pCL-Eco

pCL-Eco

pCL-Eco逆病毒载体基本信息：

pCL-Eco载体质粒图谱和多克隆位点信息：

pCL-Eco载体简介：

逆病毒包装载体pCL-Eco的使用方法

293T Growth Medium:

   450 ml DMEM (JRH Biosciences)

   50 ml FBS (JRH Sciences)

   5 ml Glutamine (200 mM in saline, JRH Sciences)

   2.5 ml Gentamycin(10 mg/ml in water, Sigma)

HC11 Growth Medium:

  5 ml L-Glutamine (200 mM in saline, JRH Sciences)

  2.5 ml Bovine Insulin (1 mg/ml in 0.01 N HCL, Sigma)

  2.5 ml Gentamycin (10 mg/ml in water, Sigma)

  0.5 ml EGF (10 μg/ml in water, Gibco)

  50 ml FBS (JRH Sciences)

  440 ml RPMI (JRH Sciences)

Day 0: Split 293T cells for transfection:

  Wash cells off plate with 10 ml Hanks and put into 15 ml Falcon tube

  Add 1 ml 10X trypsin and mix by inversion for about 1-2 minutes

  Spin at 1000 rpm in clinical centrifuge for 1-2 minutes

  Resuspend cells in 10 ml growth medium

  Count cells

  Plate cells at 1X106 cells per 100 mm dish

Day 2: Transfect 293T cells:

  Plan transfection using 2 μg total DNA and 12 μl FuGene in 200 μl total

  volume per 100 mm dish

  Dilute Fugene: 12 μl per reaction in serum free DMEM for total volume of

  200 μl

  Mix DNA plasmids in polypropelene tubes (Falcon #2063):

     1 μg DNA of interest

     1 μg pCL-Eco

     Add 200 μl diluted FuGene dropwise to each DNA tube

  Incubate RT 15 min

  Feed cells with 5 ml fresh growth media and add FuGene/DNA mixture to media

Day 3: Change medium:

  • Remove viral medium and CAREFULLY feed with 6 ml growth medium

  This step may reduce a cytostatic factor (produced after transfections) that

  can inhibit growth and infection of your target cells.

  Split HC11 cells for infection

  Seed HC11 cells at 300,000 cells per 100 mm dish

  If using coverslips, incubate with FBS for at least 1 hour prior to plating

Day 4: Infect target (HC11) cells:

  • Collect virus-containing media off 293T cells

  • Syringe filter media through .45 μm filter onto target cells

   (Evaporation O/N and loss in filter leaves about 5 ml per plate)

  • Add 10 μl 5mg/ml polybrene (final conc = 10 μg polybrene/ml media)

   (Store polybrene in aliquots to decrease freeze/thaw cycles)

  • Wrap plates in parafilm

  • Spin cells in clinical centrifuge 10 min at 1800 rpm

  • Rotate plate 1/3. Spin 10 min at 1800 rpm

  • Rotate plate 1/3. Spin 10 min at 1800 rpm

  • Remove viral media and replace with fresh growth media

Day 6+: Harvest HC11 cells:

  • Wash cells with HBSS

  • Scrape remaining cells, pellet and flash freeze for protein/RNA analysis

  Stain cells for beta-gal expression:

  Fixing Solution :

    1.35 ml 37% Formaldehyde

    0.2 ml Gluteraldehyde

    1X PBS to 25 mls

  Staining Solution:

    625μl 40 mg/ml X-gal in DMF

    50 μl 1M MgCL2

    750 μl 100 mM Potassium Ferricyanide

    750 μl 100 mM Potassium Ferrocyanide

    1X PBS to 25 mls

    [100 mM Potassium Ferrocyanide = 2.11 g/50ml PBS]

    [100 mM Potassium Ferricyanide = 1.64 g/50 ml PBS]

  Store at room temperature, protect from light.

    -gal solution and warm in 37°C water bath to prevent crystallizationâ1. Make

    2. Aspirate off media

    3. Wash cells 1X PBS

    4. Fix cells 5 min at RT in fixing solution

    5. Wash 3X PBS

    6 Optional: Add PBS to cells and warm plate at 37°C 5 min (reduces

      crystallization)

    7. Stain cells with staining solution at 37°C for 1-24 hrs

    8. Store stained cells in 70% EtOH at 4°C

      (EMBO J. 5: pg 3133, 1986)

Note:

   HC11 and MEC primary culture cells are very adherent and can withstand

   the force of spinning the plates at 1800 rpm. We found that HC11 cells died at

   2200 rpm. However, this is cell type-dependent, and spinning speed should be

   optimized accordingly.

