CN113652451B - Lentiviral vector, construction method and application thereof - Google Patents

Abstract

The invention belongs to the field of biology, and particularly relates to a lentiviral vector, a construction method and application thereof. The invention discloses a preparation method of a lentiviral vector, which comprises the following steps: using LTR 1.27-GEW-based plasmid as a skeleton, adding R and U5 regions to the 5' end, and deleting 3' PBS sequence to obtain a lentiviral vector Obio-01, and replacing the 5' CMV promoter in the lentiviral vector Obio-01 with the U3 promoter of human immunodeficiency virus-1 to obtain a lentiviral vector Obio-03; the 5' CMV promoter in the lentiviral vector Obio-01 was replaced with the U3 promoter of human immunodeficiency virus-2 to obtain lentiviral vector Obio-04. The novel fourth-generation lentiviral vector prepared by the invention can have high-yield viral titer on the premise of ensuring safety, and can be applied to the fields of gene therapy and the like.

Description

Lentiviral vector, construction method and application thereof

Technical Field

The invention belongs to the field of biology, and particularly relates to a lentiviral vector, a construction method and application thereof.

Background

Lentiviral vectors are gene therapy vectors developed based on HIV-1 (human immunodeficiency type I virus). A distinction is made between retroviral vectors in general, which have the ability to infect both dividing and non-dividing cells. The research on the lentivirus vector is developed quickly and deeply, and the lentivirus vector can effectively integrate the exogenous gene onto a host chromosome so as to achieve persistent expression. Can effectively infect various types of cells such as neuron cells, liver cells, myocardial cells, tumor cells, endothelial cells, stem cells and the like in the aspect of infection capacity, thereby achieving good gene therapy effect, developing clinical research in the United states, having ideal effect and having wide application prospect.

Lentiviruses belong to the family of retroviruses, but have a complex genome structure, comprising 4 accessory genes vif, vpr, nef, vp μ and 2 regulatory genes tat and rev, in addition to 3 structural genes similar to that of a simple retrovirus, namely gag, pol and env genes. HIV-1 is the most characteristic virus of lentiviruses, and the first lentivirus vector system is constructed based on this virus. Lentiviral vectors are constructed by separating cis-acting elements (e.g., packaging signals, long terminal repeats) from sequences encoding trans-acting proteins in the HIV-1 genome. The carrier system comprises a packaging component and a carrier component: the packaging component is constructed from the HIV-1 genome with the cis-acting sequences required for packaging, reverse transcription and integration removed, and is capable of supplying in trans the proteins required for the production of viral particles; the vector component is complementary to the packaging component and contains the HIV-1 cis-acting sequences required for packaging, reverse transcription and integration. Meanwhile, the promoter has a multiple cloning site under the control of a heterologous promoter and a target gene inserted into the site. The LTR region of HIV-1 is composed of a U3 region, an R region and a U5 region, and in order to reduce the possibility of producing a replication-competent virus (RCV) by homologous recombination of the two components, the third generation vector is constructed by replacing the U3 region of 5'LTR of the packaging component with a Cytomegalovirus (CMV) immediate early promoter or RSV promoter, and by deletion-mutating the U3 region of 3' LTR and adding SV40 polyA site. The packaging components are constructed on two plasmids, one expressing genes gag and pol, the other env. Based on this principle, a three-plasmid expression system was constructed.

The three-plasmid expression system comprises a packaging plasmid, an envelope protein plasmid and a shuttle plasmid. Wherein the packaging plasmid expresses all trans-activation proteins required by HIV-1 replication under the control of a CMV promoter, but does not generate a virus envelope protein and an accessory protein vp mu; the envelope protein plasmid encodes vesicular stomatitis virus G protein (VSV-G), and the pseudomorphic lentiviral vector enveloped by the VSV-G expands the tropism range of target cells of the vector, increases the stability of the vector, allows the vector to be concentrated by high-speed centrifugation, and improves the titer; the shuttle plasmid contains cis-sequences required for packaging, reverse transcription and integration, and retains about 350bp of gag and RRE, and a target gene or a marker gene (green fluorescent protein GFP) is inserted therein. The greatest benefit of dividing the vector system into three plasmids is that the chance of overlapping sequences is greatly reduced, reducing the possibility of RCV generation during vector recombination. The virus titer can reach 10 < Lambda > 9IU/ml after ultracentrifugation by cotransfecting 293T cells with three plasmids.

To reduce the sequence homology of the HIV-1 packaging structure, and further reduce the possibility of recombination into RCV, dull et al removed the helper genes. However, since gene rev is required for the transport of gag-pol gene, a four-plasmid expression system was constructed based on the above three-plasmid system, and the addition of the plasmid containing gene rev reduced the possibility of RCV generation and had no effect on the transduction efficiency of cells in the non-dividing phase. tRNA is used as a reverse transcription primer to be combined with PBS at the 5 'end, a reverse transcription product jumps to the 3' end to continue reverse transcription, and the finally obtained reverse transcription product is an HIV genome sequence with a packaging signal (psi), RRE and the like.

Recently reported fourth generation lentivirus, 5' end R and U5 region deletion, packaging signal (. Psi.) and RRE and other sequences are put on 3' end relative to third generation viral vector, reverse transcription is directly started from 3' end during reverse transcription, and finally the obtained reverse transcription product has no packaging signal (. Psi.), RRE and other HIV genome sequences, so that the sequence integrated into the genome only contains 4.8% of the sequence of HIV-1 genome, further enhancing safety, but with the problem of serious decline of titer and yield, according to the report, only 35% of the third generation lentivirus vector can be reached, and for this reason, the fourth generation lentivirus vector has not been practically applied. tRNA is used as a reverse transcription primer to be combined with PBS at the 3 'end and is reversely transcribed to the 5' end, and finally, the obtained reverse transcription product does not have HIV genome sequences such as a packaging signal (psi) and RRE, so that the safety is further improved. The reported titers of the existing known fourth generation lentiviruses are shown in FIG. 1, and it can be seen from FIG. 1 that the existing fourth generation lentivirus titers are lower.

Disclosure of Invention

The invention aims to invent a novel lentivirus vector, which can greatly improve the virus yield without changing the safety of a fourth generation lentivirus vector and can reach the level of the virus yield of a third generation lentivirus vector.

Specifically, the technical scheme of the invention is as follows:

the first aspect of the invention discloses a method for preparing a lentiviral vector, which comprises the following steps:

the lentiviral vector Obio-01 was obtained by using LTR 1.27-GEW-based plasmid as a backbone, adding R and U5 regions to the 5 'end, and deleting the 3' PBS sequence.

The nucleotide sequence based on LTR1.27-GEW plasmid is shown in SEQ ID NO:1, in some preferred embodiments of the present invention, the nucleotide sequence of said lentiviral vector Obio-01 is as set forth in SEQ ID NO:2, respectively.

Preferably, the 5' CMV promoter in the lentiviral vector Obio-01 was replaced with the U3 promoter of human immunodeficiency virus-1 to obtain lentiviral vector Obio-03.

