CN111534541A - Eukaryotic organism CRISPR-Cas9 double gRNA vector and construction method thereof - Google Patents

Abstract

The invention relates to a eukaryotic organism CRISPR-Cas9 double gRNA vector and a construction method thereof, wherein a piggyBac transposon system is used as a delivery system, and a double gRNA vector is constructed by an enzyme digestion connection method.

Description

Eukaryotic organism CRISPR-Cas9 double gRNA vector and construction method thereof

Technical Field

The invention belongs to the technical field of eukaryotic gene knockout, and relates to a eukaryotic CRISPR-Cas9 double gRNA vector and a construction method thereof.

Background

Since the completion of human genome project, more and more model organisms and non-model organisms including mice, fruit flies, silkworms, arabidopsis thaliana, rice and the like complete whole genome sequencing, in the face of massive genome information, reading functional gene composition is an important topic of the later genome era, various genetic manipulation technologies (transgenosis, RNAi and the like) provide a basic platform for functional genome research, and among numerous genetic manipulation technologies, a gene knockout technology and a transgenic technology are two key genetic manipulation technologies for researching functional genes.

Gene editing technology is an important genetic manipulation technology developed in recent years, and four generations of genetic manipulations including meganucleases, zinc finger nucleases, transcription factor activator nucleases, CRISPR, and the like have been developed. Unlike the previous three generations of gene editing technology (relying on protein-nucleotide mutual recognition), CRISPR technology is a completely new gene editing technology based on RNA and DNA base complementary pairing. Since the invention of the CRISPR system, gene knockout has been successfully realized in many organisms including human, mouse, fruit fly, zebra fish, silkworm, Arabidopsis, tobacco, rice and the like. The efficient gene knockout cannot be separated from the efficient delivery system, the most widely applied delivery system in animals at present is a lentivirus-mediated delivery system, and the CRISPR system has been successfully delivered in mammals such as human, mice and the like. However, the lentivirus system has two significant disadvantages, one is that the lentivirus system has limited active species and is very inefficient in animals such as insects; the second is that the load bearing capacity of the lentivirus system is limited, only thousands of nucleotides, and the delivery capacity is significantly reduced with the increase of the foreign gene. In order to realize efficient gene knockout in eukaryotes, it is urgently needed to develop a system for efficiently delivering CRISPR.

The piggyBac transposon system is a type ii transposon originally found in trichoplusia ni, 2476bp in length, comprising two Inverted Terminal Repeats (ITRs) and an expression cassette encoding a transposase. The piggyBac transposable subsystem realizes transposition by adopting a shearing-sticking mode, and has high transposition efficiency. Currently, piggyBac transposable systems have been demonstrated to transpose efficiently in many species, from insects to mammals. The piggyBac transposon system has strong carrying capacity, and has been reported to carry nucleotides of more than 200kb at most, and the carrying capacity far exceeds that of a lentivirus system. Therefore, the development of the piggyBac transposon system mediated eukaryote CRISPR knockout system has wide application prospect.

On the genome, the encoded proteins only account for a small proportion, most regions are non-coding regions, and at present, researches prove that the non-coding regions of the genome have important functions, and to realize the functional deletion of the regions, the DNA sequence of the segment needs to be completely or partially deleted, so that the DNA double strand breaks at two sites on the genome are required to be simultaneously realized, and then the functional genome segment is deleted by virtue of the DNA repair channel of the cell, so that the functional deletion of the functional genome segment is realized. At present, the deletion of a DNA fragment on a genome can be realized by means of a double gRNA system, and the double gRNA system has important technical value for researching a non-coding region on a functional genome.

Disclosure of Invention

In view of this, the invention aims to provide a double gRNA vector of eukaryotic CRISPR-Cas9 and a construction method thereof.

In order to achieve the purpose, the invention provides the following technical scheme:

1. a construction method of a double gRNA vector of eukaryotic organism CRISPR-Cas9 comprises the following specific steps:

(1) constructing a piggyBac transposon system mediated eukaryote CRISPR-Cas9 double gRNA framework vector, namely pB-CRISPR, wherein the nucleotide sequence of the vector is shown as SEQ ID NO. 1; the gene element delivery system of the vector is a piggyBac transposon system, and the gene knockout system is a CRISPR/Cas9 system;

(2) constructing a template vector for providing sgRNA scaffold and U6 promoters, namely T-DGP-7, wherein the nucleotide sequence of the template vector is shown as SEQ ID NO. 2;

(3) designing a targeting site, constructing a primer pair of a double gRNA vector, and then performing PCR amplification by using the primer pair with the T-DGP-7 obtained in the step (2) as a template to obtain an amplification product named as PCR-DGP 7-XY;

(4) digesting the pB-CRISPR obtained in the step (1) by using an endonuclease AarI as a framework, digesting the PCR-DGP7-XY obtained in the step (3) by using BbsI as a fragment, and connecting the framework and the fragment to form a double gRNA vector which is named as pB-Dul-CRISPR-XY;

(5) and (3) mixedly transfecting the pB-Dul-CRISPR-XY obtained in the step (4) and a piggyBac transposon expression vector A3-helper with a nucleotide sequence shown as SEQ ID NO.3 to a eukaryotic cell, and screening to obtain the vector.

As one of the preferable technical proposal, the specific method of the step (1) is as follows:

(1-1) synthesizing a vector PUC57-IE2-Zeocin-Ser1PA containing a Zeocin resistance gene expression cassette, wherein the nucleotide sequence of the vector is shown as SEQ ID NO. 6;

(1-2) connecting a Zeocin resistance gene expression frame IE2-Zeocin-Ser1PA on a vector PUC57-IE2-Zeocin-Ser1PA to a piggyBac transposon basic vector piggyBacModify with a nucleotide sequence shown as SEQ ID No.7 to construct an intermediate vector pB-Modified { IE2-Zeocin-Ser1PA }, wherein the nucleotide sequence is shown as SEQ ID No. 8;

(1-3) amplifying an expression frame of hr3-hsp70-Cas9-sv40 from a vector pUC57-hr3-hsp70-Cas9-sv40 with the nucleotide sequence shown as SEQ ID NO. 9; then connected to AscI site of pB-Modified { IE2-Zeocin-Ser1PA } by a seamless cloning method to construct an intermediate vector pB-Modified { IE2-Zeocin-Ser1PA } { hr3-hsp70-Cas9-SV40}, and the nucleotide sequence of the intermediate vector is shown as SEQ ID NO. 11;

(1-4) amplifying U6-gRNA from a vector pUC57-U6-gRNA with the nucleotide sequence shown in SEQ ID NO.12, connecting to a vector pB-Modified { IE2-Zeocin-Ser1PA } { hr3-hsp70-Cas9-SV40} by using an enzyme digestion connection method, and constructing a eukaryotic gene knockout basic vector pB-Modified { IE2-Zeocin-Ser1PA } { U6-gRNA } { hr 3-Cas 70-Cas9-SV40}, wherein the vector is named as pB-CRISPR.

The vector map is shown in FIG. 2.

As one of the preferable technical schemes, in the step (1-3), the vector PUC57-Hr3-Hsp70-Cas9-SV40 is obtained by replacing the promoter A4 with Hsp70 from pUC57-hA4-Cas9 (the nucleotide sequence is shown in SEQ ID NO.10, PMID: 24671069).

As one of the preferred technical solutions, in step (3), based on the CRISPR/Cas9 law of action, a targeting site for realizing eukaryotic gene knockout is designed, with 23 nucleotides in total, and the following rules are provided:

5 '-NNNNNNNNNNNNNNNNNNNNNNNNN-NGG-3'; on the basis, a primer pair of the double gRNA vector is constructed, and has the following rule:

the forward primer is > X-F,

5-ACCGATCGATGAAGACAGAAGTGNNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAA, the nucleotide sequence of which is shown in SEQ ID NO. 4;

the reverse primer is > Y-R,

5-TGATGATGATGAAGACGTAAACNNNNNNNNNNNNNNNNNNNNCACTTGTAGAGCACGAT, the nucleotide sequence of which is shown in SEQ ID NO. 5;

wherein "NNNNNNNNNNNNNNNNNNNN" in > X-F and > Y-R are targeting site X and targeting site Y, respectively.

As one of the preferred technical schemes, in the step (5), pB-Dul-CRISPR-XY and piggyBactransposon expression vector A3-helper (nucleotide sequence is shown as SEQ ID NO. 3) are expressed according to a molar ratio of 1: 1, transfecting eukaryotic cells, and screening the transfected cells by Zeocin for 2 months to obtain a cell line with two genes knocked out or functional genome fragments deleted simultaneously.

As one of the preferred technical schemes, the eukaryote includes but is not limited to silkworm, fruit fly and the like.

2. The eukaryotic CRISPR-Cas double gRNA vector is constructed by the method.

3. The vector is applied to the construction of a cell line for realizing the simultaneous knockout of two genes or the deletion of functional genome segments of eukaryotes.

As one of the preferred technical schemes, the functional genome segment includes but is not limited to coding gene, non-coding RNA, genome functional element and the like.

The invention has the beneficial effects that:

the invention discloses a construction method of a piggyBac transposon system mediated eukaryote CRISPR-Cas9 double gRNA vector. The method is characterized in that by means of the ultra-large carrying capacity and wide species effectiveness of the piggyBac transposon system, a Cas9 expression frame and a double gRNA expression frame are on the same vector, the double gRNA can be efficiently and conveniently played in numerous eukaryotes, and two genes can be simultaneously knocked out or functional genome fragments (coding genes, non-coding RNAs, genome functional elements and the like) can be deleted. At present, a lentivirus system is widely applied to deliver CRISPR whole genome editing libraries to eukaryotic cells, but the lentivirus system has low efficiency in species other than mammals, and the bearing capacity is only a few kb, so that the application of the lentivirus system is limited. Because the piggyBac transposon is realized in a 'shearing-sticking' mode to realize transposition, the copy number of the exogenous gene carried by the piggyBac transposon system integrated into a host cell can be controlled by controlling the transposon concentration and the like. Compared with the lentivirus-mediated CRISPR-Cas9 single-gene knockout system widely applied at present, the method has remarkable advantages.

Drawings

In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:

FIG. 1 is a construction process of a piggyBac transposon system mediated eukaryotic CRISPR-Cas9 double gRNA vector.

FIG. 2 is a vector pB-CRISPR map comprising: piggyBacL/piggyBacR, piggyBac swivel arm; IE2, IE2 promoter; zeocin, Zeocin resistance gene; ser1PA, bombyx mori sericin 1(Ser1) gene polyA; u6, U6 promoter; gRNA, sgRNA scaffold; hr3-hsp70, the Hr3 enhancer and the hsp70 promoter; spCas9, spCas9 protein; SV40PA, SV40 polyA.

FIG. 3 is a map of vector T-DGP7, comprising: u6, U6 promoter; gRNA, sgRNA scaffold.

FIG. 4 is a schematic diagram of a double gRNA vector system for deleting specific DNA fragments of silkworm cells, wherein the targeting sites of the double gRNAs on ME1 and ME2 are both outside a green fluorescent protein (EGFP) coding sequence, and the deleted fragments are 2791bp and 1448bp respectively; the targeting sites of ME3 and ME4 were both on the green fluorescent protein (EGFP) coding sequence, and the deletion fragments were 309bp and 61bp, respectively.

Fig. 5 is a histogram of the efficiency of deleting specific DNA fragments of silkworm cells by the dual gRNA vector system, wherein the targeting sites of the dual gRNA to ME1 and ME2 are both outside the green fluorescent protein (EGFP) coding sequence, which indicates that the efficiency of deleting specific DNA fragments of silkworm cells by the dual gRNA vector system is about 20% to 25%; the targeting sites of ME3 and ME4 are both on the coding sequence of green fluorescent protein (EGFP), which can indicate that the efficiency of the double gRNA vector system for knocking out the specific DNA of silkworm cells is over 90%.

Detailed Description

Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

All the following specific experimental methods, which are not indicated, are carried out according to accepted experimental methods and conditions, for example, according to the instructions provided by the manufacturers of reagents and consumables, or according to the classic laboratory book "molecular cloning guidelines" (third edition, J. SammBruke et al).

Example (b):

the silkworm embryonic cell line (The Bombyx mori electrophoretic cell line, BmE) used in this example was a cell line commonly used in biological experiments (PMID: 17570024).

A cell line with a deleted silkworm specific DNA fragment is constructed by using a piggyBac transposon system mediated eukaryote CRISPR-Cas9 double gRNA vector system.

1. The bombyx mori embryonic cell line BmE-Mi-EGFP of which the green fluorescent protein (EGFP) is stably transfected has a DNA sequence of a stably integrated exogenous gene shown as SEQ ID No. 13.

2. A piggyBac transposon system mediated eukaryotic organism CRISPR-Cas9 double gRNA framework vector is constructed and named pB-CRISPR, and the vector map is shown in figure 2.

3. A template vector for providing sgRNA scaffold and U6 promoters was constructed and designated T-DGP-7, and the vector map is shown in FIG. 3.

4. Designing a sgRNA targeting locus pair for specifically deleting a partial DNA sequence of the BmE-Mi-EGFP cell line according to the nucleotide sequence provided by the step 1 and the action rule of spCas9,

AATGTTCGCGAAAAGAGCGCCGG/GTGTATTTAACACATCGGAAAGG, the nucleotide sequence is shown as SEQ ID NO. 14;

ACAAGCACCTTTATACTCGGTGG/CCCTTTAAGATGGGCCTAATGGG, the nucleotide sequence is shown as SEQ ID NO. 15;

GAGCTGGACGGCGACGTAAACGG/GTGAACCGCATCGAGCTGAAGGG, the nucleotide sequence is shown as SEQ ID NO. 16;

GGGCGAGGAGCTGTTCACCGGGG/GGCCACAAGTTCAGCGTGTCCGG, the nucleotide sequence is shown as SEQ ID NO. 17;

5. designing a primer pair for constructing the double gRNA vector according to the target-hitting site designed in the step 4,

>ME1-F

5-ACCGATCGATGAAGACAGAAGTGAATGTTCGCGAAAAGAGCGCGTTTTAGAGCTAGAAA, the nucleotide sequence of which is shown in SEQ ID NO. 18;

>ME1-R

5-TGATGATGATGAAGACGTAAACTTCCGATGTGTTAAATACACCACTTGTAGAGCACGAT, the nucleotide sequence of which is shown in SEQ ID NO. 19;

>ME2-F

5-ACCGATCGATGAAGACAGAAGTGACAAGCACCTTTATACTCGGGTTTTAGAGCTAGAAA, the nucleotide sequence of which is shown in SEQ ID NO. 20;

>ME2-R

5-TGATGATGATGAAGACGTAAACATTAGGCCCATCTTAAAGGGCACTTGTAGAGCACGAT, the nucleotide sequence of which is shown in SEQ ID NO. 21;

>ME3-F

5-ACCGATCGATGAAGACAGAAGTGGAGCTGGACGGCGACGTAAAGTTTTAGAGCTAGAAA, the nucleotide sequence of which is shown in SEQ ID NO. 22;

>ME3-R

5-TGATGATGATGAAGACGTAAACTTCAGCTCGATGCGGTTCACCACTTGTAGAGCACGAT, the nucleotide sequence of which is shown in SEQ ID NO. 23;

>ME4-F

5-ACCGATCGATGAAGACAGAAGTGGGGCGAGGAGCTGTTCACCGGTTTTAGAGCTAGAAA, the nucleotide sequence of which is shown in SEQ ID NO. 24;

>ME4-R

5-TGATGATGATGAAGACGTAAACGACACGCTGAACTTGTGGCCCACTTGTAGAGCACGAT, the nucleotide sequence of which is shown in SEQ ID NO.25

6. Performing PCR amplification by using the primer pair designed in the step 5 by taking T-DGP7 as a template, and respectively naming the products as: PCR-DGP7-ME1, PCR-DGP7-ME2, PCR-DGP7-ME3 and PCR-DGP7-ME 4. The PCR amplification enzyme is high-fidelity hot-start enzyme(s) ((

Max DNA Polymerase, Takara, Japan) in a total reaction system of 50. mu.l, including 1. mu.l each of primers, 1. mu.l of template, 25. mu.l of 2 × enzyme premix, and 22. mu.l of water, under the reaction conditions of pre-denaturation at 98 ℃ for 4min, denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 5s, elongation at 72 ℃ for 5s, 35 cycles, elongation at 72 ℃ for 10min, and storage at 12 ℃.