   -gal staining at the periphery of the plate. During the spinâExpect to see

   infection the media is pushed to the outside. The cells in the center of the plate

   will not be exposed to virus and will not be efficiently infected with retrovirus.

pCL-Eco载体序列：

ORIGIN

    1 TGGTAAGCCC TCCCGTATCG TAGTTATCTA CACGACGGGG AGTCAGGCAA CTATGGATGA

   61 ACGAAATAGA CAGATCGCTG AGATAGGTGC CTCACTGATT AAGCATTGGT AACTGTCAGA

  121 CCAAGTTTAC TCATATATAC TTTAGATTGA TTTAAAACTT CATTTTTAAT TTAAAAGGAT

  181 CTAGGTGAAG ATCCTTTTTG ATAATCTCAT GACCAAAATC CCTTAACGTG AGTTTTCGTT

  241 CCACTGAGCG TCAGACCCCG TAGAAAAGAT CAAAGGATCT TCTTGAGATC CTTTTTTTCT

  301 GCGCGTAATC TGCTGCTTGC AAACAAAAAA ACCACCGCTA CCAGCGGTGG TTTGTTTGCC

  361 GGATCAAGAG CTACCAACTC TTTTTCCGAA GGTAACTGGC TTCAGCAGAG CGCAGATACC

  421 AAATACTGTC CTTCTAGTGT AGCCGTAGTT AGGCCACCAC TTCAAGAACT CTGTAGCACC

  481 GCCTACATAC CTCGCTCTGC TAATCCTGTT ACCAGTGGCT GCTGCCAGTG GCGATAAGTC

  541 GTGTCTTACC GGGTTGGACT CAAGACGATA GTTACCGGAT AAGGCGCAGC GGTCGGGCTG

  601 AACGGGGGGT TCGTGCACAC AGCCCAGCTT GGAGCGAACG ACCTACACCG AACTGAGATA

  661 CCTACAGCGT GAGCTATGAG AAAGCGCCAC GCTTCCCGAA GGGAGAAAGG CGGACAGGTA

  721 TCCGGTAAGC GGCAGGGTCG GAACAGGAGA GCGCACGAGG GAGCTTCCAG GGGGAAACGC

  781 CTGGTATCTT TATAGTCCTG TCGGGTTTCG CCACCTCTGA CTTGAGCGTC GATTTTTGTG

  841 ATGCTCGTCA GGGGGGCGGA GCCTATGGAA AAACGCCAGC AACGCGGCCT TTTTACGGTT

  901 CCTGGCCTTT TGCTGGCCTT TTGCTCACAT GTTCTTTCCT GCGTTATCCC CTGATTCTGT

  961 GGATAACCGT ATTACCGCCT TTGAGTGAGC TGATACCGCT CGCCGCAGCC GAACGACCGA

 1021 GCGCAGCGAG TCAGTGAGCG AGGAAGCGGA AGAGCGCCCA ATACGCAAAC CGCCTCTCCC

 1081 CGCGCGTTGG CCGATTCATT AATGCAGCCT CCAAAAAAGC CTCCTCACTA CTTCTGGAAT

 1141 AGCTCAGAGG CCGAGGCGGC CTCGGCCTCT GCATAAATAA AAAAAATTAG TCAGCCATGG

 1201 GGCGGAGAAT GGGCGGAACT GGGCGGAGTT AGGGGCGGGA TGGGCGGAGT AAGACCCGCA

 1261 CCCGTTCCCA AACTGCTTAT TAAAGGGGGT ATGGAGGTGC TGGACCTTGT GACAGGGCCA

 1321 GACAGTGTGA CAGAAATAGA AGCAGTCCTG TTTTGACAAG TTGCCTCTGG AAGCCTCTAC

 1381 AATGCCTCTC TTCTTTTTCT CCAGAGTAAG CGGAGGCCAG GGGCCCCCGG CCTCTGCTTA

 1441 ATACTAAAAA AAACAGCTGT TGTCATAGTA ATGATTGGGT GGAAACATTC TAGGCCTGGG

 1501 TGGAGAGGCT TTTTGCTTCC TCTTGCAAAA CCACACTGCC CTCTGGAGGG CAGTTGCCTA

