increase the supply of substrate FPP.

In the present invention, after the SaSS-delta expression cassette, a P76F39V 1-delta expression cassette is also ligated; the P76F39V 1-delta expression cassette is preferably obtained by sequentially connecting an ADH2 promoter, a CYP76F39V1 gene, an ADH2 terminator and delta, and the total length is 2467bp; the sequence lengths of the ADH2 promoter, the CYP76F39v1, the ADH2 terminator and the delta second half are 569bp, 1530bp, 120bp and 248bp respectively. In the invention, the CYP76F39v1 gene is derived from Santalum album, and is synthesized by Nanjing Jinsri biotechnology Co., ltd after Saccharomyces cerevisiae type codon optimization,

GenBank: KC 533716), the target gene TA is cloned in pUC57 vector, in order to verify whether CPR homologous to Saccharomyces cerevisiae can be matched to form a complete electron transfer system in the presence of CYP76F39v1 alone. In the invention, the nucleotide sequence of the P76F39V 1-delta expression cassette is shown in SEQ ID NO: shown at 5.

In the invention, the P76F39V 1-delta expression cassette is obtained by sequentially connecting an ADH2 promoter, CYP76F39V1, an ADH2 terminator and a delta latter half, and the total length is 2467bp; the ADH2 promoter, the Sa-CYP76F39v1, the ADH2 terminator and the delta second half have lengths of 569bp, 1530bp, 120bp and 248bp respectively.

In the present invention, the two wings of the expression cassette combination contain SmaI cleavage sites. In the invention, the two wings of the SaSS-delta expression cassette contain SacI and XbaI enzyme cutting sites, and all the 3 sites can be cut by T+BSABuffer.

In the invention, a screening gene expression cassette is also connected before the mva + EGFP expression cassette; the screening gene expression cassette comprises a delta first half-URA 3 expression cassette; the nucleotide sequence of the delta first half-URA 3 expression cassette is shown in SEQ ID NO: 1.

The invention also provides a recombinant vector which comprises the expression cassette combination according to the scheme. In the present invention, the original vector of the recombinant vector is preferably a pAUR112 plasmid; the pAUR112 plasmid was linearized with SmaI.

The invention also provides a recombinant saccharomyces cerevisiae, wherein the genome of the saccharomyces cerevisiae is used as a vector, and the expression cassette combination in the scheme is inserted. In the present invention, the chassis strain of the recombinant Saccharomyces cerevisiae is preferably BY4742. In the present invention, the insertion site of the expression cassette combination on the genome of the Saccharomyces cerevisiae is preferably a delta site.

In the invention, the genome of the recombinant saccharomyces cerevisiae is also inserted with a fusion gene containing CYP736A167 and CPR genes; the fusion gene can shorten the transmission distance of electrons between two enzymes, is beneficial to the electron transmission between electron transmission chains, and improves the catalytic efficiency of an oxidation-reduction system; the nucleotide sequence of CYP736A167 is shown in SEQ ID NO:6 is shown in the figure; the CPR gene comprises Sa-CPR2 or 46tATR1; the nucleotide sequence of the Sa-CPR2 is shown as SEQ ID NO: shown in figure 7; the nucleotide sequence of the 46tATR1 is shown as SEQ ID NO: shown as 8; the P is _ERG1 The nucleotide sequence of (2) is shown as SEQ ID NO: shown as 9; the P is _HXT1 The nucleotide sequence of (2) is shown as SEQ ID NO: shown at 10.

In the invention, the insertion site of the fusion gene on the genome of the recombinant saccharomyces cerevisiae is not particularly limited, and the fusion gene is determined according to the self-screening mark of the saccharomyces cerevisiae. In the practice of the invention, the insertion site of the fusion gene on the genome of the recombinant s.cerevisiae is preferably a leucine site, a lysine site, a histidine site or a methionine site.

In the invention, the 5' end of the ERG9 gene of the recombinant Saccharomyces cerevisiae is also inserted with P _HXT1 Promoters or P _ERG1 A promoter. The 2 promoter sequences are respectively shown in SEQ ID NO:9 and SEQ ID NO:10, wherein the promoter is specifically inserted into the 5' end of the ERG9 gene of the recombinant saccharomyces cerevisiae; the 2 promoters are inducible and feedback inhibition promoters, which are beneficial to the directional flow of the intermediate metabolic flow towards the target compound santalene. The 2 promoters are weak promoters, and can be replaced by other inducible or constitutive weak promoters, so long as ERG9 expression intensity can be down-regulated, but the patent is preferentially P _HXT1 Promoters or P _ERG1 A promoter.

The invention also provides an expression cassette combination, the recombinant vector, the recombinant strain or the application of the recombinant saccharomyces cerevisiae in preparation of santalene and/or santalene alcohol.

In the present invention, the santalene preferably includes: α -santalene (α -santalene), α -bergamotene (α -bergamotene), epi- β -santalene (epi- β -santalene), and β -santalene (β -santalene), the santalol comprising: (Z) - α -santalol, (Z) - α -bergamotol, (Z) - β -santalol (7 (Z) - β -santalol); the chemical structural formulas of the alpha-santalene, the alpha-bergamotene, the epi-beta-santalene, the (Z) -alpha-santalol, the (Z) -alpha-bergamotene and the (Z) -beta-santalol are respectively shown in the formulas I-VII;

in the present invention, the method for synthesizing santalene and/or santalene preferably comprises: the recombinant saccharomyces cerevisiae takes a simple carbon source as a substrate, and is fermented to synthesize alpha-santalene, alpha-bergamotene, epi-beta-santalene, (Z) -alpha-bergamotene and (Z) -beta-santalene; the process of synthesizing the target product without adding any inducer in the fermentation process; the simple carbon source is preferably glucose and/or sucrose.

In the present invention, the method for synthesizing santalene and/or santalene preferably comprises the steps of: inoculating the recombinant saccharomyces cerevisiae into a fermentation culture medium for culture to obtain a culture product containing santalene and/or santalene; the fermentation medium preferably includes YPD (yeast extract peptone glucose medium) or SD medium (yeast basal medium); the temperature of the culture is preferably 28 ℃; the rotational speed of the culture is preferably 180rpm; the pH value of the fermentation medium is preferably 4-6; the time of the culture is preferably 48 to 168 hours.

The technical scheme of the invention can realize the good connection between the rapid growth of cells in the exponential phase and the rapid accumulation of secondary products in the platform phase, avoids using inducers and repressing factors, and has better economy and simplicity. The invention constructs the whole biosynthesis path of Santalens/Santalols and derivatives thereof in S.cerevisiae, and lays a foundation for subsequent excellent production research.

The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention.

English abbreviation and key term definition

The santalene and santalol biosynthetic pathways are shown in figure 5. In the embodiment of the invention, saSS, HMGS (mva), FPPS, P450 and CPR are selected as optimization site genes to construct an over-expression path.

Example 1: method for constructing santalene synthesis path

The schematic diagram of the synthesis of santalene and santalol according to example 1 of the present invention is shown in fig. 6. The embodiment constructs the recombinant saccharomyces cerevisiae for preparing the santalene and/or the santalene, and over-expresses several key genes such as mva, erg20, saSS and the like in a saccharomyces cerevisiae genome to promote accumulation of intermediate products FPP and promote efficient synthesis of the santalene; in addition, the enzyme is optimized and combined and screened in the key step of santalol preparation by santalone oxidation, and the screened fusion enzyme of CYP736A167-CPR2 and CYP736A167-46tATR1 can effectively oxidize santalone to obtain santalol.

Before metabolic pathway construction, small boxes and elements are obtained first, the first round of connection is completed, and the main method is completed by adopting an overlap PCR method.

Delta+ura 3 expression cassette: amplifying the delta front half and then ligating with URA3 gene, wherein the delta front half is connected with URA3 by overlapping PCR, and the URA3 box comprises delta front half +P _URA3 +URA3+T _AOX1 (252bp+216 bp+804bp+247 bp=1519 bp), the nucleotide sequence is shown in SEQ ID NO:1, P _URA3 +URA3 amplified from pRS406, T _AOX1 The structure of the amplified pPIC9K is shown in FIG. 1.

mva + EGFP expression cassette: ADH1 promoter + mva +EGFP +NOS terminator expression cassette (705bp+1281bp+720 bp+253bp=2956 bp), nucleotide sequence as shown in SEQ ID NO: 2. A315_pADH1_AD plasmid was selected, which contained the promoter P derived from the S.cerevisiae alcohol dehydrogenase 1 gene _ADH1 The method comprises the steps of carrying out a first treatment on the surface of the Selecting pEGFP-N2 plasmid to amplify green fluorescent protein EGFP gene; selection of pGUS plasmid clone T _NOS Element, P was obtained by overlap PCR amplification _ADH1 --mva--EGFP--T _NOS An expression cassette. The P is obtained by inquiring from the 315_pADH1_AD plasmid sequence _ADH1 Element sequence, on the basis of which primers are designed to be first ligated to P _ADH1 When the gene element mva and EGFP are connected into fusion expression protein, mva gene stop codon is removed to ensure protein readthrough, and the structure schematic diagram is shown in figure 2.

Construction of ERG20 expression cassette: TEF promoter+erg20+tef terminator expression cassette (344bp+1059 bp+198bp=1601 bp), nucleotide sequence as set forth in SEQ ID NO: 4. Respectively amplifying P _TEF1 、ERG20(Accession No：NM_001181600)、T _TEF1 The sequence is purified to obtain each fragment, and the overlapping PCR technology is adopted to obtain P _TEF1 ERG20 is mixed as a template FastPfu DNA Polymerase for amplification connection, and the fragment is recovered and then is connected with T _TEF1 After mixing, PCR ligation was performed, and the bands were recovered and ligated

T1 Simple Cloning Vector (TransGen Biotech), the schematic structure is shown in FIG. 3.

Construction of the SaSS-delta expression cassette: GAP promoter +SaSS +CYC1 terminator +delta latter half (644bp+1710bp+248 bp+248 bp=2850 bp), the nucleotide sequence is shown in SEQ ID NO: 3. Amplification of P426GPD vector P Using FastPfu DNA Polymerase _GAP And T _CYC1 The single band can be obtained by recycling the sequence and each fragment of the SaSS sequence, P _TDH3 Overlap PCR with SaSS and then with T _CYC1 The sequences were subjected to overlapping PCR ligation. Construction of SaSS expression cassette ligation and ligation

-T1 Simple Cloning Vector sequencing verification, schematic structure shown in fig. 4.

Construction of the P76F39V 1-delta expression cassette: ADH2 promoter+CYP 76F39V1+ADH2 terminator+delta latter half (569bp+1530bp+120bp+248 bp=2467 bp), nucleotide sequence as shown in SEQ ID NO: shown at 5. P amplification Using overlap PCR technique FastPfu DNA Polymerase, respectively _ADH2 CYP76F39V1 and T _ADH2 Fragments after delta, and purifying to obtain each fragment, and obtaining P _ADH2 Mixing CYP76F39V1 as a template for amplification connection, and recovering fragments for later use; by T _ADH2 Mixing the delta fragments as templates for amplification and connection, and recovering the fragments for later use; finally, 2 recovery fragments are mixed as templates to make overlapping PCR connection, and the mixture is connected to the mixture after the recovery of the bands

T1 clonotype vector (TransGen Biotech), and finally constructed to obtain P _ADH2 --CYP76F39V1--T _ADH2 -delta post fragment box.

Delta sequence: amplified from Saccharomyces cerevisiae BY4742, 500bp, nucleotide sequence as shown in SEQ ID NO: 11.

In view of the high-efficiency homologous recombination capability of Saccharomyces cerevisiae, a complete expression path is constructed BY using an S.cerevisiae BY4742 in vivo homologous recombination method, pAUR112 plasmids are linearized BY SmaI, amplified BY each box, glue is recovered and purified for later use, box fragments are added into 150 mu L of BY4742 competence (LiAC preparation method) and 3-4 mu L of linearized pAUR112 (the majority of fragments are added to ensure that the molar concentration of each box fragment is close), an electric rotating cup is used for 0.2cm, electric shock conversion is performed for 1.5KV for 6ms, a proper amount of YPD is used for resuscitation for 1h, water is washed for 1 time after centrifugation, the plasmid is coated on an SD-URA3 screening plate, incubation is performed for 2d at 30 ℃, and recombinant strains are screened. Thus, 3 recombinant plasmids, namely SaSS small, medium and large metabolic pathways, were obtained, and a schematic diagram of 3 synthetic pathways for producing santalene is shown in FIG. 7, and the specific connection sequence and size are as follows:

the SaSS small pathway: delta-URA 3 expression cassette-mva +EGFP expression cassette-SaSS-delta expression cassette, this pathway was ligated to pAUR112 (7325bp+7104 bp= 14429 bp), and both wings contained SmaI cleavage sites.