7. Digesting the vector pB-CRISPR by using endonuclease AarI as a framework, digesting the PCR product described in the step 6 by using BbsI, and then respectively connecting the digested product to the framework by using DNA ligase to successfully construct double gRNA vectors which are respectively named as: pB-Dul-CRISPR-ME1, pB-Dul-CRISPR-ME2, pB-Dul-CRISPR-ME3, pB-Dul-CRISPR-ME 4.

1) The digestion conditions of the vector pB-CRISPR are 14.6 mu L of nucleic-free water, 2 mu L of 10X BufferAarI, 1 mu L of DNA (0.5-1 mu g/. mu.L), 0.4 mu L of 50X oligonucleotide (0.025mM) and 2 mu L of AarI. AarI is purchased from siemer feishel.

2) The digestion of the PCR product was carried out in a 50. mu.l system containing 10. mu.l of the vector PCR product, 5. mu.l of CutSmart buffer, 1. mu.l of BbsI supplemented with double distilled water at 37 ℃ overnight, and BbsI restriction enzyme purchased from NEB.

3) The DNA ligase is T4 DNA ligase, the total ligation is 50 μ l, wherein the skeleton and the fragments are mixed according to a molar ratio of 1: 10, 2. mu.g total mass, 5. mu.l of T4 DNA ligase buffer, 1. mu.l of T4 DNA ligase, 50. mu.l of double distilled water, ligation at 16 ℃ for 4 hours, T4 DNA ligase purchased from NEB.

8. And (2) respectively carrying out the double gRNA deletion vectors pB-Dul-CRISPR-ME1, pB-Dul-CRISPR-ME2, pB-Dul-CRISPR-ME3 and pB-Dul-CRISPR-ME4 constructed in the step 7 and a piggyBac transposase expression vector A3-helper (the nucleotide sequence is shown as SEQ ID NO. 3) according to a molar ratio of 1: 1 transfection of silkworm embryonic cell line BmE-Mi-EGFP stably transfected with Green fluorescent protein (EGFP), followed by 2 months of screening with complete medium containing Zeocin (200. mu.g/ml), successfully constructing a double gRNA vector system silkworm specific DNA deletion cell line, the complete medium being Grace's medium (Seimer Feier Co.) comprising 10% fetal bovine serum (Fetalbrook serum, FBS, Seimer Feishill) and Penicillin-Streptomycin (Penicillin-Streptomyces, 20 ten thousand units/liter, Seimer Feishill Co.), the overall flow is shown in FIG. 1.

9. And (3) analyzing the condition that the double gRNA vector system deletes specific DNA fragments of the silkworm cells by using a flow cytometer.

The targeting sites of the double gRNA to ME1 and ME2 are outside the green fluorescent protein (EGFP) coding sequence, which can indicate that the efficiency of the double gRNA vector system for deleting the specific DNA fragment of the silkworm cell is about 20-25%; the targeting sites of ME3 and ME4 are both on the coding sequence of green fluorescent protein (EGFP), which can indicate that the efficiency of the double gRNA vector system for knocking out silkworm cell-specific DNA is over 90% (FIG. 4 and FIG. 5).

Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.

Sequence listing

<110> university of southwest

<120> eukaryotic organism CRISPR-Cas9 double gRNA vector and construction method

<130>2020

<160>25

<170>SIPOSequenceListing 1.0

<210>1

<211>13408

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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 ccagtgaatt ggagatcggt acttcgcgaa 420

tgcgtcgaga taagagggtt aaaaaatata ttttacgcac catatacgca tcgggttgat 480

atcgttaata tggatcaatt tgaacagttg attaacgtgt ctctgctcaa gtctttgatc 540

aaaacgcaaa tcgacgaaaa tgtgtcggac aatatcaagt cgatgagcga aaaactaaaa 600

aggctagaat acgacaatct cacagacagc gttgagatat acggtattca cgacagcagg 660

ctgaataata aaaaaattag aaactattat ttaaccctag aaagataatc atattgtgac 720

gtacgttaaa gataatcatg cgtaaaattg acgcatgtgt tttatcggtc tgtatatcga 780

ggtttattta ttaatttgaa tagatattaa gttttattat atttacactt acatactaat 840

aataaattca acaaacaatt tatttatgtt tatttattta ttaaaaaaaa acaaaaactc 900

aaaatttctt ctataaagta acaaaacttt taaacattct ctcttttaca aaaataaact 960

tattttgtac tttaaaaaca gtcatgttgt attataaaat aagtaattag cttaacttat 1020

acataataga aacaaattat acttattagt cagtcagaaa caactttggc acatatcaat 1080

attatgctct cgacaaataa cttttttgca ttttttgcac gatgcatttg cctttcgcct 1140

tattttagag gggcagtaag tacagtaagt acgttttttc attactggct cttcagtact 1200

gtcatctgat gtaccaggca cttcatttgg caaaatatta gagatattat cgcgcaaata 1260

tctcttcaaa gtaggagctt ctaaacgctt acgcataaac gatgacgtca ggctcatgta 1320

aaggtttctc ataaattttt tgcgactttg aaccttttct cccttgctac tgacattatg 1380

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cactccgcct ttagtttgat tataatacat aaccatttgc ggtttaccgg tactttcgtt 1560

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gcgactgttt ttcagtactt ccggtatctc gcgtttgttc ctgcaggatc atgatgataa 1740

acaatgtatg gtgctaatgt tgcttcaaca acaattctgt tgaactgtgt tttcatgttt 1800

gccaacaagc acctttatac tcggtggcct ccccaccacc aacttttttg cactgcaaaa 1860

aaacacgctt ttgcacgcgg gcccatacat agtacaaact ctacgtttcg tagactattt 1920

tacataaata gtctacaccg ttgtatacgc tccaaataca ctaccacaca ttgaaccttt 1980

ttgcagtgca aaaaagtacg tgtcggcagt cacgtaggcc ggccttatcg ggtcgcgtcc 2040

tgtcacgtac gaatcacatt atcggaccgg acgagtgttg tcttatcgtg acaggacgcc 2100

agcttcctgt gttgctaacc gcagccggac gcaactcctt atcggaacag gacgcgcctc 2160

catatcagcc gcgcgttatc tcatgcgcgt gaccggacac gaggcgcccg tcccgcttat 2220

cgcgcctata aatacagccc gcaacgatct ggtaaacaca gttgaacagc atctgttcga 2280

aatggccaag ttgaccagtg ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt 2340

cgagttctgg accgaccggc tcgggttctc ccgggacttc gtggaggacg acttcgccgg 2400

tgtggtccgg gacgacgtga ccctgttcat cagcgcggtc caggaccagg tggtgccgga 2460

caacaccctg gcctgggtgt gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga 2520

ggtcgtgtcc acgaacttcc gggacgcctc cgggccggcc atgaccgaga tcggcgagca 2580

gccgtggggg cgggagttcg ccctgcgcga cccggccggc aactgcgtgc acttcgtggc 2640

cgaggagcag gactaaagct ttacaactaa acacgacttg gagtattcct tgtagtgttt 2700

aagattttaa atcttactta atgacttcga acgattttaa cgataacttt ctctttgttt 2760

aactttaatc agcatacata aaaagccccg gttttgtatc gggaagaaaa aaaatgtaat 2820

tgtgttgcct agataataaa cgtattatca aagtgtgtgg ttttccttta ccaaagaccc 2880

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tttatttaat ttgttgcacc aaaactgata cattgacgaa acgcgtatgc tagcaatgaa 3060

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ttttatttaa aaaaataaac aattttttat acataaactg tttatctagt attaatattt 3180

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atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagatt atcaaaaagg 3540

atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat 3600

gagtaaactt ggtctgacag ttaccaatgc cacctgccat cacttgtaga gcacgatatt 3660

ttgtatatat acctaaaaaa actaaactat tgaaagcgtg atttacaaca acactcgact 3720

ttacaaagat tattcaaaaa gagcaaaaac tcttaacata ttctattaaa gatatataat 3780

ataattaaaa cgaaattaaa taataacaat aaaaccttta gaatttgtaa taaaatccat 3840

aaaaacaaat gaaaacagtt atggtttgta cagcgccatc tgttattact ttgacaaaat 3900

cactatgact atctgacctt gtcttacacg ttaacaattc ttattctgtc cttatctata 3960

agccaagtac caagcttaaa ttcgtatggc ttatagttga cgatttttaa attctcaagg 4020

tatgtactta tttaatatta ataagtacta attgttaaaa tcatctaaaa caattcagtg 4080

atttacaaca atgtgtacta cataacctaa tacttataaa tttattaaac tgtattgatt 4140

cttttaggtc aatcatcatg actttaggag acttggtgtc tcaggaaaaa ggaacgcaaa 4200

aagattgagg cgtttgaaat gtattgctgg agaaagctgc tacgcattcc ttggacagct 4260

tggcgcgccc agcgtcgtga aaagaggcaa tgacaaatac aaaacgacgt atgagcagac 4320

ccgtcgccaa gacgggtcta cctctaagat gatgtcattt gttttttaaa actaactcgc 4380

tttacgagta gaattctacg tgtaaaacat aatcaagaga tgatgtcatt tgtttttcaa 4440

aaccaaactc gctttacgag tagaattcta cgtgtaaaac acaatcaaaa gatgatgtca 4500

ttcgtttttc aaaaccgaat ttaagaaatg atgtcatttg tttttcaaaa ccaaactcgc 4560

tttacgagca gaattctacg tgtaaaacac aatcaagaga tgatgtcatt tgtttttcaa 4620

aactgaatga tgtcatttgt ttttcaaaac taaacttgct ttgcgagtag aattctacgt 4680

gtaaaacaca gtcaagagat gatgtcattt gtttttcaaa actgaaccgg ctttacgagt 4740

agaattctac ttgtaaaaca taatcaagag atgatgtcat ttgtttttca aaactgaact 4800

ggctttacga gtagaattct acgtgtaaaa cataatcaag agatgatgtc atcattaaac 4860

tgatgtcatt ttatacacga ttgttaacat gtttaataat gactaatttg tttttccaaa 4920

ttaaactcgc tttacgagta gaattctact tgtaacgcac gattaagtat gaatcataag 4980

ctgatgtcat ttgttttcga cataaaatgt ttatacaatg gaatcttctt gtaaattatc 5040

caaataatat aatttatccg attctacgtt acatttaaat tcgttgttat cgtacaattc 5100

ttcaggacac gccatgtatt ggtcattttt agcgtgcaac caacgattgt atttgacgcc 5160

gtcgttggat tgcgtgttca ggttggcgta cacgtgactg ggcacggctt ctttttccat 5220

gggacgtcga ccgagaaatt tctctggccg ttattcgtta ttctctcttt tctttttggg 5280

tctctccctc tctgcactaa tgctctctca ctctgtcaca cagtaaacgg catactgctc 5340

tcgttggttc gagagagcgc gcctcgaatg ttcgcgaaaa gagcgccgga gtataaatag 5400

aggcgcttcg tctacggagc gacaattcaa ttcaaacaag caaagtgaac acgtcgctaa 5460

gcgaaagcta agcaaataaa caagcgcagc tgaacaagct aaacaatctg cagtaaagtg 5520

caagttaaag tgaatcaatt aaaagtaacc agcaaccaag taaatcaact gcaactactg 5580

aaatctgcca agaagtaatt attgaataca agaagagaac tctgggggat ctctagtcca 5640

gtgtggtgga attcgccatg gccccaaaga aaaagagaaa ggttgattac aaagaccacg 5700

acggagacta caaagaccac gacattgatt ataaagatga tgatgataaa ggaacgatgg 5760

acaaaaagta tagcatcggt ctggatattg gaactaactc cgtcggctgg gctgtaatca 5820

ccgacgaata caaggtcccg tcaaaaaagt tcaaggtatt gggtaacaca gatcgtcact 5880

ctatcaaaaa gaatctcatt ggagctctgt tgttcgacag cggcgaaaca gctgaggcca 5940

ctagactgaa gcgcaccgcc agacgccgtt acacgaggag aaagaacaga atctgctact 6000

tgcaagaaat attctcaaac gagatggcca aagtggacga ttcgttcttt cataggttag 6060

aagagagttt ccttgttgaa gaggataaaa agcacgaaag acatccgata tttggaaaca 6120

tcgtggacga agttgcttat cacgagaagt accccacgat ctatcatctg cgtaaaaagt 6180

tggtggactc gacagataag gccgacctca ggttaatata ccttgcactg gcgcacatga 6240

tcaaattcag aggccatttt ctgattgaag gtgacctgaa ccctgacaat agtgatgtgg 6300

acaaactctt cattcaatta gttcagacct acaatcaact gtttgaagag aaccctatca 6360

acgcttcagg agttgacgct aaggccatcc ttagtgcgag actgagcaaa tcccgccgtc 6420

tcgaaaactt aatcgcacag ttgcctggag agaaaaagaa cggtttgttc ggaaatctca 6480

ttgcgttgtc actcggactc acgccaaact tcaagtctaa cttcgatttg gcagaagacg 6540

cgaaactgca actgagcaaa gacacatatg acgatgacct cgataacctc ttagctcaga 6600

tcggcgatca atacgccgac ttgttcctcg ctgccaaaaa tctgtcggac gctatacttc 6660

tgagtgatat cttgcgcgtc aacacagaaa ttactaaggc tcctctgtcg gccagtatga 6720

taaaacgcta tgacgaacac catcaggatt tgacattgct caaagccctc gtgcgtcaac 6780

agctcccaga aaagtacaag gagattttct ttgatcagtc caagaatggc tacgcaggtt 6840

atatagacgg tggagcgtcg caagaagagt tctacaagtt catcaagcca atattagaaa 6900

agatggacgg cacggaagag ttacttgtta agctgaatcg tgaggacctg ttgcgtaaac 6960

agaggacatt cgataacgga tcaattccgc accaaataca tcttggcgaa ctgcacgcta 7020

tcctcaggag acaagaggac ttctacccct ttttaaagga taaccgtgaa aagatcgaga 7080

aaatcctgac tttcaggatt ccttactatg tcggcccact ggctcgtggt aatagcaggt 7140

ttgcctggat gaccaggaag tccgaagaga caattactcc gtggaacttc gaagaggtgg 7200

ttgataaagg agcatcagcg cagtctttca tagaacgcat gacaaatttt gacaagaact 7260

taccgaatga gaaggtcctt cccaaacact cactcctcta cgaatacttc acagtataca 7320

acgagctcac taaagtcaag tacgtaaccg agggtatgcg caaacccgct ttcctgtctg 7380

gagagcagaa aaaggccatc gtggaccttc tgttcaagac aaaccgtaag gtcactgtaa 7440

agcaactcaa ggaagactac ttcaaaaaga tagagtgttt cgattcagtg gaaatctctg 7500

gcgttgagga cagatttaac gcttccttgg gtacttacca cgatttgctc aagatcatta 7560

aagataagga cttcctcgac aacgaagaga acgaagatat cttagaggac atagttctca 7620

cccttacgct gtttgaagat agagagatga ttgaagagcg cctgaagact tatgctcatt 7680

tgttcgatga caaagtcatg aagcaactga aacgccgtag gtacaccggc tggggtagat 7740

tatcgcgcaa acttattaat ggtataaggg acaagcagtc gggaaaaacg atattggact 7800

ttctcaagag tgatggtttc gccaacagaa attttatgca actcatacac gatgacagct 7860

taacattcaa ggaagatatc caaaaagcac aggtgtcggg acagggcgac agtttgcacg 7920

aacatattgc taacctcgcc ggctccccgg cgataaaaaa gggtatcctt cagactgtga 7980

aagtcgtaga tgaactggtg aaggttatgg gtcgtcataa acccgagaac atagttatcg 8040

aaatggctag ggagaatcaa acaactcaga agggacagaa aaactcaaga gaacgcatga 8100

agcgcattga agagggtatc aaagagcttg gcagtcaaat cctgaaggaa caccctgtcg 8160

agaacacgca acttcagaac gaaaaattgt acctctacta tctgcagaat ggtagagata 8220

tgtacgtaga ccaagaattg gatattaacc gcctctcaga ttacgacgtg gatcatatag 8280

ttccgcagtc attcttgaag gatgactcta tcgacaacaa agtcctcaca agatcagaca 8340

agaaccgcgg aaaatcagat aatgtaccct ctgaagaggt ggttaaaaag atgaaaaact 8400

actggagaca gttacttaac gctaagttga tcacgcaaag aaagttcgat aacctcacaa 8460

aggctgaacg cggcggttta agcgagcttg acaaggccgg tttcataaaa cgtcagttag 8520

tcgaaaccag gcaaattacg aaacacgtag cccaaatatt ggattcccgc atgaacacta 8580

aatacgatga aaatgacaag ctcatccgtg aggtcaaagt aattaccctg aaaagcaagt 8640

tggtgtccga cttcagaaag gatttccagt tctacaaagt tcgcgaaatc aacaactacc 8700

accatgcaca tgacgcttac ctgaacgcag tcgtaggcac tgcgttaatt aaaaagtacc 8760

ctaaactgga atctgagttc gtgtacggtg actataaagt gtacgatgtt agaaagatga 8820

tcgctaaaag cgaacaggag attggaaagg ctaccgccaa gtatttcttt tactccaaca 8880

tcatgaattt ctttaagaccgaaatcacgt tagcaaatgg cgagatacgt aaaaggccac 8940

ttatcgaaac aaacggagaa actggcgaga tagtgtggga caagggtaga gattttgcca 9000

ctgtccgcaa agtactgtcg atgccgcaag tgaatatcgt taaaaagacc gaagttcaaa 9060

cgggaggctt cagcaaagag tccatcctgc ccaagcgtaa cagtgataaa ttgatagcta 9120

ggaaaaagga ctgggatcct aaaaagtatg gtggattcga cagcccaact gtcgcatact 9180

ccgtattggt ggttgcgaaa gtcgaaaaag gaaagagcaa aaagctcaag tccgtaaaag 9240

agctgttggg cattaccata atggaaagat catctttcga gaagaatcct atcgattttc 9300

tggaagccaa gggatataaa gaggtcaaaa aggacctcat aatcaagtta ccaaaataca 9360

gtctgttcga attggagaac ggcagaaaac gcatgcttgc atcagcgggt gaactgcaaa 9420

agggaaatga gttagcactt ccttctaaat acgtcaactt cctgtatttg gcgtcacact 9480

acgaaaaact gaagggctct ccagaagata acgagcaaaa gcagttattt gtggaacagc 9540

acaaacatta ccttgacgaa attatagagc aaatctcgga gttcagtaag agagtgattt 9600

tggctgacgc caatcttgat aaagttctgt ctgcttacaa caagcaccgt gataaaccga 9660

ttagggaaca ggccgagaac atcatacatc tcttcacact cactaacctt ggtgcacccg 9720

cagcgttcaa atattttgac accacgatag atcgtaagag gtacaccagc acgaaagaag 9780

ttttggacgc gacactcatc catcaatcaa tcacgggcct gtacgagacc agaatcgacc 9840

tgtcccagct cggtggcgac tagcggccgc gactctagat cataatcagc catgcggccg 9900

cgactctaga ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc 9960

ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat 10020

aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg 10080

cattctagtt gtggtttgtc caaactcatc aatgtatctt aaagcttatc gatacgcgta 10140

cctaggccgg ccgatctcgg atctgacaat gttcagtgca gagactcggc tacgcctcgt 10200

ggactttgaa gttgaccaac aatgtttatt cttacctcta atagtcctct gtggcaaggt 10260

caagattctg ttagaagcca atgaagaacc tggttgttca ataacatttt gttcgtctaa 10320

tatttcacta ccgcttgacg ttggctgcac ttcatgtacc tcatctataa acgcttcttc 10380

tgtatcgctc tggacgtcat cttcacttac gtgatctgat atttcactgt cagaatcctc 10440

accaacaagc tcgtcatcgc tttgcagaag agcagagagg atatgctcat cgtctaaaga 10500

actacccatt ttattatata ttagtcacga tatctataac aagaaaatat atatataata 10560

agttatcacg taagtagaac atgaaataac aatataatta tcgtatgagt taaatcttaa 10620

aagtcacgta aaagataatc atgcgtcatt ttgactcacg cggtcgttat agttcaaaat 10680

cagtgacact taccgcattg acaagcacgc ctcacgggag ctccaagcgg cgactgagat 10740

gtcctaaatg cacagcgacg gattcgcgct atttagaaag agagagcaat atttcaagaa 10800

tgcatgcgtc aattttacgc agactatctt tctagggtta aaaaagattt gcgctttact 10860

cgacctaaac tttaaacacg tcatagaatc ttcgtttgac aaaaaccaca ttgtggccaa 10920

gctgtgtgac gcgacgcgcg ctaaagaatg gcaaaccaag tcgcgcgagc gtcgactcta 10980

gaggatcccc gggtaccgag ctcgaattcg taatcatggt catagctgtt tcctgtgtga 11040

aattgttatc cgctcacaat tccacacaacatacgagccg gaagcataaa gtgtaaagcc 11100

tggggtgcct aatgagtgag ctaactcaca tcggatgccg ggaccgacga gtgcagaggc 11160

gtgcaagcga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg 11220

ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa 11280

tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac 11340

ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt 11400

gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga 11460

gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca 11520

ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg 11580

ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt 11640

cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc 11700

ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct 11760

tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc 11820

gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta 11880

tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca 11940

gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag 12000

tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag 12060

ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt 12120

agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa 12180

gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg 12240

attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga 12300

agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta 12360

atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc 12420

cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg 12480

ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga 12540

agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt 12600

tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt 12660

gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc 12720

caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc 12780

ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca 12840

gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag 12900

tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg 12960

tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa 13020

cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa 13080

cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga 13140

gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga 13200

atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg 13260

agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt 13320

ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa 13380

aataggcgta tcacgaggcc ctttcgtc 13408

<210>2

<211>741

<212>DNA

<213>Artificial

<400>2

gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt 60

ggcaccgagt cggtgctttt ttatggacga gctgtacaag tgaggggcac aagcttaatt 120

gaagctgtcc aaggaatgcg tagcagcttt ctccagcaat acatttcaaa cgcctcaatc 180

tttttgcgtt cctttttcct gagacaccaa gtctcctaaa gtcatgatga ttgacctaaa 240

agaatcaata cagtttaata aatttataag tattaggtta tgtagtacac attgttgtaa 300

atcactgaat tgttttagat gattttaaca attagtactt attaatatta aataagtaca 360

taccttgaga atttaaaaat cgtcaactat aagccatacg aatttaagct tggtacttgg 420

cttatagata aggacagaat aagaattgtt aacgtgtaag acaaggtcag atagtcatag 480

tgattttgtc aaagtaataa cagatggcgc tgtacaaacc ataactgttt tcatttgttt 540

ttatggattt tattacaaat tctaaaggtt ttattgttat tatttaattt cgttttaatt 600

atattatata tctttaatag aatatgttaa gagtttttgc tctttttgaa taatctttgt 660

aaagtcgagt gttgttgtaa atcacgcttt caatagttta gtttttttag gtatatatac 720

aaaatatcgt gctctacaag t 741

<210>3

<211>6161

<212>DNA

<213>Artificial

<400>3

aaatcaactt gtgttatagt cacggatttg ccgtccaacg tgttcctcaa aaagttgaag 60

accaacaagt ttacggacac tattaattat ttgattttgc cccacttcat tttgtgggat 120

cacaattttg ttatattttt aaacaaagct tggcactggc cgtcgtttta caacgtcgtg 180

actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca 240

gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga 300

atggcgaatg gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc 360

gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac 420

acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca 480

gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga 540

aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa 600

taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt 660

gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa 720

tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta 780

ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag 840

taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca 900

gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta 960

aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc 1020

gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc 1080

ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca 1140

ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc 1200

acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca 1260

taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac 1320

tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg 1380

cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg 1440

ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg 1500

gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac 1560

gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc 1620

aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct 1680

aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc 1740

actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc 1800

gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg 1860

atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa 1920

atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc 1980

ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt 2040

gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa 2100

cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc 2160

tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc 2220

cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct 2280

ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat 2340

gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc 2400

tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg 2460

ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc 2520

gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg 2580

cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca 2640

gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact 2700

ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa 2760

acagctatga catgattacg aattcgaatt cccatccccc tagaatccca aaacaaactg 2820

gttattgtgg taggtcattt gtttggcaga aagaaaactc gagaaatttc tctggccgtt 2880

attcgttatt ctctcttttc tttttgggtc tctccctctc tgcactaatg ctctctcact 2940

ctgtcacaca gtaaacggca tactgctctc gttggttcga gagagcgcgc ctcgaatgtt 3000

cgcgaaaaga gcgccggagt ataaatagag gcgcttcgtc tacggagcga caattcaatt 3060

caaacaagca aagtgaacac gtcgctaagc gaaagctaag caaataaaca agcgcagctg 3120

aacaagctaa acaatctgca gtaaagtgca agttaaagtg aatcaattaa aagtaaccag 3180

caaccaagta aatcaactgc aactactgaa atctgccaag aagtaattat tgaatacaag 3240

aagagaactc tgggggatcc ccgtgaggcg tgcttgtcaa tgcggtaagt gtcactgatt 3300

ttgaactata acgaccgcgt gagtcaaaat gacgcatgat tatcttttac gtgactttta 3360

agatttaact catacgataa ttatattgtt atttcatgtt ctacttacgt gataacttat 3420

tatatatata ttttcttgtt atagatatcg tgactaatat ataataaaat gggtagttct 3480

ttagacgatg agcatatcct ctctgctctt ctgcaaagcg atgacgagct tgttggtgag 3540

gattctgaca gtgaaatatc agatcacgta agtgaagatg acgtccagag cgatacagaa 3600

gaagcgttta tagatgaggt acatgaagtg cagccaacgt caagcggtag tgaaatatta 3660

gacgaacaaa atgttattga acaaccaggt tcttcattgg cttctaacag aatcttgacc 3720

ttgccacaga ggactattag aggtaagaat aaacattgtt ggtcaacttc aaagtccacg 3780

aggcgtagcc gagtctctgc actgaacatt gtcagatctc aaagaggtcc gacgcgtatg 3840

tgccgcaata tatatgaccc acttttatgc ttcaaactat tttttactga tgagataatt 3900

tcggaaattg taaaatggac aaatgctgag atatcattga aacgtcggga atctatgaca 3960

ggtgctacat ttcgtgacac gaatgaagat gaaatctatg ctttctttgg tattctggta 4020

atgacagcag tgagaaaaga taaccacatg tccacagatg acctctttga tcgatctttg 4080

tcaatggtgt acgtctctgt aatgagtcgt gatcgttttg attttttgat acgatgtctt 4140

agaatggatg acaaaagtat acggcccaca cttcgagaaa acgatgtatt tactcctgtt 4200

agaaaaatat gggatctctt tatccatcag tgcatacaaa attacactcc aggggctcat 4260

ttgaccatag atgaacagtt acttggtttt agaggacggt gtccgtttag gatgtatatc 4320

ccaaacaagc caagtaagta tggaataaaa atcctcatga tgtgtgacag tggtacgaag 4380

tatatgataa atggaatgcc ttatttggga agaggaacac agaccaacgg agtaccactc 4440

ggtgaatact acgtgaagga gttatcaaag cctgtgcacg gtagttgtcg taatattacg 4500

tgtgacaatt ggttcacctc aatccctttg gcaaaaaact tactacaaga accgtataag 4560

ttaaccattg tgggaaccgt gcgatcaaac aaacgcgaga taccggaagt actgaaaaac 4620

agtcgctcca ggccagtggg aacatcgatg ttttgttttg acggacccct tactctcgtc 4680

tcatataaac cgaagccagc taagatggta tacttattat catcttgtga tgaggatgct 4740

tctatcaacg aaagtaccgg taaaccgcaa atggttatgt attataatca aactaaaggc 4800

ggagtggaca cgctagacca aatgtgttct gtgatgacct gcagtaggaa gacgaatagg 4860

tggcctatgg cattattgta cggaatgata aacattgcct gcataaattc ttttattata 4920

tacagccata atgtcagtag caagggagaa aaggttcaaa gtcgcaaaaa atttatgaga 4980

aacctttaca tgagcctgac gtcatcgttt atgcgtaagc gtttagaagc tcctactttg 5040

aagagatatt tgcgcgataa tatctctaat attttgccaa atgaagtgcc tggtacatca 5100

gatgacagta ctgaagagcc agtaatgaaa aaacgtactt actgtactta ctgcccctct 5160

aaaataaggc gaaaggcaaa tgcatcgtgc aaaaaatgca aaaaagttat ttgtcgagag 5220

cataatattg atatgtgcca aagttgtttc tgactgacta ataagtataa tttgtttcta 5280

ttatgtataa gttaagctaa ttacttattt tataatacaa catgactgtt tttaaagtac 5340

aaaataagtt tatttttgta aaagagagaa tgtttaaaag ttttgttact ttatagaaga 5400

aattttgagt ttttgttttt ttttaataaa taaataaaca taaataaatt gtttgttgaa 5460

tttattatta gtatgtaagt gtaaatataa taaaacttaa tatctattca aattaataaa 5520

taaacctcga tatacagacc gataaaacac atgcgtcaat tttacgcatg attatcttta 5580

acgtacgtca caatatgatt atctttctag ggttaaataa tagtttctaa tttttttatt 5640

attcagcctg ctgtcgtgaa taccgtatat ctcaacgctg tctgtgagat tgtcgtattc 5700

tagccttttt agtttttcgc tcatcgactt gatattgtcc gacacatttt cgtcgatttg 5760

cgttttgatc aaagacttga gcagagacac gttaatcaac tgttcaaatt gatccatatt 5820

aacgatatca acccgatgcg tatatggtgc gtaaaatata ttttttaacc ctcttatact 5880

ttgcactctg cgttaatacg cgttcgtgta cagacgtaat catgttttct tttttggata 5940

aaactcctac tgagtttgac ctcatattag accctcacaa gttgcaaaac gtggcatttt 6000

ttaccaatga agaatttaaa gttattttaa aaaatttcat cacagattta aagaagaacc 6060

aaaaattaaa ttatttcaac agtttaatcg accagttaat caacgtgtac acagacgcgt 6120

cggcaaaaaa cacgcagccc gacgtgttgg ctaaaattat t 6161

<210>4

<211>59

<212>DNA

<213>Artificial

<220>

<221>misc_feature

<222>(24)..(43)

<223>n is a, c, g, or t

<400>4

accgatcgat gaagacagaa gtgnnnnnnn nnnnnnnnnn nnngttttag agctagaaa 59

<210>5

<211>59

<212>DNA

<213>Artificial

<220>

<221>misc_feature

<222>(23)..(42)

<223>n is a, c, g, or t

<400>5

tgatgatgat gaagacgtaa acnnnnnnnn nnnnnnnnnn nncacttgta gagcacgat 59

<210>6

<211>1383

<212>DNA

<213>Artificial

<400>6

atcgatgttc ccactggcct ggagcgactg tttttcagta cttccggtat ctcgcgtttg 60

ttcctgcagg atcatgatga taaacaatgt atggtgctaa tgttgcttca acaacaattc 120

tgttgaactg tgttttcatg tttgccaaca agcaccttta tactcggtgg cctccccacc 180

accaactttt ttgcactgca aaaaaacacg cttttgcacg cgggcccata catagtacaa 240

actctacgtt tcgtagacta ttttacataa atagtctaca ccgttgtata cgctccaaat 300

acactaccac acattgaacc tttttgcagt gcaaaaaagt acgtgtcggc agtcacgtag 360

gccggcctta tcgggtcgcg tcctgtcacg tacgaatcac attatcggac cggacgagtg 420

ttgtcttatc gtgacaggac gccagcttcc tgtgttgcta accgcagccg gacgcaactc 480

cttatcggaa caggacgcgc ctccatatca gccgcgcgtt atctcatgcg cgtgaccgga 540

cacgaggcgc ccgtcccgct tatcgcgcct ataaatacag cccgcaacga tctggtaaac 600

acagttgaac agcatctgtt cgaaatggcc aagttgacca gtgccgttcc ggtgctcacc 660

gcgcgcgacg tcgccggagc ggtcgagttc tggaccgacc ggctcgggtt ctcccgggac 720

ttcgtggagg acgacttcgc cggtgtggtc cgggacgacg tgaccctgtt catcagcgcg 780

gtccaggacc aggtggtgcc ggacaacacc ctggcctggg tgtgggtgcg cggcctggac 840

gagctgtacg ccgagtggtc ggaggtcgtg tccacgaact tccgggacgc ctccgggccg 900

gccatgaccg agatcggcga gcagccgtgg gggcgggagt tcgccctgcg cgacccggcc 960

ggcaactgcg tgcacttcgt ggccgaggag caggactaaa gctttacaac taaacacgac 1020

ttggagtatt ccttgtagtg tttaagattt taaatcttac ttaatgactt cgaacgattt 1080

taacgataac tttctctttg tttaacttta atcagcatac ataaaaagcc ccggttttgt 1140

atcgggaaga aaaaaaatgt aattgtgttg cctagataat aaacgtatta tcaaagtgtg 1200

tggttttcct ttaccaaaga cccctttaag atgggcctaa tgggcttaag tcgagtcctt 1260

tccgatgtgt taaatacaca tttattacac tgatgcgtcg aatgtacact tttaatagga 1320

tagctccact aaaaattatt ttatttattt aatttgttgc accaaaactg atacattgac 1380

gaa 1383

<210>7

<211>6291

<212>DNA

<213>Artificial

<400>7

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 ccagtgaatt ggagatcggt acttcgcgaa 420

tgcgtcgaga taagagggtt aaaaaatata ttttacgcac catatacgca tcgggttgat 480

atcgttaata tggatcaatt tgaacagttg attaacgtgt ctctgctcaa gtctttgatc 540

aaaacgcaaa tcgacgaaaa tgtgtcggac aatatcaagt cgatgagcga aaaactaaaa 600

aggctagaat acgacaatct cacagacagc gttgagatat acggtattca cgacagcagg 660

ctgaataata aaaaaattag aaactattat ttaaccctag aaagataatc atattgtgac 720

gtacgttaaa gataatcatg cgtaaaattg acgcatgtgt tttatcggtc tgtatatcga 780

ggtttattta ttaatttgaa tagatattaa gttttattat atttacactt acatactaat 840

aataaattca acaaacaatt tatttatgtt tatttattta ttaaaaaaaa acaaaaactc 900

aaaatttctt ctataaagta acaaaacttt taaacattct ctcttttaca aaaataaact 960

tattttgtac tttaaaaaca gtcatgttgt attataaaat aagtaattag cttaacttat 1020

acataataga aacaaattat acttattagt cagtcagaaa caactttggc acatatcaat 1080

attatgctct cgacaaataa cttttttgca ttttttgcac gatgcatttg cctttcgcct 1140

tattttagag gggcagtaag tacagtaagt acgttttttc attactggct cttcagtact 1200

gtcatctgat gtaccaggca cttcatttgg caaaatatta gagatattat cgcgcaaata 1260

tctcttcaaa gtaggagctt ctaaacgctt acgcataaac gatgacgtca ggctcatgta 1320

aaggtttctc ataaattttt tgcgactttg aaccttttct cccttgctac tgacattatg 1380

gctgtatata ataaaagaat ttatgcaggc aatgtttatc attccgtaca ataatgccat 1440

aggccaccta ttcgtcctcc tactgcaggt catcacagaa cacatttggt ctagcgtgtc 1500

cactccgcct ttagtttgat tataatacat aaccatttgc ggtttaccgg tactttcgtt 1560

gatagaagca tcctcatcac aagatgataa taagtatacc atcttagctg gcttcggttt 1620

atatgagacg agagtaaggg gtccgtcaaa acaaaacatc gatgttccca ctggcctgga 1680

gcgactgttt ttcagtactt ccggtatctc gcgtttgttt gatcgcacgg ttcccacaat 1740

ggttaattcg agctcgcccg gggatctaat tcaattagag actaattcaa ttagagctaa 1800

ttcaattagg atccaagctt atcgatttcg aaccctcgac cgccggagta taaatagagg 1860

cgcttcgtct acggagcgac aattcaattc aaacaagcaa agtgaacacg tcgctaagcg 1920

aaagctaagc aaataaacaa gcgcagctga acaagctaaa caatcggggt accgctagag 1980

tcgacggtac cgcgggcccg ggatccaccg gtcgccacca tggtgagcaa gggcgaggag 2040

ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag 2100

ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc 2160

atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac 2220

ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc 2280

gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac 2340

aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag 2400

ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac 2460

agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag 2520

atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc 2580

cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc 2640

ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc 2700

gccgggatca ctctcggcat ggacgagctg tacaagtaac ggccgcgact ctagatcata 2760

atcagccatg cggccgcgac tctagaccac atttgtagag gttttacttg ctttaaaaaa 2820

cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt 2880

gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa 2940

agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttaaag 3000

cttatcgata cgcgtacggc gcgcctaggc cggccgatct cggatctgac aatgttcagt 3060

gcagagactc ggctacgcct cgtggacttt gaagttgacc aacaatgttt attcttacct 3120

ctaatagtcc tctgtggcaa ggtcaagatt ctgttagaag ccaatgaaga acctggttgt 3180

tcaataacat tttgttcgtc taatatttca ctaccgcttg acgttggctg cacttcatgt 3240

acctcatcta taaacgcttc ttctgtatcg ctctggacgt catcttcact tacgtgatct 3300

gatatttcac tgtcagaatc ctcaccaaca agctcgtcat cgctttgcag aagagcagag 3360

aggatatgct catcgtctaa agaactaccc attttattat atattagtca cgatatctat 3420

aacaagaaaa tatatatata ataagttatc acgtaagtag aacatgaaat aacaatataa 3480

ttatcgtatg agttaaatct taaaagtcac gtaaaagata atcatgcgtc attttgactc 3540

acgcggtcgt tatagttcaa aatcagtgac acttaccgca ttgacaagca cgcctcacgg 3600

gagctccaag cggcgactga gatgtcctaa atgcacagcg acggattcgc gctatttaga 3660

aagagagagc aatatttcaa gaatgcatgc gtcaatttta cgcagactat ctttctaggg 3720

ttaaaaaaga tttgcgcttt actcgaccta aactttaaac acgtcataga atcttcgttt 3780

gacaaaaacc acattgtggc caagctgtgt gacgcgacgc gcgctaaaga atggcaaacc 3840

aagtcgcgcg agcgtcgact ctagaggatc cccgggtacc gagctcgaat tcgtaatcat 3900

ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag 3960

ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acatcggatg 4020

ccgggaccga cgagtgcaga ggcgtgcaag cgagcttggc gtaatcatgg tcatagctgt 4080

ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa 4140

agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac 4200

tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg 4260

cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc 4320

gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat 4380

ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca 4440

ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 4500

atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 4560

aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 4620

gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta 4680

ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 4740

ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 4800

acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 4860

gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat 4920

ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 4980

ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 5040

gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 5100

ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct 5160

agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt 5220

ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc 5280

gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac 5340

catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat 5400

cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg 5460

cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata 5520

gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta 5580

tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt 5640

gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag 5700

tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa 5760

gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc 5820

gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt 5880

taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc 5940

tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta 6000

ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa 6060

taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca 6120

tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac 6180

aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta 6240

ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt c 6291

<210>8

<211>6334

<212>DNA

<213>Artificial

<400>8

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 ccagtgaatt ggagatcggt acttcgcgaa 420

tgcgtcgaga taagagggtt aaaaaatata ttttacgcac catatacgca tcgggttgat 480

atcgttaata tggatcaatt tgaacagttg attaacgtgt ctctgctcaa gtctttgatc 540

aaaacgcaaa tcgacgaaaa tgtgtcggac aatatcaagt cgatgagcga aaaactaaaa 600

aggctagaat acgacaatct cacagacagc gttgagatat acggtattca cgacagcagg 660

ctgaataata aaaaaattag aaactattat ttaaccctag aaagataatc atattgtgac 720

gtacgttaaa gataatcatg cgtaaaattg acgcatgtgt tttatcggtc tgtatatcga 780

ggtttattta ttaatttgaa tagatattaa gttttattat atttacactt acatactaat 840

aataaattca acaaacaatt tatttatgtt tatttattta ttaaaaaaaa acaaaaactc 900

aaaatttctt ctataaagta acaaaacttt taaacattct ctcttttaca aaaataaact 960

tattttgtac tttaaaaaca gtcatgttgt attataaaat aagtaattag cttaacttat 1020

acataataga aacaaattat acttattagt cagtcagaaa caactttggc acatatcaat 1080

attatgctct cgacaaataa cttttttgca ttttttgcac gatgcatttg cctttcgcct 1140

tattttagag gggcagtaag tacagtaagt acgttttttc attactggct cttcagtact 1200

gtcatctgat gtaccaggca cttcatttgg caaaatatta gagatattat cgcgcaaata 1260

tctcttcaaa gtaggagctt ctaaacgctt acgcataaac gatgacgtca ggctcatgta 1320

aaggtttctc ataaattttt tgcgactttg aaccttttct cccttgctac tgacattatg 1380

gctgtatata ataaaagaat ttatgcaggc aatgtttatc attccgtaca ataatgccat 1440

aggccaccta ttcgtcctcc tactgcaggt catcacagaa cacatttggt ctagcgtgtc 1500

cactccgcct ttagtttgat tataatacat aaccatttgc ggtttaccgg tactttcgtt 1560

gatagaagca tcctcatcac aagatgataa taagtatacc atcttagctg gcttcggttt 1620

atatgagacg agagtaaggg gtccgtcaaa acaaaacatc gatgttccca ctggcctgga 1680

gcgactgttt ttcagtactt ccggtatctc gcgtttgttc ctgcaggatc atgatgataa 1740

acaatgtatg gtgctaatgt tgcttcaaca acaattctgt tgaactgtgt tttcatgttt 1800

gccaacaagc acctttatac tcggtggcct ccccaccacc aacttttttg cactgcaaaa 1860

aaacacgctt ttgcacgcgg gcccatacat agtacaaact ctacgtttcg tagactattt 1920

tacataaata gtctacaccg ttgtatacgc tccaaataca ctaccacaca ttgaaccttt 1980

ttgcagtgca aaaaagtacg tgtcggcagt cacgtaggcc ggccttatcg ggtcgcgtcc 2040

tgtcacgtac gaatcacatt atcggaccgg acgagtgttg tcttatcgtg acaggacgcc 2100

agcttcctgt gttgctaacc gcagccggac gcaactcctt atcggaacag gacgcgcctc 2160

catatcagcc gcgcgttatc tcatgcgcgt gaccggacac gaggcgcccg tcccgcttat 2220

cgcgcctata aatacagccc gcaacgatct ggtaaacaca gttgaacagc atctgttcga 2280

aatggccaag ttgaccagtg ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt 2340

