CN109652325B - Saccharomyces cerevisiae industrial strain for delta integration and secretory expression of cellulase and application - Google Patents

Abstract

The invention discloses a Saccharomyces cerevisiae industrial strain delta integrating and secreting expression cellulase and application thereof, and relates to a Saccharomyces cerevisiae industrial strain R delta BEC with a preservation number of CGMCC NO. 16825. The application of the industrial bacterial strain R delta BEC with the preservation number of CGMCC NO.16825 in ethanol production. The strain R delta BEC of the invention has the preservation number of CGMCC NO.16825 and secretes and expresses 5 types of cellulase: trichoderma reesei CBH1, CBH2 and EG 2; aspergillus aculeatus BGL1 and CBH 1. The strain can be used for efficiently degrading cellulose by cooperating with the existing commercialized cellulase, and the ethanol yield is improved.

Description

Saccharomyces cerevisiae industrial strain for delta integration and secretory expression of cellulase and application

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to a saccharomyces cerevisiae industrial strain for secretory expression of multiple cellulases and application thereof.

Background

The development and utilization of renewable energy is an active measure currently taken by human beings facing the problems of energy, environment, food and the like, and the fuel ethanol as a main biological energy source is widely concerned due to the unique automotive property. In 2005 + 2016, the global biofuel ethanol consumption increased from 3628 ten thousand tons to 7915 ten thousand tons, which increased by 7.3% every year. At present, over 40 countries and regions promote biofuel ethanol and vehicle ethanol gasoline, and the annual consumption of ethanol gasoline is about 6 hundred million tons, which accounts for about 60 percent of the total consumption of global gasoline. In China, in order to meet increasing energy requirements and improve air quality, fifteen departments such as the national development reform Commission, the national energy agency, the department of finance and the like, which develop 9.13.2017, jointly issue an implementation scheme about expanding the production of biofuel ethanol and popularizing and using vehicle ethanol gasoline, and grain fuel ethanol is developed moderately according to basic principles such as strict total amount control and multivariate development, the grain fuel ethanol total amount is scientifically and reasonably grasped, and cellulose fuel ethanol is vigorously developed. According to the plan, the full coverage of the ethanol gasoline is basically realized nationwide by 2020. It is estimated that the utilization amount of fuel ethanol in China will reach 1570 ten thousand tons by 2020, and the capacity gap is about 1275 ten thousand tons/year according to the current ethanol supply capacity of China. On the other hand, the annual available straw and forestry waste in China is more than 4 million tons, and if 30 percent of the straw and forestry waste is utilized, 2000 million tons of fuel ethanol can be produced. The development trend of biofuel technology is to produce fuel ethanol by utilizing lignocellulose efficiently and at low cost.

The main obstacle in producing fuel ethanol by using the lignocellulose for biotransformation at present is the lack of a technology for degrading lignocellulose at low cost: the enzyme dosage of the lignocellulose ethanol is 30-50 times of that of the starch ethanol, and the cost of the cellulase accounts for 20-30% of the total cost. A Combined Bioprocess (CBP) that combines cellulase production, hydrolysis and fermentation is considered a promising technology because of its ability to reduce hydrolysis and fermentation costs. The key point of the technology is to construct a microorganism which can effectively degrade cellulose polysaccharide into fermentation sugar and produce alcohol through fermentation. One strategy for expressing cellulase in Saccharomyces cerevisiae to make it capable of producing ethanol by fermentation of cellulose.

Over the years, various cellulases have been successfully expressed in Saccharomyces cerevisiae, but most of the research was performed in Saccharomyces cerevisiae laboratory strains, while less in industrial strains. Khramtsov et al (Khramtsov N, et al, Industrial year strain engineered to transfer ethanol from ligocellulosic biology, 2011,102(17):8310-8313) integrated three cellulase genes (Trichoderma reesei cbh2, egl2 and Aspergillus aculeatus bgl1) on the chromosome of the derivative strain of the Saccharomyces cerevisiae industrial strain using delta integration strategy and rDNA homologous recombination technique, and the resulting optimal strain was screened using 96 large-throughput to ferment 10% of the pretreated corn stover and 96h ethanol concentration reached 2.6% (v/v). G418 resistance screening markers are used in the construction of the recombinant strains, and the markers are not easy to remove in the recombinant strains, so that the recombinant strains are difficult to use for multiple times; shen and Yanxishan (Shenshen, Yanxishan. method for producing ethanol by fermenting Saccharomyces cerevisiae flora displaying cellulase. CN104673689A) are respectively displayed on the surface of a Saccharomyces cerevisiae industrial strain to express Trichoderma reesei cbh2, egl2 and Aspergillus aculeus bgl1 to obtain three recombinant strains, and the three recombinant strains are mixed according to the ratio of 2:1:1 after induction culture and are used in cellulose integrated biological processing. The recombinant strain is a mixed flora, and the proportion among the strains is very easily influenced by the fluctuation of fermentation culture conditions. In addition, cellulase is expressed on the surface of yeast, and the expression level of cellulase is directly related to the cell surface, but on the contrary, there is no such limitation in secretory expression. Another disadvantage of cell surface display is that the cellulase immobilized on the cell surface has a lower diffusion rate than the secreted enzyme.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide an industrial strain of Saccharomyces cerevisiae (Saccharomyces cerevisiae) for delta integration and secretory expression of cellulase.

The second purpose of the invention is to provide the application of the Saccharomyces cerevisiae industrial strain for delta integration and secretory expression of cellulase in ethanol production.

The technical scheme of the invention is summarized as follows:

an industrial strain R delta BEC of Saccharomyces cerevisiae (Saccharomyces cerevisiae) with a preservation number of CGMCC NO.16825 for delta integration and secretion expression of cellulase.

The application of the industrial strain R delta BEC with the preservation number of CGMCC NO.16825 in the production of ethanol.

The invention has the advantages that:

an industrial strain R delta BEC of Saccharomyces cerevisiae (Saccharomyces cerevisiae) for expressing cellulase delta integration and secretion, with the preservation number CGMCC NO.16825 for expressing 5 cellulases: trichoderma reesei CBH1, CBH2 and EG 2; aspergillus aculeatus BGL1 and CBH 1. The strain can be used for efficiently degrading cellulose by cooperating with the existing commercialized cellulase, and the ethanol yield is improved.

Drawings

FIG. 1 is a schematic diagram of a cellulase gene expression cassette.

FIG. 2 is a plasmid pG δ RU map.

FIG. 3 is a schematic diagram of cellulase gene delta integration plasmid.

FIG. 4 shows the alcohol production evaluation of Saccharomyces cerevisiae strains by fermenting corn cob with acid and alkali pretreatment.

Detailed Description

The present invention will be further described with reference to the following embodiments.

Example 1

Obtaining of host bacterium Kalpha

Spore-forming culture is carried out on high-activity dry yeast of Angel brewing, haploid strains with mating types of alpha types are obtained by screening, and reverse selection is carried out on a 5-fluoroorotic acid plate to obtain host bacteria K alpha. The method comprises the following specific steps:

(a) 0.01g of Angel high-activity dry yeast is inoculated into 5ml of YPD liquid culture medium (1% yeast powder, 2% peptone and 2% glucose) and cultured at 30 ℃ and 150rpm for 20 h.

(b) A part of the bacterial liquid is dipped by an inoculating needle to streak an YPD solid culture medium (1% yeast powder, 2% peptone, 2% glucose and 1.8% agar powder) plate, the plate is cultured at 30 ℃ until the bacterial colony is 2-3mm, a single bacterial colony is selected and inoculated into 3mL of the YPD liquid culture medium, the plate is cultured for 20h at 30 ℃ and 150rpm, 50 mu l of the bacterial liquid is inoculated into 3mL of the YPD liquid culture medium, and the plate is cultured for 6h at 30 ℃ and 150 rpm.

(c) The cells were collected by centrifugation, washed three times with sterile water, inoculated into 3mL of a pre-sporulation medium (potassium acetate 1%, yeast extract 1%, peptone 2%), and cultured at 28 ℃ for 12 hours at 150 rpm.

(d) The cells were collected by centrifugation, washed three times with sterile water, resuspended in 3mL of high performance liquid sporulation medium (potassium acetate 1%, yeast extract 0.1%, glucose 0.05%), and cultured at 28 ℃ and 250rpm for 5 days.