The route in SaSS: delta-URA 3 expression cassette-mva +EGFP expression cassette-erg 20 expression cassette-SaSS-delta expression cassette, this pathway is linked to pAUR112 (8926bp+7104 bp=16030 bp), and both wings contain SmaI cleavage sites.

The large route of SaSS: delta-URA 3 expression cassette-mva +EGFP expression cassette-erg 20 expression cassette-SaSS expression cassette-p 450-delta expression cassette, this approach was ligated to pAUR112 preformed vector (11157 bp+3505bp= 14662 bp), both wings contained SmaI cleavage sites, and both wings of SaSS expression cassette contained SacI and XbaI cleavage sites.

Example 2: method for constructing synthetic pathway of santalol

2P 450 genes CYP76F39V1 and SaCYP736A167 from S.album are selected as experimental objects, 2 Sa-CPR2 and 46tATR1 respectively from S.album and A.thiana are selected to be combined with CYP76F39V1 and SaCYP736A167 to construct a fusion P450-CPR enzyme system, wherein the combination of CYP76F39V1 and Sa-CPR2 is also used for constructing a fusion mode with or without Linker, the system verifies 2P 450 enzyme activity differences, the effect is better after which CPR combination is obtained, and the Linker has an effect on the activity of the fusion enzyme, and 7P 450/CPR combinations of the transformed santalene engineering strains are shown in FIG. 8 and Table 1.

TABLE 1 combination of different P450 and CPR fusion enzymes

Injecting tpLADH1 and truncated pLADH1 plasmid; a, codon optimized, N-terminally truncated 46 amino acids A.thaliana P450 reductase 1 (46 tATR 1); c, codon optimized s.album P450 reductase 2 (CPR 2); a fusion gene of P1A, codon optimized CYP76F39v1 and A.thaliana P450 reductase 1; a fusion gene of P1C, codon optimized CYP76F39v1 and s.album P450 reductase 2 (CPR 2); LP1C, fusion gene of codon optimized CYP76F39v1 and S.album P450 reductase 2 (CPR 2), intergenic band linker (GGGGS) 4 linkage; a fusion gene of P2A, codon optimized CYP736A167 and truncated A.thiana P450 reductase 1; P2C, codon optimized CYP736a167 fusion s.album P450 reductase 2 (CPR 2).

In the present invention, the nucleotide sequence of CYP736A167 is shown in SEQ ID NO:6, the nucleotide sequence of Sa-CPR2 is shown as SEQ ID NO:7, the nucleotide sequence of 46tATR1 is shown as SEQ ID NO:8, the nucleotide sequence of CYP76F39v1 is shown in SEQ ID NO:12, the nucleotide sequence of CYP76F39v1+SaCPR2 is shown as SEQ ID NO:13, the nucleotide sequence of CYP76F39v1+ linker-SaCPR2 is shown in SEQ ID NO:14, the nucleotide sequence of CYP76F39v1+46tATR1 is shown in SEQ ID NO:15, the nucleotide sequence of CYP736A167+SaCPR2 is shown as SEQ ID NO:16, the nucleotide sequence of CYP736A167+46tATR1 is shown as SEQ ID NO: shown at 17.

Artificial synthesis of S.cerevisiae codon optimized P450 genes CYP76F39V1, sa-CYP736A167, saCPR2 from Nanjing Jinsri Biotech Co., ltd (GenScript Company Limited) from S.album, and cloning of t46ATR1 from the truncated N-terminal 46 aa of A.thaliana into vectorpUC57, a total of 2P 450 s (CYP 76F39V1, sa-CYP736a 167) and 2 CPR s (sampr 2, t46ATR 1). CYP76F39V1 was fused with SaCPR2, t46ATR1, respectively, one without Linker and one with long flexible Linker (GGGGS) ₄ Respectively introducing engineering strains for producing santalene; the Sa-CYP736A167 and 2 CPRs are fused without Linker, and are respectively introduced into engineering strains for producing santalene for screening and fermentation detection.

The detection result shows that: the fusion enzymes CYP736A167 and CPR2, 46tATR1 oxidize Santalens to obtain (Z) -alpha-santalol, (Z) -alpha-bergamotol and (Z) -beta-santalol, and the two fusion enzymes CYP736A167-46tATR1 and CYP736A167-CPR2 have activity of oxidizing Santalens, but the activity of CYP736A167-46tATR1 is higher than that of CYP736A167-CPR2, and the shake flask level is obtained to produce Santaloles 29.56mg/L. CYP76F39V1 alone is catalytically inactive in S.cerevisiae.

Example 4: method for optimizing and constructing santalene/santalene synthesis path

A graph of squalene downregulation patterns is shown in fig. 9.

The invention adopts P _ERG1 Or P _HXT1 Down-regulating recombinant Saccharomyces cerevisiae ERG9 to reduce ERG9 expression, regulating metabolic flow as much as possible in the direction of santalenes by regulating P _ERG1 Or P _HXT1 After the gene is inserted into the 5' end of ERG9 gene, YPD fermentation is carried out, the extraction product detection finds that the product yield is obviously increased and the byproduct squarene is obviously reduced, the SIM is selected from m/ z 93, 94, 105, 107, 119, 122 and 202 ions for GC-MS quantitative detection, and P is obtained by conversion according to standard curve of concentration gradient of reference substance _HXT1 After downregulation, the alpha-santalene 38.39mg/L, alpha-bergamotene 24.64mg/L, epi-beta-santalene 3.26mg/L, beta-santalene 28.36mg/L were increased to 63.91mg/L, 43.35mg/L, 5.92mg/L and 51.53mg/L, respectively, and the 4 products were increased by 66.5%, 75.9%, 81.6 and 81.7%, respectively. P (P) _HXT1 The down regulation effect is better than P _ERG1 。

Calculated santalenes product amount via P _HXT1 And P _ERG1 After downregulation, the santalenes increased from 94.65mg/L to 164.71mg/L and 106.99mg/L, respectively, by 74.0% and 13.3%, while the squarenes decreased from 28.37mg/L to12.97mg/L and 18.87mg/L, respectively, down-regulated by 54.3% and 33.5%; at the same time via P _HXT1 And P _ERG1 After downregulation of ERG9, the santalols yield increased from 29.56mg/L to 68.81mg/L and 37.12mg/L, respectively, by 132.8% and 25.6%, and the squarene yield decreased from 38.04mg/L to 18.54mg/L and 26.58mg/L, respectively, by 51.3% and 30.1%.

Example 5: prokaryotic vector construction method and yeast engineering bacteria fermentation method

The enzyme digestion and the carrier connection reaction firstly select proper restriction enzyme, the restriction enzyme digestion system is 100-200 mu L (different buffers and concentrations are selected according to different enzymes), and common enzymes such as BamH I and HindIII are 5-10 mu L respectively, and the enzyme digestion is not more than 12h at 37 ℃.

Ligation reaction system (10 μl): 1. Mu.L of vector, 7. Mu. L, ligase 1. Mu.L of exogenous gene, 1. Mu.L of 10 Xbuffer, and ligation reaction at 16℃for more than 24 hours. Sequentially adding gene fragments or plasmids into an enzyme digestion system (enzyme is added at last, and the lower part of a pipe is not needed to be taken to avoid enzyme inactivation during the addition), and then placing the gene fragments or plasmids into a constant-temperature water bath kettle for enzyme digestion at 37 ℃ for 12 hours; the enzyme cutting is used for preparing glue (with two holes) to be recovered; sample application (all adding two-hole electrophoresis) after enzyme cutting, cutting glue under ultraviolet, packaging two strips with two tubes, dissolving in water bath, and making connection reaction for use; sequentially adding a connection reaction system, uniformly mixing 1 mu L of a carrier and 7 mu L of an exogenous gene, carrying out water bath at 45 ℃ for 5min, then placing in an ice bath, sequentially adding 1 mu L of 10 Xbuffer and Ligase (the enzyme is added last time, the enzyme is not required to be kept under a tube to avoid inactivation when the enzyme is added), and carrying out overnight reaction at 16 ℃; the following day, the host cells were transformed with the cloned host DH 5. Alpha. Respectively.

Prokaryotic E.coli DH5 alpha is used as a host bacterium for constructing a vector, and is used for preserving recombinant plasmids and extracting plasmids after culture, LB culture medium (tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, agar 15g/L, pH=7.0) is used for culture, generally, the culture is carried out at 37 ℃ and 180rpm for 12-24 hours, ampicillin or kanamycin is added into the culture medium, and specific antibiotics are added according to selection markers on the plasmids. Placing 100 mu L DH5 alpha (competent cells) on ice in the conversion process of the coli DH-5 alpha, sucking 1-5 mu L of a connecting product into an aseptic EP tube on ice, rapidly sucking 50 mu L of the connecting product when the competent cells are just melted, transferring the connecting product into the EP tube on ice, slightly and uniformly mixing, and carrying out ice bath for 30-35 min; rapidly placing the EP pipe in a water bath at 42 ℃, preserving heat for about 90 seconds, and keeping the temperature for about 90 seconds without shaking; rapidly placing in an ice bath for 2-3 min after heat shock; adding 900 mu L of fresh LB culture medium, culturing for 1-2 h on a shaking table at 37 ℃ for resuscitation; after culturing, centrifuging at 10000rpm for 1min, and discarding part of supernatant; after re-suspension, a proper amount of LB culture medium is sucked and coated on an LB plate containing antibiotics, the working concentration of kanamycin is 30-50 mug/mL, and the ampicillin is 50-100 mug/mL. After coating, the culture is carried out upside down at 37 ℃ for overnight, the colony appears after overnight, namely the positive clone, and the colony is not advisable for too long, the monoclonal shake flask is selected for culture for 16 hours, and the plasmid is extracted for double enzyme digestion verification.

Plasmid extraction and shake culture of strain for 16-24 hr, centrifuging and adding ddH ₂ O, this step can also be omitted. Generally, 4-10 mL of bacterial liquid can be used as a 2mL centrifuge tube. Removing supernatant of the centrifuged bacterial liquid, adding 250 mu L of Solution I, fully suspending bacterial cells, adding 250 mu L of Solution II, and gently reversing for 4-6 times to avoid breaking plasmids, so that the bacterial liquid becomes transparent like egg white, and the step is not suitable for more than 5min. 350 mu L Solution III is added, and the mixture is gently and fully turned over and mixed up and down for 8 to 10 times and is placed for 2 to 5 minutes at room temperature. And (3) centrifuging at 12,000rpm for 5-10 min. The supernatant was transferred to a 2mL collection tube, centrifuged at 6,000rpm at room temperature for 1min, the collection tube was removed, and the waste liquid in the collection tube was discarded. 500 μLW1 Solution was added, spun at 12000rpm, and the collection tube was emptied of waste liquid by centrifugation at room temperature for 1 min. 500. Mu. LWAsh Solution was added, centrifuged at 12000rpm for 1min at room temperature, the waste liquid in the collection tube was poured off, and washing was repeated once, and the remaining eluate was removed by sufficient centrifugation (this step was not omitted). The column was opened to allow full air drying, placed in a clean 2mL centrifuge tube, and 50. Mu.L-80. Mu.L ddH at 65deg.C was added to the center of the membrane ₂ O, standing for 2min. And (3) centrifuging at 12000rpm at room temperature for 1min, wherein the liquid in the centrifuge tube is the solution containing the target plasmid (which can be eluted repeatedly for 1 time), or freezing at-20 ℃.

S.the cerevisiae engineering bacteria culture medium adopts YPD culture medium for culturing saccharomycetes, comprises 10g/L yeast extract, 20g/L peptone, 20g/L sucrose/glucose, and if solid culture medium is prepared, 1.5% agar powder is added. Step (2) dissolving 10g Yeast Extract (yeast Extract), 20g Peptone (Peptone) in 900mL water, and adding 15g agar powder into the plate; high pressure 120 ℃ for 15-20min; 100mL of a solution containing 20g of sucrose or glucose (sucrose or glucose solution added after sterilization alone) was added before solidification of the agar; glucose, yeast extract, peptone solution are sterilized respectively, and then mixed, and sucrose or glucose is sterilized at 120deg.C for 15min.

