CN113652450B - Preparation method of lentiviral vector, lentiviral vector obtained by preparation method and application of lentiviral vector - Google Patents
Preparation method of lentiviral vector, lentiviral vector obtained by preparation method and application of lentiviral vector Download PDFInfo
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- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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- A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
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Abstract
The invention belongs to the field of biology, and particularly relates to a preparation method of a lentiviral vector, the lentiviral vector and application. The invention discloses a preparation method of a lentivirus vector, which comprises the following steps: the lentiviral vector Obio-01 was obtained by adding R and U5 regions to the 5' end of LTR 1.27-GEW-based plasmid as a backbone. 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
Technical Field
The invention belongs to the field of biology, and particularly relates to a preparation method of a lentiviral vector, the obtained lentiviral vector and application.
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 study of lentiviral vectors has progressed rapidly and has been very intensive, and the development of lentiviruses is shown in FIG. 1. The vector can effectively integrate the exogenous gene onto the host chromosome, thereby achieving 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 the 3 structural genes gag, pol and env, which are similar to the simple retroviruses. HIV-1 is the most characteristic virus of the lentiviruses, and the first lentivirus vector system was 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 consists of a U3 region, an R region and a U5 region, and in order to reduce the possibility of generation of Replication Competent Virus (RCV) by homologous recombination of the two components, the U3 region of 5'LTR of the packaging component was replaced with Cytomegalovirus (CMV) immediate early promoter or RSV promoter in the third generation vector, and the U3 region of 3' LTR was subjected to deletion mutation, and SV40 polyA site was added. 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 vpu; 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, and allows the vector to be concentrated by high-speed centrifugation, thereby improving 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 10^9IU/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. The schematic diagram of the third generation lentivirus reverse transcription is shown in FIG. 2. In fig. 2: 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". As shown in figure 2, tRNA is used as reverse transcription primer to combine with PBS at 5 'end, the reverse transcription product jumps to 3' end to continue reverse transcription, and the final reverse transcription product is HIV genome sequence with packing signal (psi), RRE, etc. Recently reported fourth generation lentivirus, 5' end R and U5 region deletion, packaging signal (psi) and RRE sequence are put into 3' end relative to third generation viral vector, reverse transcription is directly started from 3' end, the final obtained reverse transcription product has no packaging signal (psi), RRE and other HIV genome sequence, the integrated sequence in genome only contains 4.8% of HIV-1 genome sequence, further enhances safety, but with the problem of serious drop of titer and yield, according to the report, only 35% of third generation lentivirus vector can be reached, and for this reason, fourth generation lentivirus vector has not been practically used. A schematic diagram of the fourth generation lentivirus reverse transcription is shown in FIG. 3. In FIG. 3: RNA genome refers to "RNA genome", minus strand extension refers to "negative strand extension", plus strand stop synthesis refers to "strong positive strand termination synthesis", plus strand transfer refers to "positive strand hopping", and dsDNAsynthesis refers to "DNA synthesis". 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 obtained reverse transcription products do not have HIV genome sequences such as packaging signals (psi) and RRE, so that the safety is further improved. The reported titers of the existing known fourth generation lentiviruses are shown in FIG. 4, and it can be seen from FIG. 4 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 adding R and U5 regions to the 5' end of LTR 1.27-GEW-based plasmid as a backbone.
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 macaque immunodeficiency virus to give the lentiviral vector Obio-02.
In some preferred embodiments of the present invention, the nucleotide sequence of said lentiviral vector Obio-02 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-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:4, 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:5, respectively.
In a second aspect of the invention, lentiviral vectors are disclosed, which are obtainable by the above method.
In a third aspect of the invention, there is disclosed a cell which has been transfected with a lentiviral vector as described above, provided that the cell does not comprise embryonic stem cells, germ cells and fertilized eggs of a human or animal.
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 by using the plasmid composition; s2, harvesting viruses; and S3, separating the supernatant to obtain a virus product.
More preferably, the step S2 includes: harvesting the virus for the first time 45-50 hours after transfection, collecting the culture medium and replacing the cells with fresh complete culture 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 an Opti-MEM culture medium, and putting the cells back to 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-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 cultured with 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 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 great deal of innovation labor, and the lentiviral vector can have high-yield viral titer on the premise of ensuring safety, can be applied to the fields of gene therapy and the like, and has wide application prospects.
Drawings
FIG. 1 is a graph showing the history of the development of lentiviruses of the present invention;
FIG. 2 is a schematic diagram of third generation lentivirus reverse transcription;
FIG. 3 is a schematic diagram of fourth generation lentiviral reverse transcription;
FIG. 4 is a schematic representation of known fourth generation lentivirus titers;
FIG. 5 is a schematic diagram of a lentiviral vector constructed in accordance with the present invention;
FIG. 6 is a schematic diagram of the process of the reverse transcription of Obio-01 in the present invention;
FIG. 7 is a plasmid map based on LTR1.27-GEW in the present invention;
FIG. 8 is a plasmid map of Obio-01 in the present invention;
FIG. 9 is a plasmid map of Obio-02 in the present invention;
FIG. 10 is a plasmid map of Obio-03 of the present invention;
FIG. 11 is a plasmid map of Obio-04 in the present invention;
FIG. 12 is a schematic view of a microscope of cells infected with different viral vectors of the present invention;
FIG. 13 is a graph showing relative titer values of different viral vectors of the present invention;
FIG. 14 is a test of WPRE element integration after infection of cells with different viral vectors in accordance with the present invention;
FIG. 15 is a graph showing the detection of integration of Psi elements following infection of cells with different viral vectors in accordance with the present invention;
FIG. 16 shows the detection of the integration of RRE element after infection of cells with different viral vectors according to the present invention;
FIG. 17 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 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 5. Where the reverse transcription process for Obio-01 is presumed to be shown in FIG. 6, it is presumed that the vector we have modified can reverse transcribe in two ways due to the addition of the R and U5 regions at the 5' end. The plasmid map of LTR1.27-GEW is shown in FIG. 7. The plasmid maps of Obio-01, obio-02, obio-03 and Obio-04 are shown in FIGS. 8-11, respectively. The nucleotide sequences of the Obio-01, obio-02, obio-03 and Obio-04 are respectively shown in SEQ ID NO: 2-5. 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 an Opti-MEM culture medium, wherein the total volume is 500 mu l, gently mixing uniformly, 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 the medium into a 50ml centrifuge tube and replace the cells with fresh complete medium. 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 method for determining the lentivirus titer by adopting the Real time quantitative PCR method comprises the following specific steps:
preparing a sample: by 1X 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; fluorescence was recorded by photographing 72h after infection, and then cells were harvested to extract genomic DNA and subjected to quantitative PCR assay 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 ℃ and increasing by 0.5 ℃ in each step, the absorbance was read for 30 s.