cgagttctgg accgaccggc tcgggttctc ccgggacttc gtggaggacg acttcgccgg 2400

tgtggtccgg gacgacgtga ccctgttcat cagcgcggtc caggaccagg tggtgccgga 2460

caacaccctg gcctgggtgt gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga 2520

ggtcgtgtcc acgaacttcc gggacgcctc cgggccggcc atgaccgaga tcggcgagca 2580

gccgtggggg cgggagttcg ccctgcgcga cccggccggc aactgcgtgc acttcgtggc 2640

cgaggagcag gactaaagct ttacaactaa acacgacttg gagtattcct tgtagtgttt 2700

aagattttaa atcttactta atgacttcga acgattttaa cgataacttt ctctttgttt 2760

aactttaatc agcatacata aaaagccccg gttttgtatc gggaagaaaa aaaatgtaat 2820

tgtgttgcct agataataaa cgtattatca aagtgtgtgg ttttccttta ccaaagaccc 2880

ctttaagatg ggcctaatgg gcttaagtcg agtcctttcc gatgtgttaa atacacattt 2940

attacactga tgcgtcgaat gtacactttt aataggatag ctccactaaa aattatttta 3000

tttatttaat ttgttgcacc aaaactgata cattgacgaa acgcgtatgc tagcaatgaa 3060

ggcgcgccta ggccggccga tctcggatct gacaatgttc agtgcagaga ctcggctacg 3120

cctcgtggac tttgaagttg accaacaatg tttattctta cctctaatag tcctctgtgg 3180

caaggtcaag attctgttag aagccaatga agaacctggt tgttcaataa cattttgttc 3240

gtctaatatt tcactaccgc ttgacgttgg ctgcacttca tgtacctcat ctataaacgc 3300

ttcttctgta tcgctctgga cgtcatcttc acttacgtga tctgatattt cactgtcaga 3360

atcctcacca acaagctcgt catcgctttg cagaagagca gagaggatat gctcatcgtc 3420

taaagaacta cccattttat tatatattag tcacgatatc tataacaaga aaatatatat 3480

ataataagtt atcacgtaag tagaacatga aataacaata taattatcgt atgagttaaa 3540

tcttaaaagt cacgtaaaag ataatcatgc gtcattttga ctcacgcggt cgttatagtt 3600

caaaatcagt gacacttacc gcattgacaa gcacgcctca cgggagctcc aagcggcgac 3660

tgagatgtcc taaatgcaca gcgacggatt cgcgctattt agaaagagag agcaatattt 3720

caagaatgca tgcgtcaatt ttacgcagac tatctttcta gggttaaaaa agatttgcgc 3780

tttactcgac ctaaacttta aacacgtcat agaatcttcg tttgacaaaa accacattgt 3840

ggccaagctg tgtgacgcga cgcgcgctaa agaatggcaa accaagtcgc gcgagcgtcg 3900

actctagagg atccccgggt accgagctcg aattcgtaat catggtcata gctgtttcct 3960

gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt 4020

aaagcctggg gtgcctaatg agtgagctaa ctcacatcgg atgccgggac cgacgagtgc 4080

agaggcgtgc aagcgagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt 4140

tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt 4200

gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg 4260

ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg 4320

cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg 4380

cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat 4440

aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc 4500

gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc 4560

tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga 4620

agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt 4680

ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg 4740

taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc 4800

gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg 4860

gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc 4920

ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg 4980

ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc 5040

gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct 5100

caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt 5160

taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa 5220

aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa 5280

tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc 5340

tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct 5400

gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca 5460

gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt 5520

aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt 5580

gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc 5640

ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc 5700

tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt 5760

atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact 5820

ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc 5880

ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt 5940

ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg 6000

atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct 6060

gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa 6120

tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt 6180

ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc 6240

acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc 6300

tataaaaata ggcgtatcac gaggcccttt cgtc 6334

<210>9

<211>5898

<212>DNA

<213>Artificial

<400>9

ggcgcgccta tactcgagca gcgtcgtgaa aagaggcaat gacaaataca aaacgacgta 60

tgagcagacc cgtcgccaag acgggtctac ctctaagatg atgtcatttg ttttttaaaa 120

ctaactcgct ttacgagtag aattctacgt gtaaaacata atcaagagat gatgtcattt 180

gtttttcaaa accaaactcg ctttacgagt agaattctac gtgtaaaaca caatcaaaag 240

atgatgtcat tcgtttttca aaaccgaatt taagaaatga tgtcatttgt ttttcaaaac 300

caaactcgct ttacgagcag aattctacgt gtaaaacaca atcaagagat gatgtcattt 360

gtttttcaaa actgaatgat gtcatttgtt tttcaaaact aaacttgctt tgcgagtaga 420

attctacgtg taaaacacag tcaagagatg atgtcatttg tttttcaaaa ctgaaccggc 480

tttacgagta gaattctact tgtaaaacat aatcaagaga tgatgtcatt tgtttttcaa 540

aactgaactg gctttacgag tagaattcta cgtgtaaaac ataatcaaga gatgatgtca 600

tcattaaact gatgtcattt tatacacgat tgttaacatg tttaataatg actaatttgt 660

ttttccaaat taaactcgct ttacgagtag aattctactt gtaacgcacg attaagtatg 720

aatcataagc tgatgtcatt tgttttcgac ataaaatgtt tatacaatgg aatcttcttg 780

taaattatcc aaataatata atttatccga ttctacgtta catttaaatt cgttgttatc 840

gtacaattct tcaggacacg ccatgtattg gtcattttta gcgtgcaacc aacgattgta 900

tttgacgccg tcgttggatt gcgtgttcag gttggcgtac acgtgactgg gcacggcttc 960

tttttccatg ggacgtcgac cgagaaattt ctctggccgt tattcgttat tctctctttt 1020

ctttttgggt ctctccctct ctgcactaat gctctctcac tctgtcacac agtaaacggc 1080

atactgctct cgttggttcg agagagcgcg cctcgaatgt tcgcgaaaag agcgccggag 1140

tataaataga ggcgcttcgt ctacggagcg acaattcaat tcaaacaagc aaagtgaaca 1200

cgtcgctaag cgaaagctaa gcaaataaac aagcgcagct gaacaagcta aacaatctgc 1260

agtaaagtgc aagttaaagt gaatcaatta aaagtaacca gcaaccaagt aaatcaactg 1320

caactactga aatctgccaa gaagtaatta ttgaatacaa gaagagaact ctgggggatc 1380

tctagtccag tgtggtggaa ttcgccatgg ccccaaagaa aaagagaaag gttgattaca 1440

aagaccacga cggagactac aaagaccacg acattgatta taaagatgat gatgataaag 1500

gaacgatgga caaaaagtat agcatcggtc tggatattgg aactaactcc gtcggctggg 1560

ctgtaatcac cgacgaatac aaggtcccgt caaaaaagtt caaggtattg ggtaacacag 1620

atcgtcactc tatcaaaaag aatctcattg gagctctgtt gttcgacagc ggcgaaacag 1680

ctgaggccac tagactgaag cgcaccgcca gacgccgtta cacgaggaga aagaacagaa 1740

tctgctactt gcaagaaata ttctcaaacg agatggccaa agtggacgat tcgttctttc 1800

ataggttaga agagagtttc cttgttgaag aggataaaaa gcacgaaaga catccgatat 1860

ttggaaacat cgtggacgaa gttgcttatc acgagaagta ccccacgatc tatcatctgc 1920

gtaaaaagtt ggtggactcg acagataagg ccgacctcag gttaatatac cttgcactgg 1980

cgcacatgat caaattcaga ggccattttc tgattgaagg tgacctgaac cctgacaata 2040

gtgatgtgga caaactcttc attcaattag ttcagaccta caatcaactg tttgaagaga 2100

accctatcaa cgcttcagga gttgacgcta aggccatcct tagtgcgaga ctgagcaaat 2160

cccgccgtct cgaaaactta atcgcacagt tgcctggaga gaaaaagaac ggtttgttcg 2220

gaaatctcat tgcgttgtca ctcggactcacgccaaactt caagtctaac ttcgatttgg 2280

cagaagacgc gaaactgcaa ctgagcaaag acacatatga cgatgacctc gataacctct 2340

tagctcagat cggcgatcaa tacgccgact tgttcctcgc tgccaaaaat ctgtcggacg 2400

ctatacttct gagtgatatc ttgcgcgtca acacagaaat tactaaggct cctctgtcgg 2460

ccagtatgat aaaacgctat gacgaacacc atcaggattt gacattgctc aaagccctcg 2520

tgcgtcaaca gctcccagaa aagtacaagg agattttctt tgatcagtcc aagaatggct 2580

acgcaggtta tatagacggt ggagcgtcgc aagaagagtt ctacaagttc atcaagccaa 2640

tattagaaaa gatggacggc acggaagagt tacttgttaa gctgaatcgt gaggacctgt 2700

tgcgtaaaca gaggacattc gataacggat caattccgca ccaaatacat cttggcgaac 2760

tgcacgctat cctcaggaga caagaggact tctacccctt tttaaaggat aaccgtgaaa 2820

agatcgagaa aatcctgact ttcaggattc cttactatgt cggcccactg gctcgtggta 2880

atagcaggtt tgcctggatg accaggaagt ccgaagagac aattactccg tggaacttcg 2940

aagaggtggt tgataaagga gcatcagcgc agtctttcat agaacgcatg acaaattttg 3000

acaagaactt accgaatgag aaggtccttc ccaaacactc actcctctac gaatacttca 3060

cagtatacaa cgagctcact aaagtcaagt acgtaaccga gggtatgcgc aaacccgctt 3120

tcctgtctgg agagcagaaa aaggccatcg tggaccttct gttcaagaca aaccgtaagg 3180

tcactgtaaa gcaactcaag gaagactact tcaaaaagat agagtgtttc gattcagtgg 3240

aaatctctgg cgttgaggac agatttaacg cttccttggg tacttaccac gatttgctca 3300

agatcattaa agataaggac ttcctcgaca acgaagagaa cgaagatatc ttagaggaca 3360

tagttctcac ccttacgctg tttgaagata gagagatgat tgaagagcgc ctgaagactt 3420

atgctcattt gttcgatgac aaagtcatga agcaactgaa acgccgtagg tacaccggct 3480

ggggtagatt atcgcgcaaa cttattaatg gtataaggga caagcagtcg ggaaaaacga 3540

tattggactt tctcaagagt gatggtttcg ccaacagaaa ttttatgcaa ctcatacacg 3600

atgacagctt aacattcaag gaagatatcc aaaaagcaca ggtgtcggga cagggcgaca 3660

gtttgcacga acatattgct aacctcgccg gctccccggc gataaaaaag ggtatccttc 3720

agactgtgaa agtcgtagat gaactggtga aggttatggg tcgtcataaa cccgagaaca 3780

tagttatcga aatggctagg gagaatcaaa caactcagaa gggacagaaa aactcaagag 3840

aacgcatgaa gcgcattgaa gagggtatca aagagcttgg cagtcaaatc ctgaaggaac 3900

accctgtcga gaacacgcaa cttcagaacg aaaaattgta cctctactat ctgcagaatg 3960

gtagagatat gtacgtagac caagaattgg atattaaccg cctctcagat tacgacgtgg 4020

atcatatagt tccgcagtca ttcttgaagg atgactctat cgacaacaaa gtcctcacaa 4080

gatcagacaa gaaccgcgga aaatcagata atgtaccctc tgaagaggtg gttaaaaaga 4140

tgaaaaacta ctggagacag ttacttaacg ctaagttgat cacgcaaaga aagttcgata 4200

acctcacaaa ggctgaacgc ggcggtttaa gcgagcttga caaggccggt ttcataaaac 4260

gtcagttagt cgaaaccagg caaattacga aacacgtagc ccaaatattg gattcccgca 4320

tgaacactaa atacgatgaa aatgacaagc tcatccgtga ggtcaaagta attaccctga 4380

aaagcaagtt ggtgtccgac ttcagaaagg atttccagtt ctacaaagtt cgcgaaatca 4440

acaactacca ccatgcacat gacgcttacc tgaacgcagt cgtaggcact gcgttaatta 4500

aaaagtaccc taaactggaa tctgagttcg tgtacggtga ctataaagtg tacgatgtta 4560

gaaagatgat cgctaaaagc gaacaggaga ttggaaaggc taccgccaag tatttctttt 4620

actccaacat catgaatttc tttaagaccg aaatcacgtt agcaaatggc gagatacgta 4680

aaaggccact tatcgaaaca aacggagaaa ctggcgagat agtgtgggac aagggtagag 4740

attttgccac tgtccgcaaa gtactgtcga tgccgcaagt gaatatcgtt aaaaagaccg 4800

aagttcaaac gggaggcttc agcaaagagt ccatcctgcc caagcgtaac agtgataaat 4860

tgatagctag gaaaaaggac tgggatccta aaaagtatgg tggattcgac agcccaactg 4920

tcgcatactc cgtattggtg gttgcgaaag tcgaaaaagg aaagagcaaa aagctcaagt 4980

ccgtaaaaga gctgttgggc attaccataa tggaaagatc atctttcgag aagaatccta 5040

tcgattttct ggaagccaag ggatataaag aggtcaaaaa ggacctcata atcaagttac 5100

caaaatacag tctgttcgaa ttggagaacg gcagaaaacg catgcttgca tcagcgggtg 5160

aactgcaaaa gggaaatgag ttagcacttc cttctaaata cgtcaacttc ctgtatttgg 5220

cgtcacacta cgaaaaactg aagggctctc cagaagataa cgagcaaaag cagttatttg 5280

tggaacagca caaacattac cttgacgaaa ttatagagca aatctcggag ttcagtaaga 5340

