CN112300952B - Yarrowia lipolytica genetically engineered bacterium for producing alpha-pinene and application thereof - Google Patents

Yarrowia lipolytica genetically engineered bacterium for producing alpha-pinene and application thereof Download PDF

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CN112300952B
CN112300952B CN202011361363.XA CN202011361363A CN112300952B CN 112300952 B CN112300952 B CN 112300952B CN 202011361363 A CN202011361363 A CN 202011361363A CN 112300952 B CN112300952 B CN 112300952B
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pinene
yarrowia lipolytica
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韦柳静
钟驭涛
花强
聂明月
陈骏
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East China University of Science and Technology
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Abstract

The invention provides a yarrowia lipolytica genetically engineered bacterium for producing alpha-pinene, which utilizes a CRISPR/Cas9 system to make genes HMG1, NDPS1 and P TEF The tPS is sequentially introduced into a chromosome of uracil and leucine auxotroph yarrowia lipolytica to construct and obtain genetic engineering bacteria YT-14 capable of producing alpha-pinene; then constructing the gene P by using the plasmid pUC19-rDNA-HisG TEF The recombinant vector of the tPS and the gene ERG8 is transformed into the genetically engineered bacterium YT-14 to obtain the genetically engineered bacterium YT-29; and then constructing a gene ERG12 containing a gene HMG1 and an MBP tag by utilizing a plasmid pINA1269, and converting into yarrowia lipolytica genetic engineering bacterium YT-29. The yarrowia lipolytica genetically engineered bacterium YT-30 obtained by the invention can produce alpha-pinene.

Description

Yarrowia lipolytica genetically engineered bacterium for producing alpha-pinene and application thereof
Technical Field
The invention belongs to the field of genetic engineering, and particularly relates to yarrowia lipolytica genetic engineering bacteria for producing alpha-pinene and application thereof.
Background
Xylose is the second highest monosaccharide content next to glucose in lignocellulosic hydrolysate, accounting for approximately 35% of all monosaccharides. However, most microorganisms cannot effectively utilize xylose due to the inhibitory effect of carbon catabolites, which largely limits the use of lignocellulose. Because the lignocellulose is obtained with the characteristics of low cost and convenience, the development of the green biomass conversion mode has great application prospect. Yarrowia lipolytica is unable to metabolize xylose naturally, mainly due to the low expression levels of key enzymes involved in the xylose metabolic pathway. Therefore, constructing a high-efficiency cell factory to utilize xylose for synthesizing target products is a feasible path.
In addition, the swill-cooked dirty oil is waste animal grease which is recovered, waste grease of kitchen and restaurant, waste grease which is separated by a sewer oil-water separator or waste grease which is produced in other food industrial production and can not be eaten, contains various harmful components, has strong carcinogenicity and brings great harm to society. But can be used as an effective carbon source for microorganism culture because of low cost and high carbon content. In recent years, there have also been many studies on the production of high value-added compounds using oleaginous substrates (e.g., soybean oil, sunflower seed oil and rapeseed oil) as a carbon source with yarrowia lipolytica as a chassis cell. This also provides a benign idea for bioconversion of swill-cooked dirty oil.
Alpha-pinene is a naturally active monoterpene compound and is widely applied to the industries of perfume, medicines and fine chemistry. Because of the compact structure and high reactive olefin functionality of alpha-pinene, it can be used as a feedstock for the production of high density renewable fuels (e.g., jet fuels). At present, the production method of the alpha-pinene mainly comprises a natural extraction method and a microbial fermentation method. But it is considered that the natural extraction method generates high energy consumption and a large amount of chemical wastes. Thus, there is an urgent need to develop a novel, green and cost-effective biotechnology process for producing α -pinene.
However, research on synthesizing alpha-pinene by using xylose and swill-cooked dirty oil by taking yarrowia lipolytica cells as a chassis has not been reported yet.
Disclosure of Invention
The invention constructs a synthetic path of utilizing xylose to alpha-pinene by yarrowia lipolytica, and the constructed genetic engineering strain contains xylose reductase gene XR and xylitol dehydrogenase gene XDH from Scheffersomyces stipites and xylose assimilating enzyme gene XK from Yarrowia lipolytica. Preferably, the recombinant strain contains a pinene synthase gene PS from Pinus taeda and an neroli diphosphate synthase 1 gene NDPS1 from Solanum lycopersicum. Preferably, the recombinant strain overexpresses HMG1, a gene for reducing HMG-CoA to mevalonate, ERG8, a gene for phosphorylating mevalonate-5-phosphate to mevalonate-5-diphosphate, and ERG12 and AMPD, a gene for phosphorylating mevalonate to mevalonate-5-phosphate, respectively. Preferably, the recombinant strain also carries out fusion expression on MBP tag and gene ERG12. The invention constructs the genetically engineered bacteria of yarrowia lipolytica, which can lay a foundation for further synthesizing alpha-pinene by utilizing renewable biomass resources.
Therefore, the first object of the invention is to overcome the defects of the prior art and provide a yarrowia lipolytica genetically engineered bacterium for producing alpha-pinene.
The second object of the invention is to provide a construction method of yarrowia lipolytica genetically engineered bacteria producing alpha-pinene.
The third object of the invention is to provide an application of yarrowia lipolytica genetically engineered bacterium for producing alpha-pinene.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
as a first aspect of the present invention, an alpha-pinene producing yarrowia lipolytica genetically engineered bacterium is prepared by using CRISPR/Cas9 system to convert 3-hydroxy-3-methylglutaryl-CoA reductase gene HMG1, optimized and truncated neroli diphosphate synthase 1 gene NDPS1 and optimized and truncated pinene synthase gene P with TEF promoter TEF The tPS is sequentially introduced into a chromosome of uracil and leucine auxotroph yarrowia lipolytica to construct and obtain a yarrowia lipolytica genetic engineering bacterium YT-14 capable of producing alpha-pinene; then utilizing yarrowia lipolytica rDNA locus multicopy integrative plasmid pUC19-rDNA-HisG to construct an optimized and truncated pinene synthase gene P containing a TEF promoter TEF -a recombinant vector of tPS and a mevalonate kinase gene ERG8, and converting the recombinant vector into yarrowia lipolytica genetic engineering bacterium YT-14 to obtain yarrowia lipolytica genetic engineering bacterium YT-29; then utilizes yarrowia lipolytica integrative plasmid pINA1269 to construct a gene ERG12 containing 3-hydroxy-3-methylglutaryl-CoA reductase gene and mevalonate kinase gene with MBP label, and converts the gene engineering bacteria YT-29 of yarrowia lipolytica to obtain the gene engineering bacteria YT-30 of yarrowia lipolytica, wherein,
the optimized and truncated pinene synthase gene P with TEF promoter TEF The nucleotide sequence of the tPS is shown as SEQ ID No. 1; the nucleotide sequence of the MBP tag is shown as SEQ ID No.2Showing; the nucleotide sequence of the plasmid pUC19-rDNA-HisG is shown as SEQ ID No. 5; the nucleotide sequence of the optimized and truncated neroli diphosphate synthase 1 gene NDPS1 is described in patent application No. 201610817882.X, plasmid pINA1269 is obtained by the preparation method described in Madzak C, tre ton B and Roland SB. Strong hybrid promoters and integrative expression/secretion vectors for quasi-constitutive expression of heterologous proteins in the yeast Yarrowia lipolytica J Mol Microbiol Biotechnol. (2000) 2 (2): 207-216.
According to the invention, the uracil and leucine auxotrophs yarrowia lipolytica is yarrowia lipolytica Po1f.
As a second aspect of the present invention, an α -pinene producing yarrowia lipolytica genetically engineered bacterium as described above, which is capable of producing α -pinene using glucose, is used in the production of α -pinene.
Further, the application of the yarrowia lipolytica genetically engineered bacterium for producing the alpha-pinene in producing the alpha-pinene by utilizing glucose is provided.
As a third aspect of the invention, a construction method of yarrowia lipolytica genetically engineered bacterium producing alpha-pinene comprises the following steps:
step one, introducing a 3-hydroxy-3-methylglutaryl-coenzyme A reductase gene HMG1 into an A08 site of a uracil and leucine auxotroph yarrowia lipolytica chromosome by using a CRISPR/Cas9 system to obtain recombinant bacteria 1; then introducing optimized and truncated neroli diphosphate synthase 1 gene NDPS1 into XPR2 locus of recombinant bacterium 1 to obtain recombinant bacterium 2; introducing optimized and truncated pinene synthase gene P with TEF promoter into D17 site of recombinant bacterium 2 TEF -tPS to obtain yarrowia lipolytica genetic engineering bacterium YT-14 for producing alpha-pinene; the optimized and truncated pinene synthase gene P with TEF promoter TEF The nucleotide sequence of the tPS is shown as SEQ ID No. 1; the nucleotide sequence of the optimized and truncated neroli diphosphate synthase 1 gene NDPS1 is disclosed in the patent application document of patent application No. 201610817882. X;
step two, utilizing the lipolytic yardsMulti-copy integrated plasmid pUC19-rDNA-HisG of rDNA site of Saccharomyces cerevisiae, construction of optimized and truncated pinene synthase Gene P with TEF promoter TEF Recombinant vector of tPS and mevalonate kinase gene ERG8, and transformed yarrowia lipolytica genetic engineering bacterium YT-14 for producing alpha-pinene to obtain yarrowia lipolytica genetic engineering bacterium YT-29, optimized and truncated pinene synthase gene P with TEF promoter TEF The nucleotide sequence of the tPS is shown as SEQ ID No.1, and the nucleotide sequence of the plasmid pUC19-rDNA-HisG is shown as SEQ ID No. 5;
thirdly, yarrowia lipolytica integrated plasmid pINA1269-HMG1-MBP-ERG12 containing a 3-hydroxy-3-methylglutaryl-CoA reductase gene HMG1 and a mevalonate kinase gene ERG12 with an MBP label is transformed into yarrowia lipolytica genetic engineering bacterium YT-29 to obtain a yarrowia lipolytica genetic engineering bacterium YT-30, wherein the nucleotide sequence of the plasmid pINA1269-HMG1-MBP-ERG12 is shown as SEQ ID No. 3.
As a third aspect of the present invention, an alpha-pinene producing yarrowia lipolytica genetically engineered bacterium is constructed using yarrowia lipolytica integrative plasmid pINA1312 to contain one copy of mevalonate kinase ERG12, one copy of AMP deaminase gene AMPD and one copy of optimized and truncated pinene synthase gene P with TEF promoter TEF And (3) a tPS recombinant vector, and converting the yarrowia lipolytica genetic engineering bacterium YT-30 to obtain the yarrowia lipolytica genetic engineering bacterium YT-31 for producing alpha-pinene by taking carbon sources such as glucose as a substrate, wherein a plasmid pINA1312 is prepared by a preparation method described by Nicaud, J.M., madzak, C., van den Broek, P., gysler, C., duboc, P., niederbeger, P., gaillidine, C.2002.protein expression and secretion in the Yeast Yarrowia lipolytica.FEMS Yeast Res,2 (3), 371-379.
Furthermore, the yarrowia lipolytica genetic engineering bacterium YT-31 for producing alpha-pinene is prepared by transforming yarrowia lipolytica genetic engineering bacterium YT-29 containing 3-hydroxy-3-methylglutaryl-CoA reductase gene HMG1 and mevalonate kinase gene ERG12 with MBP label into yarrowia lipolytica integrated plasmid pINA1269-HMG1-MBP-ERG12 and based on the strain,transformation of the gene P containing optimized and truncated pinene synthase with TEF promoter TEF Yarrowia lipolytica integrated plasmid pINA1312-P for the tPS, mevalonate kinase gene ERG12 and AMP deaminase gene AMPD TEF -tPS-ERG12-AMPD to obtain yarrowia lipolytica genetic engineering bacterium YT-31 capable of producing alpha-pinene by taking glucose as a carbon source, wherein the nucleotide sequence of the plasmid pINA1269-HMG1-MBP-ERG12 is shown in SEQ ID No.3; the nucleotide sequence of the plasmid pINA1312-PTEF-tPS-ERG12-AMPD is shown in SEQ ID No.4.
As a fourth aspect of the present invention, an α -pinene producing yarrowia lipolytica genetically engineered bacterium as described above, which is capable of producing α -pinene using glucose and swill-cooked dirty oil, is used in the production of α -pinene.
Furthermore, the genetically engineered yarrowia lipolytica strain for producing alpha-pinene is applied to producing alpha-pinene by utilizing glucose and swill-cooked dirty oil.
As a fifth aspect of the present invention, a method for constructing genetically engineered yarrowia lipolytica producing α -pinene, comprising: step four, constructing an optimized and truncated pinene synthase gene P with a copy of mevalonate laser gene ERG12, a copy of AMP deaminase gene AMPD and a copy of TEF promoter by utilizing yarrowia lipolytica integrative plasmid pINA1312 TEF Recombinant vector of tPS, and converting the yarrowia lipolytica genetic engineering bacterium YT-30 to obtain the yarrowia lipolytica genetic engineering bacterium YT-31 which takes carbon sources such as glucose as substrates to produce alpha-pinene, and optimized and truncated pinene synthase gene P with TEF promoter TEF The nucleotide sequence of tPS is shown in SEQ ID No.1, and plasmid pINA1312 is obtained by the preparation method described in Nicaud, J.M., madzak, C.Van den Broek, P.Gysler, C.Duboc, P.Niederber, P.Gaillidin, C.2002.protein expression and secretion in the Yeast Yarrowia lipolytica. FEMS Yeast Res,2 (3), 371-379.
As a sixth aspect of the invention, a yarrowia lipolytica genetically engineered bacterium for producing alpha-pinene is constructed by integrating an optimized xylose reductase gene XR, an optimized xylitol dehydrogenase gene XDH and a xylose assimilating enzyme gene XK into rDNA multicopy sites of the yarrowia lipolytica genetically engineered bacterium YT-31 to obtain a yarrowia lipolytica genetically engineered bacterium YT-33 for producing alpha-pinene by metabolizing xylose, wherein the nucleotide sequence of the optimized xylose reductase gene XR is shown as SEQ ID No. 6; the nucleotide sequence of the optimized xylitol dehydrogenase gene XDH is shown as SEQ ID No. 7; the nucleotide sequence of the xylose assimilation enzyme gene XK is shown in SEQ ID No. 8.
As seven aspects of the invention, the genetically engineered yarrowia lipolytica bacterium for producing the alpha-pinene can produce the alpha-pinene by utilizing xylose and glucose.
Further, the application of the yarrowia lipolytica genetically engineered bacterium for producing the alpha-pinene in producing the alpha-pinene by utilizing xylose and glucose is provided.
As an eighth aspect of the present invention, an α -pinene producing yarrowia lipolytica genetically engineered bacterium as described above is used in xylose metabolism.
As a ninth aspect of the present invention, a method for constructing genetically engineered yarrowia lipolytica producing α -pinene, comprising: fifthly, integrating an optimized xylose reductase gene XR, an optimized xylitol dehydrogenase gene XDH and a xylose assimilating enzyme gene XK into rDNA multicopy sites of the yarrowia lipolytica genetic engineering bacterium YT-31 to construct a yarrowia lipolytica genetic engineering bacterium YT-33 for producing alpha-pinene by metabolizing xylose, wherein the nucleotide sequence of the optimized xylose reductase gene XR is shown as SEQ ID No. 6; the nucleotide sequence of the optimized xylitol dehydrogenase gene XDH is shown as SEQ ID No. 7; the nucleotide sequence of the xylose assimilation enzyme gene XK is shown in SEQ ID No. 8.
As a tenth aspect of the present invention, a method for producing α -pinene by fermentation of α -pinene-producing yarrowia lipolytica genetically engineered bacteria as described above, comprising the steps of: culturing the yarrowia lipolytica genetically engineered bacterium for producing alpha-pinene, adding dodecane, performing two-phase fermentation to obtain fermentation liquor, and extracting the dodecane phase of the fermentation liquor.
Further, the carbon source for culturing the alpha-pinene-producing yarrowia lipolytica genetically engineered bacterium is glucose or xylose or swill-cooked dirty oil, the concentration of the glucose is 10g/L-30g/L, and the concentration is the ratio of the mass of the glucose to the volume of the culture system before adding the glucose; the concentration of the xylose is 10g/L-30g/L, and the concentration is the ratio of the mass of the xylose to the volume of the system cultured before the xylose is added; the concentration of the swill-cooked dirty oil is 2-10%, preferably 2-5%, and the percentage is the volume percentage of the swill-cooked dirty oil and the fermentation liquid before the swill-cooked dirty oil is added; and/or the concentration of the dodecane is 2-10%, preferably 6-8%, and the percentage is the volume percentage of the dodecane and the fermentation broth before the dodecane is added.
As an eleventh aspect of the present invention, a knock-in plasmid pair of genetically engineered yarrowia lipolytica for gene knock-in, which is a CRISPR/Cas9 system-based plasmid pair for gene knock-in yarrowia lipolytica, which is phr_d17_xr and pcrisryl_d17; pHR_XPR2_XDH and pCRISPRyl_XPR2; pHR_A08_ylXK and pCRISPRyl_A08, the plasmid pHR_D17_P TEF The nucleotide sequences of tPS, pHR_XPR2_NDPS1 and pHR_A08_HMG1 are shown in SEQ ID No.9, SEQ ID No.10 and SEQ ID No. 11; the plasmids pcrispryl_d17, pcrispryl_xpr2 and pcrispryl_a08 are described in Schwartz, c., shabbir-humin, m., froue, k., blenner, M.&Wheeldon, I.Standard Markerless Gene Integration for Pathway Engineering in Yarrowia lipolytica. Acs Synth Biol 6,402-409, doi:10.1021/acslynbio.6b00285 (2017).
The construction method of yarrowia lipolytica genetically engineered bacteria for producing alpha-pinene has the beneficial effects that:
(1) The invention firstly constructs a heterologous synthesis way of alpha-pinene in yarrowia lipolytica, so that engineering strains can utilize carbon sources such as glucose and the like to carry out biosynthesis of high-added-value chemicals, and the process can reduce the production cost.
(2) The invention overexpresses genes HMG1, ERG8, ERG12 and AMPD, so that carbon flux flows into the synthesis path of alpha-pinene as much as possible, waste of substrates is reduced, and the yield of alpha-pinene can be greatly improved.
(3) The invention combines the fusion expression of MBP tag and ERG12 gene, improves the soluble expression of protein, and can greatly improve the yield of alpha-pinene.
(4) The invention expresses xylose metabolism path genes including xylose reductase gene XR, xylitol dehydrogenase gene XDH and wood assimilation enzyme gene XK, so that yarrowia lipolytica engineering bacteria can metabolize xylose to produce alpha-pinene, and provides a certain insight for bioconversion of agricultural waste lignocellulose hydrolysate.
(5) The yarrowia lipolytica genetically engineered bacterium for producing the alpha-pinene can also take swill-cooked dirty oil as a carbon source to produce the alpha-pinene with high yield, and a new solution is provided for bioconversion of the swill-cooked dirty oil.
