pUMVC

pUMVC

pUMVC逆病毒载体基本信息：

pUMVC载体质粒图谱和多克隆位点信息：

pUMVC载体简介：

双质粒系统逆病毒包装质粒pUMVC使用方法

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 (pUMVC3) 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.

pUMVC载体序列：

ORIGIN

    1 CACTCAAAGG CGGTAATACG GTTATCCACA GAATCAGGGG ATAACGCAGG AAAGAACATG

   61 TGAGCAAAAG GCCAGCAAAA GGCCAGGAAC CGTAAAAAGG CCGCGTTGCT GGCGTTTTTC

  121 CATAGGCTCC GCCCCCCTGA CGAGCATCAC AAAAATCGAC GCTCAAGTCA GAGGTGGCGA

  181 AACCCGACAG GACTATAAAG ATACCAGGCG TTTCCCCCTG GAAGCTCCCT CGTGCGCTCT

  241 CCTGTTCCGA CCCTGCCGCT TACCGGATAC CTGTCCGCCT TTCTCCCTTC GGGAAGCGTG

  301 GCGCTTTCTC ATAGCTCACG CTGTAGGTAT CTCAGTTCGG TGTAGGTCGT TCGCTCCAAG

  361 CTGGGCTGTG TGCACGAACC CCCCGTTCAG CCCGACCGCT GCGCCTTATC CGGTAACTAT

  421 CGTCTTGAGT CCAACCCGGT AAGACACGAC TTATCGCCAC TGGCAGCAGC CACTGGTAAC

  481 AGGATTAGCA GAGCGAGGTA TGTAGGCGGT GCTACAGAGT TCTTGAAGTG GTGGCCTAAC

  541 TACGGCTACA CTAGAAGAAC AGTATTTGGT ATCTGCGCTC TGCTGAAGCC AGTTACCTTC

  601 GGAAAAAGAG TTGGTAGCTC TTGATCCGGC AAACAAACCA CCGCTGGTAG CGGTGGTTTT

  661 TTTGTTTGCA AGCAGCAGAT TACGCGCAGA AAAAAAGGAT CTCAAGAAGA TCCTTTGATC

  721 TTTTCTACGG GGTCTGACGC TCAGTGGAAC GAAAACTCAC GTTAAGGGAT TTTGGTCATG

  781 AGATTATCAA AAAGGATCTT CACCTAGATC CTTTTAAATT AAAAATGAAG TTTTAAATCA

  841 ATCTAAAGTA TATATGAGTA AACTTGGTCT GACAGTTACC AATGCTTAAT CAGTGAGGCA

  901 CCTATCTCAG CGATCTGTCT ATTTCGTTCA TCCATAGTTG CCTGACTCGG GGGGGGGGGC

  961 GCTGAGGTCT GCCTCGTGAA GAAGGTGTTG CTGACTCATA CCAGGCCTGA ATCGCCCCAT

 1021 CATCCAGCCA GAAAGTGAGG GAGCCACGGT TGATGAGAGC TTTGTTGTAG GTGGACCAGT

 1081 TGGTGATTTT GAACTTTTGC TTTGCCACGG AACGGTCTGC GTTGTCGGGA AGATGCGTGA

 1141 TCTGATCCTT CAACTCAGCA AAAGTTCGAT TTATTCAACA AAGCCGCCGT CCCGTCAAGT

 1201 CAGCGTAATG CTCTGCCAGT GTTACAACCA ATTAACCAAT TCTGATTAGA AAAACTCATC

 1261 GAGCATCAAA TGAAACTGCA ATTTATTCAT ATCAGGATTA TCAATACCAT ATTTTTGAAA

 1321 AAGCCGTTTC TGTAATGAAG GAGAAAACTC ACCGAGGCAG TTCCATAGGA TGGCAAGATC