S.the cerevisiae engineering bacteria are streaked on YPD plates, cultured for 48-72 hours at 28.5 ℃, single colony is picked up to 20mLYPD culture medium, cultured for 48 hours at 28.5 ℃ at 180rpm, 1mL of bacterial liquid is sucked and transferred to 50mLYPD culture medium, and cultured at 28.5 ℃ at 180rpm until OD ₆₀₀ When=2, 10% (v/v) dodecane was added, and the culture was performed for 168h with plastic film sealing; centrifuging the fermentation product, and sucking the upper dodecane extract, na ₂ SO ₄ Drying, performing GC-MS detection on the control empty yeast BY4742 and the santalol control, and comparing the corresponding retention time to confirm that the engineering bacteria fermentation product and the control show product peaks, wherein the database comparison confirms that the engineering bacteria fermentation product and the control show santalene and santalol, and the negative control shows no such compound, so that the SaSS gene can express and synthesize santalene and santalol in the host S.cerevisiae BY4742.

Example 6: method for detecting santalene/santalene engineering bacterium products

Firstly, setting standard samples of santalol reference substances (Sigma-Aldrich, alpha-/beta-isomer, analytically pure, product number: 75031, CAS number: 11031-45-1), respectively preparing 20 mg/L concentration, 50 mg/L concentration, 100 mg/L concentration, 200 mg/L concentration and 400mg/L concentration by using dodecane as a solvent, and calculating the product yield according to the peak area.

GC-MS detection method: detection was performed using a GC-MS gas chromatograph-mass spectrometer (Agilent 8890GC system,Agilent 5977B GC/MSD). The detection method comprises the following steps: capillary column HP-5MS (nonpolar, 30m 0.25mm 0.25 μm); the temperature of the sample inlet is 250 ℃; the carrier gas is high-purity helium (the purity is more than or equal to 99.999 percent), and the flow rate is 0.8mL/min; sample injection mode: split-flow sample injection, wherein the sample injection amount is 1 mu L, and the split-flow ratio is 30:1; temperature programming conditions: the initial temperature is 40 ℃ and kept for 3min, then 10 ℃/min is increased to 130 ℃, then 2 ℃/min is increased to 180 ℃, and finally 50 ℃/min is increased to 300 ℃ and kept for 10min; mass spectrometry conditions: EI+ ionization; ionization voltage: 70eV; scanning range: 40-400 amu; SIM: m/ z 93, 94, 105, 107, 119, 122, 202[ dwell time 50msec ]; ion source temperature: 200 ℃; transmission line temperature: 300 ℃; selecting m/ z 93, 94, 105, 107, 119, 122, 202 ions for mass spectrum screening; and (5) spectrogram retrieval: and searching a WILEY and NIST08 spectrum library. GC-MS detection determines mass spectra of 4 products, namely alpha-santalene, alpha-bergamotene, epi-beta-santalene and beta-santalene, respectively, see figures 10-13. Mass spectra of the products (Z) - α -santalol, (Z) - α -bergamotol and (Z) - β -santalol are shown in fig. 14 to 16, respectively.

As can be seen from the above examples, the invention develops the engineering strain of Santalens/Santalols yeast for the first time, breaks the bottleneck in metabolic pathway, uses a constitutive strong promoter to start expression of the HMGR gene of Staphylococcus aureus and the yeast ERG20 gene, and promotes FPP to improve the yield; introducing an optimally screened P450-CPR combination, and further oxidizing Santalens into Santalols, so that the Santalens can perform one-step more reaction to relieve potential pressure caused by accumulation of chassis cell intermediates; meanwhile, aiming at a decompression adaptation mechanism of excessive FPP and an anabolite Squalene/Farnesol of chassis cell shunt, a glucose-sensitive, constitutive or Squalene feedback inhibition type weak promoter is tested to replace a wild type ERG9 promoter, so that the flow of FPP to the Squalene is weakened, and the effect of inhibiting a non-target product synthesis path is achieved.

Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, according to which one can obtain other embodiments without inventiveness, these embodiments are all within the scope of the invention.

Sequence listing

<110> university of Yunnan

<120> an expression cassette composition, recombinant vector and recombinant Saccharomyces cerevisiae and use thereof