(e) The yeast spore culture was collected by centrifugation at 500rpm for 5min at room temperature and washed with demineralization buffer (10mmol/L dithiothreitol, 100mmol/L Tris-SO)₄pH9.4) resuspending the cells to OD₆₀₀Incubate at 30 ℃ for 10min, 5 ═ 5. Centrifuging at 500rpm at room temperature for 5min to collect the above yeast cells and spores, and adding protoplast buffer solution (2)1mol/L sorbitol, 10mmol/L potassium phosphate, pH7.2) resuspension to OD₆₀₀25. Adding 200mg/mL snailase to make the final concentration 0.5mg/OD₆₀₀And reacted at 30 ℃ for 30 min. The above reaction solution was centrifuged at 500rpm at room temperature for 5min to collect precipitates, which were washed once with an equal amount of 0.5% (V/V) Triton X-100, and then resuspended in 1/4, which was the total volume of the spore reaction solution. The spores were dispersed thoroughly by ultrasonic oscillation.

(f) Streaking YPD solid culture medium plate with the spore suspension obtained above, culturing at 30 deg.C to colony size of 2-3mm, randomly selecting 10 single colonies, inoculating into 3mL YPD liquid culture medium, culturing at 30 deg.C and 150rpm for 20 hr, centrifuging, collecting thallus, and extracting chromosome for identification of mating type of strain.

(g) The above chromosome is used as template, and primers MAT-F (SEQ ID NO.1) and MAT-a (SEQ ID NO.2)

And MAT-alpha (SEQ ID NO.3) as a primer to carry out PCR reaction, wherein the reaction system is as follows:

reaction conditions are as follows: 3min at 94 ℃ (30 sec at 94 ℃,30 sec at 54 ℃, 1min at 72 ℃) x 30, 10min at 72 ℃, and pause at 25 ℃.

The PCR product was subjected to gel electrophoresis to obtain a strain of the alpha mating type having a 404bp band (a haploid strain having a mating type showed a 544bp band, an alpha haploid strain showed a 404bp band, and an alpha diploid strain showed two bands of 544bp and 404 bp). In this experiment, 3 strains of the α mating type were obtained.

(h) One of the above-obtained a.alpha.mating strains was streaked on YPD solid plates, cultured at 30 ℃ to 2-3mm, and an appropriate amount of cells were picked up and applied to 5-FOA (5-fluoroorotic acid) plates (YNB (Bacto Yeast Nitrogen Base with Amino Acids (Difco)) 0.67%, 5-FOA 0.1%, glucose 2%, threonine 150mg/L, tyrosine 30mg/L, valine 150mg/L, lysine 30mg/L, glutamic acid 100mg/L, serine 150mg/L, aspartic acid 100mg/L, methionine 20mg/L, phenylalanine 50mg/L, isoleucine 30mg/L, arginine 20mg/L, adenine 100mg/L, tryptophan 200mg/L, histidine 300mg/L, leucine 400mg/L with a sterile tip, agar powder 2%), and cultured at 30 deg.C for 2 days.

(i) The single colonies obtained above were inoculated into 2mL YPD liquid medium, cultured at 30 ℃ for 20 hours, 50. mu.L each of which was inoculated into 5mL CMG medium (0.67% YNB, 2% glucose, threonine 150mg/L, tyrosine 30mg/L, valine 150mg/L, lysine 30mg/L, glutamic acid 100mg/L, serine 150mg/L, aspartic acid 100mg/L, methionine 20mg/L, phenylalanine 50mg/L, isoleucine 30mg/L, arginine 20mg/L, adenine 100mg/L, tryptophan 200mg/L, histidine 300mg/L, leucine 400mg/L, uracil 50mg/L) and 5mL CMG medium (CMG medium)^-UCulturing in CMG medium (default uracil in CMG medium) at 30 deg.C and 150rpm for 2 days^-UThose that do not grow in the medium but grow optimally in the CMG medium (OD)₆₆₀Largest) was named K α as the host strain for the subsequent experiments.

Example 2

Construction of cellulase gene expression cassette delta integration plasmid

The schematic diagram of the cellulase gene expression cassette is shown in figure 1, wherein Ptpi is a promoter (saccharomyces cerevisiae triose phosphate isomerase promoter), xyn2s (trichoderma reesei xylanase secretion peptide) is a secretion peptide, cellulose gene is a cellulase gene exon sequence, and Tadh I (saccharomyces cerevisiae alcohol dehydrogenase terminator) is a terminator. The cellulase genes include cbh1, cbh2, egl2 derived from Trichoderma reesei, and bgl1, cbh1 derived from Aspergillus aculeatus.

The above five cellulase gene expression cassettes were constructed according to our work (flood liberation, construction of cellulase-expressing Saccharomyces cerevisiae engineered strain and its research on alcohol production by lignocellulose fermentation, Phthal university proceedings 2014, Zhang Weina, construction of cellulase gene-expressing Saccharomyces cerevisiae strain and its evaluation of substrate utilization, Phthal university proceedings 2012, and less blue, construction of alcohol-producing engineered strain using lignocellulose hydrolysate, 2011 Phthal university proceedings 2011, Jiefang Hong, et al development of a cellulose hydrolysis strain and its research on alcohol production, Jiefang moisture reaction, world Journal of biology & Microbiology & technology 2014,30(11) 2985 and 2993; Zhang Weina, et al composite of cellulose hydrolysis strain and strain 409 biology & technology,2014, 11) and J2945, CN102154344A), the corresponding plasmids are pGEM-T easy-Ptpi-xyn2s-TCBHI-TadhI, pGEM-T easy-Ptpi-xyn2s-TCBHII-TadhI, YEplac195-Ptpi-xyn2s-TEGII-TadhI, pGEM-T easy-Ptpi-xyn2s-ABGL1-TadhI and pGEM-T easy-Ptpi-xyn2s-ACBH1-TadhI, the sequences of which are shown in SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8 in sequence.

Plasmid pUC18-RYUR (patent US2010/0291652A1, sequence shown in SEQ ID NO.9) was digested simultaneously with XbaI and BamHI, and subjected to agarose gel electrophoresis to recover a 0.4kb DNA fragment as insert 1.

The plasmid pBluescript II SK (+) (GenBank: X52328.1) was digested with XbaI and BamHI, and a 2.9kb DNA fragment was recovered by electrophoresis and ligated with the insert 1 as vector fragment 1 to obtain plasmid pBSKS (+) -R.

The plasmid pBSKS (+) -R was used as a template, and PR1/PR2(SEQ ID NO.10/SEQ ID NO.11) was used as a primer set to perform PCR amplification, a 0.5kb DNA fragment was recovered by agarose gel electrophoresis, and after nucleic acid precipitation, the DNA fragment was digested with HindIII and SalI, and after nucleic acid precipitation again, the DNA fragment was used as an insert 2. After the plasmid pBSKS (+) -R is subjected to double enzyme digestion by HindIII and SalI, a 3.4kb DNA fragment is recovered by electrophoresis and is connected with the insert fragment 2 as a vector fragment 2 to obtain the plasmid pBSKS (+) -R-R.

After plasmid pBSKS (+) -R-R was digested with BamHI and SmaI, 3.8kb band DNA was recovered by agarose gel electrophoresis as vector fragment 3. Plasmid pGEM-T easy-URA3 (flood release, construction of a Saccharomyces cerevisiae engineering strain for expressing cellulase and research on alcohol production by lignocellulose fermentation, doctor academic thesis of Tianjin university 2014; SEQ ID NO.12) is subjected to double enzyme digestion by BamHI and SmaI, and a 0.9kb DNA fragment is recovered by electrophoresis and is used as an insert fragment 3 and is connected with a vector fragment 3 to obtain the plasmid pBSKS (+) -R-URA 3-R.

After the plasmid pBSKS (+) -R-URA3-R was digested with XbaI and SalI, a 1.8kb DNA fragment was recovered from the agarose gel, and the end thereof was then subjected to smoothing treatment to obtain an insert 4. After SmaI treatment of plasmid pGEM-T easy-delta SEQ (flood liberation, construction of a Saccharomyces cerevisiae engineering strain for expressing cellulase and research on alcohol production by lignocellulose fermentation, Ph university of Tianjin in 2014; SEQ ID NO.13), a 3398bp fragment is recovered through electrophoresis as a vector fragment 4 and is connected with an insert fragment 4 to obtain plasmid pGEM-T easy-5 'delta-R-RA 3-R-3' delta, which is called pG delta RU for short, as shown in figure 2.