 1381 CTGGTATCGG TCTGCGATTC CGACTCGTCC AACATCAATA CAACCTATTA ATTTCCCCTC

 1441 GTCAAAAATA AGGTTATCAA GTGAGAAATC ACCATGAGTG ACGACTGAAT CCGGTGAGAA

 1501 TGGCAAAAGC TTATGCATTT CTTTCCAGAC TTGTTCAACA GGCCAGCCAT TACGCTCGTC

 1561 ATCAAAATCA CTCGCATCAA CCAAACCGTT ATTCATTCGT GATTGCGCCT GAGCGAGACG

 1621 AAATACGCGA TCGCTGTTAA AAGGACAATT ACAAACAGGA ATCGAATGCA ACCGGCGCAG

 1681 GAACACTGCC AGCGCATCAA CAATATTTTC ACCTGAATCA GGATATTCTT CTAATACCTG

 1741 GAATGCTGTT TTCCCGGGGA TCGCAGTGGT GAGTAACCAT GCATCATCAG GAGTACGGAT

 1801 AAAATGCTTG ATGGTCGGAA GAGGCATAAA TTCCGTCAGC CAGTTTAGTC TGACCATCTC

 1861 ATCTGTAACA TCATTGGCAA CGCTACCTTT GCCATGTTTC AGAAACAACT CTGGCGCATC

 1921 GGGCTTCCCA TACAATCGAT AGATTGTCGC ACCTGATTGC CCGACATTAT CGCGAGCCCA

 1981 TTTATACCCA TATAAATCAG CATCCATGTT GGAATTTAAT CGCGGCCTCG AGCAAGACGT

 2041 TTCCCGTTGA ATATGGCTCA TAACACCCCT TGTATTACTG TTTATGTAAG CAGACAGTTT

 2101 TATTGTTCAT GATGATATAT TTTTATCTTG TGCAATGTAA CATCAGAGAT TTTGAGACAC

 2161 AACGTGGCTT TCCCCCCCCC CCATTATTGA AGCATTTATC AGGGTTATTG TCTCATGAGC

 2221 GGATACATAT TTGAATGTAT TTAGAAAAAT AAACAAATAG GGGTTCCGCG CACATTTCCC

 2281 CGAAAAGTGC CACCTGACGT CTAAGAAACC ATTATTATCA TGACATTAAC CTATAAAAAT

 2341 AGGCGTATCA CGAGGCCCTT TCGTCTCGCG CGTTTCGGTG ATGACGGTGA AAACCTCTGA

 2401 CACATGCAGC TCCCGGAGAC GGTCACAGCT TGTCTGTAAG CGGATGCCGG GAGCAGACAA

 2461 GCCCGTCAGG GCGCGTCAGC GGGTGTTGGC GGGTGTCGGG GCTGGCTTAA CTATGCGGCA

 2521 TCAGAGCAGA TTGTACTGAG AGTGCACCAT ATGCGGTGTG AAATACCGCA CAGATGCGTA

 2581 AGGAGAAAAT ACCGCATCAG ATTGGCTATT GGCCATTGCA TACGTTGTAT CCATATCATA

 2641 ATATGTACAT TTATATTGGC TCATGTCCAA CATTACCGCC ATGTTGACAT TGATTATTGA

 2701 CTAGTTATTA ATAGTAATCA ATTACGGGGT CATTAGTTCA TAGCCCATAT ATGGAGTTCC

 2761 GCGTTACATA ACTTACGGTA AATGGCCCGC CTGGCTGACC GCCCAACGAC CCCCGCCCAT

 2821 TGACGTCAAT AATGACGTAT GTTCCCATAG TAACGCCAAT AGGGACTTTC CATTGACGTC

 2881 AATGGGTGGA GTATTTACGG TAAACTGCCC ACTTGGCAGT ACATCAAGTG TATCATATGC

 2941 CAAGTACGCC CCCTATTGAC GTCAATGACG GTAAATGGCC CGCCTGGCAT TATGCCCAGT