<160> 17

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1519

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 1

tgttggaata gaaatcaact atcatctact aactagtatt tacattacta gtatattatc 60

atatacggtg ttagaagatg acgcaaatga tgagaaatag tcatctaaat tagtggaagc 120

tgaaacgcaa ggattgataa tgtaatagga tcaatgaata taaacatata aaacggaatg 180

aggaataatc gtaatattag tatgtagaaa tatagattcc attttgagga ttcctatatc 240

ctcgaggaga acttcaattc atcatttttt ttttattctt ttttttgatt tcggtttctt 300

tgaaattttt ttgattcggt aatctccgaa cagaaggaag aacgaaggaa ggagcacaga 360

cttagattgg tatatatacg catatgtagt gttgaagaaa catgaaattg cccagtattc 420

ttaacccaac tgcacagaac aaaaacctgc aggaaacgaa gataaatcat gtcgaaagct 480

acatataagg aacgtgctgc tactcatcct agtcctgttg ctgccaagct atttaatatc 540

atgcacgaaa agcaaacaaa cttgtgtgct tcattggatg ttcgtaccac caaggaatta 600

ctggagttag ttgaagcatt aggtcccaaa atttgtttac taaaaacaca tgtggatatc 660

ttgactgatt tttccatgga gggcacagtt aagccgctaa aggcattatc cgccaagtac 720

aattttttac tcttcgaaga cagaaaattt gctgacattg gtaatacagt caaattgcag 780

tactctgcgg gtgtatacag aatagcagaa tgggcagaca ttacgaatgc acacggtgtg 840

gtgggcccag gtattgttag cggtttgaag caggcggcag aagaagtaac aaaggaacct 900

agaggccttt tgatgttagc agaattgtca tgcaagggct ccctatctac tggagaatat 960

actaagggta ctgttgacat tgcgaagagc gacaaagatt ttgttatcgg ctttattgct 1020

caaagagaca tgggtggaag agatgaaggt tacgattggt tgattatgac acccggtgtg 1080

ggtttagatg acaagggaga cgcattgggt caacagtata gaaccgtgga tgatgtggtc 1140

tctacaggat ctgacattat tattgttgga agaggactat ttgcaaaggg aagggatgct 1200

aaggtagagg gtgaacgtta cagaaaagca ggctgggaag catatttgag aagatgcggc 1260

cagcaaaact aatcaagagg atgtcagaat gccatttgcc tgagagatgc aggcttcatt 1320

tttgatactt ttttatttgt aacctatata gtataggatt ttttttgtca ttttgtttct 1380

tctcgtacga gcttgctcct gatcagccta tctcgcagct gatgaatatc ttgtggtagg 1440

ggtttgggaa aatcattcga gtttgatgtt tttcttggta tttcccactc ctcttcagag 1500

tacagaagat taagtgaga 1519

<210> 2

<211> 2956

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 2

atccttttgt tgtttccggg tgtacaatat ggacttcctc ttttctggca accaaaccca 60

tacatcggga ttcctataat accttcgttg gtctccctaa catgtaggtg gcggagggga 120

gatatacaat agaacagata ccagacaaga cataatgggc taaacaagac tacaccaatt 180

acactgcctc attgatggtg gtacataacg aactaatact gtagccctag acttgatagc 240

catcatcata tcgaagtttc actacccttt ttccatttgc catctattga agtaataata 300

ggcgcatgca acttcttttc tttttttttc ttttctctct cccccgttgt tgtctcacca 360

tatccgcaat gacaaaaaaa tgatggaaga cactaaagga aaaaattaac gacaaagaca 420

gcaccaacag atgtcgttgt tccagagctg atgaggggta tctcgaagca cacgaaactt 480

tttccttcct tcattcacgc acactactct ctaatgagca acggtatacg gccttccttc 540

cagttacttg aatttgaaat aaaaaaaagt ttgctgtctt gctatcaagt ataaatagac 600

ctgcaattat taatcttttg tttcctcgtc attgttctcg ttccctttct tccttgtttc 660

tttttctgca caatatttca agctatacca agcatacaat caactatgca aagtttagat 720

aagaatttcc gacatttatc tcgtcaacaa aagttacaac aattggtaga taagcaatgg 780

ttatcagaag agcaattcaa cattttattg aatcatccat taattgatga ggaagtagca 840

aatagtttaa ttgaaaatgt catcgcgcaa ggtgcattac ccgttggatt attaccgaat 900

atcattgtgg acgataaagc atatgttgta cctatgatgg tggaagagcc ttcagttgtc 960

gctgcagcta gttatggtgc aaagctagtg aatcagactg gtggatttaa aacggtatct 1020

tctgaacgta ttatgatagg tcaaatcgtc tttgatggcg ttgacgatac tgaaaaatta 1080

tcagcagaca ttaaagcttt agagaagcaa attcatcaaa ttgcggatga ggcatatcct 1140

tctattaaag cgcgtggtgg tggttaccaa cgtatagcta ttgatacatt tcctgagcaa 1200

cagttactat ctttaaaagt atttgttgat acgaaagatg ctatgggcgc taatatgctt 1260

aatacgattt tagaggctat aactgcattt ttaaaaaatg aatttccgca aagcgacatt 1320

ttaatgagta ttttatccaa tcatgcaaca gcgtccgttg ttaaagttca aggcgaaatt 1380

gatgttaaag atttagcaag gggcgagaga actggagaag aggttgccaa acgaatggaa 1440

cgtgcttctg tattggcaca agttgatatt catcgtgctg caacacataa taaaggtgtt 1500

atgaatggca tacatgccgt tgttttagca acaggaaatg atacgcgtgg tgcagaagca 1560

agtgcgcatg catacgcgag taaagatggt caatatcgtg gtattgcaac atggagatac 1620

gatcaagaac gtcaacgttt aattggtacc atagaggtgc ctatgacatt ggcaatcgtt 1680

ggcggtggta caaaagtatt accaatagct aaagcatcat tagagctact aaatgtagag 1740

tcagcgcaag aattaggtca tgtagttgct gccgttggtt tagcacaaaa ctttgcagca 1800

tgtcgcgcgc tcgtttctga aggtatccag caaggccata tgagcttgca atataaatct 1860

ttagctattg ttgtaggtgc aaaaggtgat gaaattgcgc aagtagctga agcattgaag 1920

caagaacctc gtgcgaatac acaagttgct gaacgcattt tacaagattt aagaagccaa 1980

caaatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg 2040

gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc 2100

tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc 2160

accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg 2220

aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc 2280

ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc 2340

ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg 2400

cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag 2460

aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc 2520

gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac 2580

cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg 2640

gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag 2700

taagatcgtt caaacatttg gcaataaagt ttcttaagat tgaatcctgt tgccggtctt 2760

gcgatgatta tcatataatt tctgttgaat tacgttaagc atgtaataat taacatgtaa 2820

tgcatgacgt tatttatgag atgggttttt atgattagag tcccgcaatt atacatttaa 2880

tacgcgatag aaaacaaaat atagcgcgca aactaggata aattatcgcg cgcggtgtca 2940

tctatgttac tagatc 2956

<210> 3

<211> 2850

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

tcattatcaa tactcgccat ttcaaagaat acgtaaataa ttaatagtag tgattttcct 60

aactttattt agtcaaaaaa ttagcctttt aattctgctg taacccgtac atgcccaaaa 120

tagggggcgg gttacacaga atatataaca tcgtaggtgt ctgggtgaac agtttattcc 180

tggcatccac taaatataat ggagcccgct ttttaagctg gcatccagaa aaaaaaagaa 240

tcccagcacc aaaatattgt tttcttcacc aaccatcagt tcataggtcc attctcttag 300

cgcaactaca gagaacaggg gcacaaacag gcaaaaaacg ggcacaacct caatggagtg 360

atgcaacctg cctggagtaa atgatgacac aaggcaattg acccacgcat gtatctatct 420

cattttctta caccttctat taccttctgc tctctctgat ttggaaaaag ctgaaaaaaa 480

aggttgaaac cagttccctg aaattattcc cctacttgac taataagtat ataaagacgg 540

taggtattga ttgtaattct gtaaatctat ttcttaaact tcttaaattc tacttttata 600

gttagtcttt tttttagttt taaaacacca gaacttagtt tcgaatggac tcctctaccg 660

ccactgctat gactgcccct ttcattgacc ctacagacca cgtaaactta aaaacagaca 720

cagacgcctc agaaaacaga agaatgggta actacaagcc atctatctgg aactacgatt 780

tcttgcaatc attagcaact catcacaaca tagtcgaaga aagacatttg aaattggccg 840

aaaagttgaa gggtcaagta aagtttatgt tcggtgcacc aatggaacct ttagccaaat 900

tagaattggt tgacgttgtc caaagattgg gtttgaacca cttgttcgaa acagaaatca 960

aagaagcttt attctccatc tataaggatg gtagtaacgg ttggtggttt ggtcatttgc 1020

acgcaacctc tttgagattc agattgttga gacaatgtgg tttgttcatt cctcaagatg 1080

tctttaaaac attccaaaat aagaccggtg aattcgatat gaagttgtgc gacaacgtta 1140

agggtttgtt gtctttgtac gaagcttcat acttaggttg gaagggtgaa aacatcttgg 1200

atgaagccaa ggcttttact acaaagtgtt tgaagtctgc ttgggaaaac atatcagaaa 1260

agtggttggc aaagagagtt aagcatgctt tggccttgcc attacactgg agagtcccta 1320

gaatagaagc aagatggttt atcgaagctt acgaacaaga agcaaacatg aacccaacat 1380

tgttgaagtt ggccaagttg gatttcaaca tggtacaatc tatccatcaa aaggaaatcg 1440

gtgaattggc aagatggtgg gttactactg gtttggacaa gttggctttc gcaagaaaca 1500

acttgttgca atcttatatg tggtcatgcg ccattgcttc agatcctaaa ttcaagttgg 1560

ctagagaaac tattgttgaa ataggttccg tcttaacagt agttgatgac ggttatgacg 1620

tttacggtag tattgatgaa ttggacttat acacttcttc agtagaaaga tggtcctgtg 1680

ttgaaatcga taagttgcca aacaccttga agttgatctt tatgtctatg ttcaataaga 1740

ctaacgaagt cggtttgaga gtacaacatg aaagaggtta caactctatc cctactttta 1800

ttaaggcctg ggttgaacaa tgcaaatcat accaaaagga agctagatgg ttccatggtg 1860

gtcacactcc acctttggaa gaatactcct tgaacggttt agttagtata ggtttcccat 1920

tgttgttgat tacaggttac gtagcaatag ccgaaaacga agctgcattg gacaaagttc 1980

atccattacc tgatttgttg cactattcca gtttgttgtc cagattgatc aatgatattg 2040

gtacaagtcc tgacgaaatg gctagaggtg acaatttgaa gtctatccat tgttacatga 2100

acgaaaccgg tgcctcagaa gaagtcgcta gagaacacat caagggtgta atagaagaaa 2160

actggaagat cttgaaccaa tgttgctttg atcaatcaca attccaagaa ccttttatta 2220

cttttaattt gaactccgtt agaggtagtc atttctttta tgaattcggt gacggtttcg 2280

gtgtcactga ttcttggacc aaggttgata tgaagtcagt tttgatagac ccaatcccat 2340

taggtgaaga ataatcatgt aattagttat gtcacgctta cattcacgcc ctccccccac 2400

atccgctcta accgaaaagg aaggagttag acaacctgaa gtctaggtcc ctatttattt 2460

ttttatagtt atgttagtat taagaacgtt atttatattt caaatttttc ttttttttct 2520

gtacagacgc gtgtacgcat gtaacattat actgaaaacc ttgcttgaga aggttttggg 2580

acgctcgaag gctttaattt gcttctagta tattctgtat acctaatatt atagccttta 2640

tcaacaatgg aatcccaaca attatctcaa cattcaccca tttctcatgg tagcgcctgt 2700

gcttcggtta cttctaagga agtccacaca aatcaagatc cgttagacgt ttcagcttcc 2760

aaaacagaag aatgtgagaa ggcttccact aaggctaact ctcaacagac aacaacacct 2820

gcttcatcag ctgttccaga gaacccccat 2850

<210> 4

<211> 1601

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

gacatggagg cccagaatac cctccttgac agtcttgacg tgcgcagctc aggggcatga 60

tgtgactgtc gcccgtacat ttagcccata catccccatg tataatcatt tgcatccata 120

cattttgatg gccgcacggc gcgaagcaaa aattacggct cctcgctgca gacctgcgag 180

cagggaaacg ctcccctcac agacgcgttg aattgtcccc acgccgcgcc cctgtagaga 240

aatataaaag gttaggattt gccactgagg ttcttctttc atatacttcc ttttaaaatc 300

ttgctaggat acagttctca catcacatcc gaacataaac aaccatggct tcagaaaaag 360

aaattaggag agagagattc ttgaacgttt tccctaaatt agtagaggaa ttgaacgcat 420

cgcttttggc ttacggtatg cctaaggaag catgtgactg gtatgcccac tcattgaact 480

acaacactcc aggcggtaag ctaaatagag gtttgtccgt tgtggacacg tatgctattc 540

tctccaacaa gaccgttgaa caattggggc aagaagaata cgaaaaggtt gccattctag 600

gttggtgcat tgagttgttg caggcttact tcttggtcgc cgatgatatg atggacaagt 660

ccattaccag aagaggccaa ccatgttggt acaaggttcc tgaagttggg gaaattgcca 720

