pCMV-Gag-Pol

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相关技术服务:质粒构建    基因合成    质粒大提

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载体基本信息

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:
载体抗性: Ampicillin
筛选标记:
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-VSVGpCMV-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

载体图谱质粒图谱和多克隆位点信息

pCMV-Gag-Pol载体图谱


pCMV-Gag-Pol载体图谱

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.

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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