 1561 GCAACTAATT AAAAGAGGAT GTCGCACGGC CAGCTGCGGT CAGTTAGTCA CTTCCTGCTT

 1621 AACTGACTTG ACATTTTCTA TTTTAAGAGT CGGGAGGAAA ATTACTGTGT TGGAGGCCCT

 1681 CCGCCATCTT CTGAAGCTGA TCCAGACATG ATAAGATACA TTGATGAGTT TGGACAAACC

 1741 ACAACTAGAA TGCAGTGAAA AAAATGCTTT ATTTGTGAAA TTTGTGATGC TATTGCTTTA

 1801 TTTGTAACCA TTATAAGCTG CAATAAACAA GTTAACAACA ACAATTGCAT TCATTTTATG

 1861 TTTCAGGTTC AGGGGGAGGT GTGGGAGGTT TTTTAAAGCA AGTAAAACCT CTACAAATGT

 1921 GGTATGGCTG ATTATGATCT CTAGTCAAGG CACTATACAT CAAATATTTT TCCATAATTT

 1981 TCTTGTATAG CAGTGCAGCT TTTTCCTTTG TGGTGTAAAT AGCAAAGCAA GCAAGAGTTC

 2041 TATTACTAAA CACAGCATGA CTCAAAAAAC TTAGCAATTC TGAAGGAAAG TCCTTGGGGT

 2101 CTTCTACCTT TCTCTTCTTT TTTGGAGGAG TAGAATGTTG AGAGTCAGCA GTAGCCTCAT

 2161 CATCACTAGA TGGCATTTCT TCTGAGCAAA ACAGGTTTTC CTCATTAAAG GCATTCCACC

 2221 ACTGCTCCCA TTCATCAGTT CCATAGGTTG GAATCTAAAA TACACAAACA ATTAGAATCA

 2281 GTAGTTTAAC ACATTATACA CTTAAAAATT TTATATTTAC CTTAGAGCTT TAAATCTCTG

 2341 TAGGTAGTTT GTCCAATTAT GTCACACCAC AGAAGTAAGG TTCCTTCACA AAGATCCTCT

 2401 AGCTACCTAG CTAGATACAC TCCGCTATCG CTACGTGACT GGGTCATGGC TGCGCCCCGA

 2461 CACCCGCCAA CACCCGCTGA CGCGCCCTGA CGGGCTTGTC TGCTCCCGGC ATCCGCTTAC

 2521 AGACAAGCTG TGACCGTCTC CGGGAGCTGC ATGTGTCAGA GGTTTTCACC GTCATCACCG

 2581 AAACGCGCGA GGCAGCCAGC TTACCTCCCG GTGGTGGGTC GGTGGTCCCT GGGCAGGGGT

 2641 CTCCCGATCC CGGACGAGCC CCCAAATGAA AGACCCCCGC TGACGGGTAG TCAATCACTC

 2701 AGAGGAGACC CTCCCAAGGA ACAGCGAGAC CACAAGTCGG ATGCAACTGC AAGAGGGTTT

 2761 ATTGGATACA CGGGTACCCG GGCGACTCAG TCAATCGGAG GACTGGCGCC CCGAGTGAGG

 2821 GGTTGTGGGC TCTTTTATTG AGCTCGGGGA GCAGAAGCGC GCGAACAGAA GCGAGAAGCG

 2881 AACTGATTGG TTAGTTCAAA TAAGGCACAG GGTCATTTCA GGTCCTTGGG GCACCCTGGA

 2941 AACATCTGAT GGTTCTCTAG CTTGCCAAAC CTACAGGTGG GGTCTTTCAT TCCCCCCTTT

 3001 TTCTGGAGAC TAAATAAAAT CTTTTATTTT ATCTATGGCT CGTACTCTAT AGGCTTCAGC

 3061 TGGTGATATT GTTGAGTCAA AACTAGAGCC TGGACCACTG ATATCCTGTC TTTAACAAAT

 3121 TGGACTAATC GATTAAGAAT GCAGGGTCCG AAGAGCAAAA TCATTAGGAG TACAATGAGG

 3181 GGTCCCATAA TGGTAGATAT CAAGGTGGTA AACCAAGGGG ATCTGTTAAA CAGTCCCTCA

 3241 AACCATCCTT GAGTTGACTC AAACAGTTTC TGTCTCTGAT TAAGCCTCTC TCTCAATTTG

 3301 GCCATGCTGT CTCTCACTAG TCCTGTGTGG TCCGCATAGA AGCAACATTC TTCTTTTAGA