After the plasmid pG delta RU obtained by the construction is subjected to enzyme digestion by SfiI, a 5.2kb DNA fragment is recovered by electrophoresis, and then the DNA fragment obtained after the end smoothing treatment and the nucleic acid purification is used as a vector fragment V.

After the plasmid pGEM-T easy-Ptpi-xyn2s-ABGL1-TadhI is subjected to double enzyme digestion by XhoI and ScaI, a 3.7kb DNA fragment is recovered by agarose gel electrophoresis, then the tail end is smoothed, and nucleic acid is purified and then is connected with a vector fragment V, so that a cellulase gene expression cassette delta integrated plasmid pGEM-T easy-5 'delta-R-RA 3-R-AaBGLI-3' delta, which is marked as delta P1, is obtained.

The plasmid YEplac195-Ptpi-xyn2s-TEGII-TadhI is subjected to HindIII and EcoRI double enzyme digestion, a 2.3kb DNA fragment is recovered by agarose gel electrophoresis, then the tail end is subjected to smoothing treatment, nucleic acid is purified and then is connected with a vector fragment V to obtain a cellulase gene expression cassette delta integrated plasmid pGEM-T easy-5 'delta-R-RA 3-R-TEGII-3' delta, and the integrated plasmid is marked as delta P2.

After StuI and ScaI double digestion of plasmid pGEM-T easy-Ptpi-xyn2s-TCBHI-TadhI, a 2.6kb DNA fragment is recovered by agarose gel electrophoresis, then the end is smoothed, and nucleic acid is connected with a vector fragment V after purification to obtain a cellulase gene expression cassette delta integrated plasmid pGEM-T easy-5 'delta-R-RA 3-R-TCBHI-3' delta which is marked as delta P3.

Plasmid pGEM-T easy-Ptpi-xyn2s-TCBHII-TadhI is subjected to double enzyme digestion after StuI and ScaI, a 2.6kb DNA fragment is recovered by agarose electrophoresis, then the end is subjected to smoothing treatment, nucleic acid is connected with a vector fragment V after purification, and a cellulase gene expression cassette delta integrated plasmid pGEM-T easy-5 'delta-R-RA 3-R-TCBHII-3' delta is obtained and is marked as delta P4.

Plasmid pGEM-T easy-Ptpi-xyn2s-ACBHI-TadhI is subjected to MluI and ScaI double enzyme digestion, a 2.8kb DNA fragment is recovered through electrophoresis, then the tail end is subjected to smoothing treatment, nucleic acid is purified and then is connected with a vector fragment V, and a cellulase gene expression cassette delta integration plasmid pGEM-T easy-5 'delta-R-RA 3-R-ACBHI-3' delta is obtained and is marked as delta P5.

The schematic diagram of the cellulase gene delta integration plasmid constructed as described above is shown in FIG. 3.

Example 3

Yeast transformation and selection

After the plasmid delta P1 is subjected to NotI enzyme digestion and nucleic acid purification, a host bacterium K alpha is transformed by a lithium acetate method, a minimal medium plate with default uracil is used for screening a transformant, and a 5-fluoroorotic acid plate is used for reverse selection to obtain the transformant K alpha delta 1 with the URA3 screening marker removed.

After the plasmid delta P2 is subjected to NotI enzyme digestion and nucleic acid purification, a host bacterium K alpha delta 1 is transformed by a lithium acetate method, transformants are screened by a minimal medium plate with default uracil, and a transformant K alpha delta 2 with a URA3 screening marker removed is obtained by reverse selection by a 5-fluoroorotic acid plate.

After the plasmid IP3 is subjected to NotI enzyme digestion and nucleic acid purification, a host bacterium K alpha delta 2 is transformed by a lithium acetate method, a minimal medium plate with default uracil is used for screening a transformant, and a 5-fluoroorotic acid plate is used for reverse selection to obtain the transformant K alpha delta 3 with the URA3 screening marker removed.

After the plasmid IP4 is subjected to NotI enzyme digestion and nucleic acid purification, a host bacterium K alpha delta 3 is transformed by a lithium acetate method, a minimal medium plate with default uracil is used for screening a transformant, and a 5-fluoroorotic acid plate is used for reverse selection to obtain the transformant K alpha delta 4 with the URA3 screening marker removed.

After the plasmid IP5 is subjected to NotI enzyme digestion and nucleic acid purification, a host bacterium K alpha delta 4 is transformed by a lithium acetate method, a minimal medium plate with default uracil is used for screening a transformant, and a 5-fluoroorotic acid plate is used for reverse selection to obtain the transformant K alpha delta 5 with the URA3 screening marker removed.

The activity of the recombinant strain cellulase is determined according to the literature (flood liberation, construction of a saccharomyces cerevisiae engineering strain expressing cellulase and research on alcohol production by fermentation of lignocellulose, doctor academic thesis of Tianjin university 2014), and through detection, the strain K alpha delta 1 has BGL activity, and the strains K alpha delta 2-K alpha delta 5 have BGL, EG, CMCase and FPase activity.

After the recombinant strains K alpha delta 1-K alpha delta 5 and the original strain K alpha are cultured by YPD liquid culture medium (30 ℃, 150rpm, 20 hours), 5mL of bacterial liquid is taken and added into a 50mL shake flask with a screw opening (4g of acid-base pretreated corncob, 11mL of dilute sulfuric acid solution, pH4.0), then 10FPU/g of cellulose material and muramyl cellulase (and muramyl biotechnology, Inc.) are added, and fermentation is carried out for 10 days at 30 ℃ and 150 rpm. The fermentation broth was subjected to HPLC to determine the ethanol concentration, and the results are shown in FIG. 4.

As can be seen in fig. 4: compared with the original strain K alpha, the recombinant strains K alpha delta 1-K alpha delta 5 can improve the degradation capability of the commercial cellulase and improve the ethanol concentration of the fermentation liquor. The K α δ 5 strain with the best fermentation performance was renamed to R δ BEC and deposited with the following information:

saccharomyces cerevisiae (Saccharomyces cerevisiae) code number R delta BEC

The preservation organization: china general microbiological culture Collection center

Address: xilu No.1 Hospital No.3 of Beijing market facing Yang district

Preservation time: 11/26/2018

The preservation number is: CGMCC No.16825

The plasmid construction process comprises molecular operations such as PCR amplification, DNA fragment and plasmid restriction endonuclease digestion, DNA fragment recovery by agarose gel electrophoresis, DNA fragment end smoothing, ligation, transformation and the like, yeast sporulation and spore screening, yeast transformation, transformant screening, yeast strain enzyme activity determination, fermented cellulose alcohol production performance evaluation and the like, and can be referred to documents (Kyoho release, construction of a saccharomyces cerevisiae engineering strain for expressing cellulase, and research on alcohol production by fermentation of lignocellulose, Ph Dai university in 2014, Yangtze university, Zhou Lang, construction of an engineering strain for producing alcohol by lignocellulose hydrolysate, and research on alcohol production by Ph Dai university in 2011), which are familiar to the professional.

