| 出品公司: | Cell Biolabs, Inc. |
|---|---|
| 载体名称: | pCMV-Gag-Pol, pCMV Gag Pol |
| 质粒类型: | 哺乳动物载体；逆病毒包装；双质粒包装系统 |
| 高拷贝/低拷贝: | 高拷贝 |
| 克隆方法: | 限制性内切酶，多克隆位点 |
| 启动子: | CMV |
| 载体大小: | 11 kb |
| 5' 测序引物及序列: | CMV fwd 5’CGCAAATGGGCGGTAGGCGTG 3’ |
| 3' 测序引物及序列: | BGH-R: TAGAAGGCACAGTCGAGG |
| 载体标签: | 无 |
| 载体抗性: | Ampicillin |
| 筛选标记: | 无 |
| 克隆菌株: | DH5α,HB101等 |
| 宿主细胞（系）: | 包装细胞系如293T |
| 备注: |
pCMV-Gag-Pol载体在CMV启动子作用下表达逆病毒蛋白， Gag基因编码了病毒的壳蛋白，pol基因编码了逆病毒的反转录酶和基因整合酶。
逆病毒包装载体pCMV-gag-pol需要与信封载体 pCMV-VSVG 、pCMV-VSV-G 共同使
用，使用方法见下文。
|
| 产品目录号: | RV-111 |
| 稳定性: | 瞬表达 |
| 组成型: | 组成型 |
| 病毒/非病毒: | 非病毒 |
| 载体信息: | pCMV-Gag-Pol载体序列 |
买家导航
pCMV-Gag-Pol载体质粒基本信息
pCMV-Gag-Pol质粒图谱载体图谱和pCMV-Gag-Pol载体序列质粒序列多克隆位点信息
pCMV-Gag-Pol质粒载体简介
双质粒系统逆病毒包装质粒pCMV-Gag-Pol使用方法——逆转录病毒包装与转染方法
Day 1
1. Plate 5 x 105 293T cells in 6 cm2 dishes containing 5 mL of media. (This can be scaled up if
desired)
Day 2
1. Set up (use polypropylene tubes for this; polystyrene
tubes DO NOT work!):
1 µg retroviral DNA encoding gene X
1 µg packaging plasmid Mix
the packaging plasmid (pCMV-Gag-Pol) at a 8:1 ratio with the envelope plasmid (pCVM-VSV-
G)-a total of 1µg
DME without serum to 94μL total
6 μL Fugene
Mix and wait 15 to 30 minutes at room temperature
Add to 293T cells without touching the sides of the dish (DO NOT CHANGE MEDIA)
If you are using amphotropic virus then move immediately to BL2+ in a secondary container,
which has an absorbent material.
(This does not mean a couple of hours; it means Immediately!). The rest of this protocol is
the same for all viruses---the BL2+ safety practices are in place if you are using amphotropic
viruses
Day 3
1. Change the media to whatever media you wish to use when infecting target cells. 293T cells
are easily detached so remember not to put the media directly onto to cells, but rather
“run” it down the side of the dish. Remember that you will get the highest titer virus
when your cells are “happy.”
2. Plate out your target cells
Day 4
1. Remove the medium from the 293T cells and use a 0.45 u syringe filter to remove any 293T
cells. DO NOT use the 0.2 u filter, as it is likely to shear the envelope from your virus
making it noninfectious
Note: After filtering, the filter should be removed and placed in the biohazard bag in the
hood and the syringe rinsed with bleach and decontaminated for a minimum of 20 min. It is useful to
place a plastic beaker with bleach in the hood in advance
2. Add 8 to 10 μg/ml of polybrene (Hexadimethrine bromide) or protamine sulfate to the target
cells
3. Carry out infection for 1 to 4 hours. Remove virus and replace with fresh media
Note: If you wish to do a second infection the following day, it is important to put fresh
media on the cells and not let the virus remain on the cells overnight. The media contains huge
amounts of envelope, both associated and unassociated with viral particles, which will bind all the
cell surface receptors required for virus adsorption, resulting in their down-regulation. Hence, if
you don’t change the media after the initial infection, very few receptors will be available
for the next round of infection. In addition, very few cells tolerate the presence of high levels of
the VSV-G envelope for extended periods of time (i.e. a lot of your cells may die)
Day 5 +
1. Allow the cells to recover and begin to express the virus-encoded genes. The cells usually
require 48 hours for this to occur
2. Add drug if you are scoring for the presence of a vector that carries a drug resistance
marker. Prior to this step it is advisable that you titrate the drug to be used for selection in
order to know precisely how much to add. In addition, it is necessary to bring an extra plate of
uninfected cells (often referred to as “canaries”) which will function as a positive
control in the kill assay. Add drug to both plates. When your canaries are dead, you can remove the
drug.