tcaatgacgc attcatgtta gaggctgcta tctacaagct tttgaaatct cacttcagaa 780

acgaaaaata ctacatagat atcaccgaat tgttccatga ggtcaccttc caaaccgaat 840

tgggccaatt gatggactta atcactgcac ctgaagacaa agtcgacttg agtaagttct 900

ccctaaagaa gcactccttc atagttactt tcaagactgc ttactattct ttctacttgc 960

ctgtcgcatt ggccatgtac gttgccggta tcacggatga aaaggatttg aaacaagcca 1020

gagatgtctt gattccattg ggtgaatact tccaaattca agatgactac ttagactgct 1080

tcggtacccc agaacagatc ggtaagatcg gtacagatat ccaagataac aaatgttctt 1140

gggtaatcaa caaggcattg gaacttgctt ccgcagaaca aagaaagact ttagacgaaa 1200

attacggtaa gaaggactca gtcgcagaag ccaaatgcaa aaagattttc aatgacttga 1260

aaattgaaca gctataccac gaatatgaag agtctattgc caaggatttg aaggccaaaa 1320

tttctcaggt cgatgagtct cgtggcttca aagctgatgt cttaactgcg ttcttgaaca 1380

aagtttacaa gagaagcaaa tagactgaca ataaaaagat tcttgttttc aagaacttgt 1440

catttgtata gtttttttat attgtagttg ttctatttta atcaaatgtt agcgtgattt 1500

atattttttt tcgcctcgac atcatctgcc cagatgcgaa gttaagtgcg cagaaagtaa 1560

tatcatgcgt caatcgtatg tgaatgctgg tcgctatact g 1601

<210> 5

<211> 2467

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

gcaaaacgta ggggcaaaca aacggaaaaa tcgtttctca aattttctga tgccaagaac 60

tctaaccagt cttatctaaa aattgcctta tgatccgtct ctccggttac agcctgtgta 120

actgattaat cctgcctttc taatcaccat tctaatgttt taattaaggg attttgtctt 180

cattaacggc tttcgctcat aaaaatgtta tgacgttttg cccgcaggcg ggaaaccatc 240

cacttcacga gactgatctc ctctgccgga acaccgggca tctccaactt ataagttgga 300

gaaataagag aatttcagat tgagagaatg aaaaaaaaaa aaaaaaaaaa ggcagaggag 360

agcatagaaa tggggttcac tttttggtaa agctatagca tgcctatcac atataaatag 420

agtgccagta gcgacttttt tcacactcga aatactctta ctactgctct cttgttgttt 480

ttatcacttc ttgtttcttc ttggtaaata gaatatcaag ctacaaaaag catacaatca 540

actatcaact attaactata tcgtaataca tggatttctt gtcttgtatc ttgttcgtat 600

tattcgcatg ggcattagtc agagcattac caactttgag tagaggttct aaggcagcct 660

caggtagatt gccacctggt ccagttcctt ggccagtagt cggtaatttg ttaaagttgg 720

gtaacaagcc tcataagagt ttagctgaat tagcaaaaag ttatggtcca attatgtgtt 780

taaaattggg tcacatgacc acaattgtta tctctacccc aacagttgcc aaggaagtct 840

tgcaaaaaca agatgttgcc ttttctaata gaacaacccc agacgccgtt agagcacacg 900

gtcacgactt gtactctatg gcatggttac cagtttcaac cagatggaga acattgagaa 960

agatcagtaa ttcacatatc tttacttcac aaagattaga cgaaaatcat cacttaagaa 1020

gaagaaagtt ggacgaatta ttggccagag ttgccgaatc ttccttggtt ggtgctgtag 1080

ttgacatggg tgctgttgct ttcttgacca gtttgaactt gttatctaac accgttttct 1140

caaaagactt ggttgaacct ggtttaggtg ccgttcaaga aaccaaagaa gtagtctggg 1200

gtatgatgga agaagccggt agacctaact tagttgatta ttttcctgtc ttaagaagat 1260

tggacccaca aggtatcaga agaagaatga ctggttactt cggtaaaatg ttagaagttt 1320

tcggtgacat aatagacgaa agattggaat ggagaaagca acaatccgat ggtgactccc 1380

cagcaggtac aactaacgac gtattagatg tcttgttaaa catcatcgaa gatgccgaaa 1440

tagaagaaaa gccaaataga actgatgttg aacacttctt gttagatttg tttgcagctg 1500

gttctgatac cacatcctcc acagtcgaat gggccatgac agaattattg agaaaaccag 1560

aaacattaga aagagcaaga tccgaattac atgaaactat tggtcctgaa aatttggttc 1620

aagaagctga cttgcctaga ttaccatact tgcaagccgt cgttaaggaa acttttagat 1680

tgcatccacc agtcccattg ttattgccta gaaccgctga aaaggatgcc gaattatgcg 1740

gtttcactgt tccagctggt gcacaaatta tggttaacgc ttgggcaata ggtagagacc 1800

caggtacttg ggaagatcca gaatcctttt tgccagaaag attcttaggt tcagacgtcg 1860

atgtcaaagg tagatccttt gaattgatcc catttggtgg tggtagaaga atttgtcctg 1920

gtttaccatt ggccatcaga atggttcatt tgatgttagg tagtttgatt cacggtttta 1980

gatggaaagt agatgatgat ggtatgggtt caccagaaac agctatggac atggacgaaa 2040

agttcggtat cacattacaa aaggcaaagc cattatgcgc cgtccctatc agaggttaac 2100

cgatgccttc acgatttata gttttcatta tcaagtatgc ctatattagt atatagcatc 2160

tttagatgac agtgttcgaa gtttcacgaa taaaagataa tattctactt tttgctccct 2220

tctagtatat tctgtatacc taatattata gcctttatca acaatggaat cccaacaatt 2280

atctcaacat tcacccattt ctcatggtag cgcctgtgct tcggttactt ctaaggaagt 2340

ccacacaaat caagatccgt tagacgtttc agcttccaaa acagaagaat gtgagaaggc 2400

ttccactaag gctaactctc aacagacaac aacacctgct tcatcagctg ttccagagaa 2460

cccccat 2467

<210> 6

<211> 1503

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

atgtctccag ctactgctgt tattttgaca ttgttagttg ctttgggttt atcaattttg 60

ttgagaagga gacaaaagag aaataatttg ccaccaggtc caccagcttt gccaatcatc 120

ggtaacatcc atattttggg tactttgcca catcaatctt tgtacaattt ggctaagaaa 180

tacggtccaa ttatgtcaat gagattgggt ttagttccag cagttgttat ttcttcacca 240

gaagctgctg aattggtttt gaaaactcat gatattgttt ttgcttcaag accaagattg 300

caagttgcag attacttcca ttacggtaca aagggtgtta ttttgactga atacggtaca 360

tactggagaa atatgagaag attgtgtact gttaaattgt tgaacacagt taagatcgat 420

tctttcgctg gtactagaaa gaaagaagtt gcatcttttg ttcaatcatt aaaggaagca 480

tctgttgcac ataagatggt taatttgtca gctagagttg caaacgttat cgaaaacatg 540

gtttgtttga tggttattgg tagatcatct gatgaaagat tcaaattgaa ggaagttatt 600

caagaagctg ctcaattggc tggtgctttt aatatcggtg actacgttcc atttttaatg 660

ccattggatt tgcaaggttt gacaagaaga attaaatctg gttctaaagc atttgatgat 720

attttggaag ttattatcga tgaacatgtt caagatatta aagatcatga tgatgaacaa 780

catggtgact tcatcgatgt tttgttggct atgatgaata agccaatgga ttcaagagaa 840

ggtttgtcaa tcatcgatag aactaacatc aaagctattt tggttgatat gattggtgct 900

gcaatggata cttctacatc aggtgttgaa tgggcaattt ctgaattaat taaacatcca 960

agagttatga agaaattgca agatgaagtt aaaacagtta ttggtatgaa cagaatggtt 1020

gaagaagctg atttgccaaa attgccatac ttggatatgg ttgttaagga aactatgaga 1080

ttacatccac caggtccatt gttagttcca agagaatcta tggaagatat tacaattaat 1140

ggttactaca ttccaaagaa atcaagaatt attgttaatg cttgggcaat tggtagagat 1200

actaatgctt ggtctaataa tgcacatgaa tttttcccag aaagattcat gtcatctaat 1260

gttgatttgc aaggtcaaga ttttcaattg attccatttg gttcaggtag aagaggttgt 1320

ccaggtatga gattgggttt aactacagtt agattggttt tagctcaatt gatccattgt 1380

ttcgatttgg aattaccaaa gggtactgtt gcaactgatt tggatatgtc tgaaaagttc 1440

ggtttggcta tgccaagagc acaacatttg ttggcttttc caacatacag attggaatca 1500

taa 1503

<210> 7

<211> 2122

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

atgcaattat cttcagttaa attgatccca ttagatttga tgacagctat tttcaatggt 60

ggtggttctc cagcaggttc tggtgaagca ttgtctatgt tgttagaaaa cagagaagtt 120

gttgttgcat tgactacatc attagctgtt ttgatcggtt gtgtttttgc ttatttgtgg 180

agattttctt catctcaaaa agcagttgct gctgctaaag gtgttgaagt tgctagaaaa 240

ccagttattg gtaaagaatc tgaagcagct gaagttgatg atggtaaaaa gaaagttact 300

attttctttg gtactcaaac aggtactgct gaaggttttg caaaagcatt ggttgaagaa 360

gcaaaagcta gatacgaaaa agctattttt aaattagttg atttggatga ttatgcagct 420

gaagatgatg aatacgaaga aaaattgaag aaagaaaagt tcgcattgtt tttcttggct 480

acttatggtg acggtgaacc aactgataac gcagctagat tctacaagtg gttcacagaa 540

gaaaacgaat ctggtgaatg gttacaaaaa ttgcaattcg gtgtttttgg tttaggtaat 600

agacaatacg aacatttcaa taaggttgca aaagttgttg atgaaatttt ggctgaacaa 660

ggtggtaaaa gattagttcc agttggtttg ggtgacgatg atcaatgtat cgaagatgat 720

ttcactgctt ggagagaatt agtttggcca gaattggata aattgttatt ggatgaagat 780

gatgcaacag tttctactcc atatacagct tcagttccag aatacagagt tgtttttcat 840

gattctccag atgattattt gcagaaaaat tcttctaatg ctaacggtca ttcaatgcat 900

gatgctcaac atccatgtag agcaaatgtt gctgttagaa gagaattaca ttctccattg 960

tcagatagat catgtactca tttggaattt gatattgcag gtacaggttt ggcttatgaa 1020

actggtgacc atgttggtgt ttgttgtgaa aatttgccag aagttgttga agaagcagaa 1080

agagttttag gtttgtctcc aggtatctat ttctcaattc atgctgataa ggaagatggt 1140

actccattgg gttcatcttt accaccattg tttccaccat gtactttaag aacagcattg 1200

actcaacatg ctgatttgtt gtcttttcca aagaaagcag ctttattggc attggcagct 1260

catgcttcag atccatctga agctgataga ttgaaatatt tggcatctcc agctggtaaa 1320

gatgaatacg cacaatgggt tgttgcttca caaagatcat tgttggaagt tatggcagaa 1380

tttccatctg ctaaaccacc attaggtgtt ttgtttgcag ctgttgctcc aagattacaa 1440

ccaagattct actctatttc atcttcacca aagattgcac catctaggat tcatgttaca 1500

tgtgctttgg tttacgataa aacaccaact ggtagaatac ataaaggtgt ttgttctact 1560

tggatgaaaa atgcaatgcc aagagaagaa tcacatgatt gttcttgggc tccaatcttc 1620

gttagacaat caaacttcaa attgccatca aatacatctg ttccagttat tatgattggt 1680

ccaggtactg gtttagctcc ttttagaggt ttcttgcaag aaagattagc attgaaagaa 1740

gctggtgttg aattaggtcc agcaatcttg tttttcggtt gtagaaacag aaagatggat 1800

tacatctatg aagatgaatt ggctcatttt gttgaagcag gtgctttatc tgaattgatc 1860

gttgcatttt caagagaagg tccagctaag caatacgttc aacataagat gatggaaaaa 1920

gcatctgaaa tttggaacat gatttcagat ggtggttatg tttacgtttg tggtgacgca 1980

aagggtatgg ctaaggatgt tcatagagct ttacatacaa ttgttcatga acaaggttca 2040

ttggataatt ctaaaactga atctatggtt aaaaatttgc aaatgaatgg tagatacttg 2100

agagatgttt ggtaagagct cg 2122

<210> 8

<211> 1941

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

tggaagaaaa ctacagctga tagatctggt gaattgaagc cattgatgat cccaaagtca 60

ttgatggcta aagatgaaga tgatgatttg gatttgggtt ctggtaaaac tagagtttca 120

attttctttg gtactcaaac aggtactgct gaaggtttcg ctaaggcatt gtctgaagaa 180

attaaagcaa gatacgaaaa ggctgcagtt aaagttattg atttggatga ttatgctgca 240

gatgatgatc aatacgaaga aaagttgaag aaagaaactt tggctttctt ttgtgttgca 300

acttatggtg acggtgaacc aacagataac gctgcaagat tctacaagtg gttcacagaa 360

gaaaacgaaa gagatatcaa gttgcaacaa ttagcttatg gtgtttttgc attaggtaat 420

agacaatacg aacatttcaa taagatcggt atcgttttgg atgaagaatt gtgtaagaaa 480

ggtgctaaga gattgattga agttggtttg ggtgacgatg atcaatctat cgaagatgat 540

ttcaacgctt ggaaggaatc tttgtggtca gaattggata agttgttgaa ggatgaagat 600

gataagtctg ttgctacacc atatactgca gttattccag aatacagagt tgttactcat 660

gatccaagat tcactacaca aaaatctatg gaatcaaacg ttgctaacgg taacactaca 720

atcgatatcc atcatccatg tagagttgat gttgctgttc aaaaggaatt gcatacacat 780

gaatctgata gatcatgtat ccatttggaa ttcgatatct ctagaacagg tatcacttat 840

gaaacaggtg accatgttgg tgtttacgct gaaaatcatg ttgaaattgt tgaagaagct 900

ggtaaattgt taggtcattc tttggatttg gttttctcta ttcatgctga taaagaagat 960