 3001 ACATGACCTT ATGGGACTTT CCTACTTGGC AGTACATCTA CGTATTAGTC ATCGCTATTA

 3061 CCATGGTGAT GCGGTTTTGG CAGTACATCA ATGGGCGTGG ATAGCGGTTT GACTCACGGG

 3121 GATTTCCAAG TCTCCACCCC ATTGACGTCA ATGGGAGTTT GTTTTGGCAC CAAAATCAAC

 3181 GGGACTTTCC AAAATGTCGT AACAACTCCG CCCCATTGAC GCAAATGGGC GGTAGGCGTG

 3241 TACGGTGGGA GGTCTATATA AGCAGAGCTC GTTTAGTGAA CCGTCAGATC GCCTGGAGAC

 3301 GCCATCCACG CTGTTTTGAC CTCCATAGAA GACACCGGGA CCGATCCAGC CTCCGCGGCC

 3361 GGGAACGGTG CATTGGAACG CGGATTCCCC GTGCCAAGAG TGACGTAAGT ACCGCCTATA

 3421 GACTCTATAG GCACACCCCT TTGGCTCTTA TGCATGCTAT ACTGTTTTTG GCTTGGGGCC

 3481 TATACACCCC CGCTTCCTTA TGCTATAGGT GATGGTATAG CTTAGCCTAT AGGTGTGGGT

 3541 TATTGACCAT TATTGACCAC TCCAACGGTG GAGGGCAGTG TAGTCTGAGC AGTACTCGTT

 3601 GCTGCCGCGC GCGCCACCAG ACATAATAGC TGACAGACTA ACAGACTGTT CCTTTCCATG

 3661 GGTCTTTTCT GCAGTCACCG TCGTCGACGG TATCGATAAG CTTGATATCG AATTCGCCGC

 3721 CACCATGGGC CAGACTGTTA CCACTCCCTT AAGTTTGACC TTAGGTCACT GGAAAGATGT

 3781 CGAGCGGATC GCTCACAACC AGTCGGTAGA TGTCAAGAAG AGACGTTGGG TTACCTTCTG

 3841 CTCTGCAGAA TGGCCAACCT TTAACGTCGG ATGGCCGCGA GACGGCACCT TTAACCGAGA

 3901 CCTCATCACC CAGGTTAAGA TCAAGGTCTT TTCACCTGGC CCGCATGGAC ACCCAGACCA

 3961 GGTCCCCTAC ATCGTGACCT GGGAAGCCTT GGCTTTTGAC CCCCCTCCCT GGGTCAAGCC

 4021 CTTTGTACAC CCTAAGCCTC CGCCTCCTCT TCCTCCATCC GCCCCGTCTC TCCCCCTTGA

 4081 ACCTCCTCGT TCGACCCCGC CTCGATCCTC CCTTTATCCA GCCCTCACTC CTTCTCTAGG

 4141 CGCCAAACCT AAACCTCAAG TTCTTTCTGA CAGTGGGGGG CCGCTCATCG ACCTACTTAC

 4201 AGAAGACCCC CCGCCTTATA GGGACCCAAG ACCACCCCCT TCCGACAGGG ACGGAAATGG

 4261 TGGAGAAGCG ACCCCTGCGG GAGAGGCACC GGACCCCTCC CCAATGGCAT CTCGCCTACG

 4321 TGGGAGACGG GAGCCCCCTG TGGCCGACTC CACTACCTCG CAGGCATTCC CCCTCCGCGC

 4381 AGGAGGAAAC GGACAGCTTC AATACTGGCC GTTCTCCTCT TCTGACCTTT ACAACTGGAA

 4441 AAATAATAAC CCTTCTTTTT CTGAAGATCC AGGTAAACTG ACAGCTCTGA TCGAGTCTGT

 4501 CCTCATCACC CATCAGCCCA CCTGGGACGA CTGTCAGCAG CTGTTGGGGA CTCTGCTGAC