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.
FIG. 12 is a schematic diagram showing a microscope of cells infected with different viral vectors of the present invention, and FIG. 13 is a schematic diagram showing relative titer values of different viral vectors of the present invention. It can be seen that the titers of Obio-01, obio-02, obio-03 and Obio-04 are all significantly higher than those based on LTR1.27-GEW vector, while Obio-02 has reached the same titer level as pCLenti-CMV-EGFP-WPRE (third generation lentiviral vector).
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-02, 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 post-transcriptional regulatory sequence (WPRE), the packaging signal (Psi ), the Rev Response Element (RRE) and the promoters at the U5-terminus of the respective vectors (CMV, macaque immunodeficiency virus (SIV) and human immunodeficiency virus (HIV 1, HIV 2)). The method comprises the following specific steps:
1. infection of cells
Plating 293T cells in 24-well plates; the second day, pCLenti-CMV-EGFP-WPRE (third generation lentivirus vector), lentivirus packaged based on LTR1.27-GEW, obio-01, obio-02, obio-03 and Obio-04, were added to the mixture at 37 ℃ and 5% CO 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 in Table 1 for pCLenti-CMV-EGFP-WPRE (third generation lentiviral vector) based on LTR1.27-GEW, obio-01, obio-02, obio-03, and Obio-04 plasmids and cell genomic DNA. The PCR reaction system (50. Mu.l) was 1. 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-Jose-assist organism, shanghai) 2 O22 mu l; PCR conditions were 95 ℃,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. 14-17; 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 post-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-02, obio-03 and Obio-04 relative to pCLenti-CMV-EGFP-WPRE (third generation lentiviral vector); the packaging signal (psi), rev Response Element (RRE) and U5 promoter (CMV, macaque immunodeficiency virus (SIV) and human immunodeficiency virus (HIV 1, HIV 2) of respective vectors can not be detected, and these elements can not be integrated on cell genome after infection, and the safety is proved to be better based on LTR1.27-GEW, obio-01, obio-02, obio-03 and Obio-04 than pCLenti-CMV-EGFP-WPRE (third generation lentivirus vector). The above embodiment is the preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above embodiment, and any other changes, modifications, substitutions, combinations and simplifications which do not depart from the spirit and principle of the present invention are all equivalent substitutions and are included in the protection scope of the present invention.
Sequence listing
<110> and Yuan Biotechnology (Shanghai) Ltd
Preparation method of <120> lentiviral vector, obtained lentiviral vector and application
<160> 21
<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> 9979
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc 60
caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact 120
ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt 180
gggaggtcta tataagcagc gcgttttgcc tgtactgggt ctctctggtt agaccagatc 240
tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg 300
ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc 360
ctcagaccct tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga 420
aagcgaaagg gaaaccagag gagaacacag gtaagtgccg tgtgtggttc ccgcgggcct 480
ggcctcttta cgggttatgg cccttgcgtg ccttgaatta cttccacctg gctccagtac 540
gtgattcttg atcccgagct ggagccaggg gcgggccttg cgctttagga gccccttcgc 600
ctcgtgcttg agttgaggcc tggcctgggc gctggggccg ccgcgtgcga atctggtggc 660
accttcgcgc ctgtctcgct gctttcgata agtctctagc catttaaaat ttttgatgac 720
ctgctgcgac gctttttttc tggcaagata gtcttgtaaa tgcgggccag gatctgcaca 780
ctggtatttc ggtttttggg gccgcgggcg gcgacggggc ccgtgcgtcc cagcgcacat 840
gttcggcgag gcggggcctg cgagcgcggc caccgagaat cggacggggg tcggacgggg 900
gtagtctcaa gctggccggc ctgctctggt gcctggcctc gcgccgccgt gtatcgcccc 960
gccctgggcg gcaaggctgg cccggtcggc accagttgcg tgagcggaaa gatggccgct 1020
tcccggccct gctccagggg gctcaaaatg gaggacgcgg cgctcgggag agcgggcggg 1080
tgagtcaccc acacaaagga aaggggcctt tccgtcctca gccgtcgctt catgtgactc 1140
cacggagtac cgggcgccgt ccaggcacct cgattagttc tggagctttt ggagtacgtc 1200
gtctttaggt tggggggagg ggttttatgc gatggagttt ccccacactg agtgggtgga 1260
gactgaagtt aggccagctt ggcacttgat gtaattctcc ttggaatttg ccctttttga 1320
gtttggatct tggttcattc tcaagcctca gacagtggtt caaagttttt ttcttccatt 1380
tcaggtgtcg tgaggcactg cgtgcgccaa ttctgcagac aaatggcagt attcatccac 1440
aattttaaaa gaaaaggggg gattgggggg tacagtgcag gggaaagaat agtagacata 1500
atagcaacag acatacaaac taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt 1560
cgggtttatt acagggacag cagagatcca gtttggttaa ttaaggtacc gaattcacgc 1620
gtggagctag ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg 1680
agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc 1740
gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt 1800
gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc 1860
atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg 1920
cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 1980
ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact 2040
cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa 2100
atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta 2160
ggcgtgtacg gtgggaggtc tatataagca gagctcgttt agtgaaccgt cagatcgcct 2220
ggagacgcca tccacgctgt tttgacctcc atagaagaca ccgggaccga tccagcctcc 2280
ggtaccgaaa accccggtcc ggctagcgcc accggatccg gcggatctgg catggtgagc 2340
aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta 2400
aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg 2460
accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc 2520
accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac 2580
ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac 2640
gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc 2700
atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag 2760
tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag 2820
gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac 2880
cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc 2940
acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag 3000
ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagta aaccggtggt 3060
tatcgataat caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta 3120
tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc 3180
ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga 3240
ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac 3300
ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc 3360
cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc 3420
tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg 3480
gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc 3540
ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc 3600
gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcatcg 3660
ataccgctcg agctttaaga ccaatgactt acaaggcagc tgtagatctt agccactttt 3720
taaaagaaaa ggggggactg gaagggctaa ttcactccca acgaagacaa gatctgcttt 3780
ttgcttgtac tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac 3840
tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg 3900
cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga 3960
aaatctctag cagtggcgcc cgaacaggga cttgaaagcg aaagggaaac cagaggagct 4020
ctctcgacgc aggactcggc ttgctgaagc gcgcacggca agaggcgagg ggcggcgact 4080
ggtgagtacg ccaaaaattt tgactagcgg aggctagaag gagagagatg ggtgcgagag 4140
cgtcagtatt aagcggggga gaattagatc gcgatgggaa aaaattcggt taaggccagg 4200
gggaaagaaa aaatataaat taaaacatat agtatgggca agcagggagc tagaacgatt 4260
cgcagttaat cctggcctgt tagaaacatc agaaggctgt agacaaatac tgggacagct 4320
acaaccatcc cttcagacag gatcagaaga acttagatca ttatataata cagtagcaac 4380
cctctattgt gtgcatcaaa ggatagagat aaaagacacc aaggaagctt tagacaagat 4440
agaggaagag caaaacaaaa gtaagaccac cgcacagcaa gcggccgctg atcttcagac 4500
ctggaggagg agatatgagg gacaattgga gaagtgaatt atataaatat aaagtagtaa 4560
aaattgaacc attaggagta gcacccacca aggcaaagag aagagtggtg cagagagaaa 4620
aaagagcagt gggaatagga gctttgttcc ttgggttctt gggagcagca ggaagcacta 4680
tgggcgcagc gtcaatgacg ctgacggtac aggccagaca attattgtct ggtatagtgc 4740
agcagcagaa caatttgctg agggctattg aggcgcaaca gcatctgttg caactcacag 4800
tctggggcat caagcagctc caggcaagaa tcctggctgt ggaaagatac ctaaaggatc 4860
aacagctcct ggggatttgg ggttgctctg gaaaactcat ttgcaccact gctgtgcctt 4920
ggaatgctag ttggagtaat aaatctctgg aacagatttg gaatcacacg acctggatgg 4980
agtgggacag agaaattaac aattacacaa gcttaataca ctccttaatt gaagaatcgc 5040
aaaaccagca agaaaagaat gaacaagaat tattggaatt agataaatgg gcaagtttgt 5100
ggaattggtt taacataaca aattggctgt ggtatataaa attattcata atgatagtag 5160
gaggcttggt aggtttaaga atagtttttg ctgtactttc tatagtgaat agagttaggc 5220
agggatattc accattatcg tttcagaccc acctcccaac cccgagggga cccgacaggc 5280
ccgaaggaat agaagaagaa ggtggagaga gagacagaga cagatccatt cgattagtga 5340
acggatcggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca 5400
tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc 5460
ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg 5520
gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct 5580
ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctctaggggg 5640
tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc 5700
gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt 5760
ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc 5820
cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt 5880
agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt 5940
aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt 6000
gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa 6060
aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag 6120
gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg 6180
gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag 6240
caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc 6300
attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg 6360
cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa 6420
agctcccggg agcttgtata tccattttcg gatctgatca gcacgtgttg acaattaatc 6480
atcggcatag tatatcggca tagtataata cgacaaggtg aggaactaaa ccatggccaa 6540
gttgaccagt gccgttccgg tgctcaccgc gcgcgacgtc gccggagcgg tcgagttctg 6600
gaccgaccgg ctcgggttct cccgggactt cgtggaggac gacttcgccg gtgtggtccg 6660
ggacgacgtg accctgttca tcagcgcggt ccaggaccag gtggtgccgg acaacaccct 6720
ggcctgggtg tgggtgcgcg gcctggacga gctgtacgcc gagtggtcgg aggtcgtgtc 6780
cacgaacttc cgggacgcct ccgggccggc catgaccgag atcggcgagc agccgtgggg 6840
gcgggagttc gccctgcgcg acccggccgg caactgcgtg cacttcgtgg ccgaggagca 6900
ggactgacac gtgctacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt 6960
cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga 7020
gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag 7080
catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa 7140
actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta 7200
atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat 7260
acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt 7320
aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta 7380
atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc 7440
gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa 7500
ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa 7560
aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct 7620
ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac 7680
aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc 7740
gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc 7800
tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg 7860
tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga 7920
gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag 7980
cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta 8040
cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag 8100
agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg 8160
caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac 8220
ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc 8280
aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag 8340
tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc 8400
agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac 8460
gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc 8520
accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg 8580
tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag 8640
tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc 8700
acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac 8760
atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag 8820
aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac 8880
tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg 8940
agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc 9000
gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact 9060
ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg 9120
atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa 9180
tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt 9240
tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg 9300
tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga 9360
cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac aatctgctct 9420
gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag 9480
tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat 9540
ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga 9600
cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca 9660
tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac 9720
gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact 9780
ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa 9840
gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg 9900
cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta 9960
gtcatcgcta ttaccatgg 9979
<210> 3
<211> 10298
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca 60
ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg 120
tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc 180
agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagaac acaggtaagt 240
gccgtgtgtg gttcccgcgg gcctggcctc tttacgggtt atggcccttg cgtgccttga 300
attacttcca cctggctcca gtacgtgatt cttgatcccg agctggagcc aggggcgggc 360
cttgcgcttt aggagcccct tcgcctcgtg cttgagttga ggcctggcct gggcgctggg 420
gccgccgcgt gcgaatctgg tggcaccttc gcgcctgtct cgctgctttc gataagtctc 480