gagtgatttt ggctgacgcc aatcttgata aagttctgtc tgcttacaac aagcaccgtg 5400

ataaaccgat tagggaacag gccgagaaca tcatacatct cttcacactc actaaccttg 5460

gtgcacccgc agcgttcaaa tattttgaca ccacgataga tcgtaagagg tacaccagca 5520

cgaaagaagt tttggacgcg acactcatcc atcaatcaat cacgggcctg tacgagacca 5580

gaatcgacct gtcccagctc ggtggcgact agcggccgcg actctagatc ataatcagcc 5640

atgcggccgc gactctagac cacatttgta gaggttttac ttgctttaaa aaacctccca 5700

cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt 5760

gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt 5820

ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta aagcttatcg 5880

atacgcgtac ggcgcgcc 5898

<210>10

<211>6247

<212>DNA

<213>Artificial

<400>10

ggcgcgccta tactcgagca gcgtcgtgaa aagaggcaat gacaaataca aaacgacgta 60

tgagcagacc cgtcgccaag acgggtctac ctctaagatg atgtcatttg ttttttaaaa 120

ctaactcgct ttacgagtag aattctacgt gtaaaacata atcaagagat gatgtcattt 180

gtttttcaaa accaaactcg ctttacgagt agaattctac gtgtaaaaca caatcaaaag 240

atgatgtcat tcgtttttca aaaccgaatt taagaaatga tgtcatttgt ttttcaaaac 300

caaactcgct ttacgagcag aattctacgt gtaaaacaca atcaagagat gatgtcattt 360

gtttttcaaa actgaatgat gtcatttgtt tttcaaaact aaacttgctt tgcgagtaga 420

attctacgtg taaaacacag tcaagagatg atgtcatttg tttttcaaaa ctgaaccggc 480

tttacgagta gaattctact tgtaaaacat aatcaagaga tgatgtcatt tgtttttcaa 540

aactgaactg gctttacgag tagaattcta cgtgtaaaac ataatcaaga gatgatgtca 600

tcattaaact gatgtcattt tatacacgat tgttaacatg tttaataatg actaatttgt 660

ttttccaaat taaactcgct ttacgagtag aattctactt gtaacgcacg attaagtatg 720

aatcataagc tgatgtcatt tgttttcgac ataaaatgtt tatacaatgg aatcttcttg 780

taaattatcc aaataatata atttatccga ttctacgtta catttaaatt cgttgttatc 840

gtacaattct tcaggacacg ccatgtattg gtcattttta gcgtgcaacc aacgattgta 900

tttgacgccg tcgttggatt gcgtgttcag gttggcgtac acgtgactgg gcacggcttc 960

tttttccatg ggacgtcgac tcatcttgtc acacctacat cttactaatt tcgtaagtag 1020

attttttttt acacgtataa tgtatgtatt ctttccttaa ttaacttatt ttgaaacgaa 1080

ataaataggc tattaatatt tggaactagg ttgcggtcaa tgtcaatgtc tgtctcaact 1140

ttaattcaga atgccttgtg ttccgtagat gctataaatc aatcaagatg catcttggat 1200

tgttgccaac tcgcagctac aaaatttgtt tccaagccta agcatagtgc tgtacccgtt 1260

cccgtgtatt caaatcccgt ataatagtat aatatactcc gtaaatgtag tgtcactgct 1320

tgctgaaatg atattgcaag ttccgttggg aatcttgccg ttatcaagca atgcgatatt 1380

agcggtatgg cgggaggggg acgcgcagac tccctctgct gtattaccat atatggacac 1440

aaaacttcgt gtattgtacc ctagcgcgcg attggaggag agtctgcggc ggcggggcag 1500

gggcgccccg ataaccggcc tcatttatat agtccgccaa gcgcactcac caacattcca 1560

cgaagtgagc ttgggtcgtt gcgttgtaca gcaataacga agctgtgcaa tagcaagtta 1620

atttatttat ttataataga actatttaat taaaagtaag ttattttcat tgtgtcttca 1680

aatatattaa gtgattgtga taacggttaa cggttgttag aggattggta ctagtccagt 1740

gtggtggaat tcgccatggc cccaaagaaa aagagaaagg ttgattacaa agaccacgac 1800

ggagactaca aagaccacga cattgattat aaagatgatg atgataaagg aacgatggac 1860

aaaaagtata gcatcggtct ggatattgga actaactccg tcggctgggc tgtaatcacc 1920

gacgaataca aggtcccgtc aaaaaagttc aaggtattgg gtaacacaga tcgtcactct 1980

atcaaaaaga atctcattgg agctctgttg ttcgacagcg gcgaaacagc tgaggccact 2040

agactgaagc gcaccgccag acgccgttac acgaggagaa agaacagaat ctgctacttg 2100

caagaaatat tctcaaacga gatggccaaa gtggacgatt cgttctttca taggttagaa 2160

gagagtttcc ttgttgaaga ggataaaaag cacgaaagac atccgatatt tggaaacatc 2220

gtggacgaag ttgcttatca cgagaagtac cccacgatct atcatctgcg taaaaagttg 2280

gtggactcga cagataaggc cgacctcagg ttaatatacc ttgcactggc gcacatgatc 2340

aaattcagag gccattttct gattgaaggt gacctgaacc ctgacaatag tgatgtggac 2400

aaactcttca ttcaattagt tcagacctac aatcaactgt ttgaagagaa ccctatcaac 2460

gcttcaggag ttgacgctaa ggccatcctt agtgcgagac tgagcaaatc ccgccgtctc 2520

gaaaacttaa tcgcacagtt gcctggagag aaaaagaacg gtttgttcgg aaatctcatt 2580

gcgttgtcac tcggactcac gccaaacttc aagtctaact tcgatttggc agaagacgcg 2640

aaactgcaac tgagcaaaga cacatatgac gatgacctcg ataacctctt agctcagatc 2700

ggcgatcaat acgccgactt gttcctcgct gccaaaaatc tgtcggacgc tatacttctg 2760

agtgatatct tgcgcgtcaa cacagaaatt actaaggctc ctctgtcggc cagtatgata 2820

aaacgctatg acgaacacca tcaggatttg acattgctca aagccctcgt gcgtcaacag 2880

ctcccagaaa agtacaagga gattttcttt gatcagtcca agaatggcta cgcaggttat 2940

atagacggtg gagcgtcgca agaagagttc tacaagttca tcaagccaat attagaaaag 3000

atggacggca cggaagagtt acttgttaag ctgaatcgtg aggacctgtt gcgtaaacag 3060

aggacattcg ataacggatc aattccgcac caaatacatc ttggcgaact gcacgctatc 3120

ctcaggagac aagaggactt ctaccccttt ttaaaggata accgtgaaaa gatcgagaaa 3180

atcctgactt tcaggattcc ttactatgtc ggcccactgg ctcgtggtaa tagcaggttt 3240

gcctggatga ccaggaagtc cgaagagaca attactccgt ggaacttcga agaggtggtt 3300

gataaaggag catcagcgca gtctttcata gaacgcatga caaattttga caagaactta 3360

ccgaatgaga aggtccttcc caaacactca ctcctctacg aatacttcac agtatacaac 3420

gagctcacta aagtcaagta cgtaaccgag ggtatgcgca aacccgcttt cctgtctgga 3480

gagcagaaaa aggccatcgt ggaccttctg ttcaagacaa accgtaaggt cactgtaaag 3540

caactcaagg aagactactt caaaaagata gagtgtttcg attcagtgga aatctctggc 3600

gttgaggaca gatttaacgc ttccttgggt acttaccacg atttgctcaa gatcattaaa 3660

gataaggact tcctcgacaa cgaagagaac gaagatatct tagaggacat agttctcacc 3720

cttacgctgt ttgaagatag agagatgatt gaagagcgcc tgaagactta tgctcatttg 3780

ttcgatgacaaagtcatgaa gcaactgaaa cgccgtaggt acaccggctg gggtagatta 3840

tcgcgcaaac ttattaatgg tataagggac aagcagtcgg gaaaaacgat attggacttt 3900

ctcaagagtg atggtttcgc caacagaaat tttatgcaac tcatacacga tgacagctta 3960

acattcaagg aagatatcca aaaagcacag gtgtcgggac agggcgacag tttgcacgaa 4020

catattgcta acctcgccgg ctccccggcg ataaaaaagg gtatccttca gactgtgaaa 4080

gtcgtagatg aactggtgaa ggttatgggt cgtcataaac ccgagaacat agttatcgaa 4140

atggctaggg agaatcaaac aactcagaag ggacagaaaa actcaagaga acgcatgaag 4200

cgcattgaag agggtatcaa agagcttggc agtcaaatcc tgaaggaaca ccctgtcgag 4260

aacacgcaac ttcagaacga aaaattgtac ctctactatc tgcagaatgg tagagatatg 4320

tacgtagacc aagaattgga tattaaccgc ctctcagatt acgacgtgga tcatatagtt 4380

ccgcagtcat tcttgaagga tgactctatc gacaacaaag tcctcacaag atcagacaag 4440

aaccgcggaa aatcagataa tgtaccctct gaagaggtgg ttaaaaagat gaaaaactac 4500

tggagacagt tacttaacgc taagttgatc acgcaaagaa agttcgataa cctcacaaag 4560

gctgaacgcg gcggtttaag cgagcttgac aaggccggtt tcataaaacg tcagttagtc 4620

gaaaccaggc aaattacgaa acacgtagcc caaatattgg attcccgcat gaacactaaa 4680

tacgatgaaa atgacaagct catccgtgag gtcaaagtaa ttaccctgaa aagcaagttg 4740

gtgtccgact tcagaaagga tttccagttc tacaaagttc gcgaaatcaa caactaccac 4800

catgcacatg acgcttacct gaacgcagtc gtaggcactg cgttaattaa aaagtaccct 4860

aaactggaat ctgagttcgt gtacggtgac tataaagtgt acgatgttag aaagatgatc 4920

gctaaaagcg aacaggagat tggaaaggct accgccaagt atttctttta ctccaacatc 4980

atgaatttct ttaagaccga aatcacgtta gcaaatggcg agatacgtaa aaggccactt 5040

atcgaaacaa acggagaaac tggcgagata gtgtgggaca agggtagaga ttttgccact 5100

gtccgcaaag tactgtcgat gccgcaagtg aatatcgtta aaaagaccga agttcaaacg 5160

ggaggcttca gcaaagagtc catcctgccc aagcgtaaca gtgataaatt gatagctagg 5220

aaaaaggact gggatcctaa aaagtatggt ggattcgaca gcccaactgt cgcatactcc 5280

gtattggtgg ttgcgaaagt cgaaaaagga aagagcaaaa agctcaagtc cgtaaaagag 5340

ctgttgggca ttaccataat ggaaagatca tctttcgaga agaatcctat cgattttctg 5400

gaagccaagg gatataaaga ggtcaaaaag gacctcataa tcaagttacc aaaatacagt 5460

ctgttcgaat tggagaacgg cagaaaacgc atgcttgcat cagcgggtga actgcaaaag 5520

ggaaatgagt tagcacttcc ttctaaatac gtcaacttcc tgtatttggc gtcacactac 5580

gaaaaactga agggctctcc agaagataac gagcaaaagc agttatttgt ggaacagcac 5640

aaacattacc ttgacgaaat tatagagcaa atctcggagt tcagtaagag agtgattttg 5700

gctgacgcca atcttgataa agttctgtct gcttacaaca agcaccgtga taaaccgatt 5760

agggaacagg ccgagaacat catacatctc ttcacactca ctaaccttgg tgcacccgca 5820

gcgttcaaat attttgacac cacgatagat cgtaagaggt acaccagcac gaaagaagtt 5880

ttggacgcga cactcatcca tcaatcaatc acgggcctgt acgagaccag aatcgacctg 5940

tcccagctcg gtggcgacta gcggccgcga ctctagatca taatcagcca tgcggccgcg 6000

actctagacc acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct 6060

gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa 6120

tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca 6180

ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa agcttatcga tacgcgtacg 6240

gcgcgcc 6247

<210>11

<211>12207

<212>DNA

<213>Artificial

<400>11

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 ccagtgaatt ggagatcggt acttcgcgaa 420

tgcgtcgaga taagagggtt aaaaaatata ttttacgcac catatacgca tcgggttgat 480

atcgttaata tggatcaatt tgaacagttg attaacgtgt ctctgctcaa gtctttgatc 540

aaaacgcaaa tcgacgaaaa tgtgtcggac aatatcaagt cgatgagcga aaaactaaaa 600

aggctagaat acgacaatct cacagacagc gttgagatat acggtattca cgacagcagg 660

ctgaataata aaaaaattag aaactattat ttaaccctag aaagataatc atattgtgac 720

gtacgttaaa gataatcatg cgtaaaattg acgcatgtgt tttatcggtc tgtatatcga 780

ggtttattta ttaatttgaa tagatattaa gttttattat atttacactt acatactaat 840

aataaattca acaaacaatt tatttatgtt tatttattta ttaaaaaaaa acaaaaactc 900

aaaatttctt ctataaagta acaaaacttt taaacattct ctcttttaca aaaataaact 960

tattttgtac tttaaaaaca gtcatgttgt attataaaat aagtaattag cttaacttat 1020

acataataga aacaaattat acttattagt cagtcagaaa caactttggc acatatcaat 1080

attatgctct cgacaaataa cttttttgca ttttttgcac gatgcatttg cctttcgcct 1140

tattttagag gggcagtaag tacagtaagt acgttttttc attactggct cttcagtact 1200

gtcatctgat gtaccaggca cttcatttgg caaaatatta gagatattat cgcgcaaata 1260

tctcttcaaa gtaggagctt ctaaacgctt acgcataaac gatgacgtca ggctcatgta 1320

aaggtttctc ataaattttt tgcgactttg aaccttttct cccttgctac tgacattatg 1380

gctgtatata ataaaagaat ttatgcaggc aatgtttatc attccgtaca ataatgccat 1440

aggccaccta ttcgtcctcc tactgcaggt catcacagaa cacatttggt ctagcgtgtc 1500

cactccgcct ttagtttgat tataatacat aaccatttgc ggtttaccgg tactttcgtt 1560

gatagaagca tcctcatcac aagatgataa taagtatacc atcttagctg gcttcggttt 1620

atatgagacg agagtaaggg gtccgtcaaa acaaaacatc gatgttccca ctggcctgga 1680

gcgactgttt ttcagtactt ccggtatctc gcgtttgttc ctgcaggatc atgatgataa 1740

acaatgtatg gtgctaatgt tgcttcaaca acaattctgt tgaactgtgt tttcatgttt 1800

gccaacaagc acctttatac tcggtggcct ccccaccacc aacttttttg cactgcaaaa 1860

aaacacgctt ttgcacgcgg gcccatacat agtacaaact ctacgtttcg tagactattt 1920

tacataaata gtctacaccg ttgtatacgc tccaaataca ctaccacaca ttgaaccttt 1980

ttgcagtgca aaaaagtacg tgtcggcagt cacgtaggcc ggccttatcg ggtcgcgtcc 2040

tgtcacgtac gaatcacatt atcggaccgg acgagtgttg tcttatcgtg acaggacgcc 2100

agcttcctgt gttgctaacc gcagccggac gcaactcctt atcggaacag gacgcgcctc 2160

catatcagcc gcgcgttatc tcatgcgcgt gaccggacac gaggcgcccg tcccgcttat 2220

cgcgcctata aatacagccc gcaacgatct ggtaaacaca gttgaacagc atctgttcga 2280

aatggccaag ttgaccagtg ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt 2340

cgagttctgg accgaccggc tcgggttctc ccgggacttc gtggaggacg acttcgccgg 2400

tgtggtccgg gacgacgtga ccctgttcat cagcgcggtc caggaccagg tggtgccgga 2460

caacaccctg gcctgggtgt gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga 2520