 4561 CGGAGAAGAA AAACAACGGG TGCTCTTAGA GGCTAGAAAG GCGGTGCGGG GCGATGATGG

 4621 GCGCCCCACT CAACTGCCCA ATGAAGTCGA TGCCGCTTTT CCCCTCGAGC GCCCAGACTG

 4681 GGATTACACC ACCCAGGCAG GTAGGAACCA CCTAGTCCAC TATCGCCAGT TGCTCCTAGC

 4741 GGGTCTCCAA AACGCGGGCA GAAGCCCCAC CAATTTGGCC AAGGTAAAAG GAATAACACA

 4801 AGGGCCCAAT GAGTCTCCCT CGGCCTTCCT AGAGAGACTT AAGGAAGCCT ATCGCAGGTA

 4861 CACTCCTTAT GACCCTGAGG ACCCAGGGCA AGAAACTAAT GTGTCTATGT CTTTCATTTG

 4921 GCAGTCTGCC CCAGACATTG GGAGAAAGTT AGAGAGGTTA GAAGATTTAA AAAACAAGAC

 4981 GCTTGGAGAT TTGGTTAGAG AGGCAGAAAA GATCTTTAAT AAACGAGAAA CCCCGGAAGA

 5041 AAGAGAGGAA CGTATCAGGA GAGAAACAGA GGAAAAAGAA GAACGCCGTA GGACAGAGGA

 5101 TGAGCAGAAA GAGAAAGAAA GAGATCGTAG GAGACATAGA GAGATGAGCA AGCTATTGGC

 5161 CACTGTCGTT AGTGGACAGA AACAGGATAG ACAGGGAGGA GAACGAAGGA GGTCCCAACT

 5221 CGATCGCGAC CAGTGTGCCT ACTGCAAAGA AAAGGGGCAC TGGGCTAAAG ATTGTCCCAA

 5281 GAAACCACGA GGACCTCGGG GACCAAGACC CCAGACCTCC CTCCTGACCC TAGATGACTA

 5341 GGGAGGTCAG GGTCAGGAGC CCCCCCCTGA ACCCAGGATA ACCCTCAAAG TCGGGGGGCA

 5401 ACCCGTCACC TTCCTGGTAG ATACTGGGGC CCAACACTCC GTGCTGACCC AAAATCCTGG

 5461 ACCCCTAAGT GATAAGTCTG CCTGGGTCCA AGGGGCTACT GGAGGAAAGC GGTATCGCTG

 5521 GACCACGGAT CGCAAAGTAC ATCTAGCTAC CGGTAAGGTC ACCCACTCTT TCCTCCATGT

 5581 ACCAGACTGT CCCTATCCTC TGTTAGGAAG AGATTTGCTG ACTAAACTAA AAGCCCAAAT

 5641 CCACTTTGAG GGATCAGGAG CTCAGGTTAT GGGACCAATG GGGCAGCCCC TGCAAGTGTT

 5701 GACCCTAAAT ATAGAAGATG AGTATCGGCT ACATGAGACC TCAAAAGAGC CAGATGTTTC

 5761 TCTAGGGTCC ACATGGCTGT CTGATTTTCC TCAGGCCTGG GCGGAAACCG GGGGCATGGG

 5821 ACTGGCAGTT CGCCAAGCTC CTCTGATCAT ACCTCTGAAA GCAACCTCTA CCCCCGTGTC

 5881 CATAAAACAA TACCCCATGT CACAAGAAGC CAGACTGGGG ATCAAGCCCC ACATACAGAG

 5941 ACTGTTGGAC CAGGGAATAC TGGTACCCTG CCAGTCCCCC TGGAACACGC CCCTGCTACC

 6001 CGTTAAGAAA CCAGGGACTA ATGATTATAG GCCTGTCCAG GATCTGAGAG AAGTCAACAA

 6061 GCGGGTGGAA GACATCCACC CCACCGTGCC CAACCCTTAC AACCTCTTGA GCGGGCTCCC

 6121 ACCGTCCCAC CAGTGGTACA CTGTGCTTGA TTTAAAGGAT GCCTTTTTCT GCCTGAGACT