The yarrowia lipolytica genetically engineered bacterium for producing alpha-pinene has the beneficial effects that: the engineering bacterium can produce alpha-pinene by taking xylose and swill-cooked dirty oil as raw materials, and can provide a new and extremely competitive sustainable development way for synthesizing terpene compounds by taking non-grain crops as raw materials.
Drawings
FIG. 1 is a metabolic pathway of heterologous biosynthesis of alpha-pinene by yarrowia lipolytica.
FIG. 2 is a graph showing the results of shake flask fermentation of strains YT-14, YT-29, YT-30, YT-31 using glucose as the sole carbon source to produce alpha-pinene.
FIG. 3 is a graph showing the results of producing alpha-pinene by the strain YT-31 using different oily substrates as the sole carbon source.
FIG. 4 is a graph showing the results of shake flask fermentation of strain YT-31 using glucose or xylose as the sole carbon source to produce α -pinene.
Detailed Description
The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications. For example, CRISPR/Cas9 systems in embodiments of the invention employ the preparation methods described in Schwartz, C., shbir-Hussain, M., frogue, K., blnner, M. & Wheeldon, I.Standard Markerless Gene Integration for Pathway Engineering in Yarrowia lipotics. Acs Synth Biol 6,402-409, doi:10.1021/acsynbio.6b00285 (2017). The method for measuring the alpha-pinene in the embodiment of the invention comprises the following steps: detection using gas chromatography-mass spectrometry (GC-MS). The inlet temperature is 280 ℃, the interface temperature is 250 ℃, and the temperature of the four-stage rod is 200 ℃. The analytical capillary column model was HP-5 (30 m. Times.0.25 cm,0.25 μm film thickness). The separation carrier gas was helium at a flow rate of 1ml/min. The mass spectrometer is set in a full scanning mode, the solvent is delayed for 3min, the initial temperature is 100 ℃ and is waited for 1min, then the temperature is raised to 102 ℃ at the speed of 0.5 ℃/min, the operation is carried out for 5min after the final temperature is 280 ℃, the sample injection amount is 1 μl, and the split ratio is 20:1.
1. The strain and plasmid sources involved in the invention are as follows:
1. yarrowia lipolytica (Yarrowia lipolytica) starting strain Po1f, available from American Type Culture Collection (ATCC) under accession number
Figure BDA0002804058880000051
MYA-2613 TM 。
It should be noted that, although the strain Po1f was used as the starting strain in the examples of the present invention, conventional uracil and leucine auxotroph yarrowia lipolytica (Yarrowia lipolytica) were used as the starting strain for transformation to perform the experiments in the examples of the present invention.
2. Plasmid pINA1312: obtained by the preparation method described in Nicaud, j.m., madzak, c., van den Broek, p., gysler, c., duboc, p., niederbeger, p., gaillardin, c.2002.protein expression and secretion in the Yeast Yarrowia lipolytica.fems Yeast Res,2 (3), 371-379.
3. Plasmid pINA1269: obtained by the preparation method described in Madzak C, tre ton B and Roland SB. Strong hybrid promoters and integrative expression/secretion vectors for quasi-constitutive expression of heterologous proteins in the yeast Yarrowia lipolytica. J Mol Microbiol Biotechnol. (2000) 2 (2): 207-216.
4. Plasmids pHR_D17_hrGFP, pHR_XPR2_hrGFP and pHR_A08_hrGFP were prepared as described in Schwartz, C.Shbir-Hussain, M.Frogue, K.Blhener, M.Wheeldon, I.Standard Markerless Gene Integration for Pathway Engineering in Yarrowia lipotics. Acsynth Biol 6,402-409, doi:10.1021/acssynbio.6b00285 (2017).
5. Construction of the obtained plasmid pHR_D17_P TEF The nucleotide sequences of the-tPS, the plasmid pHR_XPR2_NDPS1, the plasmid pHR_A08_HMG1 and the plasmid pINA1269-HMG1-MBP-ERG12 are shown as SEQ ID No.9, SEQ ID No.10, SEQ ID No.11 and SEQ ID No.3 respectively.
6. Plasmid pINA1312-P TEF The nucleotide sequence of the-tPS-ERG 12-AMPD is shown as SEQ ID No. 4; the nucleotide sequence of the plasmid pUC19-rDNA-HisG is shown in SEQ ID No. 5.
7. Plasmid pCRISPRyl_D17, plasmid pCRISPRyl_XPR2 and plasmid pCRISPRyl_A08 are prepared by the preparation method of Schwartz, C.Shbir-Hussain, M.Frogue, K.Blnner, M.Wheeldon, I.Standard Markerless Gene Integration for Pathway Engineering in Yarrowia Lipolytica. Acs Synth Biol 6,402-409, doi: 10.1021/acslynbio.6b00285 (2017).
2. The reagents and materials used in the present invention are commercially available. Wherein, the kit Frozen EZ Yeast Transformation II for transformation TM (available from Zymo Research) was performed according to the method described in the kit.
3. The genes related to the invention are as follows:
1. xylose reductase gene XR: the nucleotide sequence is shown as SEQ ID No. 6.
2. Xylitol dehydrogenase gene XDH: the nucleotide sequence is shown as SEQ ID No. 7.
3. Xylose assimilating enzyme gene XK: the nucleotide sequence is shown as SEQ ID No. 8.
4. The optimized and truncated pinene synthase gene PTEF-tPS with the TEF promoter has the nucleotide sequence shown in SEQ ID No. 1.
5. Optimized MBP tag gene: the nucleotide sequence is shown as SEQ ID No. 2.
6. The nucleotide sequence of the optimized and truncated neroli diphosphate synthase 1 gene NDPS1 is disclosed in the patent application publication No. 201610817882. X.
4. The invention relates to a culture medium and preparation
1. YPD medium: 10-15g/L yeast extract powder, 20-25g/L peptone, 20-25g/L glucose, and deionized water as solvent; preparing: dissolving the above components in deionized water, stirring for dissolving, and sterilizing;
YPX medium: 10-15g/L yeast extract powder, 20-25g/L peptone and 20-25g/L xylose; preparing: dissolving the above components in deionized water, stirring for dissolving, and sterilizing.
5. A schematic diagram of the heterologous biosynthesis pathway of yarrowia lipolytica alpha-pinene constructed in the present invention is shown in FIG. 1. Wherein, the English abbreviations of FIG. 1 are:
PS: alpha-pinene synthase;
NDPS1: neroli diphosphate synthase 1;
AMPD: AMP deaminase;
ERG10: acetyl-coa acetyltransferase;
ERG13: hydroxymethylglutaryl-coa synthase;
HMG1: 3-hydroxy-3-methylglutaryl-coa reductase;
ERG12: mevalonate kinase;
ERG8: phosphomevalonate kinase;
IDI1: isopentenyl diphosphate delta-isomerase;
ERG19: mevalonate diphosphate decarboxylase.
HMG-CoA: 3-hydroxy-3-methylglutaryl-CoA
MVA: mevalonic acid
MVA-5-P: mevalonic acid-5-phosphoric acid
MVAPP: mevalonic acid 5-diphosphate
IPP: isopentenyl pyrophosphate
DMAPP: dimethallyl diphosphate
NPP: neroli diphosphate
Example 1: construction of heterologous synthetic pathway of alpha-pinene in yarrowia lipolytica
(1) Since yarrowia lipolytica is not capable of synthesizing alpha-pinene by itself, in order to be able to synthesize alpha-pinene, this example introduced an optimized and truncated pinene synthase gene P derived from Pinus taeda with a TEF promoter TEF -tPS and optimized and truncated neryl diphosphate synthase 1 gene NDPS1 from Solanum lycopersicum and overexpresses the 3-hydroxy-3-methylglutaryl coa reductase gene HMG1 from Yarrowia lipolytica.
(2) Optimized and truncated pinene synthase gene P derived from Pinus taeda with TEF promoter TEF Construction of the-tPS by means of the double cleavage sites SpeI and AvRII on plasmid pHR_D17_hrGFP, resulting in plasmid pHR_D17_P TEF -tPS. The optimized and truncated neroli diphosphate synthase 1 gene NDPS1 from Solanum lycopersicum was constructed to plasmid phr_xpr2_hrgfp by double cleavage sites PteI and NheI, resulting in plasmid phr_xpr2_ndps1. The 3-hydroxy-3-methylglutaryl-CoA reductase gene HMG1 derived from Yarrowia lipolytica was constructed into the plasmid pHR_A08_hrGFP by double cleavage sites PteI and NheI, resulting in plasmid pHR_A08_HMG1.
(3) Plasmid pair pHR_D17_P using CRISPR/Cas9 system TEF -tPS (obtained in step (2)) and pcr ispinyl_d17 were transformed into yarrowia lipolytica Po1f to obtain strain 1. It was verified that the D17 site of yarrowia lipolytica Po1f was introduced with the TEF promoter derived from Pinus taeda and truncated pinene synthase gene P TEF -tPS。
The plasmid pairs pHR_XPR2_NDPS1 (obtained in step (2)) and pCRISPRyl_XPR2 were transformed into yarrowia lipolytica strain 1 using the CRISPR/Cas9 system to obtain strain 2. It was verified that the XPR2 site of strain 1 was introduced with the optimized and truncated neroli diphosphate synthase 1 gene NDPS1 from Solanum lycopersicum.
And then converting the plasmid pair pHR_A08_HMG1 (obtained in the step (2)) and pCRISPRyl_A08 into the strain 2 by using the CRISPR/Cas9 system to obtain the yarrowia lipolytica strain YT-14 producing alpha-pinene. It was confirmed that the A08 site of the strain YT-14 was introduced with 3-hydroxy-3-methylglutaryl-CoA reductase gene HMG1 derived from Yarrowia lipolytica. The obtained yarrowia lipolytica strain YT-14 is preserved with 20% (V/V) glycerol to obtain a glycerol-preserved bacterial liquid of the recombinant strain YT-14.
(4) The recombinant strain YT-14 obtained is subjected to shake flask fermentation, and the specific shake flask fermentation method is as follows: 50mL of YPD medium was loaded in 250 mL. Taking 100-200 mu L of glycerol-preserving bacterial liquid of recombinant strain YT-14, inoculating into a test tube containing 2mL of YPD culture medium, culturing at 28-30deg.C for 16-20 hr, inoculating into YPD culture medium, adding 1-5mL dodecane for two-phase fermentation, and initiating OD 600 Culturing at 28-30deg.C for 72 hr at 0.01. After the fermentation, the yield (Titer), the weight of stem cells (DCW) and the yield (Content) of alpha-pinene of the strain YT-14 subjected to shake flask fermentation in YPD medium were respectively determined according to a standard alpha-pinene determination method, and the results are shown in FIG. 2.
As can be seen from FIG. 2, the strain YT-14 can synthesize 0.805mg/L of alpha-pinene by using glucose as a carbon source.
Example 2: metabolic engineering strategy for modifying yarrowia lipolytica to produce alpha-pinene in high yield
(1) Optimized and truncated pinene synthase gene P derived from Pinus taeda with TEF promoter TEF the-tPS is constructed on the plasmid pUC19-rDNA-HisG through the enzyme cutting site EcoRI to obtain the plasmid pUC19-rDNA-HisG-P TEF -tPS, the phosphomevalonate kinase gene ERG8 from Yarrowia lipolytica was constructed on plasmid pINA1312 by cleavage site PmlI to give plasmid pINA1312-ERG8.
(2) Ligating the expression cassette of ERG8 gene in the plasmid pINA1312-ERG8 obtained in step (1) to the plasmid pUC19-rDNA-HisG-P obtained in step (1) through the cleavage site EcoRI TEF Obtaining the gene P on tPS TEF Plasmid pUC19-rDNA-HisG-P of-tPS and ERG8 TEF -tPS-ERG8。
(3) The plasmid pUC19-rDNA-HisG-P obtained in step (2) is subjected to a transformation procedure TEF Linearization of-tPS-ERG 8 transformation into the strain YT-14 obtained in example 1 by homologous recombinationObtaining yarrowia lipolytica genetic engineering bacterium YT-29.
(4) The 3-hydroxy-3-methylglutaryl-CoA reductase gene HMG1 derived from Yarrowia lipolytica and the mevalonate kinase gene ERG12 derived from Yarrowia lipolytica, which have MBP tags, were constructed to the plasmid pINA1269 through the cleavage site PmlI, respectively, to give plasmids pINA1269-HMG1 and pINA1269-MBP-ERG12.
(5) The expression cassette of HMG1 gene in the plasmid pINA1269-HMG1 obtained in the step (4) is connected to the plasmid pINA1269-MBP-ERG12 obtained in the step (4) through the cleavage site SpeI to obtain the plasmid pINA1269-HMG1-MBP-ERG12 containing the genes HMG1 and MBP-ERG12.
(6) And (3) linearizing the plasmid pINA1269-HMG1-MBP-ERG12, and then transforming the linearized plasmid into the strain YT-29 obtained in the step (3) by using a homologous recombination method to obtain the strain YT-30.
(7) Optimized and truncated pinene synthase gene P derived from Pinus taeda with TEF promoter TEF the-tPS, the mevalonate kinase gene ERG12 from Yarrowia lipolytica and the AMP deaminase gene AMPD from Yarrowia lipolytica are constructed to a plasmid pINA1312 through a cleavage site PmlI to obtain a plasmid pINA1312-P TEF -tPS, pINA1312-ERG12 and pINA1312-AMPD.
(8) Ligating the expression cassette of ERG12 gene in the plasmid pINA1312-ERG12 obtained in the step (7) to the plasmid pINA1312-P obtained in the step (7) through the cleavage site StuI TEF Obtaining the gene P on tPS TEF Plasmid pINA1312-P of tPS and ERG12 TEF -tPS-ERG12。
(9) Then the expression cassette of the AMPD gene in the plasmid pINA1312-AMPD obtained in the step (7) is connected to the plasmid pINA1312-P obtained in the step (8) through an enzyme cleavage site ClaI TEF Obtaining the gene P on-tPS-ERG 12 TEF Plasmids pINA1312-P for tPS, ERG12 and AMPD TEF -tPS-ERG12-AMPD。
(10) The plasmid pINA1312-P obtained in step (9) is subjected to TEF Linearization of tPS-ERG12-AMPD the strain YT-30 obtained in step (6) was transformed by homologous recombination to obtain the yarrowia lipolytica engineering strain YT-31 capable of producing alpha-pinene. The yarrowia lipolytica strain YT-31 thus obtained was usedAnd (3) preserving 20% (V/V) of glycerol to obtain a glycerol-preserved bacterial liquid of the recombinant strain YT-31.
(11) The recombinant strains YT-29, YT-30 and YT-31 thus obtained were subjected to shake flask fermentation, and the specific shake flask fermentation method was as described in example 1. After the fermentation, the yield (Titer), the weight of stem cells (DCW) and the yield (Content) of alpha-pinene of the strain YT-31 subjected to shake flask fermentation in YPD medium were respectively determined according to a standard alpha-pinene determination method, and the results are shown in FIG. 2.
As can be seen from FIG. 2, strains YT-29, YT-30 and YT-31 can produce 2.765mg/L, 9.245mg/L and 19.615mg/L of alpha-pinene, respectively, using glucose as a carbon source.
Example 3: production of alpha-pinene by using swill-cooked dirty oil
The swill-cooked dirty oil of this embodiment is at least one of recovered waste animal fat, waste fat in kitchen and restaurant, waste fat separated by a sewer oil-water separator, or waste fat which is produced in other food industry and cannot be eaten.
To evaluate the feasibility of using swill-cooked dirty oil for production of alpha-pinene by yarrowia lipolytica YT-31, the present example was run on the market with swill-cooked dirty oil, sunflower seed oil, rapeseed oil and soybean oil.
The strain YT-31 is subjected to shake flask fermentation, and the specific shake flask fermentation method comprises the following steps: 50mL YPF culture medium (10-15 g/L yeast extract, 20-25g/L peptone, 2-5% swill-cooked dirty oil; preparation, dissolving each component in deionized water, stirring to dissolve, and sterilizing). The cultivation method was as described in example 1. After the fermentation, the yield (Titer), the weight of stem cells (DCW) and the yield (Content) of alpha-pinene of the strain YT-31 subjected to shake flask fermentation in a YPW culture medium are respectively measured according to a standard alpha-pinene measuring method. The results are shown in FIG. 3.
As can be seen from FIG. 3, the strain YT-31 can produce 33.835mg/L, 33.215mg/L, 28.875mg/L and 36.56mg/L of alpha-pinene, respectively, using swill-cooked dirty oil, sunflower seed oil, rapeseed oil and soybean oil as carbon sources.
Conclusion: the swill-cooked dirty oil can be used as a proper substitute carbon source for producing alpha-pinene by yarrowia lipolytica, and the yield of the alpha-pinene is extremely high, thereby providing an important reference for further reducing the production cost.
Example 4: construction of xylose metabolism pathway in yarrowia lipolytica
(1) Since yarrowia lipolytica itself is only able to metabolize a small amount of xylose, in order to be able to synthesize α -pinene using xylose, this example introduced the optimized xylose reductase gene XR and xylitol dehydrogenase gene XDH derived from Scheffersomyces stipites, and overexpressed the xylose assimilating enzyme gene XK derived from Yarrowia lipolytica.
(2) The expression cassettes of the optimized xylose reductase gene XR and xylitol dehydrogenase gene XDH from Scheffersomyces stipites and xylose assimilating enzyme gene XK from Yarrowia lipolytica were constructed to the plasmid pUC19-rDNA-HisG through the cleavage site EcoRI in sequence, to obtain the plasmid pUC19-rDNA-HisG-XR-XDH-XK.
(3) The plasmid pUC19-rDNA-HisG-XR-XDH-XK obtained in the step (2) was transformed into the strain YT-31 obtained in the example 2 by a method of homologous recombination to obtain a yarrowia lipolytica engineering strain YT-33 capable of metabolizing xylose to produce alpha-pinene. The obtained yarrowia lipolytica strain YT-33 is preserved with 20% (V/V) glycerol to obtain a glycerol-preserved bacterial liquid of the recombinant strain YT-33.
(4) The recombinant strain YT-33 obtained is subjected to shake flask fermentation, and the specific shake flask fermentation method is as follows: 50mL of YPX medium was loaded in 250 mL. Taking 100-200 mu L of glycerol-preserving bacterial liquid of recombinant strain YT-33, inoculating into a test tube containing 2mL of YPD culture medium, culturing at 28-30deg.C for 16-20 hr, inoculating into YPD culture medium, adding 1-5mL dodecane for two-phase fermentation, and initiating OD 600 Culturing at 28-30deg.C for 72 hr at 0.01. The yield of alpha-pinene (Titer), the weight of stem cells (DCW) and the yield of alpha-pinene (Content) of the strain YT-33 subjected to shake flask fermentation in YPD medium were determined by standard alpha-pinene determination methods, respectively, and the results are shown in FIG. 4.
As can be seen from FIG. 4, the strain YT-31 produces 19.52mg/L of alpha-pinene by using glucose as a carbon source; the strain YT-31 does not obtain alpha-pinene by using xylose as a carbon source.