 3361 GCAGCACACA GCCCTCCTTC TTTTAGAAAT AACAAGTCTA GGCCCCTTCG ATTCTGTAGG

 3421 ACAACTTCAG ACAGGGAAGT GAGAGACTTT TCTAGGTTAG AGATTGATTT TTCAACCTCC

 3481 CTGAGATCAT CCTGTACTGC GGCTTGGAGC TGCTGGAATT GCTGAGTGGC CATTAGAGCA

 3541 GTAGTCCCTG TTCCTATTCC AGCGGCAATT CCCCCCATGG TTAGTCCACC CAATAATAGG

 3601 GCCAGGGTTA ACGACACCGG TTCTCTTTTG TGTCGGTTGG ATCTCTCAAA CAGGCCGTAA

 3661 ACATAGCTGG GGGAATGATA GGTGACTCTT GGCCAGAGTT CGACAAGAAC ACAATAATCA

 3721 GTGGTAAGGT TCAGTATGGT GGTGGAGATG CATGGAGTAA GCCCGGTACT ACAAGCCCAC

 3781 ATGGTACCTG TAGGGGCAAC TAGATAATAG GACCCTCGAC TGCTTGTCTG GGTGGTATTA

 3841 CATAGGGCCT GATGTGTTTT GGGAACTGCT CCTATGCAGA GTCCCTGTCC GGTCACTTCG

 3901 GACAGGGTCA ACTTGTGTTG GGAGGCCACG GAGCAGTTGG CTGGAGCAGA GGTATGGTTG

 3961 GAGTAGGTAC CCAGGACGGC AACCCCTTCG TAGTAGGGGG GTCCCGCTAC TAGACACAAC

 4021 CAGCACTCTT GGGTTTTGTC AGGACTGGTG AGGTTGAGGG CTTGGTAGGC TCCGTCTACT

 4081 AAGTTTAGCA GCCTATTTTC CGTTCCCGCC GGTGGAAGTT GGGTAGGGGA GAGAGGAGTC

 4141 CCACTGGGTG GTTTGGTGAC TGAAGGCGAC TTAACAGGTT TGGGCTTGGA GAGTGGCTGT

 4201 TGGTCTGCCA GAACGGGGTT TGGCCCTATT GGGACGCGGG GTCCTAGATT TTGGTATCTG

 4261 AGTCGGATCC CAAATGTAAG CCCTGGATCT TGTCCGGAGA CATACAAACG TAAGCCCCAG

 4321 TAATGTCCTG TGGTCCAGGA AGTAACCCGT CTCCCGGCGT CTGTAAACCG AATAACTAAG

 4381 GGGTTGCACC ACTTATTATC TTTGCATACC TGGACAGCCT GGTCAGAGGT GAGATTGTTG

 4441 TTTACTGTGA TGAAATCCCA TGATGAGGAG GGCTTCCAGT AAGCTCTACC GGTTGTCTCA

 4501 CAGCCCCAAT AGGCACAGTA GAAGGAGTCT GGACCCCCAC ATGACTTGGA TTCTCGGGGG

 4561 CGGTGGGGCC CGGGGCAAAC ATAAAATCCC TCATTTGATT TATGAGTTGT CTGGTCTAGC

 4621 TTGAGTCTGT TCCAGGCAGT GTTGCACCGA GGGGTGAGGG AGGTTAAAGG TTCTTCGCAG

 4681 TCTCTGGAAC AGCCTGGGCT GCTGCCCCCT GAGCAACAAG GGGGCCCCGG GGGAGAAGAA

 4741 AAAGGGGATT GATATTCTAG CCCCCAATAA GATGGTCCAT GGTGGGCTAA CATACATAAA

 4801 TCTGGGGTAA GGTCAGGCCA CCAGGTCCAC AGAGGGTGGT TGCCAGAAGT TGCCCATACC

 4861 GTCTCCCGAT CTCCATTGGT TACCTCCCAG GTGATATTAT AGACTTGATG AGGACTGGAG

 4921 CCGGGCGAAG CAGTACTGAC CCCTCTGAGC ATCAGAAGAA TTAAGGGGAT TAGGGGGCCT

 4981 CGCGGGTTAA CCTTATTTTT AAGGGGTTTT GAGAGCGTTG AACGCGCCAT GTCAGTCTAG

 5041 AGGATGGTCC ACCCCCGGGG TCGGCAGCCT TCACGTGGGC GGCGTGTATC CAAGCTGCGA

 5101 TGCCGTCTAC TTTGAGGGCG GTGGGGGTGG TCAGCAGGAC TGTGTAAGGT CCTTTCCAGC