Sequence listing

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ggttttaaaa cgaaaattct tattcttgag taactctttc ctgtaggtca ggttgctttc 2520

tcaggtatag catgaggtcg ctcttattga ccacacctct accggcatgc cgagcaaatg 2580

cctgcaaatc gctccccatt tcacccaatt gtagatatgc taactccagc aatgagttga 2640

tgaatctcgg tgtgtatttt atgtcctcag aggacaacac ctgttgtaat cgttcttcca 2700

cacggatagt actgactatc gtcggccgcc taggccatgg tacaacatca ctagtgaatt 2760

cgcggccgcc tgcaggtcga ccatatggga gagctcccaa cgcgttggat gcatagcttg 2820

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aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt 3360

ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg 3420

tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc 3480

agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc 3540

gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta 3600

tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct 3660

acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc 3720

tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa 3780

caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa 3840

aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa 3900

aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt 3960

ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac 4020

agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc 4080

atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc 4140

cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata 4200

aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc 4260

cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc 4320

aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca 4380

ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa 4440

gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca 4500

ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt 4560

tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt 4620

tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg 4680

ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga 4740

tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc 4800

agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg 4860

acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag 4920

ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg 4980

gttccgcgca catttccccg aaaagtgcca cctgatgcgg tgtgaaatac cgcacagatg 5040

cgtaaggaga aaataccgca tcaggaaatt gtaagcgtta atattttgtt aaaattcgcg 5100

ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct 5160

tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt 5220

ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat 5280

ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca 5340

ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac 5400

gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta 5460

gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg 5520

tccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc 5580

tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag 5640

ggttttccca gtcacgacgt tgtaaaacga cggccagtga attgtaatac gactcactat 5700

a 5701

<210> 5

<211> 5551

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gggcgaattg ggcccgacgt cgcatgctcc cggccgccat ggcggccgcg ggaattcgat 60