 6181 CCACCCCACC AGTCAGCCTC TCTTCGCCTT TGAGTGGAGA GATCCAGAGA TGGGAATCTC

 6241 AGGACAATTG ACCTGGACCA GACTCCCACA GGGTTTCAAA AACAGTCCCA CCCTGTTTGA

 6301 TGAGGCACTG CACAGAGACC TAGCAGACTT CCGGATCCAG CACCCAGACT TGATCCTGCT

 6361 ACAGTACGTG GATGACTTAC TGCTGGCCGC CACTTCTGAG CTAGACTGCC AACAAGGTAC

 6421 TCGGGCCCTG TTACAAACCC TAGGGAACCT CGGGTATCGG GCCTCGGCCA AGAAAGCCCA

 6481 AATTTGCCAG AAACAGGTCA AGTATCTGGG GTATCTTCTA AAAGAGGGTC AGAGATGGCT

 6541 GACTGAGGCC AGAAAAGAGA CTGTGATGGG GCAGCCTACT CCGAAGACCC CTCGACAACT

 6601 AAGGGAGTTC CTAGGGACGG CAGGCTTCTG TCGCCTCTGG ATCCCTGGGT TTGCAGAAAT

 6661 GGCAGCCCCC TTGTACCCTC TCACCAAAAC GGGGACTCTG TTTAATTGGG GCCCAGACCA

 6721 ACAAAAGGCC TATCAAGAAA TCAAGCAAGC TCTTCTAACT GCCCCAGCCC TGGGGTTGCC

 6781 AGATTTGACT AAGCCCTTTG AACTCTTTGT CGACGAGAAG CAGGGCTACG CCAAAGGTGT

 6841 CCTAACGCAA AAACTGGGAC CTTGGCGTCG GCCGGTGGCC TACCTGTCCA AAAAGCTAGA

 6901 CCCAGTAGCA GCTGGGTGGC CCCCTTGCCT ACGGATGGTA GCAGCCATTG CCGTACTGAC

 6961 AAAGGATGCA GGCAAGCTAA CCATGGGACA GCCACTAGTC ATTCTGGCCC CCCATGCAGT

 7021 AGAGGCACTA GTCAAACAAC CCCCCGACCG CTGGCTTTCC AACGCCCGGA TGACTCACTA

 7081 TCAGGCCTTG CTTTTGGACA CGGACCGGGT CCAGTTCGGA CCGGTGGTAG CCCTGAACCC

 7141 GGCTACGCTG CTCCCACTGC CTGAGGAAGG GCTGCAACAC AACTGCCTTG ATATCCTGGC

 7201 CGAAGCCCAC GGAACCCGAC CCGACCTAAC GGACCAGCCG CTCCCAGACG CCGACCACAC

 7261 CTGGTACACG GATGGAAGCA GTCTCTTACA AGAGGGACAG CGTAAGGCGG GAGCTGCGGT

 7321 GACCACCGAG ACCGAGGTAA TCTGGGCTAA AGCCCTGCCA GCCGGGACAT CCGCTCAGCG

 7381 GGCTGAACTG ATAGCACTCA CCCAGGCCCT AAAGATGGCA GAAGGTAAGA AGCTAAATGT

 7441 TTATACTGAT AGCCGTTATG CTTTTGCTAC TGCCCATATC CATGGAGAAA TATACAGAAG

 7501 GCGTGGGTTG CTCACATCAG AAGGCAAAGA GATCAAAAAT AAAGACGAGA TCTTGGCCCT

 7561 ACTAAAAGCC CTCTTTCTGC CCAAAAGACT TAGCATAATC CATTGTCCAG GACATCAAAA

 7621 GGGACACAGC GCCGAGGCTA GAGGCAACCG GATGGCTGAC CAAGCGGCCC GAAAGGCAGC

 7681 CATCACAGAG ACTCCAGACA CCTCTACCCT CCTCATAGAA AATTCATCAC CCTACACCTC