 5161 GAGGTTCTAG GTTCTTAGTC TGGTGTCGGC GGACCCACAC TGTGTCGCCG ACTCGGTAAG

 5221 GGTGAGGTAC CACCGGTCGG TCCAGTTGTT CTTGGTAGGC TGCCGCCAGA GGTCTCCAGA

 5281 CTTCGTGCTG GACTAAGTAG AGAGCCTGTA AGTGAGCTTG GAGAGAGGGG CTGTTAGTAA

 5341 CTCTTGTCAT GTCAGGGTCA GGGAAGTTTA CAAGGGGCGG GGGTGCCCCA TATAAGATCT

 5401 CATATGGGGT GAGGCCATGG GGGCCCGGCG TGTTGCGGGC TCGGTACAGG GCTAAGGGGA

 5461 GTAGGAGCAC CCAGTCTCTA GAGCCAGTTG CAAGCGTTAA TTTAGTTAAA GTCTCCTTGA

 5521 TGGTTCTATT CATTCTTTCT ACCTGGCCTG AGCTTTGGGG TCTGTATGCA CAATGTAATT

 5581 TCCAATCAAT CCCCAACAGA TCGGCCACTG TCTGACTCAC CTTGGAGACG AAGGCAGGCC

 5641 CATTGTCAGT TCCCAATACC TGAGGCATGC CGAACCTGGG GAAGATCTCC TCTAGTAGCT

 5701 TCTTGGTTAC GACCTTGGCG GTTTCTTTCT TGGTTGGGAA GGCTTCTATC CAGCCAGAAA

 5761 AGGTATCTAT AAAAACTAGA AGATATTTAT AGCCATACAA TCCGGGCTTT ATCTCGGTGA

 5821 AATCGATCTC CCAATGAGTG CCGGGCCGAT GCCCGCGGAC CCTAGTTCCC TGTTTAACGG

 5881 CAGACTTGCT GGCGTTGACT TGTGCACAAG CTTTGCAGGT CTCAGTGATA TTTTTGAGTG

 5941 TTCGATCCCG GTTCAGCATG TAGTAGGGAC TGTGGCTTCT CTCTAGGAGA GCCTTCATTT

 6001 TTGAGAAGCT GAGGTGAGTC AGCTGATGAA GAAAGTCTAA TAATTCAAAA GTAAACTGGT

 6061 CAGGCATCAC AGGTTTTCCT TGGTAGACCC AATACTTCTT TGTTTTATCA TAAATGGCCC

 6121 CCAACTTGGT TAGGTCCTTT ATATCAGTCA CTGTGTAATG AAAATGTTCT GAGGTGTAGG

 6181 GTGATGAATT TTCTATGAGG AGGGTAGAGG TGTCTGGAGT CTCTGTGATG GCTGCCTTTC

 6241 GGGCCGCTTG GTCAGCCATC CGGTTGCCTC TAGCCTCGGC GCTGTGTCCC TTTTGATGTC

 6301 CTGGACAATG GATTATGCTA AGTCTTTTGG GCAGAAAGAG GGCTTTTAGT AGGGCCAAGA

 6361 TCTCGTCTTT ATTTTTGATC TCTTTGCCTT CTGATGTGAG CAACCCACGC CTTCTGTATA

 6421 TTTCTCCATG GATATGGGCA GTAGCAAAAG CATAACGGCT ATCAGTATAA ACATTTAGCT

 6481 TCTTACCTTC TGCCATCTTT AGGGCCTGGG TGAGTGCTAT CAGTTCAGCC CGCTGAGCGG

 6541 ATGTCCCGGC TGGCAGGGCT TTAGCCCAGA TTACCTCGGT CTCGGTGGTC ACCGCAGCTC

 6601 CCGCCTTACG CTGTCCCTCT TGTAAGAGAC TGCTTCCATC CGTGTACCAG GTGTGGTCGG

 6661 CGTCTGGGAG CGGCTGGTCC GTTAGGTCGG GTCGGGTTCC GTGGGCTTCG GCCAGGATAT

 6721 CAAGGCAGTT GTGTTGCAGC CCTTCCTCAG GCAGTGGGAG CAGCGTAGCC GGGTTCAGGG

 6781 CTACCACCGG TCCGAACTGG ACCCGGTCCG TGTCCAAAAG CAAGGCCTGA TAGTGAGTCA

 6841 TCCGGGCGTT GGAAAGCCAG CGGTCGGGGG GTTGTTTGAC TAGTGCCTCT ACTGCATGGG

 6901 GGGCCAGAAT GACTAGTGGC TGTCCCATGG TTAGCTTGCC TGCATCCTTT GTCAGTACGG