pCMV-Gag-Pol质粒序列载体序列
LOCUS pCMV-Gag-Pol 10698 bp ds-DNA circular SYN 30-JUN-2022
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 10698)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Thursday, Jun 30, 2022 from SnapGene Viewer 5.1.4.1 to
Vector NTI(R) format
https://www.biofeng.com
COMMENT VNTDATE|989625600|
COMMENT VNTDBDATE|989625600|
COMMENT VNTNAME|pCMV-Gag-Pol
FEATURES Location/Qualifiers
enhancer 130..509
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 510..713
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
promoter 758..776
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 825..6044
/codon_start=1
/label=Gag-pol
CDS 828..1244
/label=gag (truncated)
/note="truncated Moloney murine leukemia virus (MMLV) gag
gene lacking the start codon"
misc_feature 6087..6660
/label=IRES
/note="internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)"
polyA_signal 8086..8310
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
primer_bind complement(8436..8452)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 8460..8476
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
promoter complement(8484..8514)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 8529..8550
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(8838..9426)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(9597..10457)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
promoter complement(10458..10562)
/gene="bla"
/label=AmpR promoter
ORIGIN
1 tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt
61 gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat
121 atacgcgttg acattgatta ttgactagtt attaatagta atcaattacg gggtcattag
181 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
241 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
301 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
361 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
421 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
481 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc
541 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga
601 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
661 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctctggc
721 taactagaga acccactgct tactggctta tcgaaattaa tacgactcac tatagggaga
781 cccaagcttg gtaccgagct cggatcgata tctgcggccg caccatgggc cagactgtta
841 ccactccctt aagtttgacc ttaggtcact ggaaagatgt cgagcggatc gctcacaacc
901 agtcggtaga tgtcaagaag agacgttggg ttaccttctg ctctgcagaa tggccaacct
961 ttaacgtcgg atggccgcga gacggcacct ttaaccgaga cctcatcacc caggttaaga
1021 tcaaggtctt ttcacctggc ccgcatggac acccagacca ggtcccctac atcgtgacct
1081 gggaagcctt ggcttttgac ccccctccct gggtcaagcc ctttgtacac cctaagcctc
1141 cgcctcctct tcctccatcc gccccgtctc tcccccttga acctcctcgt tcgaccccgc
1201 ctcgatcctc cctttatcca gccctcactc cttctctagg cgccaaacct aaacctcaag
1261 ttctttctga cagtgggggg ccgctcatcg acctacttac agaagacccc ccgccttata
1321 gggacccaag accaccccct tccgacaggg acggaaatgg tggagaagcg acccctgcgg
1381 gagaggcacc ggacccctcc ccaatggcat ctcgcctacg tgggagacgg gagccccctg
1441 tggccgactc cactacctcg caggcattcc ccctccgcgc aggaggaaac ggacagcttc
1501 aatactggcc gttctcctct tctgaccttt acaactggaa aaataataac ccttcttttt
1561 ctgaagatcc aggtaaactg acagctctga tcgagtctgt cctcatcacc catcagccca
1621 cctgggacga ctgtcagcag ctgttgggga ctctgctgac cggagaagaa aaacaacggg
1681 tgctcttaga ggctagaaag gcggtgcggg gcgatgatgg gcgccccact caactgccca