In some preferred embodiments of the present invention, the nucleotide sequence of said lentiviral vector Obio-03 is as set forth in SEQ ID NO:3, respectively.

Preferably, the 5' CMV promoter in the lentiviral vector Obio-01 was replaced with the U3 promoter of human immunodeficiency virus-2 to obtain lentiviral vector Obio-04.

In some preferred embodiments of the present invention, the nucleotide sequence of said lentiviral vector Obio-04 is as set forth in SEQ ID NO:4, respectively.

In a second aspect, the invention discloses a lentiviral vector obtained by the above method.

In a third aspect, the invention discloses a cell infected with a lentiviral vector of the invention. The cells do not include embryonic stem cells, germ cells, and fertilized eggs of humans or animals.

In a fourth aspect of the invention, there is disclosed a method of producing a viral product by contacting a cell with an effective amount of a lentiviral vector as described above to produce the viral product.

Preferably, the method for preparing a viral product comprises:

s1, co-transfecting cells with the lentiviral vector; s2, harvesting viruses; and S3, separating the supernatant to obtain a virus product.

More preferably, the step S2 includes: after 45-50 hours of transfection, the virus was harvested for the first time, the medium was collected and the cells were replaced with fresh complete medium; after 70-75 hours of transfection, the virus was harvested a second time, the medium was collected and the cells discarded.

In some embodiments of the invention, the step S2 comprises: 48 hours after transfection, the virus was harvested for the first time, the medium was collected and the cells were replaced with fresh complete medium; 72 hours after transfection, the virus was harvested a second time, the medium was collected and the cells were discarded.

Preferably, the method for viral packaging using the above lentiviral vector comprises:

the day before transfection, 293T cells were seeded into culture dishes; taking out the cell culture dish one hour before transfection, removing the original cell culture medium, adding the Opti-MEM culture medium, and putting the cells back into the incubator; preparing a complex of a transfection reagent and a plasmid comprising the steps of:

a. dissolving virus vector plasmids (skeleton plasmid pCAG-gagpol-Tat, envelope protein plasmid pHCMV-VSVG and shuttle plasmid) to be transfected into an Opti-MEM culture medium, gently mixing uniformly, and standing to obtain a plasmid diluent; the shuttle plasmid is LTR 1.27-GEW-based plasmid or Obio-01, obio-02-Obio-04.

b. Dissolving the transfection reagent in an Opti-MEM culture medium, gently mixing uniformly, and standing to obtain a transfection reagent diluent;

c. dripping the transfection reagent diluent into the plasmid diluent, gently mixing while adding, and then placing at room temperature for 15-25min to ensure that the DNA and the transfection reagent are fully combined to form a stable transfection complex; taking out the cell culture dish, adding the prepared DNA-transfection reagent complex into the cell culture dish, and returning the cell culture dish to the incubator; after 5-8h, the medium was aspirated, washed with PBS solution, and then cultured by adding fresh complete medium. In some embodiments of the invention, the backbone plasmid pCAG-gagpol-Tat, the envelope protein plasmid pHCMV-VSVG and the shuttle plasmid are present in a mass ratio of 2:1:3.

in a fifth aspect, the invention discloses the use of the above method, the above lentiviral vector, the above cell or the above method in the field of gene therapy.

On the basis of the common general knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily without departing from the concept and the protection scope of the invention.

Compared with the prior art, the invention has the following remarkable advantages and effects:

the inventor invents a novel fourth-generation lentiviral vector through a large amount of innovative labor, and the lentiviral vector can have high yield of viral titer on the premise of ensuring safety, can be applied to the fields of gene therapy and the like, and has wide application prospect.

Drawings

FIG. 1 is a schematic representation of known fourth generation lentivirus titers;

FIG. 2 is a schematic diagram of a lentiviral vector constructed in the present invention;

FIG. 3 is a schematic diagram of the process of Obio-01 reverse transcription in the present invention;

FIG. 4 is a plasmid map based on LTR1.27-GEW in the present invention;

FIG. 5 is a plasmid map of Obio-01 in the present invention;

FIG. 6 is a plasmid map of Obio-03 of the present invention;

FIG. 7 is a plasmid map of Obio-04 in the present invention;

FIG. 8 is a schematic view of a microscope showing cells infected with different viral vectors of the present invention;

FIG. 9 is a graph showing relative titer values of different viral vectors of the present invention;

FIG. 10 is a test of WPRE element integration after infection of cells with different viral vectors in accordance with the present invention;

FIG. 11 is a graph showing the detection of integration of Psi elements after infection of cells with different viral vectors according to the present invention;

FIG. 12 is a graph showing the detection of integration of RRE element after infection of cells with different viral vectors according to the present invention;

FIG. 13 shows the detection of the integration of promoter elements at the 5' end of cells infected with different viral vectors of the present invention.

Detailed Description

The technical solutions of the present invention are described in detail below with reference to the drawings and the embodiments, but the present invention is not limited to the scope of the embodiments.

The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions. The reagents and starting materials used in the present invention are commercially available.

Example 1

The invention takes LTR1.27-GEW as a framework to construct a series of vectors, as shown in figure 2. The reverse transcription process of Obio-01 is presumed to be shown in FIG. 3, in FIG. 3: RNA genome refers to "RNA genome", minus strand stop synthesis refers to "negative strand strong termination synthesis", minus strand transfer refers to "negative strand hopping", minus strand extension refers to "negative strand extension", plus strand stop synthesis refers to "positive strand strong termination synthesis", plus strand transfer refers to "positive strand hopping", and dsDNA synthesis refers to "DNA synthesis". The plasmid map of LTR1.27-GEW is shown in FIG. 4. The plasmid maps of Obio-01, obio-03 and Obio-04 are shown in FIGS. 5-7, respectively. The nucleotide sequences of the Obio-01, obio-03 and Obio-04 are respectively shown in SEQ ID NO: 2-4. The nucleotide sequence based on LTR1.27-GEW is shown in SEQ ID NO:1 is shown. The present embodiment discloses a method for virus packaging using the above-constructed plasmid, specifically as follows:

the day before transfection, 293T cells were seeded into 100mm dishes; taking out the cell culture dish one hour before transfection, removing the original cell culture medium, adding 10ml of Opti-MEM culture medium, and putting the cells back into the incubator; preparing a complex of a transfection reagent and a plasmid comprising the steps of:

a. dissolving 32 mu g of a virus vector plasmid to be transfected (a skeleton plasmid pCAG-gagpol-Tat: an envelope protein plasmid pHCMV-VSVG: a shuttle plasmid in a mass ratio of 2; the shuttle plasmid is LTR 1.27-GEW-based plasmid or Obio-01-Obio-04.

b. Dissolving the transfection reagent in Opti-MEM culture medium with the total volume of 500. Mu.l, gently mixing, and standing for 5 minutes;

c. and dripping the transfection reagent diluent into the plasmid diluent, gently mixing while adding, and then placing at room temperature for 20min to ensure that the DNA and the transfection reagent are fully combined to form a stable transfection complex. Taking out the cell culture dish, adding the prepared DNA-transfection reagent complex into the cell culture dish, and returning the cell culture dish to the incubator; after 6h the medium was aspirated, washed once with PBS, and incubated with 10mL of fresh complete medium.