 6961 CAATGGCTGC TACCATCCGT AGGCAAGGGG GCCACCCAGC TGCTACTGGG TCTAGCTTTT

 7021 TGGACAGGTA GGCCACCGGC CGACGCCAAG GTCCCAGTTT TTGCGTTAGG ACACCTTTGG

 7081 CGTAGCCCTG CTTCTCGTCG ACAAAGAGTT CAAAGGGCTT AGTCAAATCT GGCAACCCCA

 7141 GGGCTGGGGC AGTTAGAAGA GCTTGCTTGA TTTCTTGATA GGCCTTTTGT TGGTCTGGGC

 7201 CCCAATTAAA CAGAGTCCCC GTTTTGGTGA GAGGGTACAA GGGGGCTGCC ATTTCTGCAA

 7261 ACCCAGGGAT CCAGAGGCGA CAGAAGCCTG CCGTCCCTAG GAACTCCCTT AGTTGTCGAG

 7321 GGGTCTTCGG AGTAGGCTGC CCCATCACAG TCTCTTTTCT GGCCTCAGTC AGCCATCTCT

 7381 GACCCTCTTT TAGAAGATAC CCCAGATACT TGACCTGTTT CTGGCAAATT TGGGCTTTCT

 7441 TGGCCGAGGC CCGATACCCG AGGTTCCCTA GGGTTTGTAA CAGGGCCCGA GTACCTTGTT

 7501 GGCAGTCTAG CTCAGAAGTG GCGGCCAGCA GTAAGTCATC CACGTACTGT AGCAGGATCA

 7561 AGTCTGGGTG CTGGATCCGG AAGTCTGCTA GGTCTCTGTG CAGTGCCTCA TCAAACAGGG

 7621 TGGGACTGTT TTTGAAACCC TGTGGGAGTC TGGTCCAGGT CAATTGTCCT GAGATTCCCA

 7681 TCTCTGGATC TCTCCACTCA AAGGCGAAGA GAGGCTGACT GGTGGGGTGG AGTCTCAGGC

 7741 AGAAAAAGGC ATCCTTTAAA TCAAGCACAG TGTACCACTG GTGGGACGGT GGGAGCCCGC

 7801 TCAAGAGGTT GTAAGGGTTG GGCACGGTGG GGTGGATGTC TTCCACCCGC TTGTTGACTT

 7861 CTCTCAGATC CTGGACAGGC CTATAATCAT TAGTCCCTGG TTTCTTAACG GGTAGCAGGG

 7921 GCGTGTTCCA GGGGGACTGG CAGGGTACCA GTATTCCCTG GTCCAACAGT CTCTGTATGT

 7981 GGGGCTTGAT CCCCAGTCTG GCTTCTTGTG ACATGGGGTA TTGTTTTATG GACACGGGGG

 8041 TAGAGGTTGC TTTCAGAGGT ATGATCAGAG GAGCTTGGCG AACTGCCAGT CCCATGCCCC

 8101 CGGTTTCCGC CCAGGCCTGA GGAAAATCAG ACAGCCATGT GGACCCTAGA GAAACATCTG

 8161 GCTCTTTTGA GGTCTCATGT AGCCGATACT CATCTTCTAT ATTTAGGGTC AACACTTGCA

 8221 GGGGCTGCCC CATTGGTCCC ATAACCTGAG CTCCTGATCC CTCAAAGTGG ATTTGGGCTT

 8281 TTAGTTTAGT CAGCAAATCT CTTCCTAACA GAGGATAGGG ACAGTCTGGT ACATGGAGGA

 8341 AAGAGTGGGT GACCTTACCG GTAGCTAGAT GTACTTTGCG ATCCGTGGTC CAGCGATACC

 8401 GCTTTCCTCC AGTAGCCCCT TGGACCCAGG CAGACTTATC ACTTAGGGGT CCAGGATTTT

 8461 GGGTCAGCAC GGAGTGTTGG GCCCCAGTAT CTACCAGGAA GGTGACGGGT TGCCCCCCGA

 8521 CTTTGAGGGT TATCCTGGGT TCAGGGGGGG GCTCCTGACC CTGACCTCCC TAGTCATCTA

 8581 GGGTCAGGAG GGAGGTCTGG GGTCTTGGTC CCCGAGGTCC TCGTGGTTTC TTGGGACAAT

 8641 CTTTAGCCCA GTGCCCCTTT TCTTTGCAGT AGGCACACTG GTCGCGATCG AGTTGGGACC

 8701 TCCTTCGTTC TCCTCCCTGT CTATCCTGTT TCTGTCCACT AACGACAGTG GCCAATAGCT