1741 atgaagtcga tgccgctttt cccctcgagc gcccagactg ggattacacc acccaggcag
1801 gtaggaacca cctagtccac tatcgccagt tgctcctagc gggtctccaa aacgcgggca
1861 gaagccccac caatttggcc aaggtaaaag gaataacaca agggcccaat gagtctccct
1921 cggccttcct agagagactt aaggaagcct atcgcaggta cactccttat gaccctgagg
1981 acccagggca agaaactaat gtgtctatgt ctttcatttg gcagtctgcc ccagacattg
2041 ggagaaagtt agagaggtta gaagatttaa aaaacaagac gcttggagat ttggttagag
2101 aggcagaaaa gatctttaat aaacgagaaa ccccggaaga aagagaggaa cgtatcagga
2161 gagaaacaga ggaaaaagaa gaacgccgta ggacagagga tgagcagaaa gagaaagaaa
2221 gagatcgtag gagacataga gagatgagca agctattggc cactgtcgtt agtggacaga
2281 aacaggatag acagggagga gaacgaagga ggtcccaact cgatcgcgac cagtgtgcct
2341 actgcaaaga aaaggggcac tgggctaaag attgtcccaa gaaaccacga ggacctcggg
2401 gaccaagacc ccagacctcc ctcctgaccc tagatgacta gggaggtcag ggtcaggagc
2461 ccccccctga acccaggata accctcaaag tcggggggca acccgtcacc ttcctggtag
2521 atactggggc ccaacactcc gtgctgaccc aaaatcctgg acccctaagt gataagtctg
2581 cctgggtcca aggggctact ggaggaaagc ggtatcgctg gaccacggat cgcaaagtac
2641 atctagctac cggtaaggtc acccactctt tcctccatgt accagactgt ccctatcctc
2701 tgttaggaag agatttgctg actaaactaa aagcccaaat ccactttgag ggatcaggag
2761 ctcaggttat gggaccaatg gggcagcccc tgcaagtgtt gaccctaaat atagaagatg
2821 agtatcggct acatgagacc tcaaaagagc cagatgtttc tctagggtcc acatggctgt
2881 ctgattttcc tcaggcctgg gcggaaaccg ggggcatggg actggcagtt cgccaagctc
2941 ctctgatcat acctctgaaa gcaacctcta cccccgtgtc cataaaacaa taccccatgt
3001 cacaagaagc cagactgggg atcaagcccc acatacagag actgttggac cagggaatac
3061 tggtaccctg ccagtccccc tggaacacgc ccctgctacc cgttaagaaa ccagggacta
3121 atgattatag gcctgtccag gatctgagag aagtcaacaa gcgggtggaa gacatccacc
3181 ccaccgtgcc caacccttac aacctcttga gcgggctccc accgtcccac cagtggtaca
3241 ctgtgcttga tttaaaggat gcctttttct gcctgagact ccaccccacc agtcagcctc
3301 tcttcgcctt tgagtggaga gatccagaga tgggaatctc aggacaattg acctggacca
3361 gactcccaca gggtttcaaa aacagtccca ccctgtttga tgaggcactg cacagagacc
3421 tagcagactt ccggatccag cacccagact tgatcctgct acagtacgtg gatgacttac
3481 tgctggccgc cacttctgag ctagactgcc aacaaggtac tcgggccctg ttacaaaccc
3541 tagggaacct cgggtatcgg gcctcggcca agaaagccca aatttgccag aaacaggtca
3601 agtatctggg gtatcttcta aaagagggtc agagatggct gactgaggcc agaaaagaga
3661 ctgtgatggg gcagcctact ccgaagaccc ctcgacaact aagggagttc ctagggacgg
3721 caggcttctg tcgcctctgg atccctgggt ttgcagaaat ggcagccccc ttgtaccctc
3781 tcaccaaaac ggggactctg tttaattggg gcccagacca acaaaaggcc tatcaagaaa
3841 tcaagcaagc tcttctaact gccccagccc tggggttgcc agatttgact aagccctttg
3901 aactctttgt cgacgagaaa cagggctacg ccaaaggcgt cctaacgcaa aaactgggac
3961 cttggcgtcg gccggtggcc tacctgtcta aaaagctaga cccagtggca gctggctggc
4021 ccccttgcct acggatggtg gcagccattg cagttctgac aaaagatgct ggtaagctca
4081 ctatgggaca gccattagtc attctggccc cccatgccgt agaggcacta gttaagcaac
4141 cccctgatcg ctggctctcc aacgcccgga tgacccatta ccaagccctg ctcctggaca
4201 cggaccgggt ccagttcggg ccagtagtgg ccctaaatcc agctacgctg ctccctctgc
4261 ctaaggaggg gctgcaacat gactgtcttg acatcttggc tgaagcccac ggaactagat
4321 cagatcttac ggaccagccc ctcccagacg ccgaccacac ctggtacacg gatgggagca
4381 gcttcctgca agaagggcag cgcaaggccg gagcagcggt gaccaccgag actgaggtaa
4441 tctgggccag ggcattgcca gccgggacat cggcccaaag agctgaactg atagcgctca
4501 cccaagccct aaagatggca gaaggtaaga agctaaatgt ttatactgat agccgttatg
4561 cttttgccac cgctcatatt catggagaaa tatacagaag acgcgggttg ctcacatcag