 7741 AGAACATTTT CATTACACAG TGACTGATAT AAAGGACCTA ACCAAGTTGG GGGCCATTTA

 7801 TGATAAAACA AAGAAGTATT GGGTCTACCA AGGAAAACCT GTGATGCCTG ACCAGTTTAC

 7861 TTTTGAATTA TTAGACTTTC TTCATCAGCT GACTCACCTC AGCTTCTCAA AAATGAAGGC

 7921 TCTCCTAGAG AGAAGCCACA GTCCCTACTA CATGCTGAAC CGGGATCGAA CACTCAAAAA

 7981 TATCACTGAG ACCTGCAAAG CTTGTGCACA AGTCAACGCC AGCAAGTCTG CCGTTAAACA

 8041 GGGAACTAGG GTCCGCGGGC ATCGGCCCGG CACTCATTGG GAGATCGATT TCACCGAGAT

 8101 AAAGCCCGGA TTGTATGGCT ATAAATATCT TCTAGTTTTT ATAGATACCT TTTCTGGCTG

 8161 GATAGAAGCC TTCCCAACCA AGAAAGAAAC CGCCAAGGTC GTAACCAAGA AGCTACTAGA

 8221 GGAGATCTTC CCCAGGTTCG GCATGCCTCA GGTATTGGGA ACTGACAATG GGCCTGCCTT

 8281 CGTCTCCAAG GTGAGTCAGA CAGTGGCCGA TCTGTTGGGG ATTGATTGGA AATTACATTG

 8341 TGCATACAGA CCCCAAAGCT CAGGCCAGGT AGAAAGAATG AATAGAACCA TCAAGGAGAC

 8401 TTTAACTAAA TTAACGCTTG CAACTGGCTC TAGAGACTGG GTGCTCCTAC TCCCCTTAGC

 8461 CCTGTACCGA GCCCGCAACA CGCCGGGCCC CCATGGCCTC ACCCCATATG AGATCTTATA

 8521 TGGGGCACCC CCGCCCCTTG TAAACTTCCC TGACCCTGAC ATGACAAGAG TTACTAACAG

 8581 CCCCTCTCTC CAAGCTCACT TACAGGCTCT CTACTTAGTC CAGCACGAAG TCTGGAGACC

 8641 TCTGGCGGCA GCCTACCAAG AACAACTGGA CCGACCGGTG GTACCTCACC CTTACCGAGT

 8701 CGGCGACACA GTGTGGGTCC GCCGACACCA GACTAAGAAC CTAGAACCTC GCTGGAAAGG

 8761 ACCTTACCCA GTCCTGCTGA CCACCCCCAC CGCCCTCAAA GTAGACGGCA TCGCAGCTTG

 8821 GATACACGCC GCCCACGTGA AGGCTGCCGA CCCCGGGGGT GGACCATCCT CTAGACTGAC

 8881 ATGGCGCGTT CAACGCTCTC AAAACCCCTT AAAAATAAGG TTAACCCGCG AGGCCCCCTA

 8941 ATCCCCTTAA TTCTTCTGAT GCTCAGAGGG GTCAGTGTGG GCCCGGTACC GTATACTCTA

 9001 GAGCGGCCGC GGATCCAGAT CTTTTTCCCT CTGCCAAAAA TTATGGGGAC ATCATGAAGC

 9061 CCCTTGAGCA TCTGACTTCT GGCTAATAAA GGAAATTTAT TTTCATTGCA ATAGTGTGTT

 9121 GGAATTTTTT GTGTCTCTCA CTCGGAAGGA CATATGGGAG GGCAAATCAT TTAAAACATC

 9181 AGAATGAGTA TTTGGTTTAG AGTTTGGCAA CATATGCCCA TTCTTCCGCT TCCTCGCTCA

 9241 CTGACTCGCT GCGCTCGGTC GTTCGGCTGC GGCGAGCGGT ATCAGCT

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