The strain YT-33 can respectively produce 15.44mg/L and 2.71mg/L of alpha-pinene by using glucose and xylose as carbon sources.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that the above-mentioned preferred embodiment should not be construed as limiting the invention, and the scope of the invention should be defined by the appended claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and such modifications and adaptations are intended to be comprehended within the scope of the invention.
Sequence listing
<110> university of Industy of Huadong
<120> genetically engineered yarrowia lipolytica strain producing alpha-pinene and application thereof
<130> 201139
<141> 2020-11-27
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2694
<212> DNA
<213> Artificial sequence (Artificial Sequence)
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agagaccggg ttggcggcgc atttgtgtcc caaaaaacag ccccaattgc cccaattgac 60
cccaaattga cccagtagcg ggcccaaccc cggcgagagc ccccttctcc ccacatatca 120
aacctccccc ggttcccaca cttgccgtta agggcgtagg gtactgcagt ctggaatcta 180
cgcttgttca gactttgtac tagtttcttt gtctggccat ccgggtaacc catgccggac 240
gcaaaataga ctactgaaaa tttttttgct ttgtggttgg gactttagcc aagggtataa 300
aagaccaccg tccccgaatt acctttcctc ttcttttctc tctctccttg tcaactcaca 360
cccgaaatcg ttaagcattt ccttctgagt ataagaatca ttcaaacacg tgatgctgcg 420
acgaactctg ttcggattct ctcacgaact caaggctatc cactccactg tgcccaacct 480
gggcatgtgc cgaggcggaa agtctattgc tccttctatg tctatgtcct ctactacttc 540
tgtgtccaac gaggacggcg tgccccgacg aatcgccggt caccactcta acctttggga 600
cgacgactct atcgcttctc tgtctacttc ctacgaggcc ccttcttacc gaaagcgagc 660
tgacaagctc atcggtgagg ttaagaacat tttcgacctt atgtccgttg aggacggcgt 720
tttcacttcc cctctgtctg acctgcacca ccgactgtgg atggttgact ccgttgagcg 780
actgggtatt gaccgacact tcaaggacga gatcaactcc gccctggacc atgtatactc 840
ctactggact gagaagggta ttggtcgagg tcgagagtcc ggcgtcactg accttaactc 900
taccgctctg ggtctgcgaa ccctgcgact gcacggatac actgtgtctt ctcacgttct 960
tgaccacttc aagaacgaga agggtcagtt cacttgctcc gccatccaga ctgagggtga 1020
gattcgagac gtgcttaacc tgttccgagc ctctcttatc gctttccccg gtgagaagat 1080
catggaggcc gctgagattt tctctactat gtacctaaaa gacgctttgc aaaagatccc 1140
cccttccggt ctgtcccaag aaatcgagta cctgctggag ttcggttggc acactaacct 1200
gcctcggatg gagacccgaa tgtacattga cgtgttcggt gaggacacca ctttcgagac 1260
tccttacctt attcgagaga agctgctgga actggccaag ctggagttca acattttcca 1320
ctctcttgtt aagcgagagc tacaatctct gtcccgatgg tggaaggact acggattccc 1380
tgagattact ttctcccgac accgacacgt tgagtactac accctggccg cttgcatcgc 1440
taacgaccct aagcactccg ctttccgact tggtttcgga aagatttccc acatgattac 1500
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cgctttcaag cgatgggacc cctcttccat tgagtgcctg cctgactaca tgaagggcgt 1620
ttacatggcc gtgtacgaca acatcaacga gatggcccgc gaggcacaga agattcaggg 1680
ttgggacacc gtgtcctacg ctcgaaagtc ttgggaggct ttcatcggcg cctacatcca 1740
agaggccaag tggatctcct ccggatacct gcctactttc gacgagtacc tggagaacgg 1800
aaaggtttcc ttcggatctc gaattaccac ccttgagcct atgcttaccc ttggtttccc 1860
cctgcctcct aggattctgc aagagattga cttcccttct aagttcaacg acctcatttg 1920
cgccattctg cgactgaagg gtgacaccca gtgctacaag gctgaccgag cccgaggtga 1980
agaggcttcc gctgtgtctt gctacatgaa ggaccacccc ggtattactg aggaagatgc 2040
cgttaaccag gtgaacgcta tggtggacaa cctcactaag gaactgaact gggagctgct 2100
gcgacctgac tccggcgtgc ccatctctta caagaaggtg gctttcgaca tttgccgagt 2160
gttccactac ggatacaagt accgagacgg attctccgtc gcctccattg agattaagaa 2220
cctcgtcacc cgaaccgtcg ttgagaccgt gcctctttag ggaacccgaa actaaggatc 2280
cggtacctcc atggcctgtc cccacgttgc cggtcttgcc tcctactacc tgtccatcaa 2340
tgacgaggtt ctcacccctg cccaggtcga ggctcttatt actgagtcca acaccggtgt 2400
tcttcccacc accaacctca agggctctcc caacgctgtt gcctacaacg gtgttggcat 2460
ttaggcaatt aacagatagt ttgccggtga taattctctt aacctcccac actcctttga 2520
cataacgatt tatgtaacga aactgaaatt tgaccagata ttgttgtaaa tagaaaatct 2580
ggcttgtagg tggcaaaatc ccgtctttgt tcatcaattc cctctgtgac tactcgtcat 2640
ccctttatgt tcgactgtcg tatttttatt ttccatacat acgcaagtga gatg 2694
<210> 2
<211> 1161
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
atgaagatcg aggaaggcaa gctcgtcatc tggattaacg gcgacaaggg ttacaacgga 60
ctggccgaag tgggcaagaa gttcgagaag gacaccggta ttaaggtcac cgtggagcac 120
cccgacaagc tggaggagaa gttcccccag gtggctgcta ccggtgacgg acccgacatc 180
attttctggg ctcacgaccg attcggcggt tacgctcagt ctggactgct cgctgagatc 240
acccccgaca aggctttcca ggacaagctc taccccttca cctgggacgc cgtccgatac 300
aacggcaagc tgatcgctta ccccattgct gtggaggccc tgtctctcat ctacaacaag 360
gacctgctcc ccaaccctcc caagacctgg gaggagattc ccgccctcga caaggagctg 420
aaggccaagg gcaagtccgc tctcatgttc aacctgcagg agccctactt cacctggccc 480
ctgattgctg ctgacggagg ctacgccttc aagtacgaga acggaaagta cgacattaag 540
gacgtgggtg tggacaacgc tggagctaag gctggcctga ccttcctcgt cgacctgatc 600
aagaacaagc acatgaacgc cgacaccgac tactctattg ctgaggccgc tttcaacaag 660
ggagagaccg ccatgaccat caacggaccc tgggcttggt ctaacattga cacctccaag 720
gtgaactacg gcgtcaccgt gctccccacc ttcaagggtc agccctccaa gcccttcgtc 780
ggtgtgctgt ctgccggaat caacgccgct tcccccaaca aggagctcgc taaggagttc 840
ctggagaact acctgctcac cgacgaggga ctcgaggccg tcaacaagga caagcccctc 900
ggcgctgtgg ccctgaagtc ctacgaggaa gagctggcca aggacccccg aatcgccgct 960
accatggaga acgctcagaa gggagagatc atgcccaaca ttccccagat gtctgctttc 1020
tggtacgccg tccgaaccgc tgtgattaac gccgcttccg gacgacagac cgtggacgag 1080
gccctcaagg acgctcagac caactcctct tccaataata acaataataa caacaacaac 1140
aacctgggca ttgagggtcg a 1161
<210> 3
<211> 13910
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
gaattccgtc gtcgcctgag tcatcattta tttaccagtt ggccacaaac ccttgacgat 60
ctcgtatgtc ccctccgaca tactcccggc cggctgggta cgttcgatag cgctatcggc 120
atcgacaagg tttgggtccc tagccgatac cgcactacct gagtcacaat cttcggaggt 180
ttagtcttcc acatagcacg ggcaaaagtg cgtatatata caagagcgtt tgccagccac 240
agattttcac tccacacacc acatcacaca tacaaccaca cacatccaca atggaacccg 300
aaactaagaa gaccaagact gactccaaga agattgttct tctcggcggc gacttctgtg 360
gccccgaggt gattgccgag gccgtcaagg tgctcaagtc tgttgctgag gcctccggca 420
ccgagtttgt gttcgaggac cgactcattg gaggagctgc cattgagaag gagggcgagc 480
ccatcaccga cgctactctc gacatctgcc gaaaggctga ctctattatg ctcggtgctg 540
tcggaggcgc tgccaacacc gtatggacca ctcccgacgg acgaaccgac gtgcgacccg 600
agcagggtct cctcaagctg cgaaaggacc tgaacctgta cgccaacctg cgaccctgcc 660
agctgctgtc gcccaagctc gccgatctct cccccatccg aaacgttgag ggcaccgact 720
tcatcattgt ccgagagctc gtcggaggta tctactttgg agagcgaaag gaggatgacg 780
gatctggcgt cgcttccgac accgagacct actccgttcc tgaggttgag cgaattgccc 840
gaatggccgc cttcctggcc cttcagcaca acccccctct tcccgtgtgg tctcttgaca 900
aggccaacgt gctggcctcc tctcgacttt ggcgaaagac tgtcactcga gtcctcaagg 960
acgaattccc ccagctcgag ctcaaccacc agctgatcga ctcggccgcc atgatcctca 1020
tcaagcagcc ctccaagatg aatggtatca tcatcaccac caacatgttt ggcgatatca 1080
tctccgacga ggcctccgtc atccccggtt ctctgggtct gctgccctcc gcctctctgg 1140
cttctctgcc cgacaccaac gaggcgttcg gtctgtacga gccctgtcac ggatctgccc 1200
ccgatctcgg caagcagaag gtcaacccca ttgccaccat tctgtctgcc gccatgatgc 1260
tcaagttctc tcttaacatg aagcccgccg gtgacgctgt tgaggctgcc gtcaaggagt 1320
ccgtcgaggc tggtatcact accgccgata tcggaggctc ttcctccacc tccgaggtcg 1380
gagacttgtt gccaacaagg tcaaggagct gctcaagaag gagtaagtcg tttctacgac 1440
gcattgatgg aaggagcaaa ctgacgcgcc tgcgggttgg tctaccggca gggtccgcta 1500
gtgtataaga ctctataaaa agggccctgc cctgctaatg aaatgatgat ttataattta 1560
ccggtgtagc aaccttgact agaagaagca gattgggtgt gtttgtagtg gaggacagtg 1620
gtacgttttg gaaacagtct tcttgaaagt gtcttgtcta cagtatattc actcataacc 1680
tcaatagcca agggtgtagt cggtttatta aaggaaggga gttgtggctg atgtggatag 1740
atatctttaa gctggcgact gcacccaacg agtgtggtgg tagcttgtta ctgtatattc 1800
ggtaagatat attttgtggg gttttagtgg tgtttggtag gttagtgctt ggtatatgag 1860
ttgtaggcat gacaatttgg aaaggggtgg actttgggaa tattgtggga tttcaatacc 1920
ttagtttgta cagggtaatt gttacaaatg atacaaagaa ctgtatttct tttcatttgt 1980
tttaattggt tgtatatcaa gtccgttaga cgagctcagt gccatggctt ttggcactgt 2040
atttcatttt tagaggtaca ctacattcag tgaggtatgg taaggttgag ggcataatga 2100
aggcaccttg tactgacagt cacagacctc tcaccgagaa ttttatgaga tatactcggg 2160
ttcattttag gctccgattc gattcaaatt attactgtcg aaatcggttg agcatccgtt 2220
gatttccgaa cagatctcgg cagtctctcg gatgtagaat taggtttcct tgaggcgaag 2280
atcggtttgt gtgacatgaa ttcttgaaga cgaaagggcc tcgtgatacg cctattttta 2340
taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat 2400
gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg 2460
agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa 2520
catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac 2580
ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac 2640
atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt 2700
ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tgttgacgcc 2760
gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca 2820
ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc 2880
ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag 2940
gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa 3000
ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg 3060
gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa 3120
ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg 3180
gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt 3240
gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt 3300
caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag 3360
cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat 3420
ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct 3480
taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct 3540
tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca 3600
gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc 3660
agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc 3720
aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct 3780
gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag 3840
gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc 3900
tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg 3960
agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag 4020
cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt 4080
gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac 4140
gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg 4200
ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc 4260
cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg 4320
cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatggtg cactctcagt 4380
acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact 4440
gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc 4500
tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga 4560
ggttttcacc gtcatcaccg aaacgcgcga ggcagcagat ccactagtga ggcagcagat 4620
ccactagtgg tgcatgctga ggtgtctcac aagtgccgtg cagtcccgcc cccacttgct 4680
tctctttgtg tgtagtgtac gtacattatc gagaccgttg ttcccgccca cctcgatccg 4740
gcatgctgag gtgtctcaca agtgccgtgc agtcccgccc ccacttgctt ctctttgtgt 4800
gtagtgtacg tacattatcg agaccgttgt tcccgcccac ctcgatccgg catgctgagg 4860
tgtctcacaa gtgccgtgca gtcccgcccc cacttgcttc tctttgtgtg tagtgtacgt 4920
acattatcga gaccgttgtt cccgcccacc tcgatccggc atgctgaggt gtctcacaag 4980
tgccgtgcag tcccgccccc acttgcttct ctttgtgtgt agtgtacgta cattatcgag 5040
accgttgttc ccgcccacct cgatccggca tgcactgatc acgggcaaaa gtgcgtatat 5100
atacaagagc gtttgccagc cacagatttt cactccacac accacatcac acatacaacc 5160
acacacatcc acgtgatgct acaagcagct attggaaaga ttgtgggatt tgcggtcaac 5220
cgacccatcc acacagttgt cctgacgtcc atcgtggcgt caaccgcata cctcgccatc 5280
ctcgacattg ccatcccggg tttcgagggc acacaaccca tctcatacta ccaccctgca 5340
gcaaaatctt acgacaaccc tgctgattgg acccacattg cagaggccga catcccttca 5400
gacgcctacc gacttgcatt tgcccagatc cgtgtcagtg atgttcaggg cggagaggcc 5460
cccaccatcc ctggcgccgt ggccgtgtct gatctcgacc acagaatcgt catggactac 5520
aaacagtggg ccccctggac cgccagcaac gagcagatcg cctcggagaa ccacatctgg 5580
aagcactcct tcaaggacca cgtggccttc agctggatca agtggttccg atgggcctac 5640
ctgcgtttgt ccactctcat ccagggggca gacaacttcg acattgccgt ggtcgccctt 5700
ggctatcttg ccatgcacta caccttcttc agtctcttcc gatccatgcg aaaggttggc 5760
tcgcactttt ggcttgcctc catggctctg gtctcttcca ccttcgcttt cctgcttgcg 5820
gtggtggctt cctctagcct gggttaccga cctagcatga tcaccatgtc cgagggcctg 5880
cccttcctcg tggtcgccat tggctttgac cgaaaggtca acctggctag cgaggtgctc 5940
acatccaaga gcagccagct cgctcccatg gtgcaggtga tcacaaagat cgcctccaag 6000
gcgctgtttg agtacagcct tgaggtggcc gccctgtttg ctggcgccta taccggagtt 6060
cctcgactgt cccagttttg cttcttatct gcttggatcc tcatcttcga ctacatgttt 6120
ttgctgacct tctactctgc tgtccttgct atcaagtttg agatcaatca cattaagcga 6180
aaccgaatga tccaggatgc tctcaaggag gatggtgtat ctgctgctgt tgccgagaag 6240
gtagccgact cttctcccga cgccaagctc gaccgaaagt ccgacgtttc tctttttgga 6300
gcctctggcg ccattgcggt gttcaagatc ttcatggtcc ttgggttcct tggtctcaac 6360
ctcatcaacc tgactgccat ccctcacctt ggcaaggcgg ccgccgctgc ccagtctgtg 6420
actcccatca ccctctcccc cgagcttctc catgccatcc ccgcctctgt gcccgttgtt 6480
gtcacctttg tgcccagcgt tgtgtacgag cactcccagc tcattctgca gctggaggac 6540
gccctcacta ccttcctggc tgcctgctcc aaaactattg gtgaccccgt catctccaag 6600
tacatcttcc tgtgcctgat ggtctccacc gccctgaacg tctacctgtt tggagccacc 6660
cgagaagttg tgcgaaccca gtctgtgaag gtggttgaga agcacgttcc tatcgtcatt 6720
gagaagccca gcgagaagga ggaggacacc tcttctgaag actccattga gctgactgtc 6780
ggaaagcagc ccaagcccgt gaccgagacc cgttctctgg acgacctaga ggctatcatg 6840
aaggcaggta agaccaagct tctggaggac cacgaggttg tcaagctctc tctcgagggc 6900
aagcttcctt tgtatgctct tgagaagcag cttggtgaca acacccgagc tgttggcatc 6960
cgacgatcta tcatctccca gcagtctaat accaagactt tagagacctc aaagcttcct 7020
tacctgcact acgactacga ccgtgttttt ggagcctgtt gcgagaacgt tattggttac 7080
atgcctctcc ccgttggtgt tgctggcccc atgaacattg atggcaagaa ctaccacatt 7140
cctatggcca ccactgaggg ttgtcttgtt gcctcaacca tgcgaggttg caaggccatc 7200
aacgccggtg gcggtgttac cactgtgctt actcaggacg gtatgacacg aggtccttgt 7260
gtttccttcc cctctctcaa gcgggctgga gccgctaaga tctggcttga ttccgaggag 7320
ggtctcaagt ccatgcgaaa ggccttcaac tccacctctc gatttgctcg tctccagtct 7380
cttcactcta cccttgctgg taacctgctg tttattcgat tccgaaccac cactggtgat 7440
gccatgggca tgaacatgat ctccaagggc gtcgaacact ctctggccgt catggtcaag 7500
gagtacggct tccctgatat ggacattgtg tctgtctcgg gtaactactg cactgacaag 7560
aagcccgcag cgatcaactg gatcgaaggc cgaggcaaga gtgttgttgc cgaagccacc 7620
atccctgctc acattgtcaa gtctgttctc aaaagtgagg ttgacgctct tgttgagctc 7680
aacatcagca agaatctgat cggtagtgcc atggctggct ctgtgggagg tttcaatgca 7740
cacgccgcaa acctggtgac cgccatctac cttgccactg gccaggatcc tgctcagaat 7800
gtcgagtctt ccaactgcat cacgctgatg agcaacgtcg acggtaacct gctcatctcc 7860
gtttccatgc cttctatcga ggtcggtacc attggtggag gtactatttt ggagccccag 7920
ggggctatgc tggagatgct tggcgtgcga ggtcctcaca tcgagacccc cggtgccaac 7980
gcccaacagc ttgctcgcat cattgcttct ggagttcttg cagcggagct ttcgctgtgt 8040