 8761 TGCTCATCTC TCTATGTCTC CTACGATCTC TTTCTTTCTC TTTCTGCTCA TCCTCTGTCC

 8821 TACGGCGTTC TTCTTTTTCC TCTGTTTCTC TCCTGATACG TTCCTCTCTT TCTTCCGGGG

 8881 TTTCTCGTTT ATTAAAGATC TTTTCTGCCT CTCTAACCAA ATCTCCAAGC GTCTTGTTTT

 8941 TTAAATCTTC TAACCTCTCT AACTTTCTCC CAATGTCTGG GGCAGACTGC CAAATGAAAG

 9001 ACATAGACAC ATTAGTTTCT TGCCCTGGGT CCTCAGGGTC ATAAGGAGTG TACCTGCGAT

 9061 AGGCTTCCTT AAGTCTCTCT AGGAAGGCCG AGGGAGACTC ATTGGGCCCT TGTGTTATTC

 9121 CTTTTACCTT GGCCAAATTG GTGGGGCTTC TGCCCGCGTT TTGGAGACCC GCTAGGAGCA

 9181 ACTGGCGATA GTGGACTAGG TGGTTCCTAC CTGCCTGGGT GGTGTAATCC CAGTCTGGGC

 9241 GCTCGAGGGG AAAAGCGGCA TCGACTTCAT TGGGCAGTTG AGTGGGGCGC CCATCATCGC

 9301 CCCGCACCGC CTTTCTAGCC TCTAAGAGCA CCCGTTGTTT TTCTTCTCCG GTCAGCAGAG

 9361 TCCCCAACAG CTGCTGACAG TCGTCCCAGG TGGGCTGATG GGTGATGAGG ACAGACTCGA

 9421 TCAGAGCTGT CAGTTTACCT GGATCTTCAG AAAAAGAAGG GTTATTATTT TTCCAGTTGT

 9481 AAAGGTCAGA AGAGGAGAAC GGCCAGTATT GAAGCTGTCC GTTTCCTCCT GCGCGGAGGG

 9541 GGAATGCCTG CGAGGTAGTG GAGTCGGCCA CAGGGGGCTC CCGTCTCCCA CGTAGGCGAG

 9601 ATGCCATTGG GGAGGGGTCC GGTGCCTCTC CCGCAGGGGT CGCTTCTCCA CCATTTCCGT

 9661 CCCTGTCGGA AGGGGGTGGT CTTGGGTCCC TATAAGGCGG GGGGTCTTCT GTAAGTAGGT

 9721 CGATGAGCGG CCCCCCACTG TCAGAAAGAA CTTGAGGTTT AGGTTTGGCG CCTAGAGAAG

 9781 GAGTGAGGGC TGGATAAAGG GAGGATCGAG GCGGGGTCGA ACGAGGAGGT TCAAGGGGGA

 9841 GAGACGGGGC GGATGGAGGA AGAGGAGGCG GAGGCTTAGG GTGTACAAAG GGCTTGACCC

 9901 AGGGAGGGGG GTCAAAAGCC AAGGCTTCCC AGGTCACGAT GTAGGGGACC TGGTCTGGGT

 9961 GTCCATGCGG GCCAGGTGAA AAGACCTTGA TCTTAACCTG GGTGATGAGG TCTCGGTTAA

10021 AGGTGCCGTC TCGCGGCCAT CCGACGTTAA AGGTTGGCCA TTCTGCAGAG CAGAAGGTAA

10081 CCCAACGTCT CTTCTTGACA TCTACCGACT GGTTGTGAGC GATCCGCTCG ACATCTTTCC

10141 AGTGACCTAA GGTCAAACTT AAGGGAGTGG TAACAGTCTG GCCCATATTT TCAGACAAAT

10201 ACAGAAACAC AGTCAGACAG AGACAACACA GAACGATGCC CAAGCTTGGC CAGCTTACCT

10261 CCCGGTGGTG GGTCGGTGGT CCCTGGGCAG GGGTCTCCCG ATCCCGGACG AGCCCCCAAA

10321 TGAAAGACCC CCGCTGACGG GTAGTCAATC ACTCAGAGGA GACCCTCCCA AGGAACAGCG

10381 AGACCACAAG TCGGATGCAA CTGCAAGAGG GTTTATTGGA TACACGGGTA CCCGGGCGAC

10441 TCAGTCAATC GGAGGACTGG CGCGCCGAGT GAGGGGTTGT GGGCTCTTTT ATTGAGCTCT

10501 GCTTATATAG ACCTCCCACC GTACACGCCT ACCGCCCATT TGCGTCAATG GGGCGGAGTT