Example 2

The embodiment discloses a method for purifying viruses, which specifically comprises the following steps:

48 hours after transfection, first harvest, collect media into 50ml centrifuge tubes and replace cells with fresh complete media. After 72 hours of transfection, a second harvest was performed, the medium was collected into a 50ml centrifuge tube and the cells discarded.

Centrifuging the collected culture supernatant at 3500rpm for 10min at room temperature, and pouring the supernatant into a new 50ml centrifuge tube; after centrifugation at 30,000rpm at 4 ℃ for 2h in an ultracentrifuge, the supernatant was carefully discarded, the tube was inverted onto sterilized absorbent paper, and DPBS was added to resuspend the pellet, which was collected in a 1.5ml EP tube and stored in a freezer at-80 ℃.

Example 3

The embodiment discloses a titer detection method, which specifically comprises the following steps:

the lentivirus titer is determined by adopting a Real time quantitative PCR method, and the specific steps are as follows:

preparing a sample: by 1 × 10 ⁵ Cells were seeded 293T cells per well in 24-well plates; adding the virus the next day, and replacing the fresh culture medium after 12-20 h; photographing 72h after infection to record fluorescence, and collecting cellsTaking genome DNA and making quantitative PCR experiment to determine titer.

Real-time PCR was performed on an ABI7500 instrument. The reagent SYBR Master mix was used from TAKARA.

1. The reaction system is prepared according to the following proportion:

SYBR premix ex taq：10μl；

ROX：0.4μl；

upstream primer (25 μ M): 0.5 mul;

downstream primer (25 μ M): 0.5 mul;

Genomic DNA：2.0μl；

6.6 mul of water;

2. the procedure was set to two step Real-Time quantitation. Pre-denaturation 95 ℃ for 15S, followed by denaturation 95 ℃ for 5S, annealing extension 60 ℃ for 34S in each step, for a total of 40 cycles. Each time reading the absorbance value during the extension phase.

The PCR procedure was:

Cycle 1：(1X)

Step 1：95.0℃for 00：15；

Cycle 2：(40X)

Step 1：95.0℃for 00：05

Step 2：60.0℃for 00：34；

data collection and real-time analysis are enabled.

3. A melting curve was prepared. After the PCR was completed, the mixture was denatured at 95 ℃ for 1min. Then cooled to 55 ℃ to allow the DNA double strands to be fully bound. Starting at 55 ℃ to 95 ℃, each step was increased by 0.5 ℃ and held for 30S while absorbance was read.

The dissolution curve procedure was:

Cycle 3：(1X)

Step 1：95.0℃for 01:00；

Cycle 4：(1X)

Step 1：55.0℃for 01:00；

Cycle 5：(81X)

Step 1：55.0℃-95.0℃for 00:30

the set point temperature was increased by 0.5 ℃ after 2 cycles.

The 293T cells are respectively infected by lentiviruses packaged by pCLenti-CMV-EGFP-WPRE (third generation lentivirus vector) based on LTR1.27-GEW, obio-01, obio-03 and Obio-04 according to the volumes of 0.1ul, 1ul and 10ul, and fluorescence pictures are recorded after infection for 72h, wherein the fluorescence results are shown in figure 8, and the fluorescence brightness of the lentivirus vectors Obio-01, obio03 and Obio04 is better than that of the lentivirus vectors based on LTR1.27-GEW, which indicates that the titer is better. Meanwhile, infected cells are collected, cell genome DNA is extracted, quantitative PCR experiments are carried out to determine the lentivirus titer, the titer statistical result is shown in figure 9, and a virus titer histogram shows that the titer of lentivirus vectors Obio-01, obio03 and Obio04 is superior to that of lentivirus vectors based on LTR 1.27-GEW.

Example 4

The embodiment discloses a method for detecting the safety of lentiviruses, which specifically comprises the following steps:

respectively infecting 293T cells with pCLenti-CMV-EGFP-WPRE (third generation lentivirus vector) and lentiviruses packaged based on LTR1.27-GEW, obio-01, obio-03 and Obio-04, extracting cell genome DNA after 48 hours, and respectively detecting related elements of plasmids and cell genome DNA by using a PCR method: the woodchuck hepatitis virus posttranscriptional regulatory sequence (WPRE), the packaging signal (Psi ), the Rev Response Element (RRE) and the promoters at the U5-terminus of the respective vectors (CMV, and human immunodeficiency viruses (HIV 1, HIV 2)). The method comprises the following specific steps:

1. infection of cells

Plating 293T cells in 24-well plates; adding pCLenti-CMV-EGFP-WPRE (third generation lentivirus vector) and lentivirus packaged based on LTR1.27-GEW, obio-01, obio-03 and Obio-04 on the following day, adding CO at 37 deg.C and 5% ₂ Culturing in an incubator; collecting cells after 48h of infection;

2. extraction of genomic DNA from cells

Extracting the genomic DNA of the cells collected in the step 1 by using a genomic DNA small extraction kit (Axygen, AP-MN-MS-GDNA-250G);

3. PCR detection

PCR was performed using the primers shown in Table 1 for pCLenti-CMV-EGFP-WPRE (third generation lentiviral vector) based on LTR1.27-GEW, obio-01, obio-03 and Obio-04 plasmids and cell genomic DNA. PCR (polymerase chain reaction) specific reaction systemMu.l (50. Mu.l) of DNA template, 1. Mu.l each of forward and reverse amplification primers (F/R), 25. Mu.l of 2XHieFF PCR mix (san-Ile-assist organisms in Shanghai), ddH ₂ O22 mu l; PCR conditions were 95 ℃ for 5min,1cycle;95 ℃,30s, 55 ℃,30s, 72 ℃ 40s 35cycles; 5min at 72 ℃,1cycle;4 ℃ for 5min,1cycle.

The corresponding primer list is shown in table 1 below:

TABLE 1

Detecting the PCR product by electrophoresis with 1.5% agarose gel, the specific result is shown in FIGS. 10-13; wherein the plasmid uses shuttle plasmid of each virus vector as a template as a positive control, and the genome uses 293T cell genome after infection of each virus vector as a template for identifying whether corresponding elements in the virus vectors are integrated on the 293T cell genome. The results show that the transcriptional regulatory sequences (WPRE) of the woodchuck hepatitis virus can be integrated on the cell genome after infection based on LTR1.27-GEW, obio-01, obio-03 and Obio-04 relative to pCLenti-CMV-EGFP-WPRE (third generation lentivirus vector); the above examples are preferred embodiments of the present invention, but the embodiments are not limited to the above examples, and any other changes, modifications, substitutions, combinations, simplifications, and substitutions which are made without departing from the spirit and principles of the present invention are all equivalent substitutions and are included within the scope of the present invention.