ggttctccat tggaatctgc tgttccacca ccatttccag gtccatgtac tttgggtaca 1020

ggtttagcta gatatgcaga tttgttaaat ccaccaagaa aatctgcttt ggttgcatta 1080

gctgcatacg ctactgaacc atcagaagca gaaaaattga aacatttgac atctccagat 1140

ggtaaagatg aatattcaca atggattgtt gcttctcaaa gatcattgtt agaagttatg 1200

gctgcatttc catctgctaa accaccattg ggtgttttct ttgctgcaat tgcaccaaga 1260

ttgcaaccaa gatactactc aatctcttca tctccaagat tggctccatc tagagttcat 1320

gttacttcag cattagttta cggtccaaca ccaactggta gaattcataa aggtgtttgt 1380

tctacatgga tgaaaaatgc tgttccagca gaaaaatctc atgaatgttc aggtgctcca 1440

attttcatta gagcatcaaa cttcaagttg ccatctaacc catcaactcc aattgttatg 1500

gttggtccag gtacaggttt agctcctttt agaggtttct tgcaagaaag aatggcatta 1560

aaagaagatg gtgaagaatt gggttcatct ttgttgtttt tcggttgtag aaatagacaa 1620

atggatttca tctatgaaga tgaattgaac aactttgttg atcaaggtgt tatttctgaa 1680

ttgatcatgg ctttttcaag agaaggtgca caaaaggaat acgttcaaca taagatgatg 1740

gaaaaagctg cacaagtttg ggatttgatt aaagaagaag gttatttgta cgtttgtggt 1800

gacgctaagg gtatggcaag agatgttcat agaactttgc atacaatcgt tcaagaacaa 1860

gaaggtgttt catcttcaga agctgaagca attgttaaga aattgcaaac tgaaggtaga 1920

tatttgagag atgtttggta a 1941

<210> 9

<211> 808

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

tgtcgaatac tactatgacc gctttttaga atcgtacgac aacggtgacc acttgattgg 60

tctgggggtc ctacaacttg attttatcgt tgaaaacaag aatatagaca gccttcttgc 120

caactcttat ttgcaccagc aaagaggcgg tgcaatcatc agtaatacag gacttgtctc 180

gcaagatacg accaagccgt actacgttcg ggatttaatc ttctcgcagt ctgcaggcgc 240

cttgagattt gcgttcggcc taaacgtttg ctccacaaac gtgaatggta tgaacatgga 300

catgagcgtg gttcagggca ctctacggga tcgtggcgaa tgggaatcgt tctgcaagct 360

cttctaccaa accatcggcg aatttgcgtc gctttaatgc gatactgccg tagcgggcct 420

tcgtatagct cggccgagct cgtacaaaag gcaagcagtg tatcggacag agctgatata 480

acacaatacg ctcgtagtcg atgcatgccg tggctgctct cggtcgggta taagtcttag 540

acaatagtct tacctcgcat gtataataaa tcttttgtat ttaatctatt atatgtttct 600

atgctttttt ttcctattgt tgtttgcttt tccttttcct tatttctttc tagcttctaa 660

ttttctttct tttttttttt ttttcattga aaattatata tatatatata tatcagaaca 720

attgtccagt attgaacaat acaggttatt tcgaacaatt gaaaaaaaaa aatcacagaa 780

aaacatatcg agaaaagggt cataagct 808

<210> 10

<211> 323

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

tcaagtgctg atagaagaat accactcata tgacgtgggc agaagacagc aaacgtaaac 60

atgagctgct gcgacatttg atggctttta tccgacaagc caggaaactc caccattatc 120

taatgtagca aaatatttct taacacccga agttgcgtgt ccccctcacg tttttaatca 180

tttgaattag tatattgaaa ttatatataa aggcaacaat gtccccataa tcaattccat 240

ctggggtctc atgttctttc cccaccttaa aatctataaa gatatcataa tcgtcaacta 300

gttgatatac gtaaaatcaa gct 323

<210> 11

<211> 512

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

aagctttgtt ggaatagaaa tcaactatca tctactaact agtatttaca ttactagtat 60

attatcatat acggtgttag aagatgacgc aaatgatgag aaatagtcat ctaaattagt 120

ggaagctgaa acgcaaggat tgataatgta ataggatcaa tgaatataaa catataaaac 180

ggaatgagga ataatcgtaa tattagtatg tagaaatata gattccattt tgaggattcc 240

tatatcctcg aggagaactt ctagtatatt ctgtatacct aatattatag cctttatcaa 300

caatggaatc ccaacaatta tctcaacatt cacccatttc tcatggtagc gcctgtgctt 360

cggttacttc taaggaagtc cacacaaatc aagatccgtt agacgtttca gcttccaaaa 420

cagaagaatg tgagaaggct tccactaagg ctaactctca acagacaaca acacctgctt 480

catcagctgt tccagagaac ccccatgaat tc 512

<210> 12

<211> 1539

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

ccgctcgaga tggatttctt gtcttgtatc ttgttcgtat tattcgcatg ggcattagtc 60

agagcattac caactttgag tagaggttct aaggcagcct caggtagatt gccacctggt 120

ccagttcctt ggccagtagt cggtaatttg ttaaagttgg gtaacaagcc tcataagagt 180

ttagctgaat tagcaaaaag ttatggtcca attatgtgtt taaaattggg tcacatgacc 240

acaattgtta tctctacccc aacagttgcc aaggaagtct tgcaaaaaca agatgttgcc 300

ttttctaata gaacaacccc agacgccgtt agagcacacg gtcacgactt gtactctatg 360

gcatggttac cagtttcaac cagatggaga acattgagaa agatcagtaa ttcacatatc 420

tttacttcac aaagattaga cgaaaatcat cacttaagaa gaagaaagtt ggacgaatta 480

ttggccagag ttgccgaatc ttccttggtt ggtgctgtag ttgacatggg tgctgttgct 540

ttcttgacca gtttgaactt gttatctaac accgttttct caaaagactt ggttgaacct 600

ggtttaggtg ccgttcaaga aaccaaagaa gtagtctggg gtatgatgga agaagccggt 660

agacctaact tagttgatta ttttcctgtc ttaagaagat tggacccaca aggtatcaga 720

agaagaatga ctggttactt cggtaaaatg ttagaagttt tcggtgacat aatagacgaa 780

agattggaat ggagaaagca acaatccgat ggtgactccc cagcaggtac aactaacgac 840

gtattagatg tcttgttaaa catcatcgaa gatgccgaaa tagaagaaaa gccaaataga 900

actgatgttg aacacttctt gttagatttg tttgcagctg gttctgatac cacatcctcc 960

acagtcgaat gggccatgac agaattattg agaaaaccag aaacattaga aagagcaaga 1020

tccgaattac atgaaactat tggtcctgaa aatttggttc aagaagctga cttgcctaga 1080

ttaccatact tgcaagccgt cgttaaggaa acttttagat tgcatccacc agtcccattg 1140

ttattgccta gaaccgctga aaaggatgcc gaattatgcg gtttcactgt tccagctggt 1200

gcacaaatta tggttaacgc ttgggcaata ggtagagacc caggtacttg ggaagatcca 1260

gaatcctttt tgccagaaag attcttaggt tcagacgtcg atgtcaaagg tagatccttt 1320

gaattgatcc catttggtgg tggtagaaga atttgtcctg gtttaccatt ggccatcaga 1380

atggttcatt tgatgttagg tagtttgatt cacggtttta gatggaaagt agatgatgat 1440

ggtatgggtt caccagaaac agctatggac atggacgaaa agttcggtat cacattacaa 1500

aaggcaaagc cattatgcgc cgtccctatc agaggttaa 1539

<210> 13

<211> 3659

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

ccgctcgaga tggatttctt gtcttgtatc ttgttcgtat tattcgcatg ggcattagtc 60

agagcattac caactttgag tagaggttct aaggcagcct caggtagatt gccacctggt 120

ccagttcctt ggccagtagt cggtaatttg ttaaagttgg gtaacaagcc tcataagagt 180

ttagctgaat tagcaaaaag ttatggtcca attatgtgtt taaaattggg tcacatgacc 240

acaattgtta tctctacccc aacagttgcc aaggaagtct tgcaaaaaca agatgttgcc 300

ttttctaata gaacaacccc agacgccgtt agagcacacg gtcacgactt gtactctatg 360

gcatggttac cagtttcaac cagatggaga acattgagaa agatcagtaa ttcacatatc 420

tttacttcac aaagattaga cgaaaatcat cacttaagaa gaagaaagtt ggacgaatta 480

ttggccagag ttgccgaatc ttccttggtt ggtgctgtag ttgacatggg tgctgttgct 540

ttcttgacca gtttgaactt gttatctaac accgttttct caaaagactt ggttgaacct 600

ggtttaggtg ccgttcaaga aaccaaagaa gtagtctggg gtatgatgga agaagccggt 660

agacctaact tagttgatta ttttcctgtc ttaagaagat tggacccaca aggtatcaga 720

agaagaatga ctggttactt cggtaaaatg ttagaagttt tcggtgacat aatagacgaa 780

agattggaat ggagaaagca acaatccgat ggtgactccc cagcaggtac aactaacgac 840

gtattagatg tcttgttaaa catcatcgaa gatgccgaaa tagaagaaaa gccaaataga 900

actgatgttg aacacttctt gttagatttg tttgcagctg gttctgatac cacatcctcc 960

acagtcgaat gggccatgac agaattattg agaaaaccag aaacattaga aagagcaaga 1020

tccgaattac atgaaactat tggtcctgaa aatttggttc aagaagctga cttgcctaga 1080

ttaccatact tgcaagccgt cgttaaggaa acttttagat tgcatccacc agtcccattg 1140

ttattgccta gaaccgctga aaaggatgcc gaattatgcg gtttcactgt tccagctggt 1200

gcacaaatta tggttaacgc ttgggcaata ggtagagacc caggtacttg ggaagatcca 1260

gaatcctttt tgccagaaag attcttaggt tcagacgtcg atgtcaaagg tagatccttt 1320

gaattgatcc catttggtgg tggtagaaga atttgtcctg gtttaccatt ggccatcaga 1380

atggttcatt tgatgttagg tagtttgatt cacggtttta gatggaaagt agatgatgat 1440

ggtatgggtt caccagaaac agctatggac atggacgaaa agttcggtat cacattacaa 1500

aaggcaaagc cattatgcgc cgtccctatc agaggtatgc aattatcttc agttaaattg 1560

atcccattag atttgatgac agctattttc aatggtggtg gttctccagc aggttctggt 1620

gaagcattgt ctatgttgtt agaaaacaga gaagttgttg ttgcattgac tacatcatta 1680

gctgttttga tcggttgtgt ttttgcttat ttgtggagat tttcttcatc tcaaaaagca 1740

gttgctgctg ctaaaggtgt tgaagttgct agaaaaccag ttattggtaa agaatctgaa 1800

gcagctgaag ttgatgatgg taaaaagaaa gttactattt tctttggtac tcaaacaggt 1860

actgctgaag gttttgcaaa agcattggtt gaagaagcaa aagctagata cgaaaaagct 1920

atttttaaat tagttgattt ggatgattat gcagctgaag atgatgaata cgaagaaaaa 1980

ttgaagaaag aaaagttcgc attgtttttc ttggctactt atggtgacgg tgaaccaact 2040

gataacgcag ctagattcta caagtggttc acagaagaaa acgaatctgg tgaatggtta 2100

caaaaattgc aattcggtgt ttttggttta ggtaatagac aatacgaaca tttcaataag 2160

gttgcaaaag ttgttgatga aattttggct gaacaaggtg gtaaaagatt agttccagtt 2220

ggtttgggtg acgatgatca atgtatcgaa gatgatttca ctgcttggag agaattagtt 2280

tggccagaat tggataaatt gttattggat gaagatgatg caacagtttc tactccatat 2340

acagcttcag ttccagaata cagagttgtt tttcatgatt ctccagatga ttatttgcag 2400

aaaaattctt ctaatgctaa cggtcattca atgcatgatg ctcaacatcc atgtagagca 2460

aatgttgctg ttagaagaga attacattct ccattgtcag atagatcatg tactcatttg 2520

gaatttgata ttgcaggtac aggtttggct tatgaaactg gtgaccatgt tggtgtttgt 2580

tgtgaaaatt tgccagaagt tgttgaagaa gcagaaagag ttttaggttt gtctccaggt 2640

atctatttct caattcatgc tgataaggaa gatggtactc cattgggttc atctttacca 2700

ccattgtttc caccatgtac tttaagaaca gcattgactc aacatgctga tttgttgtct 2760

tttccaaaga aagcagcttt attggcattg gcagctcatg cttcagatcc atctgaagct 2820

gatagattga aatatttggc atctccagct ggtaaagatg aatacgcaca atgggttgtt 2880

gcttcacaaa gatcattgtt ggaagttatg gcagaatttc catctgctaa accaccatta 2940

ggtgttttgt ttgcagctgt tgctccaaga ttacaaccaa gattctactc tatttcatct 3000

tcaccaaaga ttgcaccatc taggattcat gttacatgtg ctttggttta cgataaaaca 3060

ccaactggta gaatacataa aggtgtttgt tctacttgga tgaaaaatgc aatgccaaga 3120

gaagaatcac atgattgttc ttgggctcca atcttcgtta gacaatcaaa cttcaaattg 3180

ccatcaaata catctgttcc agttattatg attggtccag gtactggttt agctcctttt 3240

agaggtttct tgcaagaaag attagcattg aaagaagctg gtgttgaatt aggtccagca 3300

atcttgtttt tcggttgtag aaacagaaag atggattaca tctatgaaga tgaattggct 3360

cattttgttg aagcaggtgc tttatctgaa ttgatcgttg cattttcaag agaaggtcca 3420

gctaagcaat acgttcaaca taagatgatg gaaaaagcat ctgaaatttg gaacatgatt 3480

tcagatggtg gttatgttta cgtttgtggt gacgcaaagg gtatggctaa ggatgttcat 3540

agagctttac atacaattgt tcatgaacaa ggttcattgg ataattctaa aactgaatct 3600

atggttaaaa atttgcaaat gaatggtaga tacttgagag atgtttggta aggatcccg 3659

<210> 14

<211> 3719

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