tgaccacata tggccgccta ggcctcgagc tgcagtaacg ggagcgtaat ggtgatggaa 120

ctggacgaat ccatcaatag atacgtcctg aggaccgtgc tacccaaatg gactgattgt 180

gagggagacc taactacata gtgtttaaag attacggata tttaacttac ttagaataat 240

gccatttttt tgagttataa taatcctacg ttagtgtgag cgggatttaa actgtgagga 300

ccttaataca ttcagacact tctgcggtat caccctactt attcccttcg agattatatc 360

taggaaccca tcaggttggt ggaagattac ccgttctaag acttttcagc ttcctctatt 420

gatgttacac ctggacaccc cttttctggc atccagtttt taatcttcag tggcatgtga 480

gattctccga aattaattaa agcaatcaca caattctctc ggataccacc tcggttgaaa 540

ctgacaggtg gtttgttacg catgctaatg caaaggagcc tatatacctt tggctcggct 600

gctgtaacag ggaatataaa gggcagcata atttaggagt ttagtgaact tgcaacattt 660

actattttcc cttcttacgt aaatattttt ctttttaatt ctaaatcaat ctttttcaat 720

tttttgtttg tattcttttc ttgcttaaat ctataactac aaaaaacaca tacataaact 780

aaaaatggtc tccttcacct ccctcctcgc cggcgtcgcc gccatctcgg gcgtcttggc 840

cgctcccgcc gccgaggtcg aatccgtggc tgtggagaag cgccaagctt gctcaagcgt 900

ctggggccaa tgtggtggcc agaattggtc gggtccgact tgctgtgctt ccggaagcac 960

atgcgtctac tccaacgact attactccca gtgtcttccc ggcgctgcaa gctcaagctc 1020

gtccacgcgc gccgcgtcga cgacttctcg agtatccccc acaacatccc ggtcgagctc 1080

cgcgacgcct ccacctggtt ctactactac cagagtacct ccagtcggat cgggaaccgc 1140

tacgtattca ggcaaccctt ttgttggggt cactccttgg gccaatgcat attacgcctc 1200

tgaagttagc agcctcgcta ttcctagctt gactggagcc atggccactg ctgcagcagc 1260

tgtcgcaaag gttccctctt ttatgtggct agatactctt gacaagaccc ctctcatgga 1320

gcaaaccttg gccgacatcc gcaccgccaa caagaatggc ggtaactatg ccggacagtt 1380

tgtggtgtat gacttgccgg atcgcgattg cgctgccctt gcctcgaatg gcgaatactc 1440

tattgccgat ggtggcgtcg ccaaatataa gaactatatc gacaccattc gtcaaattgt 1500

cgtggaatat tccgatatcc ggaccctcct ggttattgag cctgactctc ttgccaacct 1560

ggtgaccaac ctcggtactc caaagtgtgc caatgctcag tcagcctacc ttgagtgcat 1620

caactacgcc gtcacacagc tgaaccttcc aaatgttgcg atgtatttgg acgctggcca 1680

tgcaggatgg cttggctggc cggcaaacca agacccggcc gctcagctat ttgcaaatgt 1740

ttacaagaat gcatcgtctc cgagagctct tcgcggattg gcaaccaatg tcgccaacta 1800

caacgggtgg aacattacca gccccccatc gtacacgcaa ggcaacgctg tctacaacga 1860

gaagctgtac atccacgcta ttggacctct tcttgccaat cacggctggt ccaacgcctt 1920

cttcatcact gatcaaggtc gatcgggaaa gcagcctacc ggacagcaac agtggggaga 1980

ctggtgcaat gtgatcggca ccggatttgg tattcgccca tccgcaaaca ctggggactc 2040

gttgctggat tcgtttgtct gggtcaagcc aggcggcgag tgtgacggca ccagcgacag 2100

cagtgcgcca cgatttgact cccactgtgc gctcccagat gccttgcaac cggcgcctca 2160

agctggtgct tggttccaag cctactttgt gcagcttctc acaaacgcaa acccatcgtt 2220

cctgtaagcg aatttcttat gatttatgat ttttattatt aaataagtta taaaaaaaat 2280

aagtgtatac aaattttaaa gtgactctta ggttttaaaa cgaaaattct tattcttgag 2340

taactctttc ctgtaggtca ggttgctttc tcaggtatag catgaggtcg ctcttattga 2400

ccacacctct accggcatgc cgagcaaatg cctgcaaatc gctccccatt tcacccaatt 2460

gtagatatgc taactccagc aatgagttga tgaatctcgg tgtgtatttt atgtcctcag 2520

aggacaacac ctgttgtaat cgttcttcca cacggatagt actgactatc gtcggccgcc 2580

taggccatgg tacaacatca ctagtgaatt cgcggccgcc tgcaggtcga ccatatggga 2640

gagctcccaa cgcgttggat gcatagcttg agtattctat agtgtcacct aaatagcttg 2700

gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac 2760

aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc 2820

acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg 2880

cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct 2940

tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac 3000

tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga 3060

gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat 3120

aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac 3180

ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct 3240

gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg 3300

ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg 3360

ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt 3420

cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg 3480

attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac 3540

ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga 3600

aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt 3660

gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt 3720

tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga 3780

ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc 3840

taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct 3900

atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata 3960

actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca 4020

cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga 4080

agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga 4140

gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg 4200

gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga 4260

gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt 4320

gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct 4380

cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca 4440

ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat 4500

accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga 4560

aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc 4620

aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg 4680

caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc 4740

ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt 4800

gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca 4860

cctgatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggaaatt 4920

gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt 4980

aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg 5040

ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc 5100

aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca 5160

agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga 5220

tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa 5280

ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc 5340

gccgcgctta atgcgccgct acagggcgcg tccattcgcc attcaggctg cgcaactgtt 5400

gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg 5460

ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga 5520

cggccagtga attgtaatac gactcactat a 5551

<210> 6

<211> 7534

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 60

cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct 120

cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat 180

tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcttgg 240

ccgcctaggc ctaacgggag cgtaatggtg atggaactgg acgaatccat caatagatac 300

gtcctgagga ccgtgctacc caaatggact gattgtgagg gagacctaac tacatagtgt 360

ttaaagatta cggatattta acttacttag aataatgcca tttttttgag ttataataat 420

cctacgttag tgtgagcggg atttaaactg tgaggacctt aatacattca gacacttctg 480

cggtatcacc ctacttattc ccttcgagat tatatctagg aacccatcag gttggtggaa 540

gattacccgt tctaagactt ttcagcttcc tctattgatg ttacacctgg acaccccttt 600

tctggcatcc agtttttaat cttcagtggc atgtgagatt ctccgaaatt aattaaagca 660

atcacacaat tctctcggat accacctcgg ttgaaactga caggtggttt gttacgcatg 720

ctaatgcaaa ggagcctata tacctttggc tcggctgctg taacagggaa tataaagggc 780

agcataattt aggagtttag tgaacttgca acatttacta ttttcccttc ttacgtaaat 840

atttttcttt ttaattctaa atcaatcttt ttcaattttt tgtttgtatt cttttcttgc 900

ttaaatctat aactacaaaa aacacataca taaactaaaa atggtctcct tcacctccct 960

cctcgccggc gtcgccgcca tctcgggcgt cttggccgct cccgccgccg aggtcgaatc 1020

cgtggctgtg gagaagcgcc agcagactgt ctggggccag tgtggaggta ttggttggag 1080

cggacctacg aattgtgctc ctggctcagc ttgttcgacc ctcaatcctt attatgcgca 1140

atgtattccg ggagccacta ctatcaccac ttcgacccgg ccaccatccg gtccaaccac 1200

caccaccagg gctacctcaa caagctcatc aactccaccc acgagctctg gggtccgatt 1260

tgccggcgtt aacatcgcgg gttttgactt tggctgtacc acagatggca cttgcgttac 1320

ctcgaaggtt tatcctccgt tgaagaactt caccggctca aacaactacc ccgatggcat 1380

cggccagatg cagcacttcg tcaacgagga cgggatgact attttccgct tacctgtcgg 1440

atggcagtac ctcgtcaaca acaatttggg cggcaatctt gattccacga gcatttccaa 1500

gtatgatcag cttgttcagg ggtgcctgtc tctgggcgca tactgcatcg tcgacatcca 1560

caattatgct cgatggaacg gtgggatcat tggtcagggc ggccctacta atgctcaatt 1620

cacgagcctt tggtcgcagt tggcatcaaa gtacgcatct cagtcgaggg tgtggttcgg 1680

catcatgaat gagccccacg acgtgaacat caacacctgg gctgccacgg tccaagaggt 1740

tgtaaccgca atccgcaacg ctggtgctac gtcgcaattc atctctttgc ctggaaatga 1800

ttggcaatct gctggggctt tcatatccga tggcagtgca gccgccctgt ctcaagtcac 1860

gaacccggat gggtcaacaa cgaatctgat ttttgacgtg cacaaatact tggactcaga 1920

caactccggt actcacgccg aatgtactac aaataacatt gacggcgcct tttctccgct 1980

tgccacttgg ctccgacaga acaatcgcca ggctatcctg acagaaaccg gtggtggcaa 2040

cgttcagtcc tgcatacaag acatgtgcca gcaaatccaa tatctcaacc agaactcaga 2100

tgtctatctt ggctatgttg gttggggtgc cggatcattt gatagcacgt atgtcctgac 2160

ggaaacaccg actagcagtg gtaactcatg gacggacaca tccttggtca gctcgtgtct 2220

cgcaagaaag taggcgaatt tcttatgatt tatgattttt attattaaat aagttataaa 2280

aaaaataagt gtatacaaat tttaaagtga ctcttaggtt ttaaaacgaa aattcttatt 2340

cttgagtaac tctttcctgt aggtcaggtt gctttctcag gtatagcatg aggtcgctct 2400

tattgaccac acctctaccg gcatgccgag caaatgcctg caaatcgctc cccatttcac 2460

ccaattgtag atatgctaac tccagcaatg agttgatgaa tctcggtgtg tattttatgt 2520

cctcagagga caacacctgt tgtaatcgtt cttccacacg gatggccgcc taggccgaat 2580

tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat 2640

cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat 2700

cgcccttccc aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg gtattttctc 2760