4621 aaggaaaaga aatcaagaac aagggcgaga tcttagccct actaaaggct ctcttcttgc
4681 ccaaaagact tagcataatt cattgcccgg ggcatcaaaa aggaaacagc gcagaggcca
4741 ggggcaaccg gatggccgac caagcggccc gagaagtagc cactagagaa actccagaca
4801 cttccacact tctgatagaa aattcaaccc cctataccca tgaacacttt cactatacag
4861 taactgatat aaaagatctg accaaactag gagccactta tgacagtgca aagaaatatt
4921 gggtctatca aggaaagcct gttatgcctg atcaattcac ctttgggtta ctagactttc
4981 ttcatcaaat gacccacctc agcttctcaa aaacaaaggc tctcctagag agaagcccca
5041 gtccctacta catgctgaac cgagatcgaa cactcaaaaa tatcactgag acctgcaaag
5101 cttgtgccca agtcaatgcc agcaagtctg ccgtcaagca aggaactagg gtccgcgggc
5161 accggcctgg cacacattgg gagattgatt ttaccgaagt aagacctgga ttgtatggct
5221 ataaatatct tttagttttc gtagatactt tctctggctg ggtagaagct ttcccaacca
5281 agaaagaaac cgccaaggtc gtaaccaaga aactgttaga ggagatcttc cccaggttcg
5341 gcatgcctca ggtgttggga actgacaatg ggcctgcctt cgtctccaag gtgagtcaga
5401 cagtggccga tctgctgggg attgattgga aattacattg tgcgtaccga ccccaaagct
5461 caggtcaggt agaaagaatg aataggacca tcaaggagac tttaactaaa ttaacgcttg
5521 caactggcac tagagactgg gtactcctac ttcccttagc cctctaccga gcccgcaaca
5581 ctccgggccc ccatggactc actccgtatg aaatcctgta tggggcgccc ccgccccttg
5641 ttaatttcca tgatcctgaa atgtcaaagt ttactaatag cccctctctc caagctcact
5701 tacaggccct ccaagcagta caacgagagg tctggaagcc actggccgct gcctatcagg
5761 accagctgga ccagccagtg ataccacacc ccttccgtgt cggcgacacc gtgtgggtac
5821 gccggcacca gactaagaac ttggaacctc gctggaaagg accctacacc gtcctgctga
5881 ccacccccac cgctctcaaa gtagacggca tcgctgcgtg gatccacgcc gctcacgtaa
5941 aggcggcgac aacccctccg gccggaacag catcaggacc gacatggaag gtccagcgtt
6001 ctcaaaaccc cttaaagata agattaaccc gtggaagccc ttaatagatg catctagggc
6061 ggccaattcc gcccctctcc cccccccccc tctccctccc ccccccctaa cgttactggc
6121 cgaagccgct tggaataagg ccggtgtgcg tttgtctata tgttattttc caccatattg
6181 ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg tcttcttgac gagcattcct
6241 aggggtcttt cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt gaaggaagca
6301 gttcctctgg aagcttcttg aagacaaaca acgtctgtag cgaccctttg caggcagcgg
6361 aaccccccac ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata agatacacct
6421 gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga tagttgtgga aagagtcaaa
6481 tggctctcct caagcgtatt caacaagggg ctgaaggatg cccagaaggt accccattgt
6541 atgggatctg atctggggcc tcggtgcaca tgctttacat gtgtttagtc gaggttaaaa
6601 aaacgtctag gccccccgaa ccacggggac gtggttttcc tttgaaaaac acgatgataa
6661 gcttgccaca acccacaagg agacgacctt ccatgaccga gtacaagccc acggtgcgcc
6721 tcgccacccg cgacgacgtc ccccgggccg tacggagctt caataccctg attgactgga
6781 acagctgtag ccctgaacag cagcgtgcgc tgctgacgcg tccggcgatt tccgcctctg
6841 acagtattac ccggacggtc agcgatattc tggataatgt aaaaacgcgc ggtgacgatg
6901 ccctgcgtga atacagcgct aaatttgata aaacagaagt gacagcgcta cgcgtcaccc
6961 ctgaagagat cgccgccgcc ggcgcgcgtc tgagcgacga attaaaacag gcgatgaccg
7021 ctgccgtcaa aaatattgaa acgttccatt ccgcgcagac gctaccgcct gtagatgtgg
7081 aaacccagcc aggcgtgcgt tgccagcagg ttacgcgtcc cgtctcgtct gtcggtctgt
7141 atattcccgg cggctcggct ccgctcttct caacggtgct gatgctggcg acgccggcgc
7201 gcattgcggg atgccagaag gtggttctgt gctcgccgcc gcccatcgct gatgaaatcc
7261 tctatgcggc gcaactgtgt ggcgtgcagg aaatctttaa cgtcggcggc gcgcaggcga
7321 ttgccgctct ggccttcggc agcgagtccg taccgaaagt ggataaaatt tttggccccg
7381 gcaacgcctt tgtaaccgaa gccaaacgtc aggtcagcca gcgtctcgac ggcgcggcta