ccgctcgaga tggatttctt gtcttgtatc ttgttcgtat tattcgcatg ggcattagtc 60

agagcattac caactttgag tagaggttct aaggcagcct caggtagatt gccacctggt 120

ccagttcctt ggccagtagt cggtaatttg ttaaagttgg gtaacaagcc tcataagagt 180

ttagctgaat tagcaaaaag ttatggtcca attatgtgtt taaaattggg tcacatgacc 240

acaattgtta tctctacccc aacagttgcc aaggaagtct tgcaaaaaca agatgttgcc 300

ttttctaata gaacaacccc agacgccgtt agagcacacg gtcacgactt gtactctatg 360

gcatggttac cagtttcaac cagatggaga acattgagaa agatcagtaa ttcacatatc 420

tttacttcac aaagattaga cgaaaatcat cacttaagaa gaagaaagtt ggacgaatta 480

ttggccagag ttgccgaatc ttccttggtt ggtgctgtag ttgacatggg tgctgttgct 540

ttcttgacca gtttgaactt gttatctaac accgttttct caaaagactt ggttgaacct 600

ggtttaggtg ccgttcaaga aaccaaagaa gtagtctggg gtatgatgga agaagccggt 660

agacctaact tagttgatta ttttcctgtc ttaagaagat tggacccaca aggtatcaga 720

agaagaatga ctggttactt cggtaaaatg ttagaagttt tcggtgacat aatagacgaa 780

agattggaat ggagaaagca acaatccgat ggtgactccc cagcaggtac aactaacgac 840

gtattagatg tcttgttaaa catcatcgaa gatgccgaaa tagaagaaaa gccaaataga 900

actgatgttg aacacttctt gttagatttg tttgcagctg gttctgatac cacatcctcc 960

acagtcgaat gggccatgac agaattattg agaaaaccag aaacattaga aagagcaaga 1020

tccgaattac atgaaactat tggtcctgaa aatttggttc aagaagctga cttgcctaga 1080

ttaccatact tgcaagccgt cgttaaggaa acttttagat tgcatccacc agtcccattg 1140

ttattgccta gaaccgctga aaaggatgcc gaattatgcg gtttcactgt tccagctggt 1200

gcacaaatta tggttaacgc ttgggcaata ggtagagacc caggtacttg ggaagatcca 1260

gaatcctttt tgccagaaag attcttaggt tcagacgtcg atgtcaaagg tagatccttt 1320

gaattgatcc catttggtgg tggtagaaga atttgtcctg gtttaccatt ggccatcaga 1380

atggttcatt tgatgttagg tagtttgatt cacggtttta gatggaaagt agatgatgat 1440

ggtatgggtt caccagaaac agctatggac atggacgaaa agttcggtat cacattacaa 1500

aaggcaaagc cattatgcgc cgtccctatc agaggtggtg gtggtggttc tggtggtggt 1560

ggttctggtg gtggtggttc tggtggtggt ggttctatgc aattatcttc agttaaattg 1620

atcccattag atttgatgac agctattttc aatggtggtg gttctccagc aggttctggt 1680

gaagcattgt ctatgttgtt agaaaacaga gaagttgttg ttgcattgac tacatcatta 1740

gctgttttga tcggttgtgt ttttgcttat ttgtggagat tttcttcatc tcaaaaagca 1800

gttgctgctg ctaaaggtgt tgaagttgct agaaaaccag ttattggtaa agaatctgaa 1860

gcagctgaag ttgatgatgg taaaaagaaa gttactattt tctttggtac tcaaacaggt 1920

actgctgaag gttttgcaaa agcattggtt gaagaagcaa aagctagata cgaaaaagct 1980

atttttaaat tagttgattt ggatgattat gcagctgaag atgatgaata cgaagaaaaa 2040

ttgaagaaag aaaagttcgc attgtttttc ttggctactt atggtgacgg tgaaccaact 2100

gataacgcag ctagattcta caagtggttc acagaagaaa acgaatctgg tgaatggtta 2160

caaaaattgc aattcggtgt ttttggttta ggtaatagac aatacgaaca tttcaataag 2220

gttgcaaaag ttgttgatga aattttggct gaacaaggtg gtaaaagatt agttccagtt 2280

ggtttgggtg acgatgatca atgtatcgaa gatgatttca ctgcttggag agaattagtt 2340

tggccagaat tggataaatt gttattggat gaagatgatg caacagtttc tactccatat 2400

acagcttcag ttccagaata cagagttgtt tttcatgatt ctccagatga ttatttgcag 2460

aaaaattctt ctaatgctaa cggtcattca atgcatgatg ctcaacatcc atgtagagca 2520

aatgttgctg ttagaagaga attacattct ccattgtcag atagatcatg tactcatttg 2580

gaatttgata ttgcaggtac aggtttggct tatgaaactg gtgaccatgt tggtgtttgt 2640

tgtgaaaatt tgccagaagt tgttgaagaa gcagaaagag ttttaggttt gtctccaggt 2700

atctatttct caattcatgc tgataaggaa gatggtactc cattgggttc atctttacca 2760

ccattgtttc caccatgtac tttaagaaca gcattgactc aacatgctga tttgttgtct 2820

tttccaaaga aagcagcttt attggcattg gcagctcatg cttcagatcc atctgaagct 2880

gatagattga aatatttggc atctccagct ggtaaagatg aatacgcaca atgggttgtt 2940

gcttcacaaa gatcattgtt ggaagttatg gcagaatttc catctgctaa accaccatta 3000

ggtgttttgt ttgcagctgt tgctccaaga ttacaaccaa gattctactc tatttcatct 3060

tcaccaaaga ttgcaccatc taggattcat gttacatgtg ctttggttta cgataaaaca 3120

ccaactggta gaatacataa aggtgtttgt tctacttgga tgaaaaatgc aatgccaaga 3180

gaagaatcac atgattgttc ttgggctcca atcttcgtta gacaatcaaa cttcaaattg 3240

ccatcaaata catctgttcc agttattatg attggtccag gtactggttt agctcctttt 3300

agaggtttct tgcaagaaag attagcattg aaagaagctg gtgttgaatt aggtccagca 3360

atcttgtttt tcggttgtag aaacagaaag atggattaca tctatgaaga tgaattggct 3420

cattttgttg aagcaggtgc tttatctgaa ttgatcgttg cattttcaag agaaggtcca 3480

gctaagcaat acgttcaaca taagatgatg gaaaaagcat ctgaaatttg gaacatgatt 3540

tcagatggtg gttatgttta cgtttgtggt gacgcaaagg gtatggctaa ggatgttcat 3600

agagctttac atacaattgt tcatgaacaa ggttcattgg ataattctaa aactgaatct 3660

atggttaaaa atttgcaaat gaatggtaga tacttgagag atgtttggta aggatcccg 3719

<210> 15

<211> 3485

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

ccgctcgaga tggatttctt gtcttgtatc ttgttcgtat tattcgcatg ggcattagtc 60

agagcattac caactttgag tagaggttct aaggcagcct caggtagatt gccacctggt 120

ccagttcctt ggccagtagt cggtaatttg ttaaagttgg gtaacaagcc tcataagagt 180

ttagctgaat tagcaaaaag ttatggtcca attatgtgtt taaaattggg tcacatgacc 240

acaattgtta tctctacccc aacagttgcc aaggaagtct tgcaaaaaca agatgttgcc 300

ttttctaata gaacaacccc agacgccgtt agagcacacg gtcacgactt gtactctatg 360

gcatggttac cagtttcaac cagatggaga acattgagaa agatcagtaa ttcacatatc 420

tttacttcac aaagattaga cgaaaatcat cacttaagaa gaagaaagtt ggacgaatta 480

ttggccagag ttgccgaatc ttccttggtt ggtgctgtag ttgacatggg tgctgttgct 540

ttcttgacca gtttgaactt gttatctaac accgttttct caaaagactt ggttgaacct 600

ggtttaggtg ccgttcaaga aaccaaagaa gtagtctggg gtatgatgga agaagccggt 660

agacctaact tagttgatta ttttcctgtc ttaagaagat tggacccaca aggtatcaga 720

agaagaatga ctggttactt cggtaaaatg ttagaagttt tcggtgacat aatagacgaa 780

agattggaat ggagaaagca acaatccgat ggtgactccc cagcaggtac aactaacgac 840

gtattagatg tcttgttaaa catcatcgaa gatgccgaaa tagaagaaaa gccaaataga 900

actgatgttg aacacttctt gttagatttg tttgcagctg gttctgatac cacatcctcc 960

acagtcgaat gggccatgac agaattattg agaaaaccag aaacattaga aagagcaaga 1020

tccgaattac atgaaactat tggtcctgaa aatttggttc aagaagctga cttgcctaga 1080

ttaccatact tgcaagccgt cgttaaggaa acttttagat tgcatccacc agtcccattg 1140

ttattgccta gaaccgctga aaaggatgcc gaattatgcg gtttcactgt tccagctggt 1200

gcacaaatta tggttaacgc ttgggcaata ggtagagacc caggtacttg ggaagatcca 1260

gaatcctttt tgccagaaag attcttaggt tcagacgtcg atgtcaaagg tagatccttt 1320

gaattgatcc catttggtgg tggtagaaga atttgtcctg gtttaccatt ggccatcaga 1380

atggttcatt tgatgttagg tagtttgatt cacggtttta gatggaaagt agatgatgat 1440

ggtatgggtt caccagaaac agctatggac atggacgaaa agttcggtat cacattacaa 1500

aaggcaaagc cattatgcgc cgtccctatc agaggttgga agaaaactac agctgataga 1560

tctggtgaat tgaagccatt gatgatccca aagtcattga tggctaaaga tgaagatgat 1620

gatttggatt tgggttctgg taaaactaga gtttcaattt tctttggtac tcaaacaggt 1680

actgctgaag gtttcgctaa ggcattgtct gaagaaatta aagcaagata cgaaaaggct 1740

gcagttaaag ttattgattt ggatgattat gctgcagatg atgatcaata cgaagaaaag 1800

ttgaagaaag aaactttggc tttcttttgt gttgcaactt atggtgacgg tgaaccaaca 1860

gataacgctg caagattcta caagtggttc acagaagaaa acgaaagaga tatcaagttg 1920

caacaattag cttatggtgt ttttgcatta ggtaatagac aatacgaaca tttcaataag 1980

atcggtatcg ttttggatga agaattgtgt aagaaaggtg ctaagagatt gattgaagtt 2040

ggtttgggtg acgatgatca atctatcgaa gatgatttca acgcttggaa ggaatctttg 2100

tggtcagaat tggataagtt gttgaaggat gaagatgata agtctgttgc tacaccatat 2160

actgcagtta ttccagaata cagagttgtt actcatgatc caagattcac tacacaaaaa 2220

tctatggaat caaacgttgc taacggtaac actacaatcg atatccatca tccatgtaga 2280

gttgatgttg ctgttcaaaa ggaattgcat acacatgaat ctgatagatc atgtatccat 2340

ttggaattcg atatctctag aacaggtatc acttatgaaa caggtgacca tgttggtgtt 2400

tacgctgaaa atcatgttga aattgttgaa gaagctggta aattgttagg tcattctttg 2460

gatttggttt tctctattca tgctgataaa gaagatggtt ctccattgga atctgctgtt 2520

ccaccaccat ttccaggtcc atgtactttg ggtacaggtt tagctagata tgcagatttg 2580

ttaaatccac caagaaaatc tgctttggtt gcattagctg catacgctac tgaaccatca 2640

gaagcagaaa aattgaaaca tttgacatct ccagatggta aagatgaata ttcacaatgg 2700

attgttgctt ctcaaagatc attgttagaa gttatggctg catttccatc tgctaaacca 2760

ccattgggtg ttttctttgc tgcaattgca ccaagattgc aaccaagata ctactcaatc 2820

tcttcatctc caagattggc tccatctaga gttcatgtta cttcagcatt agtttacggt 2880

ccaacaccaa ctggtagaat tcataaaggt gtttgttcta catggatgaa aaatgctgtt 2940

ccagcagaaa aatctcatga atgttcaggt gctccaattt tcattagagc atcaaacttc 3000

aagttgccat ctaacccatc aactccaatt gttatggttg gtccaggtac aggtttagct 3060

ccttttagag gtttcttgca agaaagaatg gcattaaaag aagatggtga agaattgggt 3120

tcatctttgt tgtttttcgg ttgtagaaat agacaaatgg atttcatcta tgaagatgaa 3180

ttgaacaact ttgttgatca aggtgttatt tctgaattga tcatggcttt ttcaagagaa 3240

ggtgcacaaa aggaatacgt tcaacataag atgatggaaa aagctgcaca agtttgggat 3300

ttgattaaag aagaaggtta tttgtacgtt tgtggtgacg ctaagggtat ggcaagagat 3360

gttcatagaa ctttgcatac aatcgttcaa gaacaagaag gtgtttcatc ttcagaagct 3420

gaagcaattg ttaagaaatt gcaaactgaa ggtagatatt tgagagatgt ttggtaagga 3480

tcccg 3485

<210> 16

<211> 3623

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

atgtctccag ctactgctgt tattttgaca ttgttagttg ctttgggttt atcaattttg 60

ttgagaagga gacaaaagag aaataatttg ccaccaggtc caccagcttt gccaatcatc 120

ggtaacatcc atattttggg tactttgcca catcaatctt tgtacaattt ggctaagaaa 180

tacggtccaa ttatgtcaat gagattgggt ttagttccag cagttgttat ttcttcacca 240

gaagctgctg aattggtttt gaaaactcat gatattgttt ttgcttcaag accaagattg 300

caagttgcag attacttcca ttacggtaca aagggtgtta ttttgactga atacggtaca 360

tactggagaa atatgagaag attgtgtact gttaaattgt tgaacacagt taagatcgat 420

tctttcgctg gtactagaaa gaaagaagtt gcatcttttg ttcaatcatt aaaggaagca 480

tctgttgcac ataagatggt taatttgtca gctagagttg caaacgttat cgaaaacatg 540

gtttgtttga tggttattgg tagatcatct gatgaaagat tcaaattgaa ggaagttatt 600

caagaagctg ctcaattggc tggtgctttt aatatcggtg actacgttcc atttttaatg 660

ccattggatt tgcaaggttt gacaagaaga attaaatctg gttctaaagc atttgatgat 720

attttggaag ttattatcga tgaacatgtt caagatatta aagatcatga tgatgaacaa 780

catggtgact tcatcgatgt tttgttggct atgatgaata agccaatgga ttcaagagaa 840

ggtttgtcaa tcatcgatag aactaacatc aaagctattt tggttgatat gattggtgct 900