cttacgcatc tgtgcggtat ttcacaccgc atatatcgga tcgtacttgt tacccatcat 2820

tgaattttga acatccgaac ctgggagttt tccctgaaac agatagtata tttgaacctg 2880

tataataata tatagtctag cgctttacgg aagacaatgt atgtatttcg gttcctggag 2940

aaactattgc atctattgca taggtaatct tgcacgtcgc atccccggtt cattttctgc 3000

gtttccatct tgcacttcaa tagcatatct ttgttaacga agcatctgtg cttcattttg 3060

tagaacaaaa atgcaacgcg agagcgctaa tttttcaaac aaagaatctg agctgcattt 3120

ttacagaaca gaaatgcaac gcgaaagcgc tattttacca acgaagaatc tgtgcttcat 3180

ttttgtaaaa caaaaatgca acgcgagagc gctaattttt caaacaaaga atctgagctg 3240

catttttaca gaacagaaat gcaacgcgag agcgctattt taccaacaaa gaatctatac 3300

ttcttttttg ttctacaaaa atgcatcccg agagcgctat ttttctaaca aagcatctta 3360

gattactttt tttctccttt gtgcgctcta taatgcagtc tcttgataac tttttgcact 3420

gtaggtccgt taaggttaga agaaggctac tttggtgtct attttctctt ccataaaaaa 3480

agcctgactc cacttcccgc gtttactgat tactagcgaa gctgcgggtg cattttttca 3540

agataaaggc atccccgatt atattctata ccgatgtgga ttgcgcatac tttgtgaaca 3600

gaaagtgata gcgttgatga ttcttcattg gtcagaaaat tatgaacggt ttcttctatt 3660

ttgtctctat atactacgta taggaaatgt ttacattttc gtattgtttt cgattcactc 3720

tatgaatagt tcttactaca atttttttgt ctaaagagta atactagaga taaacataaa 3780

aaatgtagag gtcgagttta gatgcaagtt caaggagcga aaggtggatg ggtaggttat 3840

atagggatat agcacagaga tatatagcaa agagatactt ttgagcaatg tttgtggaag 3900

cggtattcgc aatattttag tagctcgtta cagtccggtg cgtttttggt tttttgaaag 3960

tgcgtcttca gagcgctttt ggttttcaaa agcgctctga agttcctata ctttctagct 4020

agagaatagg aacttcggaa taggaacttc aaagcgtttc cgaaaacgag cgcttccgaa 4080

aatgcaacgc gagctgcgca catacagctc actgttcacg tcgcacctat atctgcgtgt 4140

tgcctgtata tatatataca tgagaagaac ggcatagtgc gtgtttatgc ttaaatgcgt 4200

acttatatgc gtctatttat gtaggatgaa aggtagtcta gtacctcctg tgatattatc 4260

ccattccatg cggggtatcg tatgcttcct tcagcactac cctttagctg ttctatatgc 4320

tgccactcct caattggatt agtctcatcc ttcaatgcta tcatttcctt tgatattgga 4380

tcgatccgat gataagctgt caaacatgag aattgggtaa taactgatat aattaaattg 4440

aagctctaat ttgtgagttt agtatacatg catttactta taatacagtt ttttagtttt 4500

gctggccgca tcttctcaaa tatgcttccc agcctgcttt tctgtaacgt tcaccctcta 4560

ccttagcatc ccttcccttt gcaaatagtc ctcttccaac aataataatg tcagatcctg 4620

tagagaccac atcatccacg gttctatact gttgacccaa tgcgtctccc ttgtcatcta 4680

aacccacacc gggtgtcata atcaaccaat cgtaaccttc atctcttcca cccatgtctc 4740

tttgagcaat aaagccgata acaaaatctt tgtcgctctt cgcaatgtca acagtaccct 4800

tagtatattc tccagtagat agggagccct tgcatgacaa ttctgctaac atcaaaaggc 4860

ctctaggttc ctttgttact tcttctgccg cctgcttcaa accgctaaca atacctgggc 4920

ccaccacacc gtgtgcattc gtaatgtctg cccattctgc tattctgtat acacccgcag 4980

agtactgcaa tttgactgta ttaccaatgt cagcaaattt tctgtcttcg aagagtaaaa 5040

aattgtactt ggcggataat gcctttagcg gcttaactgt gccctccatg gaaaaatcag 5100

tcaagatatc cacatgtgtt tttagtaaac aaattttggg acctaatgct tcaactaact 5160

ccagtaattc cttggtggta cgaacatcca atgaagcaca caagtttgtt tgcttttcgt 5220

gcatgatatt aaatagcttg gcagcaacag gactaggatg agtagcagca cgttccttat 5280

atgtagcttt cgacatgatt tatcttcgtt tcctgcatgt ttttgttctg tgcagttggg 5340

ttaagaatac tgggcaattt catgtttctt caacactaca tatgcgtata tataccaatc 5400

taagtctgtg ctccttcctt cgttcttcct tctgttcgga gattaccgaa tcaaaaaaat 5460

ttcaaagaaa ccgaaatcaa aaaaaagaat aaaaaaaaaa tgatgaattg aattgaaaag 5520

ctaattcttg aagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga 5580

taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta 5640

tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat 5700

aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc 5760

ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga 5820

aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca 5880

acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt 5940

ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg 6000

gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc 6060

atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata 6120

acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt 6180

tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag 6240

ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca 6300

aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg 6360

aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg 6420

ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag 6480

atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg 6540

aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag 6600

accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga 6660

tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt 6720

tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc 6780

tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc 6840

cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac 6900

caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac 6960

cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt 7020

cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct 7080

gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat 7140

acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt 7200

atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg 7260

cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt 7320

gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt 7380

tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg 7440

tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg 7500

agcgcagcga gtcagtgagc gaggaagcgg aaga 7534

<210> 7

<211> 6655

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

tatagtgagt cgtattacaa ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac 60

cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat 120

agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg 180

acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg 240

ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca 300

cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta 360

gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc 420

catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg 480

gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat 540

aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta 600

acgcgaattt taacaaaata ttaacgctta caatttcctg atgcggtatt ttctccttac 660

gcatctgtgc ggtatttcac accgcatcag gtggcacttt tcggggaaat gtgcgcggaa 720

cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac 780

cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg 840

tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc 900

tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg 960

atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga 1020

gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc 1080

aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag 1140

aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga 1200

gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg 1260

cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga 1320

atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt 1380

tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact 1440

ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt 1500

ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg 1560

ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta 1620

tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac 1680

tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta 1740

aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt 1800

tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt 1860

tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt 1920

gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc 1980

agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg 2040

tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg 2100

ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt 2160

cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac 2220

tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg 2280

acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg 2340

gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat 2400

ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt 2460

tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg 2520

attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa 2580

cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc 2640

ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga 2700

aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg 2760

ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc 2820

acacaggaaa cagctatgac catgattacg ccaagctatt taggtgacac tatagaatac 2880

tcaagctatg catccaacgc gttgggagct ctcccatatg gccgcctagg cctcgagctg 2940

cagtaacggg agcgtaatgg tgatggaact ggacgaatcc atcaatagat acgtcctgag 3000

gaccgtgcta cccaaatgga ctgattgtga gggagaccta actacatagt gtttaaagat 3060

tacggatatt taacttactt agaataatgc catttttttg agttataata atcctacgtt 3120

agtgtgagcg ggatttaaac tgtgaggacc ttaatacatt cagacacttc tgcggtatca 3180

ccctacttat tcccttcgag attatatcta ggaacccatc aggttggtgg aagattaccc 3240

gttctaagac ttttcagctt cctctattga tgttacacct ggacacccct tttctggcat 3300

ccagttttta atcttcagtg gcatgtgaga ttctccgaaa ttaattaaag caatcacaca 3360

attctctcgg ataccacctc ggttgaaact gacaggtggt ttgttacgca tgctaatgca 3420

aaggagccta tatacctttg gctcggctgc tgtaacaggg aatataaagg gcagcataat 3480

ttaggagttt agtgaacttg caacatttac tattttccct tcttacgtaa atatttttct 3540

ttttaattct aaatcaatct ttttcaattt tttgtttgta ttcttttctt gcttaaatct 3600

ataactacaa aaaacacata cataaactaa aaatggtctc cttcacctcc ctcctcgccg 3660

gcgtcgccgc catctcgggc gtcttggccg ctcccgccgc cgaggtcgaa tccgtggctg 3720

tggagaagcg cgatgaactg gcgttctctc ctccattcta tccctcccct tgggccaatg 3780

gccagggaga gtgggcggaa gcctaccagc gtgcagtggc cattgtctcc cagatgactc 3840

tggatgagaa ggtcaacctt accaccggaa ctggatggga gctggagaag tgtgtcggtc 3900

agactggtgg tgttccaaga ctgaacatcg gtggcatgtg tctccaggac agtcccttgg 3960

gaattcgtga cagtgactac aattcggctt tccctgctgg tgtcaacgtt gctgcgacat 4020

gggacaagaa cctggcttat ctccgtggcc aggctatggg tcaagaattt agtgacaaag 4080

gaatcgatgt tcaattgggc ccggccgcgg gtcccctcgg caggagtcct gatggaggtc 4140

gtaactggga aggtttctct ccagacccgg ctcttactgg cgtgctcttt gcggagacga 4200

tcaagggtat tcaagatgct ggtgtcgtgg caacagccaa gcattacatt ctcaatgagc 4260

aagagcattt ccgccaggtc tcagagtctg cgggctacgg attcaatatc tccgacacgg 4320

tcagctcgaa tgttgatgac aagaccattc atgaaatgta cctctggccc ttcgcggatg 4380

ccgttcgcgc aggcgttggc gccatcatgt gttcctacaa ccagatcaac aacagctacg 4440

gctgccagaa cagttacacg ctgaacaagc ttctgaaggc cgagctcggc ttccagggct 4500

tcgtgatgtc tgactggggt gctcaccaca gtggtgttgg ttctgctttg gccggcctgg 4560

atatgtcaat gcctggtgat atcaccttcg attctgccac tagtttctgg ggtaccaacc 4620

tgaccattgc tgttctcaac ggcaccgtcc cgcagtggcg cgttgacgac atggctgttc 4680

gtatcatggc tgcctactac aaggttggcc gcgaccgcct gtaccagccg cctaacttca 4740

gctcttggac tcgcgatgaa tacggcttca agtattttta ccctcaggaa ggtccctatg 4800

agaaggtcaa tcactttgtc aatgtgcagc gcaaccacag cgaggttatt cgcaagttgg 4860

gagcagacag cactgttcta ctgaagaaca acaatgccct gcctctgacc ggaaaggagc 4920

gcaaagttgc gatcctgggt gaagacgccg gatccaactc gtacggtgcc aatggctgct 4980

ctgaccgcgg ctgtgacaac ggtactcttg ccatggcttg gggtagtggc actgccgaat 5040

tcccatacct cgtgacccct gagcaggcta ttcaagctga ggtgctcaag cacaagggca 5100

gcgtctatgc catcactgac aactgggcgc tgagccaggt ggagaccctc gctaaacaag 5160

ccagtgtctc tcttgtgttc gtcaactcgg actcgggaga gggctatatc tctgtggacg 5220

gaaacgaggg cgaccgcaac aacctcaccc tctggaagaa tggcgacaac ctcatcaagg 5280

ctgctgcgaa caactgcaac aacacaatcg ttgtcatcca ctccgttgga ccggttttgg 5340

tcgacgagtg gtatgaccac cccaatgtca ctgccatcct ctgggccggt ttgcctggcc 5400

aggagtctgg caactccctg gctgatgtgc tctacggccg cgtcaacccg ggcgccaaat 5460

cgccattcac ctggggcaag acgagggagg cgtacgggga ttaccttgtc cgcgaactca 5520

acaacggcaa cggggctccc caagacgact tctcggaagg tgttttcatt gactaccgcg 5580

gattcgacaa gcgcaatgag accccgatct acgagttcgg acatggtctg agctacacca 5640

ctttcaacta ctctggcctt cacatccagg ttctcaacgc ttcgtccaac gctcaagtag 5700

ccactgagac tggcgctgct cccacattcg gacaagtcgg caatgcctcg gactatgtgt 5760

accctgaggg attgaccaga atcagcaagt tcatctaccc ctggcttaac tccaccgacc 5820

tcaaggcctc atctggcgac ccgtactatg gtgtcgacac cgcggagcac gtccccgagg 5880

gtgctaccga tggctctccc cagcccgttc tgcccgcggg tggtggcttc ggtggtaacc 5940

cccgcctcta cgatgagttg atccgcgttt cggtgacagt caagaacact ggtcgtgttg 6000

ctggtgatgc tgtacctcaa ctgtatgttt cccttggtgg acccaatgag cccagggttg 6060

tgttgcgcaa attcgaccgc cttaccctca agccctccga ggagacggtg tggacgacta 6120

ccctgactcg tcgcgatctg tccaactggg acgttgcggc tcaggattgg gtcattactt 6180

cttacccgaa gaaggtccat gttggtagct cttcgcgtca gctgcccctt cacgcagcgc 6240

tcccgaaggt gcaatgagcg aatttctatg atttatgatt tttattatta aataagttat 6300

aaaaaaaata agtgtataca aattttaaag tgactcttag gttttaaaac gaaaattctt 6360

attcttgagt aactctttcc tgtaggtcag gttgctttct caggtatagc atgaggtcgc 6420

tcttattgac cacacctcta ccggcatgcc gagcaaatgc ctgcaaatcg ctccccattt 6480

cacccaattg tagatatgtt aactccagca atgagttgat gaatctcggt gtgtatttta 6540

tgtcctcaga ggacaacacc tgttgtaatc gttcttccac acggatagta ctgactatcg 6600

tcggccgcct aggccatggc ggccgggagc atgcgacgtc gggcccaatt cgccc 6655

<210> 8

<211> 5725

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gggcgaattg ggccctgatt agttgtacca tggcctaggc ggccgacgat agtcagtact 60