tctgctcttg ctgccggcca tcttgtgcaa agtcatatga cccacaaccg gtcccaggct 8100
cctactccgg ccaagcagtc tcaggccgat ctgcagcgtc tacaaaacgg ttcgaatatt 8160
tgcatacggt catagcacgt gggaacccga aactaaggat ccaactacgg aacttgtgtt 8220
gatgtctttg cccccggctc cgatatcatc tctgcctctt accagtccga ctctggtact 8280
ttggtctact ccggtacctc catggcctgt ccccacgttg ccggtcttgc ctcctactac 8340
ctgtccatca atgacgaggt tctcacccct gcccaggtcg aggctcttat tactgagtcc 8400
aacaccggtg ttcttcccac caccaacctc aagggctctc ccaacgctgt tgcctacaac 8460
ggtgttggca tttaggcaat taacagatag tttgccggtg ataattctct taacctccca 8520
cactcctttg acataacgat ttatgtaacg aaactgaaat ttgaccagat attgttgtaa 8580
atagaaaatc tggcttgtag gtggcaaaat gcggcgtctt tgttcatcaa ttccctctgt 8640
gactactcgt catcccttta tgttcgactg tcgtatttct tattttccat acatatgcaa 8700
gtgagatgcc cgtgtactag tggcctatgc ggccgcggat ctgctgcggt aaagctcatc 8760
agcggcctat gcggccgcgg atctgctgcg gtaaagctca tcagcgtggt cgtgaagcga 8820
ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa 8880
tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga 8940
tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag 9000
gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg 9060
taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca 9120
gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca 9180
gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg 9240
gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct 9300
tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc 9360
tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggccagg acccaacgct 9420
gcccgagatg cgccgcgtgc ggctgctgga gatggcggac gcgatggata tgttctgcca 9480
agggttggtt tgcgcattca cagttctccg caagaattga ttggctccaa ttcttggagt 9540
ggtgaatccg ttagcgaggt gccgccggct tccattcagg tcgaggtggc ccggctccat 9600
gcaccgcgac gcaacgcggg gaggcagaca aggtataggg cggcgcctac aatccatgcc 9660
aacccgttcc atgtgctcgc cgaggcggca taaatcgccg tgacgatcag cggtccagtg 9720
atcgaagtta ggctggtaag agccgcgagc gatccttgaa gctgtccctg atggtcgtca 9780
tctacctgcc tggacagcat ggcctgcaac gcgggcatcc cgatgccgcc ggaagcgaga 9840
agaatcataa tggggaaggc catccagcct cgcgtcgcga acgccagcaa gacgtagccc 9900
agcgcgtcgg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg 9960
ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg 10020
ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc 10080
ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc 10140
atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga 10200
cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg 10260
agcaccgccg ccgcaaggaa tggtgcatgc tgaggtgtct cacaagtgcc gtgcagtccc 10320
gcccccactt gcttctcttt gtgtgtagtg tacgtacatt atcgagaccg ttgttcccgc 10380
ccacctcgat ccggcatgct gaggtgtctc acaagtgccg tgcagtcccg cccccacttg 10440
cttctctttg tgtgtagtgt acgtacatta tcgagaccgt tgttcccgcc cacctcgatc 10500
cggcatgctg aggtgtctca caagtgccgt gcagtcccgc ccccacttgc ttctctttgt 10560
gtgtagtgta cgtacattat cgagaccgtt gttcccgccc acctcgatcc ggcatgctga 10620
ggtgtctcac aagtgccgtg cagtcccgcc cccacttgct tctctttgtg tgtagtgtac 10680
gtacattatc gagaccgttg ttcccgccca cctcgatccg gcatgcactg atcacgggca 10740
aaagtgcgta tatatacaag agcgtttgcc agccacagat tttcactcca cacaccacat 10800
cacacataca accacacaca tccacgtgat gaagatcgag gaaggcaagc tcgtcatctg 10860
gattaacggc gacaagggtt acaacggact ggccgaagtg ggcaagaagt tcgagaagga 10920
caccggtatt aaggtcaccg tggagcaccc cgacaagctg gaggagaagt tcccccaggt 10980
ggctgctacc ggtgacggac ccgacatcat tttctgggct cacgaccgat tcggcggtta 11040
cgctcagtct ggactgctcg ctgagatcac ccccgacaag gctttccagg acaagctcta 11100
ccccttcacc tgggacgccg tccgatacaa cggcaagctg atcgcttacc ccattgctgt 11160
ggaggccctg tctctcatct acaacaagga cctgctcccc aaccctccca agacctggga 11220
ggagattccc gccctcgaca aggagctgaa ggccaagggc aagtccgctc tcatgttcaa 11280
cctgcaggag ccctacttca cctggcccct gattgctgct gacggaggct acgccttcaa 11340
gtacgagaac ggaaagtacg acattaagga cgtgggtgtg gacaacgctg gagctaaggc 11400
tggcctgacc ttcctcgtcg acctgatcaa gaacaagcac atgaacgccg acaccgacta 11460
ctctattgct gaggccgctt tcaacaaggg agagaccgcc atgaccatca acggaccctg 11520
ggcttggtct aacattgaca cctccaaggt gaactacggc gtcaccgtgc tccccacctt 11580
caagggtcag ccctccaagc ccttcgtcgg tgtgctgtct gccggaatca acgccgcttc 11640
ccccaacaag gagctcgcta aggagttcct ggagaactac ctgctcaccg acgagggact 11700
cgaggccgtc aacaaggaca agcccctcgg cgctgtggcc ctgaagtcct acgaggaaga 11760
gctggccaag gacccccgaa tcgccgctac catggagaac gctcagaagg gagagatcat 11820
gcccaacatt ccccagatgt ctgctttctg gtacgccgtc cgaaccgctg tgattaacgc 11880
cgcttccgga cgacagaccg tggacgaggc cctcaaggac gctcagacca actcctcttc 11940
caataataac aataataaca acaacaacaa cctgggcatt gagggtcgaa tggactacat 12000
catttcggcg ccaggcaaag tgattctatt tggtgaacat gccgctgtgt ttggtaagcc 12060
tgcgattgca gcagccatcg acttgcgaac atacctgctt gtcgaaacca caacatccga 12120
caccccgaca gtcacgttgg agtttccaga catccacttg aacttcaagg tccaggtgga 12180
caagctggca tctctcacag cccagaccaa ggccgaccat ctcaattggt cgactcccaa 12240
aactctggat aagcacattt tcgacagctt gtctagcttg gcgcttctgg aagaacctgg 12300
gctcactaag gtccagcagg ccgctgttgt gtcgttcttg tacctctaca tccacctatg 12360
tcccccttct gtgtgcgaag attcatcaaa ctgggtagtt cgatcaacgc tgcctatcgg 12420
cgcgggcctg ggctcttccg catccatttg tgtctgtttg gctgcaggtc ttctggttct 12480
caacggccag ctgagcattg accaggcaag agatttcaag tccctgaccg agaagcagct 12540
gtctctggtg gacgactggt ccttcgtcgg tgaaatgtgc attcacggca acccgtcggg 12600
catcgacaat gctgtggcta ctcagggagg tgctctgttg ttccagcgac ctaacaaccg 12660
agtccctctt gttgacattc ccgagatgaa gctgctgctt accaatacga agcatcctcg 12720
atctaccgca gacctggttg gtggagtcgg agttctcact aaagagtttg gctccatcat 12780
ggatcccatc atgacttcag taggcgagat ttccaaccag gccatggaga tcatttctag 12840
aggcaagaag atggtggacc agtctaacct tgagattgag cagggtatct tgcctcaacc 12900
cacctctgag gatgcctgca acgtgatgga agatggagct actcttcaaa agttgagaga 12960
tatcggttcg gaaatgcagc atctagtgag aatcaatcac ggcctgctta tcgctatggg 13020
tgtttcccac ccgaagctcg aaatcattcg aactgcctcc attgtccaca acctgggtga 13080
gaccaagctc actggtgctg gaggaggagg ttgcgccatc actctagtca cttctaaaga 13140
caagactgcg acccagctgg aggaaaatgt cattgctttc acagaggaga tggctaccca 13200
tggcttcgag gtgcacgaga ctactattgg tgccagagga gttggtatgt gcattgacca 13260
tccctctctc aagactgttg aagccttcaa gaaggtggag cgggcggatc tcaaaaacat 13320
cggtccctgg acccattagg gatccaacta cggaacttgt gttgatgtct ttgcccccgg 13380
ctccgatatc atctctgcct cttaccagtc cgactctggt actttggtct actccggtac 13440
ctccatggcc tgtccccacg ttgccggtct tgcctcctac tacctgtcca tcaatgacga 13500
ggttctcacc cctgcccagg tcgaggctct tattactgag tccaacaccg gtgttcttcc 13560
caccaccaac ctcaagggct ctcccaacgc tgttgcctac aacggtgttg gcatttaggc 13620
aattaacaga tagtttgccg gtgataattc tcttaacctc ccacactcct ttgacataac 13680
gatttatgta acgaaactga aatttgacca gatattgttg taaatagaaa atctggcttg 13740
taggtggcaa aatgcggcgt ctttgttcat caattccctc tgtgactact cgtcatccct 13800
ttatgttcga ctgtcgtatt tcttattttc catacatatg caagtgagat gcccgtgtcc 13860
gaattctcat gtttgacagc ttatcatcga tgataagctg tcaaacatga 13910
<210> 4
<211> 13055
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
aagctagctt atcgatagag accgggttgg cggcgcattt gtgtcccaaa aaacagcccc 60
aattgcccca attgacccca aattgaccca gtagcgggcc caaccccggc gagagccccc 120
ttctccccac atatcaaacc tcccccggtt cccacacttg ccgttaaggg cgtagggtac 180
tgcagtctgg aatctacgct tgttcagact ttgtactagt ttctttgtct ggccatccgg 240
gtaacccatg ccggacgcaa aatagactac tgaaaatttt tttgctttgt ggttgggact 300
ttagccaagg gtataaaaga ccaccgtccc cgaattacct ttcctcttct tttctctctc 360
tccttgtcaa ctcacacccg aaatcgttaa gcatttcctt ctgagtataa gaatcattca 420
aacacgtgat gctgcgacga actctgttcg gattctctca cgaactcaag gctatccact 480
ccactgtgcc caacctgggc atgtgccgag gcggaaagtc tattgctcct tctatgtcta 540
tgtcctctac tacttctgtg tccaacgagg acggcgtgcc ccgacgaatc gccggtcacc 600
actctaacct ttgggacgac gactctatcg cttctctgtc tacttcctac gaggcccctt 660
cttaccgaaa gcgagctgac aagctcatcg gtgaggttaa gaacattttc gaccttatgt 720
ccgttgagga cggcgttttc acttcccctc tgtctgacct gcaccaccga ctgtggatgg 780
ttgactccgt tgagcgactg ggtattgacc gacacttcaa ggacgagatc aactccgccc 840
tggaccatgt atactcctac tggactgaga agggtattgg tcgaggtcga gagtccggcg 900
tcactgacct taactctacc gctctgggtc tgcgaaccct gcgactgcac ggatacactg 960
tgtcttctca cgttcttgac cacttcaaga acgagaaggg tcagttcact tgctccgcca 1020
tccagactga gggtgagatt cgagacgtgc ttaacctgtt ccgagcctct cttatcgctt 1080
tccccggtga gaagatcatg gaggccgctg agattttctc tactatgtac ctaaaagacg 1140
ctttgcaaaa gatcccccct tccggtctgt cccaagaaat cgagtacctg ctggagttcg 1200
gttggcacac taacctgcct cggatggaga cccgaatgta cattgacgtg ttcggtgagg 1260
acaccacttt cgagactcct taccttattc gagagaagct gctggaactg gccaagctgg 1320
agttcaacat tttccactct cttgttaagc gagagctaca atctctgtcc cgatggtgga 1380
aggactacgg attccctgag attactttct cccgacaccg acacgttgag tactacaccc 1440
tggccgcttg catcgctaac gaccctaagc actccgcttt ccgacttggt ttcggaaaga 1500
tttcccacat gattactatc cttgacgaca tctacgacac tttcggcact atggaggaac 1560
tgaagctgct caccgccgct ttcaagcgat gggacccctc ttccattgag tgcctgcctg 1620
actacatgaa gggcgtttac atggccgtgt acgacaacat caacgagatg gcccgcgagg 1680
cacagaagat tcagggttgg gacaccgtgt cctacgctcg aaagtcttgg gaggctttca 1740
tcggcgccta catccaagag gccaagtgga tctcctccgg atacctgcct actttcgacg 1800
agtacctgga gaacggaaag gtttccttcg gatctcgaat taccaccctt gagcctatgc 1860
ttacccttgg tttccccctg cctcctagga ttctgcaaga gattgacttc ccttctaagt 1920
tcaacgacct catttgcgcc attctgcgac tgaagggtga cacccagtgc tacaaggctg 1980
accgagcccg aggtgaagag gcttccgctg tgtcttgcta catgaaggac caccccggta 2040
ttactgagga agatgccgtt aaccaggtga acgctatggt ggacaacctc actaaggaac 2100
tgaactggga gctgctgcga cctgactccg gcgtgcccat ctcttacaag aaggtggctt 2160
tcgacatttg ccgagtgttc cactacggat acaagtaccg agacggattc tccgtcgcct 2220
ccattgagat taagaacctc gtcacccgaa ccgtcgttga gaccgtgcct ctttagggaa 2280
cccgaaacta aggatccggt acctccatgg cctgtcccca cgttgccggt cttgcctcct 2340
actacctgtc catcaatgac gaggttctca cccctgccca ggtcgaggct cttattactg 2400
agtccaacac cggtgttctt cccaccacca acctcaaggg ctctcccaac gctgttgcct 2460
acaacggtgt tggcatttag gcaattaaca gatagtttgc cggtgataat tctcttaacc 2520
tcccacactc ctttgacata acgatttatg taacgaaact gaaatttgac cagatattgt 2580
tgtaaataga aaatctggct tgtaggtggc aaaatcccgt ctttgttcat caattccctc 2640
tgtgactact cgtcatccct ttatgttcga ctgtcgtatt tttattttcc atacatacgc 2700
aagtgagatg cccgtgtccg aattcggtgc atgctgaggt gtctcacaag tgccgtgcag 2760
tcccgccccc acttgcttct ctttgtgtgt agtgtacgta cattatcgag accgttgttc 2820
ccgcccacct cgatccggca tgctgaggtg tctcacaagt gccgtgcagt cccgccccca 2880
cttgcttctc tttgtgtgta gtgtacgtac attatcgaga ccgttgttcc cgcccacctc 2940
gatccggcat gctgaggtgt ctcacaagtg ccgtgcagtc ccgcccccac ttgcttctct 3000
ttgtgtgtag tgtacgtaca ttatcgagac cgttgttccc gcccacctcg atccggcatg 3060
ctgaggtgtc tcacaagtgc cgtgcagtcc cgcccccact tgcttctctt tgtgtgtagt 3120
gtacgtacat tatcgagacc gttgttcccg cccacctcga tccggcatgc actgatcacg 3180
ggcaaaagtg cgtatatata caagagcgtt tgccagccac agattttcac tccacacacc 3240
acatcacaca tacaaccaca cacatccacg tgatgccgca gcaagcaatg gatatcaagg 3300
gcaaggccaa gtctgtgccc atgcccgaag aagacgacct ggactcgcat tttgtgggtc 3360
ccatctctcc ccgacctcac ggagcagacg agattgctgg ctacgtgggc tgcgaagacg 3420
acgaagacga gcttgaagaa ctgggaatgc tgggccgatc tgcgtccacc cacttctctt 3480
acgcggaaga acgccacctc atcgaggttg atgccaagta cagagctctt catggccatc 3540
tgcctcatca gcactctcag agtcccgtgt ccagatcttc gtcatttgtg cgggccgaaa 3600
tgaaccaccc ccctccccca ccctccagcc acacccacca acagccagag gacgatgacg 3660
catcttccac tcgatctcga tcgtcgtctc gagcttctgg acgcaagttc aacagaaaca 3720
gaaccaagtc tggatcttcg ctgagcaagg gtctccagca gctcaacatg accggatcgc 3780
tcgaagaaga gccctacgag agcgatgacg atgcccgact atctgcggaa gacgacattg 3840
tctatgatgc tacccagaaa gacacctgca agcccatatc tcctactctc aaacgcaccc 3900
gcaccaagga cgacatgaag aacatgtcca tcaacgacgt caaaatcacc accaccacag 3960
aagatcctct tgtggcccag gagctgtcca tgatgttcga aaaggtgcag tactgccgag 4020
acctccgaga caagtaccaa accgtgtcgc tacagaagga cggagacaac cccaaggatg 4080
acaagacaca ctggaaaatt taccccgagc ctccaccacc ctcctggcac gagaccgaaa 4140
agcgattccg aggctcgtcc aaaaaggagc accaaaagaa agacccgaca atggatgaat 4200
tcaaattcga ggactgcgaa atccccggac ccaacgacat ggtcttcaag cgagatccta 4260
cctgtgtcta tcaggtctat gaggatgaaa gctctctcaa cgaaaataag ccgtttgttg 4320
ccatcccctc aatccgagat tactacatgg atctggagga tctcattgtg gcttcgtctg 4380
acggacctgc caagtctttt gctttccgac gactgcaata tctagaagcc aagtggaacc 4440
tctactacct gctcaacgag tacacggaga caaccgagtc caagaccaac ccccatcgag 4500
acttttacaa cgtacgaaag gtcgacaccc acgttcacca ctctgcctgc atgaaccaga 4560
agcatctgct gcgattcatc aaatacaaga tgaagaactg ccctgatgaa gttgtcatcc 4620
accgagacgg tcgggagctg acactctccc aggtgtttga gtcacttaac ttgactgcct 4680
acgacctgtc tatcgatacc cttgatatgc atgctcacaa ggactcgttc catcgatttg 4740
acaagttcaa cctcaagtac aaccctgtcg gtgagtctcg actgcgagaa atcttcctaa 4800
agaccgacaa ctacatccag ggtcgatacc tagctgagat cacaaaggag gtgttccagg 4860
atctcgagaa ctcgaagtac cagatggcgg agtaccgtat ttccatctac ggtcggtcca 4920
aggacgagtg ggacaagctg gctgcctggg tgctggacaa caaactgttt tcgcccaatg 4980
ttcggtggtt gatccaggtg cctcgactgt acgacattta caagaaggct ggtctggtta 5040
acacctttgc cgacattgtg cagaacgtct ttgagcctct tttcgaggtc accaaggatc 5100
ccagtaccca tcccaagctg cacgtgttcc tgcagcgagt tgtgggcttt gactctgtcg 5160
atgacgagtc gaagctggac cgacgtttcc accgaaagtt cccaactgca gcatactggg 5220
acagcgcaca gaaccctccc tactcgtact ggcagtacta tctatacgcc aacatggcct 5280
ccatcaacac ctggagacag cgtttgggct ataatacttt tgagttgcga ccccatgctg 5340
gagaggctgg tgacccagag catcttctgt gcacttatct ggttgctcag ggtatcaacc 5400
acggtattct gttgcgaaag gtgcccttca ttcagtacct ttactacctg gaccagatcc 5460
ccattgccat gtctcctgtg tccaacaatg cgctgttcct cacgttcgac aagaacccct 5520
tctactcata cttcaagcgg ggtctcaacg tgtccttgtc atcggatgat cctctgcagt 5580
ttgcttacac taaggaggct ctgattgagg agtactctgt ggctgcgctc atttacaagc 5640
tttccaacgt ggatatgtgt gagcttgctc gaaactcggt actgcaatct ggctttgagc 5700
gaatcatcaa ggagcattgg atcggcgaaa actacgagat ccatggcccc gagggcaaca 5760
ccatccagaa gacaaacgtg cccaatgtgc gtctggcctt ccgagacgag actttgaccc 5820
acgagcttgc tctggtggac aagtacacca atcttgagga gtttgagcgg ctgcatggtt 5880
aaggatccaa ctacggaact tgtgttgatg tctttgcccc cggctccgat atcatctctg 5940
cctcttacca gtccgactct ggtactttgg tctactccgg tacctccatg gcctgtcccc 6000
acgttgccgg tcttgcctcc tactacctgt ccatcaatga cgaggttctc acccctgccc 6060
aggtcgaggc tcttattact gagtccaaca ccggtgttct tcccaccacc aacctcaagg 6120
gctctcccaa cgctgttgcc tacaacggtg ttggcattta ggcaattaac agatagtttg 6180
ccggtgataa ttctcttaac ctcccacact cctttgacat aacgatttat gtaacgaaac 6240
tgaaatttga ccagatattg ttgtaaatag aaaatctggc ttgtaggtgg caaaatgcgg 6300
cgtctttgtt catcaattcc ctctgtgact actcgtcatc cctttatgtt cgactgtcgt 6360
atttcttatt ttccatacat atgcaagtga gatgcccgtg tgaattccat gtgtaacact 6420
cgctctggag agttagtcat ccgacagggt aactctaatc tcccaacacc ttattaactc 6480
tgcgtaactg taactcttct tgccacgtcg atcttactca attttcctgc tcatcatctg 6540
ctggattgtt gtctatcgtc tggctctaat acatttattg tttattgccc aaacaacttt 6600