7441 tcgatatgcc agccgggccg tctgaagtac tggtgatcgc agacagcggc gcaacaccgg
7501 atttcgtcgc ttctgacctg ctctcccagg ctgagcacgg cccggattcc caggtgatcc
7561 tgctgacgcc tgatgctgac attgcccgca aggtggcgga ggcggtagaa cgtcaactgg
7621 cggaactgcc gcgcgcggac accgcccggc aggccctgag cgccagtcgt ctgattgtga
7681 ccaaagattt agcgcagtgc gtcgccatct ctaatcagta tgggccggaa cacttaatca
7741 tccagacgcg caatgcgcgc gatttggtgg atgcgattac cagcgcaggc tcggtatttc
7801 tcggcgactg gtcgccggaa tccgccggtg attacgcttc cggaaccaac catgttttac
7861 cgacctatgg ctatactgct acctgttcca gccttgggtt agcggatttc cagaaacgga
7921 tgaccgttca ggaactgtcg aaagcgggct tttccgctct ggcatcaacc attgaaacat
7981 tggcggcggc agaacgtctg accgcccata aaaatgccgt gaccctgcgc gtaaacgccc
8041 tcaaggagca agcatgataa tctagagctc gctgatcagc ctcgactgtg ccttctagtt
8101 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
8161 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
8221 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca
8281 ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct
8341 cgagtgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata
8401 ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
8461 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
8521 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
8581 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
8641 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
8701 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
8761 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
8821 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
8881 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
8941 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
9001 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
9061 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
9121 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
9181 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
9241 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
9301 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
9361 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
9421 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
9481 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
9541 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
9601 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
9661 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
9721 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
9781 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
9841 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
9901 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
9961 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
10021 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
10081 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
10141 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
10201 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
10261 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
10321 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
10381 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
10441 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
10501 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
10561 cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatct cccgatcccc
10621 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc
10681 ctgcttgtgt gttggagg
//
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