gcaatggata cttctacatc aggtgttgaa tgggcaattt ctgaattaat taaacatcca 960

agagttatga agaaattgca agatgaagtt aaaacagtta ttggtatgaa cagaatggtt 1020

gaagaagctg atttgccaaa attgccatac ttggatatgg ttgttaagga aactatgaga 1080

ttacatccac caggtccatt gttagttcca agagaatcta tggaagatat tacaattaat 1140

ggttactaca ttccaaagaa atcaagaatt attgttaatg cttgggcaat tggtagagat 1200

actaatgctt ggtctaataa tgcacatgaa tttttcccag aaagattcat gtcatctaat 1260

gttgatttgc aaggtcaaga ttttcaattg attccatttg gttcaggtag aagaggttgt 1320

ccaggtatga gattgggttt aactacagtt agattggttt tagctcaatt gatccattgt 1380

ttcgatttgg aattaccaaa gggtactgtt gcaactgatt tggatatgtc tgaaaagttc 1440

ggtttggcta tgccaagagc acaacatttg ttggcttttc caacatacag attggaatca 1500

atgcaattat cttcagttaa attgatccca ttagatttga tgacagctat tttcaatggt 1560

ggtggttctc cagcaggttc tggtgaagca ttgtctatgt tgttagaaaa cagagaagtt 1620

gttgttgcat tgactacatc attagctgtt ttgatcggtt gtgtttttgc ttatttgtgg 1680

agattttctt catctcaaaa agcagttgct gctgctaaag gtgttgaagt tgctagaaaa 1740

ccagttattg gtaaagaatc tgaagcagct gaagttgatg atggtaaaaa gaaagttact 1800

attttctttg gtactcaaac aggtactgct gaaggttttg caaaagcatt ggttgaagaa 1860

gcaaaagcta gatacgaaaa agctattttt aaattagttg atttggatga ttatgcagct 1920

gaagatgatg aatacgaaga aaaattgaag aaagaaaagt tcgcattgtt tttcttggct 1980

acttatggtg acggtgaacc aactgataac gcagctagat tctacaagtg gttcacagaa 2040

gaaaacgaat ctggtgaatg gttacaaaaa ttgcaattcg gtgtttttgg tttaggtaat 2100

agacaatacg aacatttcaa taaggttgca aaagttgttg atgaaatttt ggctgaacaa 2160

ggtggtaaaa gattagttcc agttggtttg ggtgacgatg atcaatgtat cgaagatgat 2220

ttcactgctt ggagagaatt agtttggcca gaattggata aattgttatt ggatgaagat 2280

gatgcaacag tttctactcc atatacagct tcagttccag aatacagagt tgtttttcat 2340

gattctccag atgattattt gcagaaaaat tcttctaatg ctaacggtca ttcaatgcat 2400

gatgctcaac atccatgtag agcaaatgtt gctgttagaa gagaattaca ttctccattg 2460

tcagatagat catgtactca tttggaattt gatattgcag gtacaggttt ggcttatgaa 2520

actggtgacc atgttggtgt ttgttgtgaa aatttgccag aagttgttga agaagcagaa 2580

agagttttag gtttgtctcc aggtatctat ttctcaattc atgctgataa ggaagatggt 2640

actccattgg gttcatcttt accaccattg tttccaccat gtactttaag aacagcattg 2700

actcaacatg ctgatttgtt gtcttttcca aagaaagcag ctttattggc attggcagct 2760

catgcttcag atccatctga agctgataga ttgaaatatt tggcatctcc agctggtaaa 2820

gatgaatacg cacaatgggt tgttgcttca caaagatcat tgttggaagt tatggcagaa 2880

tttccatctg ctaaaccacc attaggtgtt ttgtttgcag ctgttgctcc aagattacaa 2940

ccaagattct actctatttc atcttcacca aagattgcac catctaggat tcatgttaca 3000

tgtgctttgg tttacgataa aacaccaact ggtagaatac ataaaggtgt ttgttctact 3060

tggatgaaaa atgcaatgcc aagagaagaa tcacatgatt gttcttgggc tccaatcttc 3120

gttagacaat caaacttcaa attgccatca aatacatctg ttccagttat tatgattggt 3180

ccaggtactg gtttagctcc ttttagaggt ttcttgcaag aaagattagc attgaaagaa 3240

gctggtgttg aattaggtcc agcaatcttg tttttcggtt gtagaaacag aaagatggat 3300

tacatctatg aagatgaatt ggctcatttt gttgaagcag gtgctttatc tgaattgatc 3360

gttgcatttt caagagaagg tccagctaag caatacgttc aacataagat gatggaaaaa 3420

gcatctgaaa tttggaacat gatttcagat ggtggttatg tttacgtttg tggtgacgca 3480

aagggtatgg ctaaggatgt tcatagagct ttacatacaa ttgttcatga acaaggttca 3540

ttggataatt ctaaaactga atctatggtt aaaaatttgc aaatgaatgg tagatacttg 3600

agagatgttt ggtaaggatc ccg 3623

<210> 17

<211> 3441

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 17

atgtctccag ctactgctgt tattttgaca ttgttagttg ctttgggttt atcaattttg 60

ttgagaagga gacaaaagag aaataatttg ccaccaggtc caccagcttt gccaatcatc 120

ggtaacatcc atattttggg tactttgcca catcaatctt tgtacaattt ggctaagaaa 180

tacggtccaa ttatgtcaat gagattgggt ttagttccag cagttgttat ttcttcacca 240

gaagctgctg aattggtttt gaaaactcat gatattgttt ttgcttcaag accaagattg 300

caagttgcag attacttcca ttacggtaca aagggtgtta ttttgactga atacggtaca 360

tactggagaa atatgagaag attgtgtact gttaaattgt tgaacacagt taagatcgat 420

tctttcgctg gtactagaaa gaaagaagtt gcatcttttg ttcaatcatt aaaggaagca 480

tctgttgcac ataagatggt taatttgtca gctagagttg caaacgttat cgaaaacatg 540

gtttgtttga tggttattgg tagatcatct gatgaaagat tcaaattgaa ggaagttatt 600

caagaagctg ctcaattggc tggtgctttt aatatcggtg actacgttcc atttttaatg 660

ccattggatt tgcaaggttt gacaagaaga attaaatctg gttctaaagc atttgatgat 720

attttggaag ttattatcga tgaacatgtt caagatatta aagatcatga tgatgaacaa 780

catggtgact tcatcgatgt tttgttggct atgatgaata agccaatgga ttcaagagaa 840

ggtttgtcaa tcatcgatag aactaacatc aaagctattt tggttgatat gattggtgct 900

gcaatggata cttctacatc aggtgttgaa tgggcaattt ctgaattaat taaacatcca 960

agagttatga agaaattgca agatgaagtt aaaacagtta ttggtatgaa cagaatggtt 1020

gaagaagctg atttgccaaa attgccatac ttggatatgg ttgttaagga aactatgaga 1080

ttacatccac caggtccatt gttagttcca agagaatcta tggaagatat tacaattaat 1140

ggttactaca ttccaaagaa atcaagaatt attgttaatg cttgggcaat tggtagagat 1200

actaatgctt ggtctaataa tgcacatgaa tttttcccag aaagattcat gtcatctaat 1260

gttgatttgc aaggtcaaga ttttcaattg attccatttg gttcaggtag aagaggttgt 1320

ccaggtatga gattgggttt aactacagtt agattggttt tagctcaatt gatccattgt 1380

ttcgatttgg aattaccaaa gggtactgtt gcaactgatt tggatatgtc tgaaaagttc 1440

ggtttggcta tgccaagagc acaacatttg ttggcttttc caacatacag attggaatca 1500

tggaagaaaa ctacagctga tagatctggt gaattgaagc cattgatgat cccaaagtca 1560

ttgatggcta aagatgaaga tgatgatttg gatttgggtt ctggtaaaac tagagtttca 1620

attttctttg gtactcaaac aggtactgct gaaggtttcg ctaaggcatt gtctgaagaa 1680

attaaagcaa gatacgaaaa ggctgcagtt aaagttattg atttggatga ttatgctgca 1740

gatgatgatc aatacgaaga aaagttgaag aaagaaactt tggctttctt ttgtgttgca 1800

acttatggtg acggtgaacc aacagataac gctgcaagat tctacaagtg gttcacagaa 1860

gaaaacgaaa gagatatcaa gttgcaacaa ttagcttatg gtgtttttgc attaggtaat 1920

agacaatacg aacatttcaa taagatcggt atcgttttgg atgaagaatt gtgtaagaaa 1980

ggtgctaaga gattgattga agttggtttg ggtgacgatg atcaatctat cgaagatgat 2040

ttcaacgctt ggaaggaatc tttgtggtca gaattggata agttgttgaa ggatgaagat 2100

gataagtctg ttgctacacc atatactgca gttattccag aatacagagt tgttactcat 2160

gatccaagat tcactacaca aaaatctatg gaatcaaacg ttgctaacgg taacactaca 2220

atcgatatcc atcatccatg tagagttgat gttgctgttc aaaaggaatt gcatacacat 2280

gaatctgata gatcatgtat ccatttggaa ttcgatatct ctagaacagg tatcacttat 2340

gaaacaggtg accatgttgg tgtttacgct gaaaatcatg ttgaaattgt tgaagaagct 2400

ggtaaattgt taggtcattc tttggatttg gttttctcta ttcatgctga taaagaagat 2460

ggttctccat tggaatctgc tgttccacca ccatttccag gtccatgtac tttgggtaca 2520

ggtttagcta gatatgcaga tttgttaaat ccaccaagaa aatctgcttt ggttgcatta 2580

gctgcatacg ctactgaacc atcagaagca gaaaaattga aacatttgac atctccagat 2640

ggtaaagatg aatattcaca atggattgtt gcttctcaaa gatcattgtt agaagttatg 2700

gctgcatttc catctgctaa accaccattg ggtgttttct ttgctgcaat tgcaccaaga 2760

ttgcaaccaa gatactactc aatctcttca tctccaagat tggctccatc tagagttcat 2820

gttacttcag cattagttta cggtccaaca ccaactggta gaattcataa aggtgtttgt 2880

tctacatgga tgaaaaatgc tgttccagca gaaaaatctc atgaatgttc aggtgctcca 2940

attttcatta gagcatcaaa cttcaagttg ccatctaacc catcaactcc aattgttatg 3000

gttggtccag gtacaggttt agctcctttt agaggtttct tgcaagaaag aatggcatta 3060

aaagaagatg gtgaagaatt gggttcatct ttgttgtttt tcggttgtag aaatagacaa 3120

atggatttca tctatgaaga tgaattgaac aactttgttg atcaaggtgt tatttctgaa 3180

ttgatcatgg ctttttcaag agaaggtgca caaaaggaat acgttcaaca taagatgatg 3240

gaaaaagctg cacaagtttg ggatttgatt aaagaagaag gttatttgta cgtttgtggt 3300

gacgctaagg gtatggcaag agatgttcat agaactttgc atacaatcgt tcaagaacaa 3360

gaaggtgttt catcttcaga agctgaagca attgttaaga aattgcaaac tgaaggtaga 3420

tatttgagag atgtttggta a 3441

Claims (7)

1. An expression cassette combination, sequentially connected with the following components from 5 'to 3': screening the gene expression cassette, mva + EGFP expression cassette, erg20 expression cassette, saSS-delta expression cassette and P76F39V 1-delta expression cassette;

the nucleotide sequence of the mva + EGFP expression cassette is shown in SEQ ID NO:2 is shown in the figure;

the nucleotide sequence of the SaSS-delta expression cassette is shown as SEQ ID NO:3 is shown in the figure;

the nucleotide sequence of the erg20 expression cassette is shown in SEQ ID NO:4 is shown in the figure;

the nucleotide sequence of the P76F39V 1-delta expression cassette is shown in SEQ ID NO:5 is shown in the figure;

the screening gene expression cassette comprises a delta first half-URA 3 expression cassette; the nucleotide sequence of the delta first half-URA 3 expression cassette is shown in SEQ ID NO: 1.

2. A recombinant vector comprising the expression cassette combination of claim 1.

3. A recombinant saccharomyces cerevisiae having the genome of saccharomyces cerevisiae as a vector, into which the expression cassette combination of claim 1 is inserted.

4. The recombinant s.cerevisiae according to claim 3, wherein the 5' end of the ERG9 gene of the recombinant s.cerevisiae is further inserted with P _HXT1 Promoters or P _ERG1 A promoter; the P is _HXT1 The nucleotide sequence of the promoter is shown in SEQ ID NO: shown as 9; the P is _ERG1 The nucleotide sequence of the promoter is shown in SEQ ID NO: shown at 10.

5. The recombinant s.cerevisiae according to claim 3, wherein the genome of the recombinant s.cerevisiae is further inserted withCYP736A167And a fusion gene of CPR gene;

the saidCYP736A167The nucleotide sequence of (2) is shown as SEQ ID NO:6 is shown in the figure;

the CPR gene isSa-CPR2Or 46tATR1; the saidSa-CPR2Such as the nucleotide sequence of SEQ ID NO: shown in figure 7; the nucleotide sequence of the 46tATR1 is shown as SEQ ID NO: shown at 8.

6. Use of the expression cassette combination of claim 1, the recombinant vector of claim 2, the recombinant saccharomyces cerevisiae of claim 3 or 4 for the preparation of santalene.

7. The use of the recombinant s.cerevisiae according to claim 5 for the preparation of santalol.

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