cattgcacgt aagtgaattg ttttataaca gcgttcgcca attgctgcgc catcgtcgtc 6660
cggctgtcct accgttaggg tagtgtgtct cacactaccg aggttactag agttgggaaa 6720
gcgatactgc ctcggacaca ccacctggtc ttacgactgc agagagaatc ggcgttacct 6780
cctcacaaag ccctcagtgc ggccgcccgg ggtgggcgaa gaactccagc atgagatccc 6840
cgcgctggag gatcatccag ccggcgtccc ggaaaacgat tccgaagccc aacctttcat 6900
agaaggcggc ggtggaatcg aaatctcgtg atggcaggtt gggcgtcgct tggtcggtca 6960
tttcgaaccc cagagtcccg ctcagaagaa ctcgtcaaga aggcgataga aggcgatgcg 7020
ctgcgaatcg ggagcggcga taccgtaaag cacgaggaag cggtcagccc attcgccgcc 7080
aagctcttca gcaatatcac gggtagccaa cgctatgtcc tgatagcggt ccgccacacc 7140
cagccggcca cagtcgatga atccagaaaa gcggccattt tccaccatga tattcggcaa 7200
gcaggcatcg ccatgggtca cgacgagatc ctcgccgtcg ggcatgcgcg ccttgagcct 7260
ggcgaacagt tcggctggcg cgagcccctg atgctcttcg tccagatcat cctgatcgac 7320
aagaccggct tccatccgag tacgtgctcg ctcgatgcga tgtttcgctt ggtggtcgaa 7380
tgggcaggta gccggatcaa gcgtatgcag ccgccgcatt gcatcagcca tgatggatac 7440
tttctcggca ggagcaaggt gagatgacag gagatcctgc cccggcactt cgcccaatag 7500
cagccagtcc cttcccgctt cagtgacaac gtcgagcaca gctgcgcaag gaacgcccgt 7560
cgtggccagc cacgatagcc gcgctgcctc gtcctgcagt tcattcaggg caccggacag 7620
gtcggtcttg acaaaaagaa ccgggcgccc ctgcgctgac agccggaaca cggcggcatc 7680
agagcagccg attgtctgtt gtgcccagtc atagccgaat agcctctcca cccaagcggc 7740
cggagaacct gcgtgcaatc catcttgttc aatcatgcga aacgatcctc atcctgtctc 7800
ttgatcagat cttgatcccc tgcgccatca gatccttggc ggcaagaaag ccatccagtt 7860
tactttgcag ggcttcccaa ccttaccaga gggcgcccca gctggcaatt ccggttcgct 7920
tgctgtccat aaaaccgccc agtctagcta tcgccatgta agcccactgc aagctacctg 7980
ctttctcttt gcgcttgcgt tttcccttgt ccagatagcc cagtagctga cattcatccg 8040
gggtcagcac cgtttctgcg gactggcttt ctacgtgttc cgcttccttt agcagccctt 8100
gcgccctgag tgcttgcggc agcgtgaagc tagcttatgc ggtgtgaaat accgcacaga 8160
tgcgtaagga gaaaataccg catcaggcgc tcttccgctt cctcgctcac tgactcgctg 8220
cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta 8280
tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc 8340
aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 8400
catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 8460
caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 8520
ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt 8580
aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 8640
gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 8700
cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 8760
ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta 8820
tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 8880
tccggcaaac aaaccaccgc tggtagcggc ggttttttgt ttgcaagcag cagattacgc 8940
gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc ttactgaacg gtgatcccca 9000
ccggaattgc ggccgctgtc gggaaccgcg ttcaggtgga acaggacacc tcccttgcac 9060
ttcttggtat atcagtatag gctgatgtat tcatagtggg gtttttcata ataaatttac 9120
taacggcagg caacattcac tcggcttaaa cgcaaaacgg accgtcttga tatcttctga 9180
cgcattgacc accgagaaat agtgttagtt accgggtgag ttattgttct tctacacagg 9240
cgacgcccat cgtctagagt tgatgtacta actcagattt cactacctac cctatccctg 9300
gtacgcacaa agcactttgc tagatagagt cgacaaaggc ctggagggca ttttggtatc 9360
aagctagctt atcgatacgc gtgcatgctg aggtgtctca caagtgccgt gcagtcccgc 9420
ccccacttgc ttctctttgt gtgtagtgta cgtacattat cgagaccgtt gttcccgccc 9480
acctcgatcc ggcatgctga ggtgtctcac aagtgccgtg cagtcccgcc cccacttgct 9540
tctctttgtg tgtagtgtac gtacattatc gagaccgttg ttcccgccca cctcgatccg 9600
gcatgctgag gtgtctcaca agtgccgtgc agtcccgccc ccacttgctt ctctttgtgt 9660
gtagtgtacg tacattatcg agaccgttgt tcccgcccac ctcgatccgg catgctgagg 9720
tgtctcacaa gtgccgtgca gtcccgcccc cacttgcttc tctttgtgtg tagtgtacgt 9780
acattatcga gaccgttgtt cccgcccacc tcgatccggc atgcactgat cacgggcaaa 9840
agtgcgtata tatacaagag cgtttgccag ccacagattt tcactccaca caccacatca 9900
cacatacaac cacacacatc cacgtgatgg actacatcat ttcggcgcca ggcaaagtga 9960
ttctatttgg tgaacatgcc gctgtgtttg gtaagcctgc gattgcagca gccatcgact 10020
tgcgaacata cctgcttgtc gaaaccacaa catccgacac cccgacagtc acgttggagt 10080
ttccagacat ccacttgaac ttcaaggtcc aggtggacaa gctggcatct ctcacagccc 10140
agaccaaggc cgaccatctc aattggtcga ctcccaaaac tctggataag cacattttcg 10200
acagcttgtc tagcttggcg cttctggaag aacctgggct cactaaggtc cagcaggccg 10260
ctgttgtgtc gttcttgtac ctctacatcc acctatgtcc cccttctgtg tgcgaagatt 10320
catcaaactg ggtagttcga tcaacgctgc ctatcggcgc gggcctgggc tcttccgcat 10380
ccatttgtgt ctgtttggct gcaggtcttc tggttctcaa cggccagctg agcattgacc 10440
aggcaagaga tttcaagtcc ctgaccgaga agcagctgtc tctggtggac gactggtcct 10500
tcgtcggtga aatgtgcatt cacggcaacc cgtcgggcat cgacaatgct gtggctactc 10560
agggaggtgc tctgttgttc cagcgaccta acaaccgagt ccctcttgtt gacattcccg 10620
agatgaagct gctgcttacc aatacgaagc atcctcgatc taccgcagac ctggttggtg 10680
gagtcggagt tctcactaaa gagtttggct ccatcatgga tcccatcatg acttcagtag 10740
gcgagatttc caaccaggcc atggagatca tttctagagg caagaagatg gtggaccagt 10800
ctaaccttga gattgagcag ggtatcttgc ctcaacccac ctctgaggat gcctgcaacg 10860
tgatggaaga tggagctact cttcaaaagt tgagagatat cggttcggaa atgcagcatc 10920
tagtgagaat caatcacggc ctgcttatcg ctatgggtgt ttcccacccg aagctcgaaa 10980
tcattcgaac tgcctccatt gtccacaacc tgggtgagac caagctcact ggtgctggag 11040
gaggaggttg cgccatcact ctagtcactt ctaaagacaa gactgcgacc cagctggagg 11100
aaaatgtcat tgctttcaca gaggagatgg ctacccatgg cttcgaggtg cacgagacta 11160
ctattggtgc cagaggagtt ggtatgtgca ttgaccatcc ctctctcaag actgttgaag 11220
ccttcaagaa ggtggagcgg gcggatctca aaaacatcgg tccctggacc cattagggat 11280
ccggtacctc catggcctgt ccccacgttg ccggtcttgc ctcctactac ctgtccatca 11340
atgacgaggt tctcacccct gcccaggtcg aggctcttat tactgagtcc aacaccggtg 11400
ttcttcccac caccaacctc aagggctctc ccaacgctgt tgcctacaac ggtgttggca 11460
tttaggcaat taacagatag tttgccggtg ataattctct taacctccca cactcctttg 11520
acataacgat ttatgtaacg aaactgaaat ttgaccagat attgttgtaa atagaaaatc 11580
tggcttgtag gtggcaaaat cccgtctttg ttcatcaatt ccctctgtga ctactcgtca 11640
tccctttatg ttcgactgtc gtatttttat tttccataca tacgcaagtg agatgcccgt 11700
gtcaggcctg tttctcggtg tacagagctt ggtcctcctt gaagttgcga cacatgtctt 11760
gatagtatct tggcttctct ctcttgagct tttccataac aagttcttct gcctccagga 11820
agtccatggg tggtttgatc atggttttgg tgtagtggta gtgcagtggt ggtattgtga 11880
ctggggatgt agttgagaat aagtcataca caagtcagct ttcttcgagc ctcatataag 11940
tataagtagt tcaacgtatt agcactgtac ccagcatctc cgtatcgaga aacacaacaa 12000
catgccccat tggacagacc atgcggatac acaggttgtg cagtaccata catactcgat 12060
cagacaggtc gtctgaccat catacaagct gaacagcgct ccatacttgc acgctctcta 12120
tatacacagt taaattacat atccatagtc taacctctaa cagttaatct tctggtaagc 12180
ctcccagcca gccttctggt atcgcttggc ctcctcaata ggatctcggt tctggccgta 12240
cagacctcgg ccgacaatta tgatatccgt tccggtagac atgacatcct caacagttcg 12300
gtactgctgt ccgagagcgt ctcccttgtc gtcaagaccc accccggggg tcagaataag 12360
ccagtcctca gagtcgccct taggtcggtt ctgggcaatg aagccaacca caaactcggg 12420
gtcggatcgg gcaagctcaa tggtctgctt ggagtactcg ccagtggcca gagagccctt 12480
gcaagacagc tcggccagca tgagcagacc tctggccagc ttctcgttgg gagaggggac 12540
taggaactcc ttgtactggg agttctcgta gtcagagacg tcctccttct tctgttcaga 12600
gacagtttcc tcggcaccag ctcgcaggcc agcaatgatt ccggttccgg gtacaccgtg 12660
ggcgttggtg atatcggacc actcggcgat tcggtgacac cggtactggt gcttgacagt 12720
gttgccaata tctgcgaact ttctgtcctc gaacaggaag aaaccgtgct taagagcaag 12780
ttccttgagg gggagcacag tgccggcgta ggtgaagtcg tcaatgatgt cgatatgggt 12840
cttgatcatg cacacataag gtccgacctt atcggcaagc tcaatgagct ccttggtggt 12900
ggtaacatcc agagaagcac acaggttggt tttcttggct gccacgagct tgagcactcg 12960
agcggcaaag gcggacttgt ggacgttagc tcgagcttcg taggagggca ttttggtggt 13020
gaagaggaga ctgaaataaa tttagtctgc agccc 13055
<210> 5
<211> 8166
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300
tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
tttcccagtc acgacgttgt aaaacgacgg ccagtcatat gtcgatccta aggggtggca 420
taactgtcgc gtacggcccg ataagggcct tctccaaaag ggaagccggt tgaaattccg 480
gcacttggat gtggattctc cacggcaacg taactgaatg tggggacggt ggcacaagtc 540
ttggaaggag ttatcttttc tttttaacgg agtcaacacc ctggaattag tttgtctaga 600
gatagggtat cgttccggaa gaggggggca gctttgtccc ctccgatgca cttgtgacgc 660
cccttgaaaa cccgcaggaa ggaatagttt tcacgccaag tcgtactgat aaccgcagca 720
ggtctccaag gtgaacagcc tctagttgat agaataatgt agataaggga agtcggcaaa 780
atagatccgt aacttcggga taaggattgg ctctgggggt tggtggatgg aagcgtggga 840
gaccccaagg gactggcggc tgggcaactg gcagccggac ccgcggcaga cactgcgtcg 900
ctccgtccac atcatcaacc gccccagaac tggtacggac aaggggaatc tgactgtcta 960
attaaaacat agctttgcga tggttgtaaa acaatgttga cgcaaagtga tttctgccca 1020
gtgctctgaa tgtcaaagtg aagaaattca agaattccag atcttccagt ggtgcatgaa 1080
cgcatgagaa agcccccgga agatcatctt ccgggggctt tttttttggc gcgcgataca 1140
gaccggttca gacaggataa agaggaacgc agaatgttag acaacacccg cttacgcata 1200
gctattcaga aatcaggccg tttaagcgat gattcacgag aattgctggc ccgctgcggc 1260
ataaaaatta atttacacac tcagcgcctg attgcgatgg cggaaaacat gccgattgat 1320
atcctgcgcg tgcgtgatga tgacattccg ggtctggtaa tggatggcgt ggtcgatctc 1380
ggtattatcg gcgaaaacgt gctggaagaa gagctactca accgccgcgc acagggcgaa 1440
gatccacgct atttaaccct gcgccgtctt gacttcggcg gctgccgttt atcgctggca 1500
acaccggttg acgaagcctg ggacggcccg gccgcgctgg acggtaaacg tatcgctacc 1560
tcatatccgc acctcctcaa acgctacctc gaccagaaag gcgtctcttt taaatcgtgt 1620
ctgttaaatg gttctgtcga agtcgcgccg cgcgcggggc tggccgacgc tatctgcgat 1680
ttggtctcta ccggcgcgac gcttgaagct aacggcctgc gtgaagtcga agttatctac 1740
cgctctaaag cctgtctgat tcagcgcgac ggtgagatgg cacagagcaa gcaagagctg 1800
atcgataaat tgctgacccg tattcagggc gtgattcagg cgcgcgaatc gaaatacatc 1860
atgatgcacg cgccaagtga acgcctggaa gaggttatcg ccctgctgcc aggcgccgaa 1920
aggccgacaa ttctgccgct ggcaggcgag caacagcgcg tggcgatgca catggtcagc 1980
agcgaaacgt tgttctggga aaccatggag aaactgaaag cgcttggcgc cagctcgatt 2040
ctggtactgc cgatcgagaa gatgatggag tgatctgacg cctgatggcg ctgcgcttat 2100
caggcctacg taatgcgttg atattttggg ttctgtaggc cggataaggc ggaaccctgt 2160
gatggagtaa agaccatgag cttcaatacc ctgattgact ggaacagcgg taccggtgtg 2220
ttctgtggag cattctcact tttggtaaac gacattgctt caagtgcagc ggaatcaaaa 2280
agtataaagt gggcagcgag tatacctgta cagactgtag gcgataactc aatccaatta 2340
ccccccacaa catgactggc caaactgatc tcaagacttt attgaaatca gcaacaccga 2400
ttctcaatga aggcacatac ttcttctgca acattcactt gacgcctaaa gttggtgaga 2460
aatggaccga caagacatat tctgctatcc acggactgtt gcctgtgtcg gtggctacaa 2520
tacgtgagtc agaagggctg acggtggtgg ttcccaagga aaaggtcgac gagtatctgt 2580
ctgactcgtc attgccgcct ttggagtacg actccaacta tgagtgtgct tggatcactt 2640
tgacgataca ttcttcgttg gaggctgtgg gtctgacagc tgcgttttcg gcgcggttgg 2700
ccgacaacaa tatcagctgc aacgtcattg ctggctttca tcatgatcac atttttgtcg 2760
gcaaaggcga cgcccagaga gccattgacg ttctttctaa tttggaccga tagccgtata 2820
gtccagtcta tctataagtt caactaactc gtaactatta ccataacata tacttcactg 2880
ccccagataa ggttccgata aaaagttctg cagactaaat ttatttcagt ctcctcttca 2940
ccaccaaaat gccctcctac gaagctcgag ctaacgtcca caagtccgcc tttgccgctc 3000
gagtgctcaa gctcgtggca gccaagaaaa ccaacctgtg tgcttctctg gatgttacca 3060
ccaccaagga gctcattgag cttgccgata aggtcggacc ttatgtgtgc atgatcaaaa 3120
cccatatcga catcattgac gacttcacct acgccggcac tgtgctcccc ctcaaggaac 3180
ttgctcttaa gcacggtttc ttcctgttcg aggacagaaa gttcgcagat attggcaaca 3240
ctgtcaagca ccagtaccgg tgtcaccgaa tcgccgagtg gtccgatatc accaacgccc 3300
acggtgtacc cggaaccgga atcattgctg gcctgcgagc tggtgccgag gaaactgtct 3360
ctgaacagaa gaaggaggac gtctctgact acgagaactc ccagtacaag gagttcctag 3420
tcccctctcc caacgagaag ctggccagag gtctgctcat gctggccgag ctgtcttgca 3480
agggctctct ggccactggc gagtactcca agcagaccat tgagcttgcc cgatccgacc 3540
ccgagtttgt ggttggcttc attgcccaga accgacctaa gggcgactct gaggactggc 3600
ttattctgac ccccggggtg ggtcttgacg acaagggaga cgctctcgga cagcagtacc 3660
gaactgttga ggatgtcatg tctaccggaa cggatatcat aattgtcggc cgaggtctgt 3720
acggccagaa ccgagatcct attgaggagg ccaagcgata ccagaaggct ggctgggagg 3780
cttaccagaa gattaactgt tagaggttag actatggata tgtaatttaa ctgtgtatat 3840
agagagcgtg caagtatgga gcgcttgttc agcttgtatg atggtcagac gacctgtctg 3900
atcgagtatg tatgatactg cacaacctgt gtatccgcat gatctgtcca atggggcatg 3960
ttgttgtgtt tctcgatacg gagatgctgg gtacagtgct aatacgttga actacttata 4020
cttatatgag gctcgaagaa agctgacttg tgtatgactt attctcaact acatccccag 4080
tcacaatacc accactgcac taccactaca ccggatccca gatcttccag tggtgcatga 4140
acgcatgaga aagcccccgg aagatcatct tccgggggct ttttttttgg cgcgcgatac 4200
agaccggttc agacaggata aagaggaacg cagaatgtta gacaacaccc gcttacgcat 4260
agctattcag aaatcaggcc gtttaagcga tgattcacga gaattgctgg cccgctgcgg 4320
cataaaaatt aatttacaca ctcagcgcct gattgcgatg gcggaaaaca tgccgattga 4380
tatcctgcgc gtgcgtgatg atgacattcc gggtctggta atggatggcg tggtcgatct 4440
cggtattatc ggcgaaaacg tgctggaaga agagctactc aaccgccgcg cacagggcga 4500
agatccacgc tatttaaccc tgcgccgtct tgacttcggc ggctgccgtt tatcgctggc 4560
aacaccggtt gacgaagcct gggacggccc ggccgcgctg gacggtaaac gtatcgctac 4620
ctcatatccg cacctcctca aacgctacct cgaccagaaa ggcgtctctt ttaaatcgtg 4680
tctgttaaat ggttctgtcg aagtcgcgcc gcgcgcgggg ctggccgacg ctatctgcga 4740
tttggtctct accggcgcga cgcttgaagc taacggcctg cgtgaagtcg aagttatcta 4800
ccgctctaaa gcctgtctga ttcagcgcga cggtgagatg gcacagagca agcaagagct 4860
gatcgataaa ttgctgaccc gtattcaggg cgtgattcag gcgcgcgaat cgaaatacat 4920
catgatgcac gcgccaagtg aacgcctgga agaggttatc gccctgctgc caggcgccga 4980
aaggccgaca attctgccgc tggcaggcga gcaacagcgc gtggcgatgc acatggtcag 5040
cagcgaaacg ttgttctggg aaaccatgga gaaactgaaa gcgcttggcg ccagctcgat 5100
tctggtactg ccgatcgaga agatgatgga gtgatctgac gcctgatggc gctgcgctta 5160
tcaggcctac gtaatgcgtt gatattttgg gttctgtagg ccggataagg cggaaccctg 5220
tgatggagta aagaccatga gcttcaatac cctgattgac tggaacagca aattcaacca 5280
agcgcgggta aacggcggga gtaactatga ctctcttaag gtagccaaat gcctcgtcat 5340
ctaattagtg acgcgcatga atggattaac gagattccca ctgtccctat ctactatcta 5400
gcgaaaccac agccaaggga acgggcttgg cagaatcagc ggggaaagaa gaccctgttg 5460
agcttgactc tagtttgaca ttgtgaagag acataggggg tgtagaataa gtgggagctt 5520
cggcgccggt gaaataccac tacccttatc gtttctttac ttatttagta agtggaagtg 5580
gtttaacaac cattttctag cattcctttc caggctgaag acattgtcag gtggggagtt 5640
tggctggggc ggcacatctg ttaaaagata acgcagatgt cctaaggggg actcaatgag 5700
aacagaaatc tcatgtagaa caaaagggta aaagtcccct tgattttgat tttcagtgtg 5760
aatacaaacc atgaaagtgt ggcctatcga tcctttagtt gttcggagtt tgaacctaga 5820
ggtgccagaa aagttaccac agggataact ggcttgtggc agtcaagcgt tcatagcgac 5880
attgcttttt gatccttcga tgtcggctct tcctatcata ccgaagcata tgggcgtaat 5940
catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac 6000
gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa 6060
ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat 6120
gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc 6180
tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg 6240
cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag 6300
gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc 6360
gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag 6420
gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga 6480
ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc 6540
atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 6600
tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt 6660
ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca 6720
gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca 6780
ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag 6840
ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca 6900
agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg 6960
ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa 7020
aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta 7080
tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag 7140
cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga 7200
tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac 7260
cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc 7320
ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta 7380
gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac 7440
gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat 7500
gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa 7560
gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg 7620
tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag 7680
aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc 7740
cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct 7800
caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat 7860
cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg 7920
ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc 7980
aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta 8040
tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg 8100
tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct 8160
ttcgtc 8166
<210> 6
<211> 957
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
atgccttcca tcaagttgaa ctccggttac gacatgcccg ccgtcggatt cggatgctgg 60
aaggttgacg ttgacacttg ctctgagcaa atctatcgag ccatcaagac cggataccga 120
ctgttcgacg gcgctgagga ctacgctaac gagaaactgg tcggcgctgg tgtgaagaag 180
gctatcgacg agggtattgt gaagcgagag gacctgttcc ttacttctaa gctctggaac 240
aactaccacc accctgacaa cgtggagaag gccctgaacc gaaccctgtc tgacctccag 300
gttgactacg ttgacctgtt ccttattcac ttccccgtca ctttcaagtt cgtgcctctg 360
gaggaaaagt accctcccgg attctactgc gggaagggtg acaacttcga ctacgaggac 420
gtgcctattc ttgagacttg gaaggctctg gagaagctcg tcaaggccgg gaagatccga 480
tctattggtg tgtctaactt ccccggcgct ctgctgcttg accttctccg aggcgccact 540
attaagcctt ccgtgctcca agtggagcac cacccctact tgcaacagcc tcgactcatt 600
gagttcgccc agtcacgcgg aattgccgtc accgcttact cttctttcgg tcctcagtct 660
ttcgtggaat tgaaccaggg tcgagccctg aacacctctc ctctgttcga gaacgagacc 720
attaaggcta tcgccgctaa gcacggaaag tctcccgctc aggtcctgct gcgatggtct 780
tctcagcgag gtatcgccat catccccaag tccaacactg tgccccgact ccttgagaac 840
aaggacgtta actctttcga cctcgacgag caggacttcg ccgacattgc caagctggac 900
attaacctgc gattcaacga cccttgggac tgggacaaga tccccatttt cgtttag 957
<210> 7
<211> 1092
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
atgactgcta acccctccct cgtcctgaac aagattgacg acatctcttt cgagacctac 60
gacgctcctg agatttctga gcccactgac gttcttgtcc aggtgaaaaa gaccggaatt 120
tgcggctccg acatccactt ctacgctcac ggtcgaatcg gtaacttcgt tcttactaag 180
cctatggtgc tgggtcacga gtctgccggc actgttgtcc aggttggaaa gggagttacc 240
tctctgaagg ttggtgacaa cgtcgctatt gagcccggta ttccttctcg attctctgac 300
gagtacaagt ctggtcacta caacctttgc cctcacatgg ccttcgccgc cacccctaac 360
tctaaggaag gggagcctaa ccctcctggt actctttgca agtacttcaa gtctcccgag 420
gacttccttg ttaagctgcc cgaccacgtt tccctggagc ttggcgctct cgtcgagccc 480
ctgtctgtgg gtgtgcacgc ctccaagctg ggctcagtcg ccttcggcga ctacgttgct 540
gttttcggcg ccggccccgt gggtctgctt gccgctgccg tggccaagac cttcggcgcc 600
aagggcgtta tcgtggttga catcttcgac aacaagttaa aaatggctaa ggacattggc 660
gccgctactc acactttcaa ctctaagact ggcgggtcgg aggaacttat taaggctttc 720
ggtggaaacg ttcccaacgt tgtgcttgag tgcaccggtg ctgagccttg cattaagctc 780
ggtgtggacg ctatcgctcc tggaggtcga ttcgtccagg ttggtaacgc tgccggtccc 840
gtgtctttcc ccatcaccgt gttcgccatg aaagaactta ccctgttcgg atctttccga 900
tacggattca acgactacaa gactgctgtg ggcattttcg acaccaacta ccagaacggt 960
cgagagaacg ctcctatcga cttcgagcag ctcattaccc accgatacaa gttcaaggac 1020
gccattgagg cttacgacct cgtgcgagcc ggaaagggcg ctgtgaagtg ccttattgac 1080
ggacctgagt aa 1092
<210> 8
<211> 1623
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
atgtatctcg gactggatct ttcgactcaa cagctcaagg gcatcattct ggacacaaaa 60
acgctggaca cggtcacaca agtccatgtg gactttgagg acgacttgcc gcagttcaac 120
accgaaaagg gcgtctttca cagctctaca gtggccggag aaatcaatgc tcctgtggca 180
atgtgggggg cagctgtgga cttgctgata gagcgtctgt caaaggaaat agacctttcc 240
acgatcaagt ttgtgtcggg ctcgtgccag caacacggct ctgtttatct caacagcagc 300
tacaaggagg gcctgggttc tctggacaaa cacaaagact tgtctacagg agtgtcatcc 360
ttactggcgc tcgaagtcag ccccaattgg caggatgcaa gcacggagaa ggagtgtgcg 420
cagtttgagg ctgcagtcgg cggtcccgag cagctggctg agatcactgg ctctcgagca 480
catactcgtt tcaccgggcc ccagattctc aaggtcaagg aacgcaaccc caaggtattc 540
aaggccacgt cacgggtcca gctcatatcc aactttctag catctctgtt tgccggcaag 600
gcgtgcccct ttgatcttgc tgacgcctgt ggaatgaatc tgtgggacat ccagaatggc 660
cagtggtgca agaaactcac agatctcatc accgatgaca cccactcggt cgagtccctc 720
cttggagacg tggaaacaga ccccaaggct ctactgggca aaatctcgcc ctatttcgtc 780
tccaagggct tctctccctc ttgtcaggtg gcacagttca caggcgacaa cccaggcact 840
atgctggctc tccccttaca ggccaatgac gtgattgtgt ctttgggaac atctacgacc 900
gccctcgtcg taacaaacaa gtacatgccc gaccccggat accatgtgtt caaccacccc 960
atggagggat acatgggcat gctgtgctac tgcaacggag gtctagcacg agagaagatc 1020
cgagacgagc ttggaggctg ggacgagttt aatgaggcgg ccgagaccac caacacagtg 1080
tctgctgacg atgtccatgt tggcatctac tttccactac gagaaatcct tcctcgagca 1140
ggtccctttg aacgacgttt catctacaac agacaaagtg aacagcttac agagatggct 1200
tctccagagg actcactggc aaccgaacac aaaccgcagg ctcaaaatct caaggacacg 1260
tggccgccac aaatggacgc cactgccatc attcaaagcc aggccctcag tatcaaaatg 1320
agactccaac gcatgatgca tggcgatatt ggaaaggtgt attttgtggg aggcgcctcg 1380
gtcaacactg ctatctgcag cgtaatgtct gccatcttaa aaccaacaaa gggcgcttgg 1440
agatgtggtc tggaaatggc aaacgcttgt gccattggaa gtgcccatca cgcctggctt 1500
tgcgacccca acaagacagg ccaggtacag gttcacgaag aagaggtcaa atacaagaat 1560
gtggacacag acgtgctact caaggcgttc aagctggccg aaaacgcctg cctggagaaa 1620
taa 1623
<210> 9
<211> 9431
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac 60
aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg 120
catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg 180
taaggagaaa ataccgcatc aggcgccatt cgccattcag gctgcgcaac tgttgggaag 240
ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa 300
ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca 360
gtgaattcga gctcggtacc cgcttttcgt agataatgga atacaaatgg atatccagag 420
tatacacatg gatagtatac actgacacga caattctgta tctctttatg ttaactactg 480
tgaggcgtta aatagagctt gatatataaa atgttacatt tcacagtctg aacttttgca 540
gattacctaa tttggtaaga tattaattat gaactgaaag ttgatggcat ccctaaattt 600
gatgaaaggg ggatcccccg ggttcgaagg taccaaggaa gcatgcggta cagatagagt 660
ctcttcaact cgctcaaaaa acaaccgatt gctctcaatc tttggatgag gaatactggc 720
acccatgact caggtctcac cacttctgtc catcttctaa gtccactata tcacccctcc 780
aacccctccc cgttccctct cctaccccat atttccgaac ctaattacaa acaaaggtta 840
aattgagcag ctctaaattc tccacatcaa agtacaaata ccgtgcacag cctccacata 900
tgcctctgtt cagatcactg cacactctca aacgtctctc aaccatgccc acaatcactc 960
tcatccacaa ccccaagtgc tccaaatcat gtgctgctct cgagctgctg gagcagaagc 1020
aaattagccc caatgtcgtg cagtacctcg acacacctct ttccgttgct gagatttcgt 1080
ctattgttcg gaaactcgac atgaagcccg tcgagctgct cagaaagggc gagccgcaat 1140
tttctgagct caatttgggg gcggccaaca cgcccgatgc cgagatcatt gatgccatgg 1200
cgaaacaccc caatctaatc gagcggccta ttctgattgt ggatgacaag gctgtgattg 1260
gacggcccac agaaaagctc aatgacctgc tgggaaagct gtagatacgc aagtactgag 1320
taatagaaag agttgatttg gcctaaatat cgtttctaga atttttgaaa aaaagatgcc 1380
ttcgaccaga tttgaactga tgatctcagc attaagagtg ctgcgcctta ccacttggcc 1440
acgaaggctg gtttttgaaa agaagaataa ttaaaagtgt gctacgtgaa aattgaaata 1500
aatatccatg aatcattagc aaaaaaataa ataaaataaa aaatacatca ttagaataaa 1560
aaaaaaacaa gacatgccct ttatgacctc aaacaccact ccaaggcctg gatgataaac 1620
atgtttttcc tgtccaagct agtcttctat cactagtaga gaccgggttg gcggcgcatt 1680
tgtgtcccaa aaaacagccc caattgcccc aattgacccc aaattgaccc agtagcgggc 1740
ccaaccccgg cgagagcccc cttctcccca catatcaaac ctcccccggt tcccacactt 1800
gccgttaagg gcgtagggta ctgcagtctg gaatctacgc ttgttcagac tttgtactag 1860
tttctttgtc tggccatccg ggtaacccat gccggacgca aaatagacta ctgaaaattt 1920
ttttgctttg tggttgggac tttagccaag ggtataaaag accaccgtcc ccgaattacc 1980
tttcctcttc ttttctctct ctccttgtca actcacaccc gaaatcgtta agcatttcct 2040
tctgagtata agaatcattc aaacacgtga tgctgcgacg aactctgttc ggattctctc 2100
acgaactcaa ggctatccac tccactgtgc ccaacctggg catgtgccga ggcggaaagt 2160
ctattgctcc ttctatgtct atgtcctcta ctacttctgt gtccaacgag gacggcgtgc 2220
cccgacgaat cgccggtcac cactctaacc tttgggacga cgactctatc gcttctctgt 2280
ctacttccta cgaggcccct tcttaccgaa agcgagctga caagctcatc ggtgaggtta 2340
agaacatttt cgaccttatg tccgttgagg acggcgtttt cacttcccct ctgtctgacc 2400
tgcaccaccg actgtggatg gttgactccg ttgagcgact gggtattgac cgacacttca 2460
aggacgagat caactccgcc ctggaccatg tatactccta ctggactgag aagggtattg 2520
gtcgaggtcg agagtccggc gtcactgacc ttaactctac cgctctgggt ctgcgaaccc 2580
tgcgactgca cggatacact gtgtcttctc acgttcttga ccacttcaag aacgagaagg 2640
gtcagttcac ttgctccgcc atccagactg agggtgagat tcgagacgtg cttaacctgt 2700
tccgagcctc tcttatcgct ttccccggtg agaagatcat ggaggccgct gagattttct 2760
ctactatgta cctaaaagac gctttgcaaa agatcccccc ttccggtctg tcccaagaaa 2820
tcgagtacct gctggagttc ggttggcaca ctaacctgcc tcggatggag acccgaatgt 2880
acattgacgt gttcggtgag gacaccactt tcgagactcc ttaccttatt cgagagaagc 2940
tgctggaact ggccaagctg gagttcaaca ttttccactc tcttgttaag cgagagctac 3000
aatctctgtc ccgatggtgg aaggactacg gattccctga gattactttc tcccgacacc 3060
gacacgttga gtactacacc ctggccgctt gcatcgctaa cgaccctaag cactccgctt 3120
tccgacttgg tttcggaaag atttcccaca tgattactat ccttgacgac atctacgaca 3180
ctttcggcac tatggaggaa ctgaagctgc tcaccgccgc tttcaagcga tgggacccct 3240
cttccattga gtgcctgcct gactacatga agggcgttta catggccgtg tacgacaaca 3300
tcaacgagat ggcccgcgag gcacagaaga ttcagggttg ggacaccgtg tcctacgctc 3360
gaaagtcttg ggaggctttc atcggcgcct acatccaaga ggccaagtgg atctcctccg 3420
gatacctgcc tactttcgac gagtacctgg agaacggaaa ggtttccttc ggatctcgaa 3480
ttaccaccct tgagcctatg cttacccttg gtttccccct gcctcctagg attctgcaag 3540
agattgactt cccttctaag ttcaacgacc tcatttgcgc cattctgcga ctgaagggtg 3600
acacccagtg ctacaaggct gaccgagccc gaggtgaaga ggcttccgct gtgtcttgct 3660
acatgaagga ccaccccggt attactgagg aagatgccgt taaccaggtg aacgctatgg 3720
tggacaacct cactaaggaa ctgaactggg agctgctgcg acctgactcc ggcgtgccca 3780
tctcttacaa gaaggtggct ttcgacattt gccgagtgtt ccactacgga tacaagtacc 3840
gagacggatt ctccgtcgcc tccattgaga ttaagaacct cgtcacccga accgtcgttg 3900
agaccgtgcc tctttaggga acccgaaact aaggatccgg tacctccatg gcctgtcccc 3960
acgttgccgg tcttgcctcc tactacctgt ccatcaatga cgaggttctc acccctgccc 4020
aggtcgaggc tcttattact gagtccaaca ccggtgttct tcccaccacc aacctcaagg 4080
gctctcccaa cgctgttgcc tacaacggtg ttggcattta ggcaattaac agatagtttg 4140
ccggtgataa ttctcttaac ctcccacact cctttgacat aacgatttat gtaacgaaac 4200
tgaaatttga ccagatattg ttgtaaatag aaaatctggc ttgtaggtgg caaaatcccg 4260
tctttgttca tcaattccct ctgtgactac tcgtcatccc tttatgttcg actgtcgtat 4320
ttttattttc catacatacg caagtgagat gcctaggtca cctatattac ggaagcagtg 4380
gtactcaagc tcagaacgtg ctgcgacaag atcaacgaat actggctggc aagaacaagg 4440
acggcaatcg caccttcaac gacgaactca tatatcagcc tctcagagcg ggaggcattg 4500
gccttttgaa cattgtcgag caggcaaaga gtctccgtgc cacgcgagcg gctcgtttct 4560
ttcagggatg atactgctgc aagacaaaat gctatgtttc tcttttccag cgagacagta 4620
gaattgggct atgggcttga atcccaccct gctacgataa tgaacaagat tcagtggttc 4680
gagacgggaa gctggcactg gcgccacttc ctatctgact tcatggaccg atgtctagga 4740
cttgcaaggc tgtcttctat agctgtactg gcggagcctc cagccgactt gcgccagagg 4800
gcgcagtggt caagagagag gacgaaagtt gacctgttcg aacgccagcg tcgtgaaaac 4860
ccgatcgcaa ccatgtctcg ggagcaaggt gcatatctcg agtcgcaaat cgatgtccct 4920
gtcaaggtgg caaaggacag ctggcgagat cgtttcaagg ccatgaagaa caagcactgg 4980
ttggggacac tgccatccga gcttgatccg aaccacctac actggcttaa gacaaatcag 5040
ttcctgagca agcactaccg acgagatgat aagagcgctg ctacgctaca catgttgcgg 5100
ctgagccgcc tccccttcac ccggtggtac cctaaatcaa aatctgtcca gtacagagac 5160
attggatgta gttcttgtca actcttcaaa cagaagaact acctccacga gcaccttttc 5220
gtcaattgtc cagcgtccaa ggccatctgc cagacgataa gcttggcgta atcatggtca 5280
tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga 5340
agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg 5400
cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc 5460
caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac 5520
tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata 5580
cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa 5640
aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct 5700
gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa 5760
agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg 5820
cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcaatgctca 5880
cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa 5940
ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg 6000
gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg 6060
tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg 6120
acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc 6180
tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag 6240
attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac 6300
gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc 6360
ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag 6420
taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt 6480
ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag 6540
ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca 6600
gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact 6660
ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca 6720
gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg 6780
tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc 6840
atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg 6900
gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca 6960
tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt 7020
atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc 7080
agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc 7140
ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca 7200
tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa 7260
aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat 7320
tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa 7380
aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa 7440
accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc cagatcctct 7500
agagtcgaag cggccgctat gtctgataaa aggatgtaac ataggcaagc tgctcgtgag 7560
tgttgagtac gaaccttaga tccaaatcac ccgcacccac ggatatactt gcttgaatat 7620
acagtagtat gcggccgctt cgacaccata tcatataaaa ctaacaatgc agtcgacgag 7680
tatctgtctg actcgtcatt gccgcctttg gagtacgact ccaactatga gtgtgcttgg 7740
atcactttga cgatacattc ttcgttggag gctgtgggtc tgacagctgc gttttcggcg 7800
cggttggccg acaacaatat cagctgcaac gtcattgctg gctttcatca tgatcacatt 7860
tttgtcggca aaggcgacgc ccagagagcc attgacgttc tttctaattt ggaccgatag 7920
ccgtatagtc cagtctatct ataagttcaa ctaactcgta actattacca taacatatac 7980
ttcactgccc cagataaggt tccgataaaa agttctgcag actaaattta tttcagtctc 8040
ctcttcacca ccaaaatgcc ctcctacgaa gctcgagcta acgtccacaa gtccgccttt 8100
gccgctcgag tgctcaagct cgtggcagcc aagaaaacca acctgtgtgc ttctctggat 8160
gttaccacca ccaaggagct cattgagctt gccgataagg tcggacctta tgtgtgcatg 8220
atcaagaccc atatcgacat cattgacgac ttcacctacg ccggcactgt gctccccctc 8280
aaggaacttg ctcttaagca cggtttcttc ctgttcgagg acagaaagtt cgcagatatt 8340
ggcaacactg tcaagcacca gtacaagaac ggtgtctacc gaatcgccga gtggtccgat 8400
atcaccaacg cccacggtgt acccggaacc ggaatcattg ctggcctgcg agctggtgcc 8460
gaggaaactg tctctgaaca gaagaaggag gacgtctctg actacgagaa ctcccagtac 8520
aaggagttcc tggtcccctc tcccaacgag aagctggcca gaggtctgct catgctggcc 8580
gagctgtctt gcaagggctc tctggccact ggcgagtact ccaagcagac cattgagctt 8640
gcccgatccg accccgagtt tgtggttggc ttcattgccc agaaccgacc taagggcgac 8700
tctgaggact ggcttattct gacccccggg gtgggtcttg acgacaaggg agacgctctc 8760
ggacagcagt accgaactgt tgaggatgtc atgtctaccg gaacggatat cataattgtc 8820
ggccgaggtc tgtacggcca gaaccgagat cctattgagg aggccaagcg ataccagaag 8880
gctggctggg aggcttacca gaagattaac tgttagaggt tagactatgg atatgtcatt 8940
taactgtgta tatagagagc gtgcaagtat ggagcgcttg ttcagcttgt atgatggtca 9000
gacgacctgt ctgatcgagt atgtatgata ctgcacaacc tgtgtatccg catgatctgt 9060
ccaatggggc atgttgttgt gtttctcgat acggagatgc tgggtacaag tagctaatac 9120
gattgaacta cttatactta tatgaggctt gaagaaagct gacttgtgta tgacttattc 9180
tcaactacat ccccagtcac aataccacca ctgcactacc actacaccaa aaccatgatc 9240
aaaccaccca tggacttcct ggaggcagaa gaacttgtta tggaaaagct caagagagag 9300
aagccaagat actatcaaga catgtgtcgc aacttcaagg aggaccaagc tctgtacacc 9360
gagaaacagg cctttgtcga cgatctggcc ctttcgtctc gcgcgtttcg gtgatgacgg 9420
tgaaaacctc t 9431
<210> 10
<211> 8404
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac 60
aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg 120
catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg 180
taaggagaaa ataccgcatc aggcgccatt cgccattcag gctgcgcaac tgttgggaag 240
ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa 300
ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca 360
gtgaattcga gctcggtacc cgcttttcgt agataatgga atacaaatgg atatccagag 420
tatacacatg gatagtatac actgacacga caattctgta tctctttatg ttaactactg 480
tgaggcgtta aatagagctt gatatataaa atgttacatt tcacagtctg aacttttgca 540
gattacctaa tttggtaaga tattaattat gaactgaaag ttgatggcat ccctaaattt 600
gatgaaaggg ggatcccccg ggttcgaagg taccaaggaa gcatgcggta cgtaataaga 660
gcctcgacct gggcaggggt gagaacctcg tcattgatgg acaggtagta ggaggcaaga 720
ccggcaacgt ggggacaggc catggaggta ccggagtaga ccaaagtacc agagtcggac 780
tggtaagagg cagagatgat atcggagccg ggggcaaaga catcaacaca agttccgtag 840
ttggatccct gtccaccgga ccagacggag atcttatcgc tagagtcaat ggaacccaca 900
gtgatgatac cagaggtgga gcctccaatg ttaccgggag agtcgttaca ggcgtccacg 960
gcatcgtttc ccgcagcgat ggcgacaagc agaccctcct gggtagctcg agaccatagg 1020
gcgtcctggg aagcggactt gggtcctcct ccagagaagt tcagcactcc tcgaggcaga 1080
gtgtctcgct tggagatgta gtcgttgaga gcccaggtga agccctggtt gatgacggag 1140
agagctgcgg atcggcctgc aaacaccttg acggccacca gcttggtgtt ggcgtcgact 1200
ccgtatgtct ttcctcccac ggtacctgca acgtgagtgc cgtgaccgag aagatcagcg 1260
ttctgtgtgt cagcgaagtt ggctccccag acagctcggc ctccgaactc ggagtgggtg 1320
gttcggatac cagagtcaac aacgtaagaa acggtggggt gcttgccaac tgtctctcga 1380
acgtaggcgt agtttccagt ctgggccttc ttatgagaga ttctagacag gccccattga 1440
gtgacgggag tagtctggat agctcgcttg gcattagaag aagcaggaat ctcggggaga 1500
gacacaatgg tatcgggctc gaccgtcaga acagacgact ccttgagctt gtcaacgata 1560
gtggagttga actttccaac aaatccgaga aagccagatc catccgtgta gacaggaaga 1620
tcaagagcag aggtgatacc gttggacgag tactagtgca tgctgaggtg tctcacaagt 1680
gccgtgcagt cccgccccca cttgcttctc tttgtgtgta gtgtacgtac attatcgaga 1740
ccgttgttcc cgcccacctc gatccggcat gctgaggtgt ctcacaagtg ccgtgcagtc 1800
ccgcccccac ttgcttctct ttgtgtgtag tgtacgtaca ttatcgagac cgttgttccc 1860
gcccacctcg atccggcatg ctgaggtgtc tcacaagtgc cgtgcagtcc cgcccccact 1920
tgcttctctt tgtgtgtagt gtacgtacat tatcgagacc gttgttcccg cccacctcga 1980
tccggcatgc tgaggtgtct cacaagtgcc gtgcagtccc gcccccactt gcttctcttt 2040
gtgtgtagtg tacgtacatt atcgagaccg ttgttcccgc ccacctcgat ccggcatgca 2100
ctgatcacgg gcaaaagtgc gtatatatac aagagcgttt gccagccaca gattttcact 2160
ccacacacca catcacacat acaaccacac acatccacgt gatgtccgcc cgaggtctca 2220
acaaaatctc ttgctccctc aacctacaga ccgagaagct gtgctacgag gacaacgaca 2280
acgacctcga cgaagagctg atgcctaagc acatcgctct catcatggac ggaaaccgac 2340
gatgggccaa ggacaagggt ctggaggtgt acgagggcca caagcacatt attcctaagc 2400
tgaaggaaat ttgcgacatt tcttctaagc tggggataca gattattacc gctttcgctt 2460
tctctaccga gaactggaag cgatctaagg aagaggtgga cttcctgctc cagatgttcg 2520
aggaaatcta cgacgagttc tctcgatccg gcgtgcgagt gtccattatc ggatgcaagt 2580
ctgacctccc catgaccctc cagaagtgca tcgccctcac cgaagaaacc accaagggta 2640
acaagggtct gcacctcgtg attgctctga actacggcgg atactacgac attctccaag 2700
ctaccaagtc catcgtcaac aaggctatga acggactgct ggacgtggag gacattaaca 2760
agaacctgtt cgaccaagaa ttggaatcta agtgccccaa ccctgacctg ctcatccgaa 2820
ccggcggtga gcagcgagtg tctaacttcc tcctgtggca gctggcttac accgagttct 2880
acttcaccaa caccctgttc cctgacttcg gtgaagagga tctgaaggaa gctatcatga 2940
acttccagca gcgacaccga cgattcggtg gccacaccta ctagcacgtg ggaacccgaa 3000
actaaggatc cggtacctcc atggcctgtc cccacgttgc cggtcttgcc tcctactacc 3060
tgtccatcaa tgacgaggtt ctcacccctg cccaggtcga ggctcttatt actgagtcca 3120
acaccggtgt tcttcccacc accaacctca agggctctcc caacgctgtt gcctacaacg 3180
gtgttggcat ttaggcaatt aacagatagt ttgccggtga taattctctt aacctcccac 3240
actcctttga cataacgatt tatgtaacga aactgaaatt tgaccagata ttgttgtaaa 3300
tagaaaatct ggcttgtagg tggcaaaatc ccgtctttgt tcatcaattc cctctgtgac 3360
tactcgtcat ccctttatgt tcgactgtcg tatttttatt ttccatacat acgcaagtga 3420
gatgcctagg ccagagagtc cagcttctgg atttcggagg cgatctggtc aacagtagcg 3480
ctactgtcaa agacaacgat gaactgatcc ttctcatcaa gatctcgctt gtggtgctta 3540
aacttcttgg actgcttagc gaccacggac agaatagatg agtaggcagc ggcggcaggg 3600
cccgtcgatg gggtttatat cgtgtttctg gacaagatac tatccagaaa ctgctctgat 3660
tgcgtcacga aacacggctc tcgccaagac gcgccaagac gatgccctca ttatcttggt 3720
gcaatacagg tggagtgtgt gcattagttg gtgccgtgcc ttctcacgat atctgacagg 3780
ggcttgggtt tgggttggaa gtcggattcc gctgaaacat ggtgttgtgc agatgtgagt 3840
ctctcaaagt cggttcatca tactccattg acatcatggt acagtgcgta cgtaaaagat 3900
ggcctgacta caagtagtgg gatctctcta acattgtcca tcacatcgta aatcacaccg 3960
cctatccatg aaccctgatg ccgatcgtcg aagttagacc agggccagtg gtgggtagcg 4020
tggggtaggc tgccactgcc gttggaaccc cttgtgtccc tggactacaa cttaaccaac 4080
atgtcagaca aaggagagaa aagtgtccgc tccaccgaac tgacgccagg aatgtaccgt 4140
ggcgctcaag aggtcatgtg taattaagga tacaagataa gctgcgccct tgaatgcaaa 4200
aagcacgcag ctcatatgtt aatttccaat caagcttggc gtaatcatgg tcatagctgt 4260
ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa 4320
agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac 4380
tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg 4440
cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc 4500
gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat 4560
ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca 4620
ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 4680
atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 4740
aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 4800
gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta 4860
ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 4920
ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 4980
acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 5040
gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat 5100
ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 5160
ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 5220
gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 5280
ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct 5340
agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt 5400
ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc 5460
gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac 5520
catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat 5580
cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg 5640
cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata 5700
gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta 5760
tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt 5820
gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag 5880
tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa 5940
gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc 6000
gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt 6060
taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc 6120
tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta 6180
ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa 6240
taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca 6300
tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac 6360
aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta 6420
ttatcatgac attaacctat aaaaataggc gtatcacgag gcccagatcc tctagagtcg 6480
aagcggccgc tatgtctgat aaaaggatgt aacataggca agctgctcgt gagtgttgag 6540
tacgaacctt agatccaaat cacccgcacc cacggatata cttgcttgaa tatacagtag 6600
tatgcggccg cttcgacacc atatcatata aaactaacaa tgcagtcgac gagtatctgt 6660
ctgactcgtc attgccgcct ttggagtacg actccaacta tgagtgtgct tggatcactt 6720
tgacgataca ttcttcgttg gaggctgtgg gtctgacagc tgcgttttcg gcgcggttgg 6780
ccgacaacaa tatcagctgc aacgtcattg ctggctttca tcatgatcac atttttgtcg 6840
gcaaaggcga cgcccagaga gccattgacg ttctttctaa tttggaccga tagccgtata 6900
gtccagtcta tctataagtt caactaactc gtaactatta ccataacata tacttcactg 6960
ccccagataa ggttccgata aaaagttctg cagactaaat ttatttcagt ctcctcttca 7020
ccaccaaaat gccctcctac gaagctcgag ctaacgtcca caagtccgcc tttgccgctc 7080
gagtgctcaa gctcgtggca gccaagaaaa ccaacctgtg tgcttctctg gatgttacca 7140
ccaccaagga gctcattgag cttgccgata aggtcggacc ttatgtgtgc atgatcaaga 7200
cccatatcga catcattgac gacttcacct acgccggcac tgtgctcccc ctcaaggaac 7260
ttgctcttaa gcacggtttc ttcctgttcg aggacagaaa gttcgcagat attggcaaca 7320
ctgtcaagca ccagtacaag aacggtgtct accgaatcgc cgagtggtcc gatatcacca 7380
acgcccacgg tgtacccgga accggaatca ttgctggcct gcgagctggt gccgaggaaa 7440
ctgtctctga acagaagaag gaggacgtct ctgactacga gaactcccag tacaaggagt 7500
tcctggtccc ctctcccaac gagaagctgg ccagaggtct gctcatgctg gccgagctgt 7560
cttgcaaggg ctctctggcc actggcgagt actccaagca gaccattgag cttgcccgat 7620
ccgaccccga gtttgtggtt ggcttcattg cccagaaccg acctaagggc gactctgagg 7680
actggcttat tctgaccccc ggggtgggtc ttgacgacaa gggagacgct ctcggacagc 7740
agtaccgaac tgttgaggat gtcatgtcta ccggaacgga tatcataatt gtcggccgag 7800
gtctgtacgg ccagaaccga gatcctattg aggaggccaa gcgataccag aaggctggct 7860
gggaggctta ccagaagatt aactgttaga ggttagacta tggatatgtc atttaactgt 7920
gtatatagag agcgtgcaag tatggagcgc ttgttcagct tgtatgatgg tcagacgacc 7980
tgtctgatcg agtatgtatg atactgcaca acctgtgtat ccgcatgatc tgtccaatgg 8040
ggcatgttgt tgtgtttctc gatacggaga tgctgggtac aagtagctaa tacgattgaa 8100
ctacttatac ttatatgagg cttgaagaaa gctgacttgt gtatgactta ttctcaacta 8160
catccccagt cacaatacca ccactgcact accactacac caaaaccatg atcaaaccac 8220
ccatggactt cctggaggca gaagaacttg ttatggaaaa gctcaagaga gagaagccaa 8280
gatactatca agacatgtgt cgcaacttca aggaggacca agctctgtac accgagaaac 8340
aggcctttgt cgacgatctg gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac 8400
ctct 8404
<210> 11
<211> 10923
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 11
gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac 60
aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg 120
catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg 180
taaggagaaa ataccgcatc aggcgccatt cgccattcag gctgcgcaac tgttgggaag 240
ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa 300
ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca 360
gtgaattcga gctcggtacc cgcttttcgt agataatgga atacaaatgg atatccagag 420
tatacacatg gatagtatac actgacacga caattctgta tctctttatg ttaactactg 480
tgaggcgtta aatagagctt gatatataaa atgttacatt tcacagtctg aacttttgca 540
gattacctaa tttggtaaga tattaattat gaactgaaag ttgatggcat ccctaaattt 600
gatgaaaggg ggatcccccg ggttcgaagg taccaaggaa gcatgcggta ctgcataaaa 660
cccgcataca cgtgcataat acttgcataa cgatagcata taaactgcac gattgctgca 720
cttgacttta tatcaaactc aatatcagca ccacccgatt ctctgattat ttcatcaact 780
ataggagaga atgagttggt gataagacat cggtaccaat aaaaatcacg tgactattcc 840
acaggccttc tgttatatca tcagatgcct ttgaacattc tcttgtcgaa ctgagtccaa 900
tttcaacaac tctttccaca gtgcagtgcc cggttaggcc aattagggtt tggcaaaaat 960
cggcaatata agacagcaaa ctaaaattta tataaagccc atcgctgcct tgttggagcc 1020
tcgttttttc ccacaacaca agcttagcag taattgggtc ttctattctc aattgcacgc 1080
tccccccctc tccttacaat tcgctcactc tcccgactat ttaacaacgc acgacaaaca 1140
tagcttcaat tccccattat gattatctgg ttatcggtgg aggctaagat ggtgtagctt 1200
ctgcttgtcg agccgtctcg tacggcgcca agacgctgct ggtcgagttc aaggcgctgg 1260
gaggcacctg cgtcaacgtg ggctgtgccc aaaaagatca cgtggaacgc ctccgatctg 1320
gcgggccgaa tccgacaggc ccaggagtac ggcttctccg acgtggaccc caagtccgcc 1380
gacaactttg actggaccgg attcaaggcc aaacgagacg cctacgtcaa acgactcaat 1440
ggcatctacg aacgaaacct ccagaaggag ggcgtggagt acgtgtttgg ttgggccacc 1500
ctctacaagc aggagggtca ggagttcccc ctggtactgg ccaagagcga cgatggcaac 1560
gccaagttgt actccgcaaa gaagattaag attgccaccg gcggcaagcc cattctgccc 1620
gacgtgcctg gagccgagta cggcattgat tactagtggt gcatgctgag gtgtctcaca 1680
agtgccgtgc agtcccgccc ccacttgctt ctctttgtgt gtagtgtacg tacattatcg 1740
agaccgttgt tcccgcccac ctcgatccgg catgctgagg tgtctcacaa gtgccgtgca 1800
gtcccgcccc cacttgcttc tctttgtgtg tagtgtacgt acattatcga gaccgttgtt 1860
cccgcccacc tcgatccggc atgctgaggt gtctcacaag tgccgtgcag tcccgccccc 1920
acttgcttct ctttgtgtgt agtgtacgta cattatcgag accgttgttc ccgcccacct 1980
cgatccggca tgctgaggtg tctcacaagt gccgtgcagt cccgccccca cttgcttctc 2040
tttgtgtgta gtgtacgtac attatcgaga ccgttgttcc cgcccacctc gatccggcat 2100
gcactgatca cgggcaaaag tgcgtatata tacaagagcg tttgccagcc acagattttc 2160
actccacaca ccacatcaca catacaacca cacacatcca cgtgatgcta caagcagcta 2220
ttggaaagat tgtgggattt gcggtcaacc gacccatcca cacagttgtc ctgacgtcca 2280