atccgtgtgg aagaacgatt acaacaggtg ttgtcctctg aggacataaa atacacaccg 120

agattcatca actcattgct ggagttagca tatctacaat tgggtgaaat ggggagcgat 180

ttgcaggcat ttgctcggca tgccggtaga ggtgtggtca ataagagcga cctcatgcta 240

tacctgagaa agcaacctga cctacaggaa agagttactc aagaataaga attttcgttt 300

taaaacctaa gagtcacttt aaaatttgta tacacttatt ttttttataa cttatttaat 360

aataaaaatc ataaatcata agaaattcgc ttacaagcac tgcgagtaga agtcattctg 420

cttggtgcaa gtgccgctgg cgcaggtggt ggggccagtc catccctgtc caccgcactg 480

tccgtacagc tgggcaacat tcgtggacga gctcgaggtg gtggacgtct tggaagtggt 540

ggtggtggtc ttggagctag tggtggtggt cgaggccttg gtagtcaagg tggtgctgct 600

ggagctggag ccgcccgagg tcgaaccgga gtaggtcgag ttgagggtac cgaacttgat 660

gttggagtag gtgacatacg agttcgggta gttggcctca acgtaggtgg catttccggt 720

ggtggtgggg caggtaccgc gggcgacacc agggctggta gccgagctgg tggtggggta 780

gtcgctgtcg agccagagca tgtcggcggc gtggtcatcc cacagactga gcaccaggac 840

catgccgtcg ccgagggcgt cacccatggc agacagtccg ccgtgggtgt cgaagacgtt 900

ggtgtcgcca aacaaggtct tctcggcctt gcagaagctc tcggtgatcg agttgccgct 960

cgcagcagtg taggtggagg ggccgttgcc aatcacgaca ccgttctgca cgtagagacg 1020

acggatctca gtcagggtac cagtgtcagt accatcgttg gtgatgaact gcgtcacaac 1080

ggtgaagggc ttgctggtgt cgacggtctt tccgggacca tagaagttgg tgttgcccag 1140

gcggtaaggg ttgaaatcac atccatcagg atcgcaagta ccggagtaac ggtcggcagt 1200

gtcactgtaa gtgccaccgc aagaatcgcc cgtacacatg gtctgctgga cggagtcgca 1260

agggtgggcg gtgaaggcac tggagacgct gttcgcctcc cagatatcca tctcagggca 1320

gcaggagccg tggttaccag cacccgcatt gacatcggtc gaagatggct cccagccctc 1380

aatgttggct tgaccatcga tgaacttgag atcccgagga cattgggagt cgcagtatcc 1440

ggttccatac ttggcgccag ccttgttcgt tgggaagcgg gaaacgccgc cgtcagcgtc 1500

catagagacg aagtagaggg caccgttcag tccgcaggga aggttagaga cgtcgacatc 1560

gaaagtgaat tcccgattga gaagcttgaa agtctcgtag gtgctgtcgt cagccagaag 1620

atacagacgc gagccaatgt tcttctgcga cgacgtagta acgaagttga gacggagcga 1680

cgagccgctg gtggtcacac cataggtgct cgcgtaagtg gcaccctcaa gggcacattc 1740

ggacgcgcag gtggtatcgg tggagcagat cgaactgtcc caagtgttgc cgctgtagca 1800

gttggtgtag ccaccaacct cgtgcaccca acgccaattg gcgtcaatga ccacgctgcc 1860

gctggtggta gtgcagctac cactgccgct gcaggtttgc caggtcaagc tggggtgcgt 1920

ttcagcggtg taagtgccga cctgctggcg cttctccaca gccacggatt cgacctcggc 1980

ggcgggagcg gccaagacgc ccgagatggc ggcgacgccg gcgaggaggg aggtgaagga 2040

gaccattttt agtttatgta tgtgtttttt gtagttatag atttaagcaa gaaaagaata 2100

caaacaaaaa attgaaaaag attgatttag aattaaaaag aaaaatattt acgtaagaag 2160

ggaaaatagt aaatgttgca agttcactaa actcctaaat tatgctgccc tttatattcc 2220

ctgttacagc agccgagcca aaggtatata ggctcctttg cattagcatg cgtaacaaac 2280

cacctgtcag tttcaaccga ggtggtatcc gagagaattg tgtgattgct ttaattaatt 2340

tcggagaatc tcacatgcca ctgaagatta aaaactggat gccagaaaag gggtgtccag 2400

gtgtaacatc aatagaggaa gctgaaaagt cttagaacgg gtaatcttcc accaacctga 2460

tgggttccta gatataatct cgaagggaat aagtagggtg ataccgcaga agtgtctgaa 2520

tgtattaagg tcctcacagt ttaaatcccg ctcacactaa cgtaggatta ttataactca 2580

aaaaaatggc attattctaa gtaagttaaa tatccgtaat ctttaaacac tatgtagtta 2640

ggtctccctc acaatcagtc catttgggta gcacggtcct caggacgtat ctattgatgg 2700

attcgtccag ttccatcacc attacgctcc cgttactgca gctcgaggcc taggcggcca 2760

tatgtggtca atcactagtg aattcgcggc cgcctgcagg tcgaccatat gggagagctc 2820

ccaacgcgtt ggatgcatag cttgagtatt ctatagtgtc acctaaatag cttggcgtaa 2880

tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata 2940

cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta 3000

attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa 3060

tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg 3120

ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag 3180

gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa 3240

ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 3300

cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 3360

ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 3420

accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 3480

catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 3540

gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 3600

tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 3660

agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 3720

actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 3780

gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 3840

aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 3900

gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 3960

aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 4020

atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca 4080

gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg 4140

atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca 4200

ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt 4260

cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt 4320

agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca 4380

cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca 4440

tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga 4500

agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact 4560

gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga 4620

gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg 4680

ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc 4740

tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga 4800

tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat 4860

gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt 4920

caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt 4980

atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgat 5040

gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcagga aattgtaagc 5100

gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa 5160

taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt 5220

gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg 5280

cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt 5340

ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga 5400

gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg 5460

ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg 5520

cttaatgcgc cgctacaggg cgcgtccatt cgccattcag gctgcgcaac tgttgggaag 5580

ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa 5640

ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca 5700

gtgaattgta atacgactca ctata 5725

<210> 9

<211> 4839

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

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 ccagtgccaa gcttgcatgc ctgcagacgc 420