Sequence listing

<110> and Yuan Biotechnology (Shanghai) Ltd

<120> lentiviral vector, construction method and application thereof

<160> 18

<170> SIPOSequenceListing 1.0

<210> 1

<211> 9798

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 60

catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 120

acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 180

ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 240

aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 300

ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 360

tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc 420

ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 480

ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa 540

tgggcggtag gcgtgtacgg tgggaggtct atataagcag cgcgttttgc ctgtactgtg 600

gcgcccgaac agggacttga aagcgaaagg gaaaccagag gagaacacag gtaagtgccg 660

tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg ccttgaatta 720

cttccacctg gctccagtac gtgattcttg atcccgagct ggagccaggg gcgggccttg 780

cgctttagga gccccttcgc ctcgtgcttg agttgaggcc tggcctgggc gctggggccg 840

ccgcgtgcga atctggtggc accttcgcgc ctgtctcgct gctttcgata agtctctagc 900

catttaaaat ttttgatgac ctgctgcgac gctttttttc tggcaagata gtcttgtaaa 960

tgcgggccag gatctgcaca ctggtatttc ggtttttggg gccgcgggcg gcgacggggc 1020

ccgtgcgtcc cagcgcacat gttcggcgag gcggggcctg cgagcgcggc caccgagaat 1080

cggacggggg tcggacgggg gtagtctcaa gctggccggc ctgctctggt gcctggcctc 1140

gcgccgccgt gtatcgcccc gccctgggcg gcaaggctgg cccggtcggc accagttgcg 1200

tgagcggaaa gatggccgct tcccggccct gctccagggg gctcaaaatg gaggacgcgg 1260

cgctcgggag agcgggcggg tgagtcaccc acacaaagga aaggggcctt tccgtcctca 1320

gccgtcgctt catgtgactc cacggagtac cgggcgccgt ccaggcacct cgattagttc 1380

tggagctttt ggagtacgtc gtctttaggt tggggggagg ggttttatgc gatggagttt 1440

ccccacactg agtgggtgga gactgaagtt aggccagctt ggcacttgat gtaattctcc 1500

ttggaatttg ccctttttga gtttggatct tggttcattc tcaagcctca gacagtggtt 1560

caaagttttt ttcttccatt tcaggtgtcg tgaggcactg cgtgcgccaa ttctgcagac 1620

aaatggcagt attcatccac aattttaaaa gaaaaggggg gattgggggg tacagtgcag 1680

gggaaagaat agtagacata atagcaacag acatacaaac taaagaatta caaaaacaaa 1740

ttacaaaaat tcaaaatttt cgggtttatt acagggacag cagagatcca gtttggttaa 1800

ttaaggtacc gaattcacgc gtggagctag ttattaatag taatcaatta cggggtcatt 1860

agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg 1920

ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac 1980

gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt 2040

ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa 2100

atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta 2160

catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg 2220

gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg 2280

gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc 2340

attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctcgttt 2400

agtgaaccgt cagatcgcct ggagacgcca tccacgctgt tttgacctcc atagaagaca 2460

ccgggaccga tccagcctcc ggtaccgaaa accccggtcc ggctagcgcc accggatccg 2520

gcggatctgg catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg 2580

tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg 2640

atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc 2700

cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg 2760

accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc 2820

gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg 2880

gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca 2940

tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca 3000

agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg 3060

tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc 3120

ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg 3180

atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc 3240

tgtacaagta aaccggtggt tatcgataat caacctctgg attacaaaat ttgtgaaaga 3300

ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg 3360

cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc 3420

tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc 3480

actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt 3540

tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt 3600

gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg 3660

aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg 3720

tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg 3780

ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt 3840

tgggccgcct ccccgcatcg ataccgctcg agctttaaga ccaatgactt acaaggcagc 3900

tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa ttcactccca 3960

acgaagacaa gatctgcttt ttgcttgtac tgggtctctc tggttagacc agatctgagc 4020

ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg 4080

agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag 4140

acccttttag tcagtgtgga aaatctctag cagtggcgcc cgaacaggga cttgaaagcg 4200

aaagggaaac cagaggagct ctctcgacgc aggactcggc ttgctgaagc gcgcacggca 4260

agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg aggctagaag 4320

gagagagatg ggtgcgagag cgtcagtatt aagcggggga gaattagatc gcgatgggaa 4380

aaaattcggt taaggccagg gggaaagaaa aaatataaat taaaacatat agtatgggca 4440

agcagggagc tagaacgatt cgcagttaat cctggcctgt tagaaacatc agaaggctgt 4500

agacaaatac tgggacagct acaaccatcc cttcagacag gatcagaaga acttagatca 4560

ttatataata cagtagcaac cctctattgt gtgcatcaaa ggatagagat aaaagacacc 4620

aaggaagctt tagacaagat agaggaagag caaaacaaaa gtaagaccac cgcacagcaa 4680

gcggccgctg atcttcagac ctggaggagg agatatgagg gacaattgga gaagtgaatt 4740

atataaatat aaagtagtaa aaattgaacc attaggagta gcacccacca aggcaaagag 4800

aagagtggtg cagagagaaa aaagagcagt gggaatagga gctttgttcc ttgggttctt 4860

gggagcagca ggaagcacta tgggcgcagc gtcaatgacg ctgacggtac aggccagaca 4920

attattgtct ggtatagtgc agcagcagaa caatttgctg agggctattg aggcgcaaca 4980

gcatctgttg caactcacag tctggggcat caagcagctc caggcaagaa tcctggctgt 5040

ggaaagatac ctaaaggatc aacagctcct ggggatttgg ggttgctctg gaaaactcat 5100

ttgcaccact gctgtgcctt ggaatgctag ttggagtaat aaatctctgg aacagatttg 5160

gaatcacacg acctggatgg agtgggacag agaaattaac aattacacaa gcttaataca 5220

ctccttaatt gaagaatcgc aaaaccagca agaaaagaat gaacaagaat tattggaatt 5280

agataaatgg gcaagtttgt ggaattggtt taacataaca aattggctgt ggtatataaa 5340

attattcata atgatagtag gaggcttggt aggtttaaga atagtttttg ctgtactttc 5400

tatagtgaat agagttaggc agggatattc accattatcg tttcagaccc acctcccaac 5460

cccgagggga cccgacaggc ccgaaggaat agaagaagaa ggtggagaga gagacagaga 5520

cagatccatt cgattagtga acggatcggc ccgtttaaac ccgctgatca gcctcgactg 5580

tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg 5640

aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga 5700

gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg 5760

aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa 5820

ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg 5880

gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt 5940

tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc 6000

gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg 6060

attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga 6120

cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc 6180

ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa 6240

aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt 6300

agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa 6360

ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag 6420

catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct 6480

aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc 6540

agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg 6600

aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca 6660

gcacgtgttg acaattaatc atcggcatag tatatcggca tagtataata cgacaaggtg 6720

aggaactaaa ccatggccaa gttgaccagt gccgttccgg tgctcaccgc gcgcgacgtc 6780

gccggagcgg tcgagttctg gaccgaccgg ctcgggttct cccgggactt cgtggaggac 6840

gacttcgccg gtgtggtccg ggacgacgtg accctgttca tcagcgcggt ccaggaccag 6900

gtggtgccgg acaacaccct ggcctgggtg tgggtgcgcg gcctggacga gctgtacgcc 6960

gagtggtcgg aggtcgtgtc cacgaacttc cgggacgcct ccgggccggc catgaccgag 7020

atcggcgagc agccgtgggg gcgggagttc gccctgcgcg acccggccgg caactgcgtg 7080

cacttcgtgg ccgaggagca ggactgacac gtgctacgag atttcgattc caccgccgcc 7140

ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag 7200

cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat 7260

ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat 7320

tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc 7380

tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg 7440

ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa 7500

tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac 7560

ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt 7620

gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga 7680

gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca 7740

ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg 7800

ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt 7860

cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc 7920

ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct 7980

tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc 8040

gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta 8100

tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca 8160

gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag 8220

tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag 8280

ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt 8340

agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa 8400

gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg 8460

attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga 8520

agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta 8580

atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc 8640

cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg 8700

ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga 8760

agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt 8820

tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt 8880

gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc 8940

caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc 9000

ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca 9060

gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag 9120

tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg 9180

tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa 9240

cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa 9300

cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga 9360

gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga 9420

atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg 9480

agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt 9540

ccccgaaaag tgccacctga cgtcgacgga tcgggagatc tcccgatccc ctatggtgca 9600

ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg 9660

tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga 9720

ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac 9780

gggccagata tacgcgtt 9798

<210> 2

<211> 9961

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 60

catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 120

acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 180

ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 240

aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 300

ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 360

tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc 420

ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 480

ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa 540

tgggcggtag gcgtgtacgg tgggaggtct atataagcag cgcgttttgc ctgtactggg 600

tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg 660

cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt 720

gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt 780

ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagaacaca ggtaagtgcc 840

gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt 900

acttccacct ggctccagta cgtgattctt gatcccgagc tggagccagg ggcgggcctt 960

gcgctttagg agccccttcg cctcgtgctt gagttgaggc ctggcctggg cgctggggcc 1020

gccgcgtgcg aatctggtgg caccttcgcg cctgtctcgc tgctttcgat aagtctctag 1080

ccatttaaaa tttttgatga cctgctgcga cgcttttttt ctggcaagat agtcttgtaa 1140

atgcgggcca ggatctgcac actggtattt cggtttttgg ggccgcgggc ggcgacgggg 1200

cccgtgcgtc ccagcgcaca tgttcggcga ggcggggcct gcgagcgcgg ccaccgagaa 1260

tcggacgggg gtcggacggg ggtagtctca agctggccgg cctgctctgg tgcctggcct 1320

cgcgccgccg tgtatcgccc cgccctgggc ggcaaggctg gcccggtcgg caccagttgc 1380

gtgagcggaa agatggccgc ttcccggccc tgctccaggg ggctcaaaat ggaggacgcg 1440

gcgctcggga gagcgggcgg gtgagtcacc cacacaaagg aaaggggcct ttccgtcctc 1500

agccgtcgct tcatgtgact ccacggagta ccgggcgccg tccaggcacc tcgattagtt 1560

ctggagcttt tggagtacgt cgtctttagg ttggggggag gggttttatg cgatggagtt 1620

tccccacact gagtgggtgg agactgaagt taggccagct tggcacttga tgtaattctc 1680

cttggaattt gccctttttg agtttggatc ttggttcatt ctcaagcctc agacagtggt 1740

tcaaagtttt tttcttccat ttcaggtgtc gtgaggcact gcgtgcgcca attctgcaga 1800

caaatggcag tattcatcca caattttaaa agaaaagggg ggattggggg gtacagtgca 1860

ggggaaagaa tagtagacat aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa 1920

attacaaaaa ttcaaaattt tcgggtttat tacagggaca gcagagatcc agtttggtta 1980

attaaggtac cgaattcacg cgtggagcta gttattaata gtaatcaatt acggggtcat 2040

tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg 2100

gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa 2160

cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact 2220

tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta 2280

aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt 2340

acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg 2400

ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg 2460

ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc 2520

cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctcgtt 2580

tagtgaaccg tcagatcgcc tggagacgcc atccacgctg ttttgacctc catagaagac 2640

accgggaccg atccagcctc cggtaccgaa aaccccggtc cggctagcgc caccggatcc 2700

ggcggatctg gcatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg 2760

gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc 2820

gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg 2880

ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc 2940

gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag 3000

cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag 3060

ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac 3120

atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac 3180

aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc 3240

gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg 3300

cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc 3360

gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag 3420

ctgtacaagt aaaccggtgg ttatcgataa tcaacctctg gattacaaaa tttgtgaaag 3480

attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat 3540

gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc 3600

ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg 3660

cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct 3720

ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct 3780

tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg 3840

gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac 3900

gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct 3960

gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct 4020

ttgggccgcc tccccgcatc gataccgctc gagctttaag accaatgact tacaaggcag 4080

ctgtagatct tagccacttt ttaaaagaaa aggggggact ggaagggcta attcactccc 4140

aacgaagaca agatctgctt tttgcttgta ctgggtctct ctggttagac cagatctgag 4200

cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt 4260

gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca 4320

gaccctttta gtcagtgtgg aaaatctcta gcagttgaaa gcgaaaggga aaccagagga 4380

gctctctcga cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg 4440

actggtgagt acgccaaaaa ttttgactag cggaggctag aaggagagag atgggtgcga 4500

gagcgtcagt attaagcggg ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc 4560

agggggaaag aaaaaatata aattaaaaca tatagtatgg gcaagcaggg agctagaacg 4620

attcgcagtt aatcctggcc tgttagaaac atcagaaggc tgtagacaaa tactgggaca 4680

gctacaacca tcccttcaga caggatcaga agaacttaga tcattatata atacagtagc 4740

aaccctctat tgtgtgcatc aaaggataga gataaaagac accaaggaag ctttagacaa 4800

gatagaggaa gagcaaaaca aaagtaagac caccgcacag caagcggccg ctgatcttca 4860

gacctggagg aggagatatg agggacaatt ggagaagtga attatataaa tataaagtag 4920

taaaaattga accattagga gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag 4980

aaaaaagagc agtgggaata ggagctttgt tccttgggtt cttgggagca gcaggaagca 5040

ctatgggcgc agcgtcaatg acgctgacgg tacaggccag acaattattg tctggtatag 5100

tgcagcagca gaacaatttg ctgagggcta ttgaggcgca acagcatctg ttgcaactca 5160

cagtctgggg catcaagcag ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg 5220