tagccattta aaatttttga tgacctgctg cgacgctttt tttctggcaa gatagtcttg 540
taaatgcggg ccaggatctg cacactggta tttcggtttt tggggccgcg ggcggcgacg 600
gggcccgtgc gtcccagcgc acatgttcgg cgaggcgggg cctgcgagcg cggccaccga 660
gaatcggacg ggggtcggac gggggtagtc tcaagctggc cggcctgctc tggtgcctgg 720
cctcgcgccg ccgtgtatcg ccccgccctg ggcggcaagg ctggcccggt cggcaccagt 780
tgcgtgagcg gaaagatggc cgcttcccgg ccctgctcca gggggctcaa aatggaggac 840
gcggcgctcg ggagagcggg cgggtgagtc acccacacaa aggaaagggg cctttccgtc 900
ctcagccgtc gcttcatgtg actccacgga gtaccgggcg ccgtccaggc acctcgatta 960
gttctggagc ttttggagta cgtcgtcttt aggttggggg gaggggtttt atgcgatgga 1020
gtttccccac actgagtggg tggagactga agttaggcca gcttggcact tgatgtaatt 1080
ctccttggaa tttgcccttt ttgagtttgg atcttggttc attctcaagc ctcagacagt 1140
ggttcaaagt ttttttcttc catttcaggt gtcgtgaggc actgcgtgcg ccaattctgc 1200
agacaaatgg cagtattcat ccacaatttt aaaagaaaag gggggattgg ggggtacagt 1260
gcaggggaaa gaatagtaga cataatagca acagacatac aaactaaaga attacaaaaa 1320
caaattacaa aaattcaaaa ttttcgggtt tattacaggg acagcagaga tccagtttgg 1380
ttaattaagg taccgaattc acgcgtggag ctagttatta atagtaatca attacggggt 1440
cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc 1500
ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag 1560
taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc 1620
acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg 1680
gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc 1740
agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca 1800
atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca 1860
atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg 1920
ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc 1980
gtttagtgaa ccgtcagatc gcctggagac gccatccacg ctgttttgac ctccatagaa 2040
gacaccggga ccgatccagc ctccggtacc gaaaaccccg gtccggctag cgccaccgga 2100
tccggcggat ctggcatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc 2160
ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag 2220
ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc 2280
gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac 2340
cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag 2400
gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc 2460
gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc 2520
aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc 2580
gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc 2640
agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg 2700
ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag 2760
cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac 2820
gagctgtaca agtaaaccgg tggttatcga taatcaacct ctggattaca aaatttgtga 2880
aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt 2940
aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa 3000
atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt 3060
gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct 3120
cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg 3180
ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc 3240
ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg 3300
gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct 3360
gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc 3420
cctttgggcc gcctccccgc atcgataccg ctcgagcttt aagaccaatg acttacaagg 3480
cagctgtaga tcttagccac tttttaaaag aaaagggggg actggaaggg ctaattcact 3540
cccaacgaag acaagatctg ctttttgctt gtactgggtc tctctggtta gaccagatct 3600
gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc 3660
cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc 3720
tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa 3780
agcgaaaggg aaaccagagg agctctctcg acgcaggact cggcttgctg aagcgcgcac 3840
ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta gcggaggcta 3900
gaaggagaga gatgggtgcg agagcgtcag tattaagcgg gggagaatta gatcgcgatg 3960
ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat aaattaaaac atatagtatg 4020
ggcaagcagg gagctagaac gattcgcagt taatcctggc ctgttagaaa catcagaagg 4080
ctgtagacaa atactgggac agctacaacc atcccttcag acaggatcag aagaacttag 4140
atcattatat aatacagtag caaccctcta ttgtgtgcat caaaggatag agataaaaga 4200
caccaaggaa gctttagaca agatagagga agagcaaaac aaaagtaaga ccaccgcaca 4260
gcaagcggcc gctgatcttc agacctggag gaggagatat gagggacaat tggagaagtg 4320
aattatataa atataaagta gtaaaaattg aaccattagg agtagcaccc accaaggcaa 4380
agagaagagt ggtgcagaga gaaaaaagag cagtgggaat aggagctttg ttccttgggt 4440
tcttgggagc agcaggaagc actatgggcg cagcgtcaat gacgctgacg gtacaggcca 4500
gacaattatt gtctggtata gtgcagcagc agaacaattt gctgagggct attgaggcgc 4560
aacagcatct gttgcaactc acagtctggg gcatcaagca gctccaggca agaatcctgg 4620
ctgtggaaag atacctaaag gatcaacagc tcctggggat ttggggttgc tctggaaaac 4680
tcatttgcac cactgctgtg ccttggaatg ctagttggag taataaatct ctggaacaga 4740
tttggaatca cacgacctgg atggagtggg acagagaaat taacaattac acaagcttaa 4800
tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg 4860
aattagataa atgggcaagt ttgtggaatt ggtttaacat aacaaattgg ctgtggtata 4920
taaaattatt cataatgata gtaggaggct tggtaggttt aagaatagtt tttgctgtac 4980
tttctatagt gaatagagtt aggcagggat attcaccatt atcgtttcag acccacctcc 5040
caaccccgag gggacccgac aggcccgaag gaatagaaga agaaggtgga gagagagaca 5100
gagacagatc cattcgatta gtgaacggat cggcccgttt aaacccgctg atcagcctcg 5160
actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc 5220
ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt 5280
ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat 5340
tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa 5400
agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg 5460
gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct 5520
cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta 5580
aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa 5640
cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct 5700
ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc 5760
aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg 5820
ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc 5880
agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc 5940
tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc 6000
aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc 6060
ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt 6120
atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt 6180
ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg 6240
atcagcacgt gttgacaatt aatcatcggc atagtatatc ggcatagtat aatacgacaa 6300
ggtgaggaac taaaccatgg ccaagttgac cagtgccgtt ccggtgctca ccgcgcgcga 6360
cgtcgccgga gcggtcgagt tctggaccga ccggctcggg ttctcccggg acttcgtgga 6420
ggacgacttc gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg cggtccagga 6480
ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg acgagctgta 6540
cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac gcctccgggc cggccatgac 6600
cgagatcggc gagcagccgt gggggcggga gttcgccctg cgcgacccgg ccggcaactg 6660
cgtgcacttc gtggccgagg agcaggactg acacgtgcta cgagatttcg attccaccgc 6720
cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct 6780
ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta 6840
taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact 6900
gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc 6960
gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta 7020
tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc 7080
ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg 7140
aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg 7200
tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg 7260
gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa 7320
cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc 7380
gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc 7440
aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag 7500
ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct 7560
cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta 7620
ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc 7680
cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc 7740
agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt 7800
gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct 7860
gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc 7920
tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca 7980
agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta 8040
agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa 8100
atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg 8160
cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg 8220
actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc 8280
aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc 8340
cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa 8400
ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc 8460
cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg 8520
ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc 8580
cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat 8640
ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg 8700
tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc 8760
ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg 8820
aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat 8880
gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg 8940
gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg 9000
ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct 9060
catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac 9120
atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg 9180
tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct 9240
tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc 9300
ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat 9360
gtacgggcca gatatacgcg ttaacttgtt tattgcagct tataatggtt acaaataaag 9420
caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt 9480
gtccaaactc atcaatgtat cttatcatgt ctggatcaac tggataactc aagctaacca 9540
aaatcatccc aaacttccca ccccataccc tattaccact gccaattacc tagtggtttc 9600
atttactcta aacctgtgat tcctctgaat tattttcatt ttaaagaaat tgtatttgtt 9660
aaatatgtac tacaaactta gtagttttta aagaaattgt atttgttaaa tatgtactac 9720
aaacttagta gttggaaggg ctgttttaca gtgaaagaag acatagaatc ctggatatat 9780
acttagaaaa agaagaaggg ataattgcag attggcagaa ctatactagt gggccaggag 9840
taagataccc aatgttcttt gggtggctat ggaagctagt accagtagat acctcacaag 9900
agggagagga cactgagact gacactgaga ctcactgctt attacaccca gcacaaacaa 9960
gcaggcatga tgacatgcat ggggagacac tggtctggaa gtttgactcc atgctggccc 10020
ttaagtatga ggcctttact cgatacccag aagaatttgg gcacaagtca ggcctaccag 10080
aagatgagtg gaaggcgaaa ctgaaagcaa gagggatacc atttagttaa gggcaggaac 10140
aaccatacat ggccagggca ggaagtagct actgaaaaca gctgagactg cagggacttt 10200
ccagaagggg ctgtaccagg ggagggatat gggaggagct ggtggggaac gccctcatac 10260
cttctgtata aattcacccg ctgcttgcat tgtacttc 10298
<210> 4
<211> 10185
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca 60
ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg 120
tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc 180
agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagaac acaggtaagt 240
gccgtgtgtg gttcccgcgg gcctggcctc tttacgggtt atggcccttg cgtgccttga 300
attacttcca cctggctcca gtacgtgatt cttgatcccg agctggagcc aggggcgggc 360
cttgcgcttt aggagcccct tcgcctcgtg cttgagttga ggcctggcct gggcgctggg 420
gccgccgcgt gcgaatctgg tggcaccttc gcgcctgtct cgctgctttc gataagtctc 480
tagccattta aaatttttga tgacctgctg cgacgctttt tttctggcaa gatagtcttg 540
taaatgcggg ccaggatctg cacactggta tttcggtttt tggggccgcg ggcggcgacg 600
gggcccgtgc gtcccagcgc acatgttcgg cgaggcgggg cctgcgagcg cggccaccga 660
gaatcggacg ggggtcggac gggggtagtc tcaagctggc cggcctgctc tggtgcctgg 720
cctcgcgccg ccgtgtatcg ccccgccctg ggcggcaagg ctggcccggt cggcaccagt 780
tgcgtgagcg gaaagatggc cgcttcccgg ccctgctcca gggggctcaa aatggaggac 840
gcggcgctcg ggagagcggg cgggtgagtc acccacacaa aggaaagggg cctttccgtc 900
ctcagccgtc gcttcatgtg actccacgga gtaccgggcg ccgtccaggc acctcgatta 960
gttctggagc ttttggagta cgtcgtcttt aggttggggg gaggggtttt atgcgatgga 1020
gtttccccac actgagtggg tggagactga agttaggcca gcttggcact tgatgtaatt 1080
ctccttggaa tttgcccttt ttgagtttgg atcttggttc attctcaagc ctcagacagt 1140
ggttcaaagt ttttttcttc catttcaggt gtcgtgaggc actgcgtgcg ccaattctgc 1200
agacaaatgg cagtattcat ccacaatttt aaaagaaaag gggggattgg ggggtacagt 1260
gcaggggaaa gaatagtaga cataatagca acagacatac aaactaaaga attacaaaaa 1320
caaattacaa aaattcaaaa ttttcgggtt tattacaggg acagcagaga tccagtttgg 1380
ttaattaagg taccgaattc acgcgtggag ctagttatta atagtaatca attacggggt 1440
cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc 1500
ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag 1560
taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc 1620
acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg 1680
gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc 1740
agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca 1800
atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca 1860
atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg 1920
ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc 1980
gtttagtgaa ccgtcagatc gcctggagac gccatccacg ctgttttgac ctccatagaa 2040
gacaccggga ccgatccagc ctccggtacc gaaaaccccg gtccggctag cgccaccgga 2100
tccggcggat ctggcatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc 2160
ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag 2220
ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc 2280
gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac 2340
cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag 2400
gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc 2460
gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc 2520
aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc 2580
gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc 2640
agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg 2700
ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag 2760
cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac 2820
gagctgtaca agtaaaccgg tggttatcga taatcaacct ctggattaca aaatttgtga 2880
aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt 2940
aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa 3000
atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt 3060
gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct 3120
cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg 3180
ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc 3240
ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg 3300
gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct 3360
gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc 3420
cctttgggcc gcctccccgc atcgataccg ctcgagcttt aagaccaatg acttacaagg 3480
cagctgtaga tcttagccac tttttaaaag aaaagggggg actggaaggg ctaattcact 3540
cccaacgaag acaagatctg ctttttgctt gtactgggtc tctctggtta gaccagatct 3600
gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc 3660
cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc 3720
tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa 3780
agcgaaaggg aaaccagagg agctctctcg acgcaggact cggcttgctg aagcgcgcac 3840
ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta gcggaggcta 3900
gaaggagaga gatgggtgcg agagcgtcag tattaagcgg gggagaatta gatcgcgatg 3960
ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat aaattaaaac atatagtatg 4020
ggcaagcagg gagctagaac gattcgcagt taatcctggc ctgttagaaa catcagaagg 4080
ctgtagacaa atactgggac agctacaacc atcccttcag acaggatcag aagaacttag 4140
atcattatat aatacagtag caaccctcta ttgtgtgcat caaaggatag agataaaaga 4200
caccaaggaa gctttagaca agatagagga agagcaaaac aaaagtaaga ccaccgcaca 4260
gcaagcggcc gctgatcttc agacctggag gaggagatat gagggacaat tggagaagtg 4320
aattatataa atataaagta gtaaaaattg aaccattagg agtagcaccc accaaggcaa 4380
agagaagagt ggtgcagaga gaaaaaagag cagtgggaat aggagctttg ttccttgggt 4440
tcttgggagc agcaggaagc actatgggcg cagcgtcaat gacgctgacg gtacaggcca 4500
gacaattatt gtctggtata gtgcagcagc agaacaattt gctgagggct attgaggcgc 4560
aacagcatct gttgcaactc acagtctggg gcatcaagca gctccaggca agaatcctgg 4620
ctgtggaaag atacctaaag gatcaacagc tcctggggat ttggggttgc tctggaaaac 4680
tcatttgcac cactgctgtg ccttggaatg ctagttggag taataaatct ctggaacaga 4740
tttggaatca cacgacctgg atggagtggg acagagaaat taacaattac acaagcttaa 4800
tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg 4860
aattagataa atgggcaagt ttgtggaatt ggtttaacat aacaaattgg ctgtggtata 4920
taaaattatt cataatgata gtaggaggct tggtaggttt aagaatagtt tttgctgtac 4980
tttctatagt gaatagagtt aggcagggat attcaccatt atcgtttcag acccacctcc 5040
caaccccgag gggacccgac aggcccgaag gaatagaaga agaaggtgga gagagagaca 5100
gagacagatc cattcgatta gtgaacggat cggcccgttt aaacccgctg atcagcctcg 5160
actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc 5220
ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt 5280
ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat 5340
tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa 5400
agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg 5460
gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct 5520
cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta 5580
aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa 5640
cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct 5700
ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc 5760
aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg 5820
ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc 5880
agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc 5940
tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc 6000
aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc 6060
ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt 6120
atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt 6180
ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg 6240
atcagcacgt gttgacaatt aatcatcggc atagtatatc ggcatagtat aatacgacaa 6300
ggtgaggaac taaaccatgg ccaagttgac cagtgccgtt ccggtgctca ccgcgcgcga 6360
cgtcgccgga gcggtcgagt tctggaccga ccggctcggg ttctcccggg acttcgtgga 6420
ggacgacttc gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg cggtccagga 6480
ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg acgagctgta 6540
cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac gcctccgggc cggccatgac 6600
cgagatcggc gagcagccgt gggggcggga gttcgccctg cgcgacccgg ccggcaactg 6660
cgtgcacttc gtggccgagg agcaggactg acacgtgcta cgagatttcg attccaccgc 6720
cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct 6780
ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta 6840
taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact 6900
gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc 6960
gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta 7020
tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc 7080
ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg 7140
aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg 7200
tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg 7260
gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa 7320
cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc 7380
gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc 7440
aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag 7500
ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct 7560
cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta 7620
ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc 7680
cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc 7740
agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt 7800
gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct 7860
gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc 7920
tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca 7980
agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta 8040
agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa 8100
atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg 8160
cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg 8220
actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc 8280
aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc 8340
cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa 8400
ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc 8460
cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg 8520
ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc 8580
cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat 8640
ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg 8700
tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc 8760
ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg 8820
aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat 8880
gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg 8940
gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg 9000
ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct 9060
catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac 9120
atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg 9180
tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct 9240
tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc 9300
ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat 9360
gtacgggcca gatatacgcg ttaacttgtt tattgcagct tataatggtt acaaataaag 9420
caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt 9480
gtccaaactc atcaatgtat cttatcatgt ctggatcaac tggataactc aagctaacca 9540
aaatcatccc aaacttccca ccccataccc tattaccact gccaattacc tagtggtttc 9600
atttactcta aacctgtgat tcctctgaat tattttcatt ttaaagaaat tgtatttgtt 9660
aaatatgtac tacaaactta gtagttttta aagaaattgt atttgttaaa tatgtactac 9720
aaacttagta gttggaaggg ctaattcact cccaaagaag acaagatatc cttgatctgt 9780
ggatctacca cacacaaggc tacttccctg attagcagaa ctacacacca gggccagggg 9840
tcagatatcc actgaccttt ggatggtgct acaagctagt accagttgag ccagataagg 9900