ggtcgtgtcc acgaacttcc gggacgcctc cgggccggcc atgaccgaga tcggcgagca 2580

gccgtggggg cgggagttcg ccctgcgcga cccggccggc aactgcgtgc acttcgtggc 2640

cgaggagcag gactaaagct ttacaactaa acacgacttg gagtattcct tgtagtgttt 2700

aagattttaa atcttactta atgacttcga acgattttaa cgataacttt ctctttgttt 2760

aactttaatc agcatacata aaaagccccg gttttgtatc gggaagaaaa aaaatgtaat 2820

tgtgttgcct agataataaa cgtattatca aagtgtgtgg ttttccttta ccaaagaccc 2880

ctttaagatg ggcctaatgg gcttaagtcg agtcctttcc gatgtgttaa atacacattt 2940

attacactga tgcgtcgaat gtacactttt aataggatag ctccactaaa aattatttta 3000

tttatttaat ttgttgcacc aaaactgata cattgacgaa acgcgtatgc tagcaatgaa 3060

ggcgcgccca gcgtcgtgaa aagaggcaat gacaaataca aaacgacgta tgagcagacc 3120

cgtcgccaag acgggtctac ctctaagatg atgtcatttg ttttttaaaa ctaactcgct 3180

ttacgagtag aattctacgt gtaaaacata atcaagagat gatgtcattt gtttttcaaa 3240

accaaactcg ctttacgagt agaattctac gtgtaaaaca caatcaaaag atgatgtcat 3300

tcgtttttca aaaccgaatt taagaaatga tgtcatttgt ttttcaaaac caaactcgct 3360

ttacgagcag aattctacgt gtaaaacaca atcaagagat gatgtcattt gtttttcaaa 3420

actgaatgat gtcatttgtt tttcaaaact aaacttgctt tgcgagtaga attctacgtg 3480

taaaacacag tcaagagatg atgtcatttg tttttcaaaa ctgaaccggc tttacgagta 3540

gaattctact tgtaaaacat aatcaagaga tgatgtcatt tgtttttcaa aactgaactg 3600

gctttacgag tagaattcta cgtgtaaaac ataatcaaga gatgatgtca tcattaaact 3660

gatgtcattt tatacacgat tgttaacatg tttaataatg actaatttgt ttttccaaat 3720

taaactcgct ttacgagtag aattctactt gtaacgcacg attaagtatg aatcataagc 3780

tgatgtcatt tgttttcgac ataaaatgtt tatacaatgg aatcttcttg taaattatcc 3840

aaataatata atttatccga ttctacgtta catttaaatt cgttgttatc gtacaattct 3900

tcaggacacg ccatgtattg gtcattttta gcgtgcaacc aacgattgta tttgacgccg 3960

tcgttggatt gcgtgttcag gttggcgtac acgtgactgg gcacggcttc tttttccatg 4020

ggacgtcgac cgagaaattt ctctggccgt tattcgttat tctctctttt ctttttgggt 4080

ctctccctct ctgcactaat gctctctcac tctgtcacac agtaaacggc atactgctct 4140

cgttggttcg agagagcgcg cctcgaatgt tcgcgaaaag agcgccggag tataaataga 4200

ggcgcttcgt ctacggagcg acaattcaat tcaaacaagc aaagtgaaca cgtcgctaag 4260

cgaaagctaa gcaaataaac aagcgcagct gaacaagcta aacaatctgc agtaaagtgc 4320

aagttaaagt gaatcaatta aaagtaacca gcaaccaagt aaatcaactg caactactga 4380

aatctgccaa gaagtaatta ttgaatacaa gaagagaact ctgggggatc tctagtccag 4440

tgtggtggaa ttcgccatgg ccccaaagaa aaagagaaag gttgattaca aagaccacga 4500

cggagactac aaagaccacg acattgatta taaagatgat gatgataaag gaacgatgga 4560

caaaaagtat agcatcggtc tggatattgg aactaactcc gtcggctggg ctgtaatcac 4620

cgacgaatac aaggtcccgt caaaaaagtt caaggtattg ggtaacacag atcgtcactc 4680

tatcaaaaag aatctcattg gagctctgtt gttcgacagc ggcgaaacag ctgaggccac 4740

tagactgaag cgcaccgcca gacgccgtta cacgaggaga aagaacagaa tctgctactt 4800

gcaagaaata ttctcaaacg agatggccaa agtggacgat tcgttctttc ataggttaga 4860

agagagtttc cttgttgaag aggataaaaa gcacgaaaga catccgatat ttggaaacat 4920

cgtggacgaa gttgcttatc acgagaagta ccccacgatc tatcatctgc gtaaaaagtt 4980

ggtggactcg acagataagg ccgacctcag gttaatatac cttgcactgg cgcacatgat 5040

caaattcaga ggccattttc tgattgaagg tgacctgaac cctgacaata gtgatgtgga 5100

caaactcttc attcaattag ttcagaccta caatcaactg tttgaagaga accctatcaa 5160

cgcttcagga gttgacgcta aggccatcct tagtgcgaga ctgagcaaat cccgccgtct 5220

cgaaaactta atcgcacagt tgcctggaga gaaaaagaac ggtttgttcg gaaatctcat 5280

tgcgttgtca ctcggactca cgccaaactt caagtctaac ttcgatttgg cagaagacgc 5340

gaaactgcaa ctgagcaaag acacatatga cgatgacctc gataacctct tagctcagat 5400

cggcgatcaa tacgccgact tgttcctcgc tgccaaaaat ctgtcggacg ctatacttct 5460

gagtgatatc ttgcgcgtca acacagaaat tactaaggct cctctgtcgg ccagtatgat 5520

aaaacgctat gacgaacacc atcaggattt gacattgctc aaagccctcg tgcgtcaaca 5580

gctcccagaa aagtacaagg agattttctt tgatcagtcc aagaatggct acgcaggtta 5640

tatagacggt ggagcgtcgc aagaagagtt ctacaagttc atcaagccaa tattagaaaa 5700

gatggacggc acggaagagt tacttgttaa gctgaatcgt gaggacctgt tgcgtaaaca 5760

gaggacattc gataacggat caattccgca ccaaatacat cttggcgaac tgcacgctat 5820

cctcaggaga caagaggact tctacccctt tttaaaggat aaccgtgaaa agatcgagaa 5880

aatcctgact ttcaggattc cttactatgt cggcccactg gctcgtggta atagcaggtt 5940

tgcctggatg accaggaagt ccgaagagac aattactccg tggaacttcg aagaggtggt 6000

tgataaagga gcatcagcgc agtctttcat agaacgcatg acaaattttg acaagaactt 6060

accgaatgag aaggtccttc ccaaacactc actcctctac gaatacttca cagtatacaa 6120

cgagctcact aaagtcaagt acgtaaccga gggtatgcgc aaacccgctt tcctgtctgg 6180

agagcagaaa aaggccatcg tggaccttct gttcaagaca aaccgtaagg tcactgtaaa 6240

gcaactcaag gaagactact tcaaaaagat agagtgtttc gattcagtgg aaatctctgg 6300

cgttgaggac agatttaacg cttccttggg tacttaccac gatttgctca agatcattaa 6360

agataaggac ttcctcgaca acgaagagaa cgaagatatc ttagaggaca tagttctcac 6420

ccttacgctg tttgaagata gagagatgat tgaagagcgc ctgaagactt atgctcattt 6480

gttcgatgac aaagtcatga agcaactgaa acgccgtagg tacaccggct ggggtagatt 6540

atcgcgcaaa cttattaatg gtataaggga caagcagtcg ggaaaaacga tattggactt 6600

tctcaagagt gatggtttcg ccaacagaaa ttttatgcaa ctcatacacg atgacagctt 6660

aacattcaag gaagatatcc aaaaagcaca ggtgtcggga cagggcgaca gtttgcacga 6720

acatattgct aacctcgccg gctccccggc gataaaaaag ggtatccttc agactgtgaa 6780

agtcgtagat gaactggtga aggttatggg tcgtcataaa cccgagaaca tagttatcga 6840

aatggctagg gagaatcaaa caactcagaa gggacagaaa aactcaagag aacgcatgaa 6900

gcgcattgaa gagggtatca aagagcttgg cagtcaaatc ctgaaggaac accctgtcga 6960

gaacacgcaa cttcagaacg aaaaattgta cctctactat ctgcagaatg gtagagatat 7020

gtacgtagac caagaattgg atattaaccg cctctcagat tacgacgtgg atcatatagt 7080

tccgcagtca ttcttgaagg atgactctat cgacaacaaa gtcctcacaa gatcagacaa 7140

gaaccgcgga aaatcagata atgtaccctc tgaagaggtg gttaaaaaga tgaaaaacta 7200

ctggagacag ttacttaacg ctaagttgat cacgcaaaga aagttcgata acctcacaaa 7260

ggctgaacgc ggcggtttaa gcgagcttga caaggccggt ttcataaaac gtcagttagt 7320

cgaaaccagg caaattacga aacacgtagc ccaaatattg gattcccgca tgaacactaa 7380

atacgatgaa aatgacaagc tcatccgtga ggtcaaagta attaccctga aaagcaagtt 7440

ggtgtccgac ttcagaaagg atttccagtt ctacaaagtt cgcgaaatca acaactacca 7500

ccatgcacat gacgcttacc tgaacgcagt cgtaggcact gcgttaatta aaaagtaccc 7560

taaactggaa tctgagttcg tgtacggtga ctataaagtg tacgatgtta gaaagatgat 7620

cgctaaaagc gaacaggaga ttggaaaggc taccgccaag tatttctttt actccaacat 7680

catgaatttc tttaagaccg aaatcacgtt agcaaatggc gagatacgta aaaggccact 7740

tatcgaaaca aacggagaaa ctggcgagat agtgtgggac aagggtagag attttgccac 7800

tgtccgcaaa gtactgtcga tgccgcaagt gaatatcgtt aaaaagaccg aagttcaaac 7860

gggaggcttc agcaaagagt ccatcctgcc caagcgtaac agtgataaat tgatagctag 7920

gaaaaaggac tgggatccta aaaagtatgg tggattcgac agcccaactg tcgcatactc 7980

cgtattggtg gttgcgaaag tcgaaaaagg aaagagcaaa aagctcaagt ccgtaaaaga 8040

gctgttgggc attaccataa tggaaagatc atctttcgag aagaatccta tcgattttct 8100

ggaagccaag ggatataaag aggtcaaaaa ggacctcata atcaagttac caaaatacag 8160

tctgttcgaa ttggagaacg gcagaaaacg catgcttgca tcagcgggtg aactgcaaaa 8220

gggaaatgag ttagcacttc cttctaaata cgtcaacttc ctgtatttgg cgtcacacta 8280

cgaaaaactg aagggctctc cagaagataa cgagcaaaag cagttatttg tggaacagca 8340

caaacattac cttgacgaaa ttatagagca aatctcggag ttcagtaaga gagtgatttt 8400

ggctgacgcc aatcttgata aagttctgtc tgcttacaac aagcaccgtg ataaaccgat 8460

tagggaacag gccgagaaca tcatacatct cttcacactc actaaccttg gtgcacccgc 8520

agcgttcaaa tattttgaca ccacgataga tcgtaagagg tacaccagca cgaaagaagt 8580

tttggacgcg acactcatcc atcaatcaat cacgggcctg tacgagacca gaatcgacct 8640

gtcccagctc ggtggcgact agcggccgcg actctagatc ataatcagcc atgcggccgc 8700

gactctagac cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc 8760

tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata 8820

atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc 8880

attctagttg tggtttgtcc aaactcatca atgtatctta aagcttatcg atacgcgtac 8940

ctaggccggc cgatctcgga tctgacaatg ttcagtgcag agactcggct acgcctcgtg 9000

gactttgaag ttgaccaaca atgtttattc ttacctctaa tagtcctctg tggcaaggtc 9060

aagattctgt tagaagccaa tgaagaacct ggttgttcaa taacattttg ttcgtctaat 9120

atttcactac cgcttgacgt tggctgcact tcatgtacct catctataaa cgcttcttct 9180

gtatcgctct ggacgtcatc ttcacttacg tgatctgata tttcactgtc agaatcctca 9240

ccaacaagct cgtcatcgct ttgcagaaga gcagagagga tatgctcatc gtctaaagaa 9300

ctacccattt tattatatat tagtcacgat atctataaca agaaaatatatatataataa 9360

gttatcacgt aagtagaaca tgaaataaca atataattat cgtatgagtt aaatcttaaa 9420

agtcacgtaa aagataatca tgcgtcattt tgactcacgc ggtcgttata gttcaaaatc 9480

agtgacactt accgcattga caagcacgcc tcacgggagc tccaagcggc gactgagatg 9540

tcctaaatgc acagcgacgg attcgcgcta tttagaaaga gagagcaata tttcaagaat 9600

gcatgcgtca attttacgca gactatcttt ctagggttaa aaaagatttg cgctttactc 9660

gacctaaact ttaaacacgt catagaatct tcgtttgaca aaaaccacat tgtggccaag 9720

ctgtgtgacg cgacgcgcgc taaagaatgg caaaccaagt cgcgcgagcg tcgactctag 9780

aggatccccg ggtaccgagc tcgaattcgt aatcatggtc atagctgttt cctgtgtgaa 9840

attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct 9900

ggggtgccta atgagtgagc taactcacat cggatgccgg gaccgacgag tgcagaggcg 9960

tgcaagcgag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc 10020

tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat 10080

gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc 10140

tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg 10200

ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 10260

cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 10320

gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 10380

tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 10440

agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 10500

tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 10560

cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg 10620

ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 10680

ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 10740

ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 10800

ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc 10860

cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 10920

gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 10980

atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga 11040

ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa 11100

gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa 11160

tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc 11220

ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga 11280

taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa 11340

gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt 11400

gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg 11460

ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc 11520

aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg 11580

gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag 11640

cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt 11700

actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt 11760

caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac 11820

gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac 11880

ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag 11940

caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa 12000

tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga 12060

gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc 12120

cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa 12180

ataggcgtat cacgaggccc tttcgtc 12207

<210>12

<211>3947

<212>DNA

<213>Artificial

<400>12

gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt 60

cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 120

tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 180

aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt 240

ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg 300

ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga 360

tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc 420

tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac 480

actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg 540

gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca 600

acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg 660

gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg 720

acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg 780

gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag 840

ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg 900

gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct 960

cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac 1020

agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact 1080

catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga 1140

tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt 1200

cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct 1260

gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc 1320

taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc 1380

ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc 1440

tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 1500

ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt 1560

cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg 1620

agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 1680

gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt 1740

atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 1800

gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt 1860

gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta 1920

ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt 1980

cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc 2040

cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca 2100

acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc 2160

cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg 2220

accatgatta cgccaagctc gcttgcacgc ctctgcactc gtcggtcccg gcatccgatg 2280

acccaatctc gagttgctag caatgaaaga tctttatcga tttagccaaa agcaaaagct 2340

tgaccaaaaa taggataata tttgtttttt tatttaaaaa aataaacaat tttttataca 2400

taaactgttt atctagtatt aatatttatg ttaacatttg ataacgaatc aaatatattt 2460

ttaaactaat taaaaaatcc gatgtatgtt ataaaattgt tctagaaaaa aagcaccgac 2520

tcggtgccac tttttcaagt tgataacgga ctagccttat tttaacttgc tatttctagc 2580

tctaaaacac tggcaggtgt cttgacgagt tcttctgaat tattaacgct tacaatttcc 2640

tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcatc aggtggcact 2700

tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg 2760

tatccgctca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga 2820

agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgccac 2880

ctgccatcac ttgtagagca cgatattttg tatatatacc taaaaaaact aaactattga 2940

aagcgtgatt tacaacaaca ctcgacttta caaagattat tcaaaaagag caaaaactct 3000

taacatattc tattaaagat atataatata attaaaacga aattaaataa taacaataaa 3060

acctttagaa tttgtaataa aatccataaa aacaaatgaa aacagttatg gtttgtacag 3120

cgccatctgt tattactttg acaaaatcac tatgactatc tgaccttgtc ttacacgtta 3180

acaattctta ttctgtcctt atctataagc caagtaccaa gcttaaattc gtatggctta 3240

tagttgacga tttttaaatt ctcaaggtat gtacttattt aatattaata agtactaatt 3300

gttaaaatca tctaaaacaa ttcagtgatt tacaacaatg tgtactacat aacctaatac 3360

ttataaattt attaaactgt attgattctt ttaggtcaat catcatgact ttaggagact 3420

tggtgtctca ggaaaaagga acgcaaaaag attgaggcgt ttgaaatgta ttgctggaga 3480

aagctgctac gcattccttg gacagcttgg cgcgccatct cgacgcattc gcgaagtacc 3540

gatctccaat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac 3600

ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc 3660

ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg 3720

gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatggtgca ctctcagtac 3780

aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc 3840

gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg 3900

gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcga 3947

<210>13

<211>3877

<212>DNA

<213>Artificial

<400>13

aagtgcttga aatgctaaat gttttcaatt tttcgccatt aagacaagcc tacacaaatg 60

cttctataaa ttatgccaag cacgttagca gcttctacga gccccaacca ctattaattc 120

gaacagcatg ttttttttgc agtgcgcaat gtttaacaca ctatattatc aatactacta 180

aagataacac ataccaatgc atttcgtctc aaagagaatt ttattctctt cacgacgaaa 240

aaaaaagttt tgctctattt ccaacaacaa caaaaatatg agtaatttat tcaaacggtt 300

tgcttaagag ataagaaaaa agtgaccact attaattcga acgcggcgta agcttacctt 360

aatctcaaga agagcaaaac aaaagcaact aatgtaacgg aatcattatc tagttatgat 420

ctgcaaataa tgctgcagcc taggcgagaa atttctctgg ccgttattcg ttattctctc 480

ttttcttttt gggtctctcc ctctctgcac taatgctctc tcactctgtc acacagtaaa 540

cggcatactg ctctcgttgg ttcgagagag cgcgcctcga atgttcgcga aaagagcgcc 600

ggagtataaa tagaggcgct tcgtctacgg agcgacaatt caattcaaac aagcaaagtg 660

aacacgtcgc taagcgaaag ctaagcaaat aaacaagcgc agctgaacaa gctaaacaat 720

ctgcagtaaa gtgcaagtta aagtgaatca attaaaagta accagcaacc aagtaaatca 780

actgcaacta ctgaaatctg ccaagaagta attattgaat acaagaagag aactctgggg 840

gatcatgacc gaatacaaac ccacagtgag actggccact agagacgatg ttcctagagc 900

tgtcagaact ttggctgccg ctttcgccga ttacccagct actagacaca ccgttgaccc 960

ggatagacac atcgaaagag tcaccgaatt gcaggaactc ttcctgacaa gagttggtct 1020

cgacattgga aaggtctggg tggccgacga tggagccgct gttgctgtct ggacaactcc 1080

cgaatcggtg gaagccggcg ctgttttcgc cgaaataggt cctagaatgg ctgaattgtc 1140

aggttctaga ctcgccgctc aacagcaaat ggaaggactg ttggcccctc acagaccaaa 1200

agaaccggcc tggttcctcg ctactgtggg agttagccca gatcaccagg gtaaaggact 1260

gggctccgct gtggttttgc caggagtcga agctgctgaa agagccggcg tgccggcttt 1320

cttggaaacc tcagccccaa gaaacctccc gttctacgaa agactgggct tcaccgtgac 1380

agctgacgtc gaagtgcccg aaggccctag aacatggtgc atgactagaa aacctggtgc 1440

tgactacaag gacgatgacg ataaagatta taaagacgat gacgataaag actataaaga 1500

tgacgacgat aaatacccct acgacgtgcc tgattacgct cggccgcgac tctagatcat 1560

aatcagccat gcggccgcga ctctagacca catttgtaga ggttttactt gctttaaaaa 1620

acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact 1680

tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata 1740

aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttaaa 1800

gcttatcgat acgcgtagtc gaccatgatg ataaacaatg tatggtgcta atgttgcttc 1860

aacaacaatt ctgttgaact gtgttttcat gtttgccaac aagcaccttt atactcggtg 1920

gcctccccac caccaacttt tttgcactgc aaaaaaacac gcttttgcac gcgggcccat 1980

acatagtaca aactctacgt ttcgtagact attttacata aatagtctac accgttgtat 2040

acgctccaaa tacactacca cacattgaac ctttttgcag tgcaaaaaag tacgtgtcgg 2100

cagtcacgta ggccggcctt atcgggtcgc gtcctgtcac gtacgaatca cattatcgga 2160

ccggacgagt gttgtcttat cgtgacagga cgccagcttc ctgtgttgct aaccgcagcc 2220

ggacgcaact ccttatcgga acaggacgcg cctccatatc agccgcgcgt tatctcatgc 2280

gcgtgaccgg acacgaggcg cccgtcccgc ttatcgcgcc tataaataca gcccgcaacg 2340

atctggtaaa cacagttgaa cagcatctgt tcgaaaccgg tgcgatcgca tggtgagcaa 2400

gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa 2460

cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac 2520

cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac 2580

cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt 2640

cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga 2700

cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat 2760

cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta 2820

caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt 2880

gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca 2940

gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac 3000

ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt 3060

cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaaa gatctggtac 3120

ctaaagcttt acaactaaac acgacttgga gtattccttg tagtgtttaa gattttaaat 3180

cttacttaat gacttcgaac gattttaacg ataactttct ctttgtttaa ctttaatcag 3240

catacataaa aagccccggt tttgtatcgg gaagaaaaaa aatgtaattg tgttgcctag 3300

ataataaacg tattatcaaa gtgtgtggtt ttcctttacc aaagacccct ttaagatggg 3360

cctaatgggc ttaagtcgag tcctttccga tgtgttaaat acacatttat tacactgatg 3420

cgtcgaatgt acacttttaa taggatagct ccactaaaaa ttattttatt tatttaattt 3480

gttgcaccaa aactgataca ttgacgaaac gcgtatggcg cgccattaat taaattattg 3540

ttttaagtat gatagtaaat cacattacgc cgcgttcgaa ttaatagtgg tcactttttt 3600

cttatctctt aagcaaaccg tttgaataaa ttactcatat ttttgttgtt gttggaaata 3660

gagcaaaact ttttttttcg tcgtgaagag aataaaattc tctttgagac gaaatgcatt 3720

ggtatgtgtt atctttagta gtattgataa tatagtgtgt taaacattgc gcactgcaaa 3780

aaaaacatgc tgttcgaatt aatagtggtt ggggctcgta gaaaacgaaa aatatcttaa 3840

gctagcatag agaatggagc aaaactcaat ttgatgc 3877

<210>14

<211>46

<212>DNA

<213>Artificial

<400>14

aatgttcgcg aaaagagcgc cgggtgtatt taacacatcg gaaagg 46

<210>15

<211>46

<212>DNA

<213>Artificial

<400>15

acaagcacct ttatactcgg tggcccttta agatgggcct aatggg 46

<210>16

<211>46

<212>DNA

<213>Artificial

<400>16

gagctggacg gcgacgtaaa cgggtgaacc gcatcgagct gaaggg 46

<210>17

<211>46

<212>DNA

<213>Artificial

<400>17

gggcgaggag ctgttcaccg gggggccaca agttcagcgt gtccgg 46

<210>18

<211>59

<212>DNA

<213>Artificial

<400>18

accgatcgat gaagacagaa gtgaatgttc gcgaaaagag cgcgttttag agctagaaa 59

<210>19

<211>59

<212>DNA

<213>Artificial

<400>19

tgatgatgat gaagacgtaa acttccgatg tgttaaatac accacttgta gagcacgat 59

<210>20

<211>59

<212>DNA

<213>Artificial

<400>20

accgatcgat gaagacagaa gtgacaagca cctttatact cgggttttag agctagaaa 59

<210>21

<211>59

<212>DNA

<213>Artificial

<400>21

tgatgatgat gaagacgtaa acattaggcc catcttaaag ggcacttgta gagcacgat 59

<210>22

<211>59

<212>DNA

<213>Artificial

<400>22

accgatcgat gaagacagaa gtggagctgg acggcgacgt aaagttttag agctagaaa 59

<210>23

<211>59

<212>DNA

<213>Artificial

<400>23

tgatgatgat gaagacgtaa acttcagctc gatgcggttc accacttgta gagcacgat 59

<210>24

<211>59

<212>DNA

<213>Artificial

<400>24

accgatcgat gaagacagaa gtggggcgag gagctgttca ccggttttag agctagaaa 59

<210>25

<211>59

<212>DNA

<213>Artificial

<400>25

tgatgatgat gaagacgtaa acgacacgct gaacttgtgg cccacttgta gagcacgat 59

Claims (7)

1. A construction method of a double gRNA vector of eukaryotic organism CRISPR-Cas9 is characterized by comprising the following specific steps:

(1) constructing a piggyBac transposon system mediated eukaryote CRISPR-Cas9 double gRNA framework vector, namely pB-CRISPR, wherein the nucleotide sequence of the vector is shown as SEQ ID NO. 1; the gene element delivery system of the vector is a piggyBac transposon system, and the gene knockout system is a CRISPR/Cas9 system;

(2) constructing a template vector for providing sgRNA scaffold and U6 promoters, namely T-DGP-7, wherein the nucleotide sequence of the template vector is shown as SEQ ID NO. 2;

(3) designing a targeting site, constructing a primer pair of a double gRNA vector, and then performing PCR amplification by using the primer pair with the T-DGP-7 obtained in the step (2) as a template to obtain an amplification product named as PCR-DGP 7-XY;

(4) digesting the pB-CRISPR obtained in the step (1) by using an endonuclease AarI as a framework, digesting the PCR-DGP7-XY obtained in the step (3) by using BbsI as a fragment, and connecting the framework and the fragment to form a double gRNA vector which is named as pB-Dul-CRISPR-XY;

(5) and (3) mixing the pB-Dul-CRISPR-XY obtained in the step (4) with a piggyBactransposon expression vector A3-helper with the nucleotide sequence shown as SEQ ID NO.3 for transfection of eukaryotic cells, and screening to obtain the vector.

2. The construction method according to claim 1, wherein the specific method of step (1) is as follows:

(1-1) synthesizing a vector PUC57-IE2-Zeocin-Ser1PA containing a Zeocin resistance gene expression cassette, wherein the nucleotide sequence of the vector is shown as SEQ ID NO. 6;

(1-2) connecting a Zeocin resistance gene expression frame IE2-Zeocin-Ser1PA on a vector PUC57-IE2-Zeocin-Ser1PA to a piggyBac transposon basic vector piggyBacModify with a nucleotide sequence shown as SEQ ID No.7 to construct an intermediate vector pB-Modified { IE2-Zeocin-Ser1PA }, wherein the nucleotide sequence is shown as SEQ ID No. 8;

(1-3) amplifying an expression frame of hr3-hsp70-Cas9-sv40 from a vector pUC57-hr3-hsp70-Cas9-sv40 with the nucleotide sequence shown as SEQ ID NO. 9; then connected to AscI site of pB-Modified { IE2-Zeocin-Ser1PA } by a seamless cloning method to construct an intermediate vector pB-Modified { IE2-Zeocin-Ser1PA } { hr3-hsp70-Cas9-SV40}, and the nucleotide sequence of the intermediate vector is shown as SEQ ID NO. 11;

(1-4) amplifying U6-gRNA from a vector pUC57-U6-gRNA with the nucleotide sequence shown in SEQ ID NO.12, connecting to a vector pB-Modified { IE2-Zeocin-Ser1PA } { hr3-hsp70-Cas9-SV40} by using an enzyme digestion connection method, and constructing a eukaryotic gene knockout basic vector pB-Modified { IE2-Zeocin-Ser1PA } { U6-gRNA } { hr 3-Cas 70-Cas9-SV40}, wherein the vector is named as pB-CRISPR.

3. The construction method according to claim 1, wherein in step (3), based on CRISPR/Cas9 action rules, the targeting sites designed for realizing eukaryotic gene knockout are 23 nucleotides in total, and have the following rules:

5 '-NNNNNNNNNNNNNNNNNNNNNNNNN-NGG-3'; on the basis, a primer pair of the double gRNA vector is constructed, and has the following rule:

the forward primer is > X-F,

5-ACCGATCGATGAAGACAGAAGTGNNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAA, the nucleotide sequence of which is shown in SEQ ID NO. 4;

the reverse primer is > Y-R,

5-TGATGATGATGAAGACGTAAACNNNNNNNNNNNNNNNNNNNNCACTTGTAGAGCACGAT, the nucleotide sequence of which is shown in SEQ ID NO. 5;

wherein "NNNNNNNNNNNNNNNNNNNN" in > X-F and > Y-R are targeting site X and targeting site Y, respectively.

4. The method according to claim 1, wherein in the step (5), the pB-Dul-CRISPR-XY and piggyBac transposon expression vector A3-helper (nucleotide sequence shown in SEQ ID NO. 3) are expressed in a molar ratio of 1: 1, transfecting eukaryotic cells, and screening the transfected cells by Zeocin for 2 months to obtain a cell line with two genes knocked out or functional genome fragments deleted simultaneously.

5. The method of claim 1, wherein the eukaryotic organism includes, but is not limited to, bombyx mori, drosophila, and the like.

6. A eukaryotic CRISPR-Cas double gRNA vector constructed by the method of any one of claims 1-5.

7. Use of the vector of claim 6 for the construction of a cell line for the simultaneous knock-out of two genes or the deletion of a functional gene fragment in eukaryotes.

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