atcaacagct cctggggatt tggggttgct ctggaaaact catttgcacc actgctgtgc 5280

cttggaatgc tagttggagt aataaatctc tggaacagat ttggaatcac acgacctgga 5340

tggagtggga cagagaaatt aacaattaca caagcttaat acactcctta attgaagaat 5400

cgcaaaacca gcaagaaaag aatgaacaag aattattgga attagataaa tgggcaagtt 5460

tgtggaattg gtttaacata acaaattggc tgtggtatat aaaattattc ataatgatag 5520

taggaggctt ggtaggttta agaatagttt ttgctgtact ttctatagtg aatagagtta 5580

ggcagggata ttcaccatta tcgtttcaga cccacctccc aaccccgagg ggacccgaca 5640

ggcccgaagg aatagaagaa gaaggtggag agagagacag agacagatcc attcgattag 5700

tgaacggatc ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag 5760

ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact 5820

gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt 5880

ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat 5940

gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg 6000

gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc 6060

agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc 6120

tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg 6180

ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca 6240

cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc 6300

tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct 6360

tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa 6420

caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc 6480

caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt 6540

gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt 6600

cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg 6660

cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct 6720

ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca 6780

aaaagctccc gggagcttgt atatccattt tcggatctga tcagcacgtg ttgacaatta 6840

atcatcggca tagtatatcg gcatagtata atacgacaag gtgaggaact aaaccatggc 6900

caagttgacc agtgccgttc cggtgctcac cgcgcgcgac gtcgccggag cggtcgagtt 6960

ctggaccgac cggctcgggt tctcccggga cttcgtggag gacgacttcg ccggtgtggt 7020

ccgggacgac gtgaccctgt tcatcagcgc ggtccaggac caggtggtgc cggacaacac 7080

cctggcctgg gtgtgggtgc gcggcctgga cgagctgtac gccgagtggt cggaggtcgt 7140

gtccacgaac ttccgggacg cctccgggcc ggccatgacc gagatcggcg agcagccgtg 7200

ggggcgggag ttcgccctgc gcgacccggc cggcaactgc gtgcacttcg tggccgagga 7260

gcaggactga cacgtgctac gagatttcga ttccaccgcc gccttctatg aaaggttggg 7320

cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct 7380

ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa 7440

tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc 7500

caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc 7560

gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa 7620

catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac 7680

attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca 7740

ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc 7800

ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc 7860

aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc 7920

aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag 7980

gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc 8040

gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt 8100

tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct 8160

ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg 8220

ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct 8280

tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat 8340

tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg 8400

ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa 8460

aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt 8520

ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc 8580

tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt 8640

atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta 8700

aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat 8760

ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac 8820

tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg 8880

ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag 8940

tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt 9000

aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt 9060

gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt 9120

tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt 9180

cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct 9240

tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt 9300

ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac 9360

cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa 9420

actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa 9480

ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca 9540

aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct 9600

ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga 9660

atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc 9720

tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag tacaatctgc 9780

tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag 9840

tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag 9900

aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag atatacgcgt 9960

t 9961

<210> 3

<211> 10167

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca 60

aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct 120

tatcatgtct ggatcaactg gataactcaa gctaaccaaa atcatcccaa acttcccacc 180

ccatacccta ttaccactgc caattaccta gtggtttcat ttactctaaa cctgtgattc 240

ctctgaatta ttttcatttt aaagaaattg tatttgttaa atatgtacta caaacttagt 300

agtttttaaa gaaattgtat ttgttaaata tgtactacaa acttagtagt tggaagggct 360

aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca cacaaggcta 420

cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac tgacctttgg 480

atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca ataaaggaga 540

gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg agagagaagt 600

gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag agctgcatcc 660

ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg ctggggactt 720

tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat cctgcatata 780

agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga gcctgggagc 840

tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc 900

aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt 960

agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag cgaaagggaa 1020

accagaggag aacacaggta agtgccgtgt gtggttcccg cgggcctggc ctctttacgg 1080

gttatggccc ttgcgtgcct tgaattactt ccacctggct ccagtacgtg attcttgatc 1140

ccgagctgga gccaggggcg ggccttgcgc tttaggagcc ccttcgcctc gtgcttgagt 1200

tgaggcctgg cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg 1260

tctcgctgct ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct 1320

ttttttctgg caagatagtc ttgtaaatgc gggccaggat ctgcacactg gtatttcggt 1380

ttttggggcc gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg 1440

gggcctgcga gcgcggccac cgagaatcgg acgggggtcg gacgggggta gtctcaagct 1500

ggccggcctg ctctggtgcc tggcctcgcg ccgccgtgta tcgccccgcc ctgggcggca 1560

aggctggccc ggtcggcacc agttgcgtga gcggaaagat ggccgcttcc cggccctgct 1620

ccagggggct caaaatggag gacgcggcgc tcgggagagc gggcgggtga gtcacccaca 1680

caaaggaaag gggcctttcc gtcctcagcc gtcgcttcat gtgactccac ggagtaccgg 1740

gcgccgtcca ggcacctcga ttagttctgg agcttttgga gtacgtcgtc tttaggttgg 1800

ggggaggggt tttatgcgat ggagtttccc cacactgagt gggtggagac tgaagttagg 1860

ccagcttggc acttgatgta attctccttg gaatttgccc tttttgagtt tggatcttgg 1920

ttcattctca agcctcagac agtggttcaa agtttttttc ttccatttca ggtgtcgtga 1980

ggcactgcgt gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa 2040

aaggggggat tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca 2100

tacaaactaa agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca 2160

gggacagcag agatccagtt tggttaatta aggtaccgaa ttcacgcgtg gagctagtta 2220

ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac 2280

ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc 2340

aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt 2400

ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac 2460

gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac 2520

cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt 2580

gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc 2640

aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt 2700

tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg 2760

ggaggtctat ataagcagag ctcgtttagt gaaccgtcag atcgcctgga gacgccatcc 2820

acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccggt accgaaaacc 2880

ccggtccggc tagcgccacc ggatccggcg gatctggcat ggtgagcaag ggcgaggagc 2940

tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt 3000

tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca 3060

tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg 3120

gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg 3180

ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca 3240

agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg 3300

gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca 3360

gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga 3420

tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc 3480

ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc 3540

tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg 3600

ccgggatcac tctcggcatg gacgagctgt acaagtaaac cggtggttat cgataatcaa 3660

cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt 3720

acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct 3780

ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc 3840

gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg 3900

ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc 3960

acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc 4020

actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt 4080

gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca 4140

gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt 4200

cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcatcgata ccgctcgagc 4260

tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg 4320

gggactggaa gggctaattc actcccaacg aagacaagat ctgctttttg cttgtactgg 4380

gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact 4440

gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg 4500

tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag 4560

ttgaaagcga aagggaaacc agaggagctc tctcgacgca ggactcggct tgctgaagcg 4620

cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt gactagcgga 4680

ggctagaagg agagagatgg gtgcgagagc gtcagtatta agcgggggag aattagatcg 4740

cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa aatataaatt aaaacatata 4800