tagaagaggc caataaagga gagaacacca gcttgttaca ccctgtgagc ctgcatggga 9960
tggatgaccc ggagagagaa gtgttagagt ggaggtttga cagccgccta gcatttcatc 10020
acgtggcccg agagctgcat ccggagtact tcaagaactg ctgatatcga gcttgctaca 10080
agggactttc cgctggggac tttccaggga ggcgtggcct gggcgggact ggggagtggc 10140
gagccctcag atcctgcata taagcagctg ctttttgcct gtact 10185
<210> 5
<211> 10298
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca 60
ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg 120
tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc 180
agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagaac acaggtaagt 240
gccgtgtgtg gttcccgcgg gcctggcctc tttacgggtt atggcccttg cgtgccttga 300
attacttcca cctggctcca gtacgtgatt cttgatcccg agctggagcc aggggcgggc 360
cttgcgcttt aggagcccct tcgcctcgtg cttgagttga ggcctggcct gggcgctggg 420
gccgccgcgt gcgaatctgg tggcaccttc gcgcctgtct cgctgctttc gataagtctc 480
tagccattta aaatttttga tgacctgctg cgacgctttt tttctggcaa gatagtcttg 540
taaatgcggg ccaggatctg cacactggta tttcggtttt tggggccgcg ggcggcgacg 600
gggcccgtgc gtcccagcgc acatgttcgg cgaggcgggg cctgcgagcg cggccaccga 660
gaatcggacg ggggtcggac gggggtagtc tcaagctggc cggcctgctc tggtgcctgg 720
cctcgcgccg ccgtgtatcg ccccgccctg ggcggcaagg ctggcccggt cggcaccagt 780
tgcgtgagcg gaaagatggc cgcttcccgg ccctgctcca gggggctcaa aatggaggac 840
gcggcgctcg ggagagcggg cgggtgagtc acccacacaa aggaaagggg cctttccgtc 900
ctcagccgtc gcttcatgtg actccacgga gtaccgggcg ccgtccaggc acctcgatta 960
gttctggagc ttttggagta cgtcgtcttt aggttggggg gaggggtttt atgcgatgga 1020
gtttccccac actgagtggg tggagactga agttaggcca gcttggcact tgatgtaatt 1080
ctccttggaa tttgcccttt ttgagtttgg atcttggttc attctcaagc ctcagacagt 1140
ggttcaaagt ttttttcttc catttcaggt gtcgtgaggc actgcgtgcg ccaattctgc 1200
agacaaatgg cagtattcat ccacaatttt aaaagaaaag gggggattgg ggggtacagt 1260
gcaggggaaa gaatagtaga cataatagca acagacatac aaactaaaga attacaaaaa 1320
caaattacaa aaattcaaaa ttttcgggtt tattacaggg acagcagaga tccagtttgg 1380
ttaattaagg taccgaattc acgcgtggag ctagttatta atagtaatca attacggggt 1440
cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc 1500
ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag 1560
taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc 1620
acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg 1680
gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc 1740
agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca 1800
atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca 1860
atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg 1920
ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc 1980
gtttagtgaa ccgtcagatc gcctggagac gccatccacg ctgttttgac ctccatagaa 2040
gacaccggga ccgatccagc ctccggtacc gaaaaccccg gtccggctag cgccaccgga 2100
tccggcggat ctggcatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc 2160
ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag 2220
ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc 2280
gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac 2340
cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag 2400
gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc 2460
gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc 2520
aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc 2580
gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc 2640
agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg 2700
ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag 2760
cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac 2820
gagctgtaca agtaaaccgg tggttatcga taatcaacct ctggattaca aaatttgtga 2880
aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt 2940
aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa 3000
atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt 3060
gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct 3120
cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg 3180
ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc 3240
ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg 3300
gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct 3360
gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc 3420
cctttgggcc gcctccccgc atcgataccg ctcgagcttt aagaccaatg acttacaagg 3480
cagctgtaga tcttagccac tttttaaaag aaaagggggg actggaaggg ctaattcact 3540
cccaacgaag acaagatctg ctttttgctt gtactgggtc tctctggtta gaccagatct 3600
gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc 3660
cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc 3720
tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa 3780
agcgaaaggg aaaccagagg agctctctcg acgcaggact cggcttgctg aagcgcgcac 3840
ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta gcggaggcta 3900
gaaggagaga gatgggtgcg agagcgtcag tattaagcgg gggagaatta gatcgcgatg 3960
ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat aaattaaaac atatagtatg 4020
ggcaagcagg gagctagaac gattcgcagt taatcctggc ctgttagaaa catcagaagg 4080
ctgtagacaa atactgggac agctacaacc atcccttcag acaggatcag aagaacttag 4140
atcattatat aatacagtag caaccctcta ttgtgtgcat caaaggatag agataaaaga 4200
caccaaggaa gctttagaca agatagagga agagcaaaac aaaagtaaga ccaccgcaca 4260
gcaagcggcc gctgatcttc agacctggag gaggagatat gagggacaat tggagaagtg 4320
aattatataa atataaagta gtaaaaattg aaccattagg agtagcaccc accaaggcaa 4380
agagaagagt ggtgcagaga gaaaaaagag cagtgggaat aggagctttg ttccttgggt 4440
tcttgggagc agcaggaagc actatgggcg cagcgtcaat gacgctgacg gtacaggcca 4500
gacaattatt gtctggtata gtgcagcagc agaacaattt gctgagggct attgaggcgc 4560
aacagcatct gttgcaactc acagtctggg gcatcaagca gctccaggca agaatcctgg 4620
ctgtggaaag atacctaaag gatcaacagc tcctggggat ttggggttgc tctggaaaac 4680
tcatttgcac cactgctgtg ccttggaatg ctagttggag taataaatct ctggaacaga 4740
tttggaatca cacgacctgg atggagtggg acagagaaat taacaattac acaagcttaa 4800
tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg 4860
aattagataa atgggcaagt ttgtggaatt ggtttaacat aacaaattgg ctgtggtata 4920
taaaattatt cataatgata gtaggaggct tggtaggttt aagaatagtt tttgctgtac 4980
tttctatagt gaatagagtt aggcagggat attcaccatt atcgtttcag acccacctcc 5040
caaccccgag gggacccgac aggcccgaag gaatagaaga agaaggtgga gagagagaca 5100
gagacagatc cattcgatta gtgaacggat cggcccgttt aaacccgctg atcagcctcg 5160
actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc 5220
ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt 5280
ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat 5340
tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa 5400
agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg 5460
gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct 5520
cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta 5580
aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa 5640
cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct 5700
ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc 5760
aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg 5820
ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc 5880
agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc 5940
tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc 6000
aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc 6060
ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt 6120
atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt 6180
ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg 6240