tcaatgttgt tcatgatgtt ttgaagtttt taaaaaggga aattcacctt atataggata 480

cgggttgaga acatgcacaa agaggtgaaa cacaatgtgg gacatgataa agaacttttt 540

cttgtttagc agcggcgttc tgcaagcaac aaccctttta ctggtgatct taatttttat 600

gtatgtcaga aaaacgaaaa agaaaaacaa ggaatcttct ggctttatgg atgataaaca 660

ttaattgtga tgggcattct tttcaaagaa tgtcttttcc tttttcatgt ttcacatgaa 720

acattagcgt cattttactt gcgatttgga cttatgttaa aatataaaag taaaatttag 780

agatgatata aaaacattcg gtgcaaagca tgttgcagcg atgaacaaaa gagcagtcgt 840

ttatgctgct cgtcgagggc gatgatgtag tttctggttt ttaaatctca cataatgaaa 900

gagagagaga gagaagcaaa caaaaaattt tcagttcaat acaacagatc acgtgatctt 960

tttgtaagat gaagttgaag tagtgttgca ccgtgccaat gcaggtggct attagattaa 1020

atatgtgatt tgttctatta agtttcctgt ataattaatg gggagcgctg attctctttt 1080

ggtacgcttc ccatccagca tttctgtatc tttcaccttc aaccttagga tctctaccct 1140

tggcgaaaag tcctctgcca acaatgatga tatctgatcc accacttaca acttcgtcga 1200

cggttctgta ctgctgaccc aatgcatcgc ctttgtcgtc taaacctaca cctggggtca 1260

tgattagcca atcaaaccct tcttctcttc ctcccatatc gttctgagca atgaacccaa 1320

taacgaaatc tttatcactc tttgcaatat caacggtacc cttagtatat tcaccgtgtg 1380

ctagagaacc cttggaagac aattcagcaa gcatcaataa tccccttggt tctttggtga 1440

cctcttgcgc accttgtttc aagccagcaa caataccagc accagtaacc ccgtgggcgt 1500

tggtgatatc agaccattct gcgatacggt aaacgcccga tgtatattgt aatttgactg 1560

tgttaccgat atcggcgaat tttctgtcct caaatatcaa gaacttgtat ttctctgcca 1620

atgctttcaa tggaacgaca gtaccctcat aactgaaatc atccaagata tcaacgtgtg 1680

ttttcaaaag gcaaatgtat ggacccaacg tttcaacaag tttcaatagc tcatcagtcg 1740

aacgaacgtc aagagaagca cacaaattgg tcttcttttc atccattaaa cgtaaaagtt 1800

tcgatgcaac cggacttgca tgagtctcag ctctactggt atatgatttt gtggacatgg 1860

tgcaactaat tgacgggagt gtattgacgc tggcgtactg gctttcacaa aatggcccaa 1920

tcacaaccac atcttagata gttgaaatga ctttagataa catcaattga gatgagctta 1980

atcatgtcaa agctaaaagt gtcaccatga acgacaattc ttaagcaaat cacgtgatat 2040

agatccacga ataaccacca tttgatgctc gaggcaagta atgtgtgtaa aaaaatgcgt 2100

taccaccatc caatgcagac cgatcttcta cccagaatca tctagaacgc tcaatgttgt 2160

tcatgatgtt ttgaagtttt taaaaaggga aattcacctt atataggata cgggttgaga 2220

acatgcacaa agaggtgaaa cacaatgtgg gacatgataa agaacttttt cttgtttagc 2280

agcggcgttc tgcaagcaac aaccctttta ctggtgatct taatttttat gtatgtcaga 2340

aaaacgaaaa agaaaaacaa ggaatcttct ggctttatgg atgataaaca ttaattgtga 2400

tgggcattct tttcaaagaa tgtcttttcc tttttcatgt ttcacatgaa acattagcgt 2460

cattttactt gcgatttgga cttatgttaa aatataaaag taaaatttag agatgatata 2520

aaaacattcg gtgcaaagca tgttgcagcg atgaacaaaa gagcagtcgt ttatgctgct 2580

cggatccccg ggtaccgagc tcgaattcgt aatcatggtc atagctgttt cctgtgtgaa 2640

attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct 2700

ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc 2760

agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg 2820

gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc 2880

ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag 2940

gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa 3000

aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc 3060

gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc 3120

ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg 3180

cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt 3240

cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc 3300

gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc 3360

cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag 3420

agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg 3480

ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa 3540

ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag 3600

gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact 3660

cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa 3720

attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt 3780

accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag 3840

ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca 3900

gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc 3960

agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt 4020

ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg 4080

ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca 4140

gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg 4200

ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca 4260

tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg 4320

tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct 4380

cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca 4440

tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca 4500

gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg 4560

tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac 4620

ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt 4680

attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc 4740

cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat 4800

taacctataa aaataggcgt atcacgaggc cctttcgtc 4839

<210> 10

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ccgcaagctt acgctcaatg ttgttcatg 29

<210> 11

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

attagtcgac gagcagcata aacgactgc 29

<210> 12

<211> 3932

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gggcgaattg ggcccgacgt cgcatgctcc cggccgccat ggcggccgcg ggaattcgat 60

gccggatcca ggaaacgaag ataaatcatg tcgaaagcta catataagga acgtgctgct 120

actcatccta gtcctgttgc tgccaagcta tttaatatca tgcacgaaaa gcaaacaaac 180

ttgtgtgctt cattggatgt tcgtaccacc aaggaattac tggagttagt tgaagcatta 240

ggtcccaaaa tttgtttact aaaaacacat gtggatatct tgactgattt ttccatggag 300

ggcacagtta agccgctaaa ggcattatcc gccaagtaca attttttact cttcgaagac 360

agaaaatttg ctgacattgg taatacagtc aaattgcagt actctgcggg tgtatacaga 420

atagcagaat gggcagacat tacgaatgca cacggtgtgg tgggcccagg tattgttagc 480

ggtttgaagc aggcggcaga agaagtaaca aaggaaccta gaggcctttt gatgttagca 540

gaattgtcat gcaagggctc cctatctact ggagaatata ctaagggtac tgttgacatt 600

gcgaagagcg acaaagattt tgttatcggc tttattgctc aaagagacat gggtggaaga 660

gatgaaggtt acgattggtt gattatgaca cccggtgtgg gtttagatga caagggagac 720

gcattgggtc aacagtatag aaccgtggat gatgtggtct ctacaggatc tgacattatt 780

attgttggaa gaggactatt tgcaaaggga agggatgcta aggtagaggg tgaacgttac 840

agaaaagcag gctgggaagc atatttgaga agatgcggcc agcaaaacta aaaaactgta 900

ttataagtaa atgcatgtat actaaactca caaattagag cttcaattta attatatcag 960

ttattacccc cgggggcatc actagtgaat tcgcggccgc ctgcaggtcg accatatggg 1020

agagctccca acgcgttgga tgcatagctt gagtattcta tagtgtcacc taaatagctt 1080

ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca 1140

caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact 1200

cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct 1260

gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc 1320

ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca 1380

ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg 1440

agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca 1500

taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa 1560

cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc 1620

tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc 1680

gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct 1740

gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg 1800

tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag 1860

gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta 1920

cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg 1980

aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt 2040

tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt 2100

ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag 2160

attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat 2220

ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc 2280

tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat 2340

aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc 2400

acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag 2460

aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag 2520

agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt 2580

ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg 2640

agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt 2700

tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc 2760

tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc 2820

attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa 2880

taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg 2940

aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc 3000

caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag 3060

gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt 3120

cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt 3180

tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc 3240

acctgatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggaaat 3300

tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt 3360

taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg 3420

gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt 3480

caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc 3540

aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg 3600

atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa 3660

aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc 3720

cgccgcgctt aatgcgccgc tacagggcgc gtccattcgc cattcaggct gcgcaactgt 3780

tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt 3840

gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg 3900

acggccagtg aattgtaata cgactcacta ta 3932

<210> 13

<211> 3398

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

gggcgaattg ggcccgacgt cgcatgctcc cggccgccat ggcggccgcg ggaattcgat 60

gccgcgtgtt ggaatagaaa tcaactatca tctactaact agtatttaca ttactagtat 120

attatcatat acggtgttag aagatgacgc aaatgatgag aaatagtcat ctaaattagt 180

ggaagctgaa acgcaaggat tgataatgta ataggatcaa tgaatataaa catggatcct 240

ctagagctag ccccgggggc cgcctaggcc ataaaatgat gataataata tttatagaat 300

tgtgtagaat tgcagattcc cttttatgga ttcctaaatc cttgaggaga acttctagta 360

tattctgtat acctaatatt atagccttta tcaacaatgg aatcccaaca attatctcaa 420

cattcaccca tttctcatgg ccgatcacta gtgaattcgc ggccgcctgc aggtcgacca 480

tatgggagag ctcccaacgc gttggatgca tagcttgagt attctatagt gtcacctaaa 540

tagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat 600

tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag 660

ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg 720

ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc 780

ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc 840

agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa 900

catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt 960

tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 1020

gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 1080

ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 1140

cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 1200

caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 1260

ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 1320

taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 1380

taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac 1440

cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg 1500

tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt 1560

gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt 1620

catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa 1680

atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga 1740

ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt 1800

gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg 1860

agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga 1920

gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga 1980

agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg 2040

catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc 2100

aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc 2160

gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca 2220

taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac 2280

caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg 2340

ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc 2400

ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg 2460

tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac 2520

aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat 2580

actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata 2640

catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa 2700

agtgccacct gatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca 2760

ggaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 2820

attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 2880

gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc 2940

caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc 3000

ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag 3060

cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa 3120

agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac 3180

cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc attcgccatt caggctgcgc 3240

aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg 3300

ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt 3360

aaaacgacgg ccagtgaatt gtaatacgac tcactata 3398

Claims (2)

1. Saccharomyces cerevisiae (delta) integrating and secreting and expressing cellulaseSaccharomyces cerevisiae) The preservation number of the industrial strain R delta BEC is CGMCC NO. 16825.

2. Use of the industrial strain R δ BEC according to claim 1 in the production of ethanol.

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