gtatgggcaa gcagggagct agaacgattc gcagttaatc ctggcctgtt agaaacatca 4860

gaaggctgta gacaaatact gggacagcta caaccatccc ttcagacagg atcagaagaa 4920

cttagatcat tatataatac agtagcaacc ctctattgtg tgcatcaaag gatagagata 4980

aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaaaag taagaccacc 5040

gcacagcaag cggccgctga tcttcagacc tggaggagga gatatgaggg acaattggag 5100

aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag cacccaccaa 5160

ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag ctttgttcct 5220

tgggttcttg ggagcagcag gaagcactat gggcgcagcg tcaatgacgc tgacggtaca 5280

ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga gggctattga 5340

ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc aggcaagaat 5400

cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg gttgctctgg 5460

aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata aatctctgga 5520

acagatttgg aatcacacga cctggatgga gtgggacaga gaaattaaca attacacaag 5580

cttaatacac tccttaattg aagaatcgca aaaccagcaa gaaaagaatg aacaagaatt 5640

attggaatta gataaatggg caagtttgtg gaattggttt aacataacaa attggctgtg 5700

gtatataaaa ttattcataa tgatagtagg aggcttggta ggtttaagaa tagtttttgc 5760

tgtactttct atagtgaata gagttaggca gggatattca ccattatcgt ttcagaccca 5820

cctcccaacc ccgaggggac ccgacaggcc cgaaggaata gaagaagaag gtggagagag 5880

agacagagac agatccattc gattagtgaa cggatcggcc cgtttaaacc cgctgatcag 5940

cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct 6000

tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc 6060

attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg 6120

aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg 6180

cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa 6240

gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc 6300

ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag 6360

ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca 6420

aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc 6480

gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa 6540

cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct 6600

attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt 6660

gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat 6720

gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag 6780

tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat 6840

cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt 6900

tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg 6960

cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg 7020

atctgatcag cacgtgttga caattaatca tcggcatagt atatcggcat agtataatac 7080

gacaaggtga ggaactaaac catggccaag ttgaccagtg ccgttccggt gctcaccgcg 7140

cgcgacgtcg ccggagcggt cgagttctgg accgaccggc tcgggttctc ccgggacttc 7200

gtggaggacg acttcgccgg tgtggtccgg gacgacgtga ccctgttcat cagcgcggtc 7260

caggaccagg tggtgccgga caacaccctg gcctgggtgt gggtgcgcgg cctggacgag 7320

ctgtacgccg agtggtcgga ggtcgtgtcc acgaacttcc gggacgcctc cgggccggcc 7380

atgaccgaga tcggcgagca gccgtggggg cgggagttcg ccctgcgcga cccggccggc 7440

aactgcgtgc acttcgtggc cgaggagcag gactgacacg tgctacgaga tttcgattcc 7500

accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg 7560

atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca 7620

gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt 7680

tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata 7740

ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat 7800

tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg 7860

ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag 7920

tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt 7980

ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 8040

ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 8100

gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 8160

gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 8220

cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 8280

ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 8340

tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg 8400

gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 8460

tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 8520

ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 8580

ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct 8640

ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 8700

accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 8760

tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca 8820

cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat 8880

taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac 8940

caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt 9000

gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt 9060

gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag 9120

ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct 9180

attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt 9240

gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc 9300

tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt 9360

agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg 9420

gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg 9480

actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct 9540

tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc 9600

attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt 9660

tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt 9720

tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg 9780

aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat 9840

tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg 9900

cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatct cccgatcccc 9960

tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc 10020

ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc 10080

aaggcttgac cgacaattgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc 10140

gcgatgtacg ggccagatat acgcgtt 10167

<210> 4

<211> 10280

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca 60

aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct 120

tatcatgtct ggatcaactg gataactcaa gctaaccaaa atcatcccaa acttcccacc 180

ccatacccta ttaccactgc caattaccta gtggtttcat ttactctaaa cctgtgattc 240

ctctgaatta ttttcatttt aaagaaattg tatttgttaa atatgtacta caaacttagt 300

agtttttaaa gaaattgtat ttgttaaata tgtactacaa acttagtagt tggaagggct 360

gttttacagt gaaagaagac atagaatcct ggatatatac ttagaaaaag aagaagggat 420

aattgcagat tggcagaact atactagtgg gccaggagta agatacccaa tgttctttgg 480

gtggctatgg aagctagtac cagtagatac ctcacaagag ggagaggaca ctgagactga 540

cactgagact cactgcttat tacacccagc acaaacaagc aggcatgatg acatgcatgg 600

ggagacactg gtctggaagt ttgactccat gctggccctt aagtatgagg cctttactcg 660

atacccagaa gaatttgggc acaagtcagg cctaccagaa gatgagtgga aggcgaaact 720

gaaagcaaga gggataccat ttagttaagg gcaggaacaa ccatacatgg ccagggcagg 780

aagtagctac tgaaaacagc tgagactgca gggactttcc agaaggggct gtaccagggg 840

agggatatgg gaggagctgg tggggaacgc cctcatacct tctgtataaa ttcacccgct 900

gcttgcattg tacttcgggt ctctctggtt agaccagatc tgagcctggg agctctctgg 960

ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt 1020

gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt 1080

gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg gaaaccagag 1140

gagaacacag gtaagtgccg tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg 1200