atcagcacgt gttgacaatt aatcatcggc atagtatatc ggcatagtat aatacgacaa 6300
ggtgaggaac taaaccatgg ccaagttgac cagtgccgtt ccggtgctca ccgcgcgcga 6360
cgtcgccgga gcggtcgagt tctggaccga ccggctcggg ttctcccggg acttcgtgga 6420
ggacgacttc gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg cggtccagga 6480
ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg acgagctgta 6540
cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac gcctccgggc cggccatgac 6600
cgagatcggc gagcagccgt gggggcggga gttcgccctg cgcgacccgg ccggcaactg 6660
cgtgcacttc gtggccgagg agcaggactg acacgtgcta cgagatttcg attccaccgc 6720
cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct 6780
ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta 6840
taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact 6900
gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc 6960
gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta 7020
tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc 7080
ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg 7140
aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg 7200
tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg 7260
gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa 7320
cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc 7380
gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc 7440
aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag 7500
ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct 7560
cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta 7620
ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc 7680
cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc 7740
agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt 7800
gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct 7860
gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc 7920
tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca 7980
agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta 8040
agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa 8100
atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg 8160
cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg 8220
actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc 8280
aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc 8340
cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa 8400
ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc 8460
cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg 8520
ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc 8580
cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat 8640
ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg 8700
tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc 8760
ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg 8820
aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat 8880
gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg 8940
gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg 9000
ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct 9060
catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac 9120
atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg 9180
tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct 9240
tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc 9300
ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat 9360
gtacgggcca gatatacgcg ttaacttgtt tattgcagct tataatggtt acaaataaag 9420
caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt 9480
gtccaaactc atcaatgtat cttatcatgt ctggatcaac tggataactc aagctaacca 9540
aaatcatccc aaacttccca ccccataccc tattaccact gccaattacc tagtggtttc 9600
atttactcta aacctgtgat tcctctgaat tattttcatt ttaaagaaat tgtatttgtt 9660
aaatatgtac tacaaactta gtagttttta aagaaattgt atttgttaaa tatgtactac 9720
aaacttagta gttggaaggg ctgttttaca gtgaaagaag acatagaatc ctggatatat 9780
acttagaaaa agaagaaggg ataattgcag attggcagaa ctatactagt gggccaggag 9840
taagataccc aatgttcttt gggtggctat ggaagctagt accagtagat acctcacaag 9900
agggagagga cactgagact gacactgaga ctcactgctt attacaccca gcacaaacaa 9960
gcaggcatga tgacatgcat ggggagacac tggtctggaa gtttgactcc atgctggccc 10020
ttaagtatga ggcctttact cgatacccag aagaatttgg gcacaagtca ggcctaccag 10080
aagatgagtg gaaggcgaaa ctgaaagcaa gagggatacc atttagttaa gggcaggaac 10140
aaccatacat ggccagggca ggaagtagct actgaaaaca gctgagactg cagggacttt 10200
ccagaagggg ctgtaccagg ggagggatat gggaggagct ggtggggaac gccctcatac 10260
cttctgtata aattcacccg ctgcttgcat tgtacttc 10298
<210> 6
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
ctatgtggat acgctgcttt a 21
<210> 7
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
cataaagaga cagcaaccag g 21
<210> 8
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
ctctctcgac gcaggactcg g 21
<210> 9
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
ccttctagcc tccgctagtc a 21
<210> 10
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
ctttgttcct tgggttcttg g 21
<210> 11
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
tagccctcag caaattgttc t 21
<210> 12
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
tggcaccaaa atcaacggga c 21
<210> 13
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
acacaacaga cgggcacaca c 21
<210> 14
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
gtaggcgtgt acggtgggag g 21
<210> 15
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
aagaggccag gcccgcggga a 21
<210> 16
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
attacagtgc aagaagacat a 21
<210> 17
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
gtaacatccc cttgtggaaa g 21
<210> 18
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
cactcccaaa gaagacaaga t 21
<210> 19
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
gccacgcctc cctggaaagt c 21
<210> 20
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
ggaagggctg ttttacagtg a 21
<210> 21
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
gcagcgggtg aatttataca g 21
Claims (8)
1. A method for producing a lentiviral vector, comprising:
taking LTR 1.27-GEW-based plasmid as a skeleton, and adding R and U5 regions at the 5' end to obtain a lentiviral vector Obio-01; the nucleotide sequence of the lentiviral vector Obio-01 is shown as SEQ ID NO:2, respectively.
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 plasmid obtainable by the method of any one of claims 1 to 3.
5. A cell that has been transfected with the lentiviral vector plasmid of claim 4, with the proviso that the cell does not comprise a human or animal embryonic stem cell, germ cell, or fertilized egg.
6. A method of producing a viral product, comprising contacting a cell with an effective amount of the lentiviral vector plasmid of claim 4 to produce the viral product.
7. The method of claim 6, comprising:
s1, co-transfecting a cell with a composition comprising the lentiviral vector plasmid of claim 4; s2, harvesting viruses; and S3, separating the supernatant to obtain a virus product.
8. The method of claim 7, wherein step S2 comprises: harvesting the virus for the first time 45-50 hours after transfection, collecting the culture medium and replacing the cells with fresh complete culture 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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