tcgtggcgtc aaccgcatac ctcgccatcc tcgacattgc catcccgggt ttcgagggca 2340
cacaacccat ctcatactac caccctgcag caaaatctta cgacaaccct gctgattgga 2400
cccacattgc agaggccgac atcccttcag acgcctaccg acttgcattt gcccagatcc 2460
gtgtcagtga tgttcagggc ggagaggccc ccaccatccc tggcgccgtg gccgtgtctg 2520
atctcgacca cagaatcgtc atggactaca aacagtgggc cccctggacc gccagcaacg 2580
agcagatcgc ctcggagaac cacatctgga agcactcctt caaggaccac gtggccttca 2640
gctggatcaa gtggttccga tgggcctacc tgcgtttgtc cactctcatc cagggggcag 2700
acaacttcga cattgccgtg gtcgcccttg gctatcttgc catgcactac accttcttca 2760
gtctcttccg atccatgcga aaggttggct cgcacttttg gcttgcctcc atggctctgg 2820
tctcttccac cttcgctttc ctgcttgcgg tggtggcttc ctctagcctg ggttaccgac 2880
ctagcatgat caccatgtcc gagggcctgc ccttcctcgt ggtcgccatt ggctttgacc 2940
gaaaggtcaa cctggctagc gaggtgctca catccaagag cagccagctc gctcccatgg 3000
tgcaggtgat cacaaagatc gcctccaagg cgctgtttga gtacagcctt gaggtggccg 3060
ccctgtttgc tggcgcctat accggagttc ctcgactgtc ccagttttgc ttcttatctg 3120
cttggatcct catcttcgac tacatgtttt tgctgacctt ctactctgct gtccttgcta 3180
tcaagtttga gatcaatcac attaagcgaa accgaatgat ccaggatgct ctcaaggagg 3240
atggtgtatc tgctgctgtt gccgagaagg tagccgactc ttctcccgac gccaagctcg 3300
accgaaagtc cgacgtttct ctttttggag cctctggcgc cattgcggtg ttcaagatct 3360
tcatggtcct tgggttcctt ggtctcaacc tcatcaacct gactgccatc cctcaccttg 3420
gcaaggcggc cgccgctgcc cagtctgtga ctcccatcac cctctccccc gagcttctcc 3480
atgccatccc cgcctctgtg cccgttgttg tcacctttgt gcccagcgtt gtgtacgagc 3540
actcccagct cattctgcag ctggaggacg ccctcactac cttcctggct gcctgctcca 3600
aaactattgg tgaccccgtc atctccaagt acatcttcct gtgcctgatg gtctccaccg 3660
ccctgaacgt ctacctgttt ggagccaccc gagaagttgt gcgaacccag tctgtgaagg 3720
tggttgagaa gcacgttcct atcgtcattg agaagcccag cgagaaggag gaggacacct 3780
cttctgaaga ctccattgag ctgactgtcg gaaagcagcc caagcccgtg accgagaccc 3840
gttctctgga cgacctagag gctatcatga aggcaggtaa gaccaagctt ctggaggacc 3900
acgaggttgt caagctctct ctcgagggca agcttccttt gtatgctctt gagaagcagc 3960
ttggtgacaa cacccgagct gttggcatcc gacgatctat catctcccag cagtctaata 4020
ccaagacttt agagacctca aagcttcctt acctgcacta cgactacgac cgtgtttttg 4080
gagcctgttg cgagaacgtt attggttaca tgcctctccc cgttggtgtt gctggcccca 4140
tgaacattga tggcaagaac taccacattc ctatggccac cactgagggt tgtcttgttg 4200
cctcaaccat gcgaggttgc aaggccatca acgccggtgg cggtgttacc actgtgctta 4260
ctcaggacgg tatgacacga ggtccttgtg tttccttccc ctctctcaag cgggctggag 4320
ccgctaagat ctggcttgat tccgaggagg gtctcaagtc catgcgaaag gccttcaact 4380
ccacctctcg atttgctcgt ctccagtctc ttcactctac ccttgctggt aacctgctgt 4440
ttattcgatt ccgaaccacc actggtgatg ccatgggcat gaacatgatc tccaagggcg 4500
tcgaacactc tctggccgtc atggtcaagg agtacggctt ccctgatatg gacattgtgt 4560
ctgtctcggg taactactgc actgacaaga agcccgcagc gatcaactgg atcgaaggcc 4620
gaggcaagag tgttgttgcc gaagccacca tccctgctca cattgtcaag tctgttctca 4680
aaagtgaggt tgacgctctt gttgagctca acatcagcaa gaatctgatc ggtagtgcca 4740
tggctggctc tgtgggaggt ttcaatgcac acgccgcaaa cctggtgacc gccatctacc 4800
ttgccactgg ccaggatcct gctcagaatg tcgagtcttc caactgcatc acgctgatga 4860
gcaacgtcga cggtaacctg ctcatctccg tttccatgcc ttctatcgag gtcggtacca 4920
ttggtggagg tactattttg gagccccagg gggctatgct ggagatgctt ggcgtgcgag 4980
gtcctcacat cgagaccccc ggtgccaacg cccaacagct tgctcgcatc attgcttctg 5040
gagttcttgc agcggagctt tcgctgtgtt ctgctcttgc tgccggccat cttgtgcaaa 5100
gtcatatgac ccacaaccgg tcccaggctc ctactccggc caagcagtct caggccgatc 5160
tgcagcgtct acaaaacggt tcgaatattt gcatacggtc atagcacgtg ggaacccgaa 5220
actaaggatc caactacgga acttgtgttg atgtctttgc ccccggctcc gatatcatct 5280
ctgcctctta ccagtccgac tctggtactt tggtctactc cggtacctcc atggcctgtc 5340
cccacgttgc cggtcttgcc tcctactacc tgtccatcaa tgacgaggtt ctcacccctg 5400
cccaggtcga ggctcttatt actgagtcca acaccggtgt tcttcccacc accaacctca 5460
agggctctcc caacgctgtt gcctacaacg gtgttggcat ttaggcaatt aacagatagt 5520
ttgccggtga taattctctt aacctcccac actcctttga cataacgatt tatgtaacga 5580
aactgaaatt tgaccagata ttgttgtaaa tagaaaatct ggcttgtagg tggcaaaatg 5640
cggcgtcttt gttcatcaat tccctctgtg actactcgtc atccctttat gttcgactgt 5700
cgtatttctt attttccata catatgcaag tgagatgccc gtgtcctagg cttctttgct 5760
ctcgagaccc agcctaagcg agtggcggtg gttggaggag gctacattga cgtggagctg 5820
gccggtgtct tccacggact caactccgaa accacccttt tctgtcgagg ccagacggtg 5880
ctccgagcgt tcgacatcat gatccaggac accatcaccg actactaaca agaagggcat 5940
caacgtgctc aagggctctg gcgtcaagaa gattgtcaag aaggataatg gcgagctcac 6000
ctacgggcag gatggcgccg agaatgatat cactctcgat tcgctcattt ggatattgga 6060
cgagagcctc tcaaggacac cctcaacctg agcgagtttg gcatcaagac caacaagcgg 6120
ggctacattg aggtcaacga gtgccagcaa tcgtctttcg acaacattta ttcgcttgga 6180
gacgtttgct gcaaggtcga gctgacccca tggctattgc tactggacga aatctgtcca 6240
accggctgtt tggtcccgct gagttcaagg atcagaagca ggactacacc gatgttcctt 6300
cggccgtatt ttcccacccc gaggttggct ccaacggcat cacggaggct gccgccaagg 6360
agcagtatgg tgaggacaac gtcaagatct acacctctaa gtttgtcgcc atgtactacg 6420
ccatgttgca ggagaaagcc cctaccgcct acaagctggt gtgtgtcgga aaggacgaga 6480
aggttgttgg tctgcacatt gttggcaccg actcttccga gattctgcag ggttttggcg 6540
tggccgttcg aatgggagct accaaggccg atttcgacaa tgttgtggct ctttgtccca 6600
cttctgccga ggagctggtg acaatgggat ataccgggag cgcggcgaga gatttctctg 6660
gtttgggcta tcgatattta atcaactaga ctccactgtt gctgtcacag ggtcagagaa 6720
gatactagag tacggaagtg cgttccgttg aagcttggcg taatcatggt catagctgtt 6780
tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa 6840
gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact 6900
gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc 6960
ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg 7020
ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc 7080
cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag 7140
gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca 7200
tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca 7260
ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg 7320
atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcaatgct cacgctgtag 7380
gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt 7440
tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca 7500
cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg 7560
cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt 7620
tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc 7680
cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg 7740
cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg 7800
gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta 7860
gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg 7920
gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg 7980
ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc 8040
atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc 8100
agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc 8160
ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag 8220
tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat 8280
ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg 8340
caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt 8400
gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag 8460
atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg 8520
accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt 8580
aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct 8640
gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac 8700
tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat 8760
aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat 8820
ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca 8880
aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat 8940
tatcatgaca ttaacctata aaaataggcg tatcacgagg cccagatcct ctagagtcga 9000
agcggccgct atgtctgata aaaggatgta acataggcaa gctgctcgtg agtgttgagt 9060
acgaacctta gatccaaatc acccgcaccc acggatatac ttgcttgaat atacagtagt 9120
atgcggccgc ttcgacacca tatcatataa aactaacaat gcagtcgacg agtatctgtc 9180
tgactcgtca ttgccgcctt tggagtacga ctccaactat gagtgtgctt ggatcacttt 9240
gacgatacat tcttcgttgg aggctgtggg tctgacagct gcgttttcgg cgcggttggc 9300
cgacaacaat atcagctgca acgtcattgc tggctttcat catgatcaca tttttgtcgg 9360
caaaggcgac gcccagagag ccattgacgt tctttctaat ttggaccgat agccgtatag 9420
tccagtctat ctataagttc aactaactcg taactattac cataacatat acttcactgc 9480
cccagataag gttccgataa aaagttctgc agactaaatt tatttcagtc tcctcttcac 9540
caccaaaatg ccctcctacg aagctcgagc taacgtccac aagtccgcct ttgccgctcg 9600
agtgctcaag ctcgtggcag ccaagaaaac caacctgtgt gcttctctgg atgttaccac 9660
caccaaggag ctcattgagc ttgccgataa ggtcggacct tatgtgtgca tgatcaagac 9720
ccatatcgac atcattgacg acttcaccta cgccggcact gtgctccccc tcaaggaact 9780
tgctcttaag cacggtttct tcctgttcga ggacagaaag ttcgcagata ttggcaacac 9840
tgtcaagcac cagtacaaga acggtgtcta ccgaatcgcc gagtggtccg atatcaccaa 9900
cgcccacggt gtacccggaa ccggaatcat tgctggcctg cgagctggtg ccgaggaaac 9960
tgtctctgaa cagaagaagg aggacgtctc tgactacgag aactcccagt acaaggagtt 10020
cctggtcccc tctcccaacg agaagctggc cagaggtctg ctcatgctgg ccgagctgtc 10080
ttgcaagggc tctctggcca ctggcgagta ctccaagcag accattgagc ttgcccgatc 10140
cgaccccgag tttgtggttg gcttcattgc ccagaaccga cctaagggcg actctgagga 10200
ctggcttatt ctgacccccg gggtgggtct tgacgacaag ggagacgctc tcggacagca 10260
gtaccgaact gttgaggatg tcatgtctac cggaacggat atcataattg tcggccgagg 10320
tctgtacggc cagaaccgag atcctattga ggaggccaag cgataccaga aggctggctg 10380
ggaggcttac cagaagatta actgttagag gttagactat ggatatgtca tttaactgtg 10440
tatatagaga gcgtgcaagt atggagcgct tgttcagctt gtatgatggt cagacgacct 10500
gtctgatcga gtatgtatga tactgcacaa cctgtgtatc cgcatgatct gtccaatggg 10560
gcatgttgtt gtgtttctcg atacggagat gctgggtaca agtagctaat acgattgaac 10620
tacttatact tatatgaggc ttgaagaaag ctgacttgtg tatgacttat tctcaactac 10680
atccccagtc acaataccac cactgcacta ccactacacc aaaaccatga tcaaaccacc 10740
catggacttc ctggaggcag aagaacttgt tatggaaaag ctcaagagag agaagccaag 10800
atactatcaa gacatgtgtc gcaacttcaa ggaggaccaa gctctgtaca ccgagaaaca 10860
ggcctttgtc gacgatctgg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc 10920
tct 10923

Claims (10)

1. A genetically engineered yarrowia lipolytica strain for producing alpha-pinene is characterized in that a CRISPR/Cas9 system is utilized to make 3-hydroxy-3-methylglutaryl-CoA reductase geneHMG1Optimized and truncated neroli diphosphate synthase 1 geneNDPS1And optimized and truncated pinene synthase genes with TEF promoterP TEF -tPSSequentially introducing uracil and leucine auxotroph yarrowia lipolytica chromosome to construct and obtain a yarrowia lipolytica genetic engineering bacterium YT-14 capable of producing alpha-pinene; then utilizing yarrowia lipolytica rDNA locus multicopy integrative plasmid pUC19-rDNA-HisG to construct optimized and truncated pinene synthase gene containing TEF promoterP TEF -tPSAnd the mevalonate kinase geneERG8And transforming the yarrowia lipolytica genetic engineering bacterium YT-14 to obtain the yarrowia lipolytica genetic engineering bacterium YT-29; then utilizes yarrowia lipolytica integrated plasmid pINA1269 to construct gene containing 3-hydroxy-3-methylglutaryl-CoA reductaseHMG1And mevalonate kinase gene with MBP labelERG12And transforming yarrowia lipolytica genetic engineering bacterium YT-29 to obtain yarrowia lipolytica genetic engineering bacterium YT-30, wherein,
the optimized and truncated pinene synthase gene with TEF promoterP TEF -tPSThe nucleotide sequence of (2) is shown as SEQ ID No. 1; the nucleotide sequence of the MBP tag is shown as SEQ ID No. 2; the nucleotide sequence of the plasmid pUC19-rDNA-HisG is shown as SEQ ID No. 5; optimized and truncated neroli diphosphate synthase 1 geneNDPS1See patent application No. 2016817882. X, plasmid pINA1269 was obtained by the preparation method described in Madzak C, traton B and Roland SB. Strong hybrid promoters and integrative expression/secretion vectors for quasi-constitutive expression of heterologous proteins in the yeast Yarrowia lipolytica. J Mol Microbiol Biotechnol. (2000) 2 (2): 207-216.
2. Use of the genetically engineered yarrowia lipolytica bacterium for producing alpha-pinene of claim 1 in the production of alpha-pinene.
3. The use of claim 2, wherein the α -pinene producing yarrowia lipolytica genetically engineered strain utilizes glucose to produce α -pinene.
4. A genetically engineered yarrowia lipolytica strain for producing alpha-pinene is characterized in that yarrowia lipolytica integrated plasmid pINA1312 is utilized to construct a mevalonate kinase gene containing one copyERG12One copy of the AMP deaminase geneAMPDAnd one copy of an optimized and truncated pinene synthase gene with a TEF promoterP TEF -tPSThe yarrowia lipolytica genetic engineering bacterium YT-30 of claim 1 is transformed to obtain the yarrowia lipolytica genetic engineering bacterium YT-31 which takes glucose as a substrate to produce alpha-pinene, wherein, plasmid pINA1312 adopts Nicaud, J.M., madzak, C., van den Broek, P., gysler, C., duboc, P., niederbeger, P., gaillidin, C.2002.Protein expression and secretion in the yeastYarrowia lipolyticaFEMS Yeast Res,2 (3), 371-379;
wherein the optimized and truncated pinene synthase gene with TEF promoterP TEF -tPSThe nucleotide sequence of (2) is shown as SEQ ID No. 1.
5. The use of the genetically engineered yarrowia lipolytica bacterium of claim 4 for producing alpha-pinene in the production of alpha-pinene.
6. The use of the genetically engineered yarrowia lipolytica strain of claim 4 for the production of alpha-pinene using glucose or swill-cooked dirty oil.
7. A genetically engineered yarrowia lipolytica strain for producing alpha-pinene is characterized in that optimized xylose reductase gene is firstly adoptedXROptimized xylitol dehydrogenase geneXDHAnd xylose assimilating enzyme geneXKThe yarrowia lipolytica gene engineering bacterium YT-33 integrated into the rDNA multicopy site of the yarrowia lipolytica gene engineering bacterium YT-31 of claim 4 to construct the yarrowia lipolytica gene engineering bacterium YT-33 which metabolizes xylose to produce alpha-pinene, the optimized xylose reductase geneXRThe nucleotide sequence of (2) is shown as SEQ ID No. 6; the optimized xylitol dehydrogenase geneXDHThe nucleotide sequence of (2) is shown as SEQ ID No. 7; the xylose assimilating enzyme geneXKThe nucleotide sequence of (2) is shown as SEQ ID No. 8.
8. Use of the genetically engineered yarrowia lipolytica bacterium of claim 7 for producing alpha-pinene in the production of alpha-pinene.
9. Use of the genetically engineered yarrowia lipolytica bacterium of claim 7 for producing alpha-pinene in the production of alpha-pinene using xylose and glucose.
10. Use of the genetically engineered yarrowia lipolytica bacterium of claim 7 for production of alpha-pinene in xylose metabolism.
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