cccttgcgtg ccttgaatta cttccacctg gctccagtac gtgattcttg atcccgagct 1260

ggagccaggg gcgggccttg cgctttagga gccccttcgc ctcgtgcttg agttgaggcc 1320

tggcctgggc gctggggccg ccgcgtgcga atctggtggc accttcgcgc ctgtctcgct 1380

gctttcgata agtctctagc catttaaaat ttttgatgac ctgctgcgac gctttttttc 1440

tggcaagata gtcttgtaaa tgcgggccag gatctgcaca ctggtatttc ggtttttggg 1500

gccgcgggcg gcgacggggc ccgtgcgtcc cagcgcacat gttcggcgag gcggggcctg 1560

cgagcgcggc caccgagaat cggacggggg tcggacgggg gtagtctcaa gctggccggc 1620

ctgctctggt gcctggcctc gcgccgccgt gtatcgcccc gccctgggcg gcaaggctgg 1680

cccggtcggc accagttgcg tgagcggaaa gatggccgct tcccggccct gctccagggg 1740

gctcaaaatg gaggacgcgg cgctcgggag agcgggcggg tgagtcaccc acacaaagga 1800

aaggggcctt tccgtcctca gccgtcgctt catgtgactc cacggagtac cgggcgccgt 1860

ccaggcacct cgattagttc tggagctttt ggagtacgtc gtctttaggt tggggggagg 1920

ggttttatgc gatggagttt ccccacactg agtgggtgga gactgaagtt aggccagctt 1980

ggcacttgat gtaattctcc ttggaatttg ccctttttga gtttggatct tggttcattc 2040

tcaagcctca gacagtggtt caaagttttt ttcttccatt tcaggtgtcg tgaggcactg 2100

cgtgcgccaa ttctgcagac aaatggcagt attcatccac aattttaaaa gaaaaggggg 2160

gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac 2220

taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag 2280

cagagatcca gtttggttaa ttaaggtacc gaattcacgc gtggagctag ttattaatag 2340

taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt 2400

acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg 2460

acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat 2520

ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct 2580

attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg 2640

gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg 2700

ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc 2760

caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa 2820

tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc 2880

tatataagca gagctcgttt agtgaaccgt cagatcgcct ggagacgcca tccacgctgt 2940

tttgacctcc atagaagaca ccgggaccga tccagcctcc ggtaccgaaa accccggtcc 3000

ggctagcgcc accggatccg gcggatctgg catggtgagc aagggcgagg agctgttcac 3060

cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt 3120

gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac 3180

caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca 3240

gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc 3300

cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg 3360

cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga 3420

cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa 3480

cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca 3540

caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg 3600

cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa 3660

agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat 3720

cactctcggc atggacgagc tgtacaagta aaccggtggt tatcgataat caacctctgg 3780

attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat 3840

gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt 3900

tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca 3960

ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg 4020

ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg 4080

aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca 4140

attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca 4200

cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc 4260

ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc 4320

agacgagtcg gatctccctt tgggccgcct ccccgcatcg ataccgctcg agctttaaga 4380

ccaatgactt acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg 4440

gaagggctaa ttcactccca acgaagacaa gatctgcttt ttgcttgtac tgggtctctc 4500

tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag 4560

cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct 4620

ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagttgaaag 4680

cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg 4740

caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga 4800

aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg 4860

aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg 4920

caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct 4980

gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat 5040

cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca 5100

ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc 5160

aagcggccgc tgatcttcag acctggagga ggagatatga gggacaattg gagaagtgaa 5220

ttatataaat ataaagtagt aaaaattgaa ccattaggag tagcacccac caaggcaaag 5280

agaagagtgg tgcagagaga aaaaagagca gtgggaatag gagctttgtt ccttgggttc 5340

ttgggagcag caggaagcac tatgggcgca gcgtcaatga cgctgacggt acaggccaga 5400

caattattgt ctggtatagt gcagcagcag aacaatttgc tgagggctat tgaggcgcaa 5460

cagcatctgt tgcaactcac agtctggggc atcaagcagc tccaggcaag aatcctggct 5520

gtggaaagat acctaaagga tcaacagctc ctggggattt ggggttgctc tggaaaactc 5580

atttgcacca ctgctgtgcc ttggaatgct agttggagta ataaatctct ggaacagatt 5640

tggaatcaca cgacctggat ggagtgggac agagaaatta acaattacac aagcttaata 5700

cactccttaa ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga attattggaa 5760

ttagataaat gggcaagttt gtggaattgg tttaacataa caaattggct gtggtatata 5820

aaattattca taatgatagt aggaggcttg gtaggtttaa gaatagtttt tgctgtactt 5880

tctatagtga atagagttag gcagggatat tcaccattat cgtttcagac ccacctccca 5940

accccgaggg gacccgacag gcccgaagga atagaagaag aaggtggaga gagagacaga 6000

gacagatcca ttcgattagt gaacggatcg gcccgtttaa acccgctgat cagcctcgac 6060

tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct 6120

ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct 6180

gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg 6240

ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag 6300

aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc 6360

gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc 6420

tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa 6480

tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact 6540

tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt 6600

gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa 6660

ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt 6720

aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag 6780

ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc 6840

aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa 6900

agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc 6960

ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat 7020

gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt 7080

ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat 7140

cagcacgtgt tgacaattaa tcatcggcat agtatatcgg catagtataa tacgacaagg 7200

tgaggaacta aaccatggcc aagttgacca gtgccgttcc ggtgctcacc gcgcgcgacg 7260

tcgccggagc ggtcgagttc tggaccgacc ggctcgggtt ctcccgggac ttcgtggagg 7320

acgacttcgc cggtgtggtc cgggacgacg tgaccctgtt catcagcgcg gtccaggacc 7380

aggtggtgcc ggacaacacc ctggcctggg tgtgggtgcg cggcctggac gagctgtacg 7440

ccgagtggtc ggaggtcgtg tccacgaact tccgggacgc ctccgggccg gccatgaccg 7500

agatcggcga gcagccgtgg gggcgggagt tcgccctgcg cgacccggcc ggcaactgcg 7560

tgcacttcgt ggccgaggag caggactgac acgtgctacg agatttcgat tccaccgccg 7620

ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc 7680

agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata 7740

atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc 7800

attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga 7860

cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc 7920

cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct 7980

aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa 8040

acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta 8100

ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc 8160

gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg 8220

caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt 8280

tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa 8340

gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct 8400

ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc 8460

cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg 8520

tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct 8580

tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag 8640

cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga 8700

agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga 8760

agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg 8820

gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag 8880

aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag 8940

ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat 9000

gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct 9060

taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac 9120

tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa 9180

tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg 9240

gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt 9300

gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca 9360

ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt 9420

cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct 9480

tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg 9540

cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg 9600

agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg 9660

cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa 9720

aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt 9780

aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt 9840

gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt 9900

gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca 9960

tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat 10020

ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tctcccgatc ccctatggtg 10080

cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg 10140

tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt 10200

gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt 10260

acgggccaga tatacgcgtt 10280

<210> 5

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ctatgtggat acgctgcttt a 21

<210> 6

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

cataaagaga cagcaaccag g 21

<210> 7

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ctctctcgac gcaggactcg g 21

<210> 8

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

ccttctagcc tccgctagtc a 21

<210> 9

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ctttgttcct tgggttcttg g 21

<210> 10

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

tagccctcag caaattgttc t 21

<210> 11

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tggcaccaaa atcaacggga c 21

<210> 12

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

acacaacaga cgggcacaca c 21

<210> 13

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

gtaggcgtgt acggtgggag g 21

<210> 14

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

aagaggccag gcccgcggga a 21

<210> 15

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

cactcccaaa gaagacaaga t 21

<210> 16

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

gccacgcctc cctggaaagt c 21

<210> 17

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

ggaagggctg ttttacagtg a 21

<210> 18

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

gcagcgggtg aatttataca g 21

Claims (8)

1. A method for producing a lentiviral vector, comprising:

the plasmid based on LTR1.27-GEW is used as a framework, R and U5 regions are added at the 5 'end, and a PBS sequence at the 3' end is deleted at the same time, so that a lentiviral vector Obio-01 is obtained, and is shown as SEQ ID NO. 2.

2. The method according to claim 1, wherein the 5' CMV promoter in the lentiviral vector Obio-01 is replaced with the U3 promoter of human immunodeficiency virus-1 to obtain the lentiviral vector Obio-03.

3. The method according to claim 1, wherein the 5' CMV promoter in the lentiviral vector Obio-01 is replaced with the U3 promoter of human immunodeficiency virus-2 to obtain lentiviral vector Obio-04.

4. A lentiviral vector obtainable by the method of any one of claims 1 to 3.

5. A cell infected with the lentiviral vector of claim 4.

6. A method of producing a viral product, comprising contacting a cell with an effective amount of the lentiviral vector of claim 4 to produce the viral product.

7. The method of claim 6, comprising:

s1, co-transfecting a cell with a vector comprising the lentivirus of claim 4;

s2, harvesting viruses;

and S3, separating the supernatant to obtain a virus product.

8. The method according to claim 7, wherein the step S2 comprises: after 45-50 hours of transfection, the virus was harvested for the first time, the medium was collected and the cells were replaced with fresh complete medium; after 70-75 hours of transfection, the virus was harvested a second time, the medium was collected and the cells discarded.

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