10561 GTTACGACAT TTTGGAAAGT CCCGTTGATT TTGGTGCCAA AACAAACTCC CATTGACGTC

10621 AATGGGGTGG AGACTTGGAA ATCCCCGTGA GTCAAACCGC TATCCACGCC CATTGATGTA

10681 CTGCCAAAAC CGCATCACCA TGGTAATAGC GATGACTAAT ACGTAGATGT ACTGCCAAGT

10741 AGGAAAGTCC CATAAGGTCA TGTACTGGGC ATAATGCCAG GCGGGCCATT TACCGTCATT

10801 GACGTCAATA GGGGGCGTAC TTGGCATATG ATACACTTGA TGTACTGCCA AGTGGGCAGT

10861 TTACCGTAAA TACTCCACCC ATTGACGTCA ATGGAAAGTC CCTATTGGCG TTACTATGGG

10921 AACATACGTC ATTATTGACG TCAATGGGCG GGGGTCGTTG GGCGGTCAGC CAGGCGGGCC

10981 ATTTACCGTA AGTTATGTAA CGCGGAACTC CATATATGGG CTATGAACTA ATGACCCCGT

11041 AATTGATTAC TATTAATAAC TAGTCAATAA TCAATGTCAA CGCGCTGGCG TAATAGCGAA

11101 GAGGCCCGCA CCGATCGCCC TTCCCAACAG TTGCGCAGCC TGAATGGCGA ATGGCGCCTG

11161 ATGCGGTATT TTCTCCTTAC GCATCTGTGC GGTATTTCAC ACCGCATATG GTGCACTCTC

11221 AGTACAATCT GCTCTGATGC CGCATAGTTA AGCCAGCCCC GACACCCGCC AACACCCGCT

11281 GACGCGCCCT GACGGGCTTG TCTGCTCCCG GCATCCGCTT ACAGACAAGC TGTGACCGTC

11341 TCCGGGAGCT GCATGTGTCA GAGGTTTTCA CCGTCATCAC CGAAACGCGC GAGACGAAAG

11401 GGCCTCGTGA TACGCCTATT TTTATAGGTT AATGTCATGA TAATAATGGT TTCTTAGACG

11461 TCAGGTGGCA CTTTTCGGGG AAATGTGCGC GGAACCCCTA TTTGTTTATT TTTCTAAATA

11521 CATTCAAATA TGTATCCGCT CATGAGACAA TAACCCTGAT AAATGCTTCA ATAATATTGA

11581 AAAAGGAAGA GTATGAGTAT TCAACATTTC CGTGTCGCCC TTATTCCCTT TTTTGCGGCA

11641 TTTTGCCTTC CTGTTTTTGC TCACCCAGAA ACGCTGGTGA AAGTAAAAGA TGCTGAAGAT

11701 CAGTTGGGTG CACGAGTGGG TTACATCGAA CTGGATCTCA ACAGCGGTAA GATCCTTGAG

11761 AGTTTTCGCC CCGAAGAACG TTTTCCAATG ATGAGCACTT TTAAAGTTCT GCTATGTGGC

11821 GCGGTATTAT CCCGTATTGA CGCCGGGCAA GAGCAACTCG GTCGCCGCAT ACACTATTCT

11881 CAGAATGACT TGGTTGAGTA CTCACCAGTC ACAGAAAAGC ATCTTACGGA TGGCATGACA

11941 GTAAGAGAAT TATGCAGTGC TGCCATAACC ATGAGTGATA ACACTGCGGC CAACTTACTT

12001 CTGACAACGA TCGGAGGACC GAAGGAGCTA ACCGCTTTTT TGCACAACAT GGGGGATCAT

12061 GTAACTCGCC TTGATCGTTG GGAACCGGAG CTGAATGAAG CCATACCAAA CGACGAGCGT

12121 GACACCACGA TGCCTGCAGC AATGGCAACA ACGTTGCGCA AACTATTAAC TGGCGAACTA

12181 CTTACTCTAG CTTCCCGGCA ACAATTAATA GACTGGATGG AGGCGGATAA AGTTGCAGGA

12241 CCACTTCTGC GCTCGGCCCT TCCGGCTGGC TGGTTTATTG CTGATAAATC TGGAGCCGGT

12301 GAGCGTGGGT CTCGCGGTAT CATTGCAGCA CTGGGGCCAG A

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