pUMVC

价格:1250元

联系方式:I47-825O-882O

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载体基本信息

出品公司: Addgene
试剂名称: pUMVC
质粒类型: 哺乳动物载体;逆病毒包装载体;双质粒包装系统
高拷贝/低拷贝: 低拷贝
克隆方法: 限制性内切酶,多克隆位点
启动子: CMV
载体大小: 9287 bp
5' 测序引物及序列: CMV fwd 5’CGCAAATGGGCGGTAGGCGTG 3’
3' 测序引物及序列: --
载体标签:
载体抗性: Kanamycin.html' target='_blank'>卡那霉素
筛选标记:
克隆菌株: DH5α等
宿主细胞(系): 包装细胞系如293T
备注: 逆病毒包装载体pUMVC需要与信封质粒 pCMV-VSV-G  或者 pCMV-VSVG 共同使用,使用方法见下文。
产品目录号: #8449
稳定性: 瞬表达
组成型/诱导型: 组成型
病毒/非病毒: 非病毒

载体质粒图谱和多克隆位点信息

pUMVC载体图谱



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.

载体序列

LOCUS       pUMVC Sequencing Result	9287 bp 	DNA	SYN
DEFINITION  pUMVC Sequencing Result
ACCESSION   
KEYWORDS    
SOURCE      
  ORGANISM  other sequences; artificial sequences; vectors.
FEATURES             Location/Qualifiers
     source          1..9287
                     /organism="pUMVC Sequencing Result"
                     /mol_type="other DNA"
     gene            complement(1246..2061)
                     /label="KanR2"
                     /gene="KanR2"
     CDS             complement(1246..2061)
                     /label="ORF frame 3"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLY
                     GKPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIP
                     GKTAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGL
                     VDASDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVG
                     RVGIADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF*"
     promoter        complement(2207..2235)
                     /label="AmpR_promoter"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLY
                     GKPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIP
                     GKTAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGL
                     VDASDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVG
                     RVGIADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF*"
     misc_feature    complement(2394..2416)
                     /label="pGEX_3_primer"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLY
                     GKPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIP
                     GKTAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGL
                     VDASDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVG
                     RVGIADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF*"
     promoter        2687..3263
                     /label="CMV_immearly_promoter"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLY
                     GKPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIP
                     GKTAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGL
                     VDASDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVG
                     RVGIADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF*"
     misc_feature    2766..3053
                     /label="CAG_enhancer"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLY
                     GKPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIP
                     GKTAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGL
                     VDASDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVG
                     RVGIADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF*"
     misc_feature    3220..3240
                     /label="CMV_fwd_primer"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLY
                     GKPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIP
                     GKTAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGL
                     VDASDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVG
                     RVGIADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF*"
     promoter        3221..3290
                     /label="CMV_promoter"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLY
                     GKPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIP
                     GKTAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGL
                     VDASDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVG
                     RVGIADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF*"
     promoter        3233..3352
                     /label="CMV2_promoter"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLY
                     GKPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIP
                     GKTAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGL
                     VDASDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVG
                     RVGIADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF*"
     misc_feature    3264..3283
                     /label="pCEP_fwd_primer"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLY
                     GKPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIP
                     GKTAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGL
                     VDASDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVG
                     RVGIADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF*"
     misc_feature    3266..3290
                     /label="LNCX_primer"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLY
                     GKPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIP
                     GKTAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGL
                     VDASDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVG
                     RVGIADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF*"
     CDS             3725..5341
                     /label="ORF frame 2"
                     /translation="MGQTVTTPLSLTLGHWKDVERIAHNQSVDVKKRRWVTFCSAEWP
                     TFNVGWPRDGTFNRDLITQVKIKVFSPGPHGHPDQVPYIVTWEALAFDPPPWVKPFVH
                     PKPPPPLPPSAPSLPLEPPRSTPPRSSLYPALTPSLGAKPKPQVLSDSGGPLIDLLTE
                     DPPPYRDPRPPPSDRDGNGGEATPAGEAPDPSPMASRLRGRREPPVADSTTSQAFPLR
                     AGGNGQLQYWPFSSSDLYNWKNNNPSFSEDPGKLTALIESVLITHQPTWDDCQQLLGT
                     LLTGEEKQRVLLEARKAVRGDDGRPTQLPNEVDAAFPLERPDWDYTTQAGRNHLVHYR
                     QLLLAGLQNAGRSPTNLAKVKGITQGPNESPSAFLERLKEAYRRYTPYDPEDPGQETN
                     VSMSFIWQSAPDIGRKLERLEDLKNKTLGDLVREAEKIFNKRETPEEREERIRRETEE
                     KEERRRTEDEQKEKERDRRRHREMSKLLATVVSGQKQDRQGGERRRSQLDRDQCAYCK
                     EKGHWAKDCPKKPRGPRGPRPQTSLLTLDD*"
     misc_feature    3731..4138
                     /label="gag"
                     /translation="MGQTVTTPLSLTLGHWKDVERIAHNQSVDVKKRRWVTFCSAEWP
                     TFNVGWPRDGTFNRDLITQVKIKVFSPGPHGHPDQVPYIVTWEALAFDPPPWVKPFVH
                     PKPPPPLPPSAPSLPLEPPRSTPPRSSLYPALTPSLGAKPKPQVLSDSGGPLIDLLTE
                     DPPPYRDPRPPPSDRDGNGGEATPAGEAPDPSPMASRLRGRREPPVADSTTSQAFPLR
                     AGGNGQLQYWPFSSSDLYNWKNNNPSFSEDPGKLTALIESVLITHQPTWDDCQQLLGT
                     LLTGEEKQRVLLEARKAVRGDDGRPTQLPNEVDAAFPLERPDWDYTTQAGRNHLVHYR
                     QLLLAGLQNAGRSPTNLAKVKGITQGPNESPSAFLERLKEAYRRYTPYDPEDPGQETN
                     VSMSFIWQSAPDIGRKLERLEDLKNKTLGDLVREAEKIFNKRETPEEREERIRRETEE
                     KEERRRTEDEQKEKERDRRRHREMSKLLATVVSGQKQDRQGGERRRSQLDRDQCAYCK
                     EKGHWAKDCPKKPRGPRGPRPQTSLLTLDD*"
     misc_feature    4074..4096
                     /label="MSCV_primer"
                     /translation="MGQTVTTPLSLTLGHWKDVERIAHNQSVDVKKRRWVTFCSAEWP
                     TFNVGWPRDGTFNRDLITQVKIKVFSPGPHGHPDQVPYIVTWEALAFDPPPWVKPFVH
                     PKPPPPLPPSAPSLPLEPPRSTPPRSSLYPALTPSLGAKPKPQVLSDSGGPLIDLLTE
                     DPPPYRDPRPPPSDRDGNGGEATPAGEAPDPSPMASRLRGRREPPVADSTTSQAFPLR
                     AGGNGQLQYWPFSSSDLYNWKNNNPSFSEDPGKLTALIESVLITHQPTWDDCQQLLGT
                     LLTGEEKQRVLLEARKAVRGDDGRPTQLPNEVDAAFPLERPDWDYTTQAGRNHLVHYR
                     QLLLAGLQNAGRSPTNLAKVKGITQGPNESPSAFLERLKEAYRRYTPYDPEDPGQETN
                     VSMSFIWQSAPDIGRKLERLEDLKNKTLGDLVREAEKIFNKRETPEEREERIRRETEE
                     KEERRRTEDEQKEKERDRRRHREMSKLLATVVSGQKQDRQGGERRRSQLDRDQCAYCK
                     EKGHWAKDCPKKPRGPRGPRPQTSLLTLDD*"
     misc_feature    4074..4096
                     /label="pLXSN_5_primer"
                     /translation="MGQTVTTPLSLTLGHWKDVERIAHNQSVDVKKRRWVTFCSAEWP
                     TFNVGWPRDGTFNRDLITQVKIKVFSPGPHGHPDQVPYIVTWEALAFDPPPWVKPFVH
                     PKPPPPLPPSAPSLPLEPPRSTPPRSSLYPALTPSLGAKPKPQVLSDSGGPLIDLLTE
                     DPPPYRDPRPPPSDRDGNGGEATPAGEAPDPSPMASRLRGRREPPVADSTTSQAFPLR
                     AGGNGQLQYWPFSSSDLYNWKNNNPSFSEDPGKLTALIESVLITHQPTWDDCQQLLGT
                     LLTGEEKQRVLLEARKAVRGDDGRPTQLPNEVDAAFPLERPDWDYTTQAGRNHLVHYR
                     QLLLAGLQNAGRSPTNLAKVKGITQGPNESPSAFLERLKEAYRRYTPYDPEDPGQETN
                     VSMSFIWQSAPDIGRKLERLEDLKNKTLGDLVREAEKIFNKRETPEEREERIRRETEE
                     KEERRRTEDEQKEKERDRRRHREMSKLLATVVSGQKQDRQGGERRRSQLDRDQCAYCK
                     EKGHWAKDCPKKPRGPRGPRPQTSLLTLDD*"
     misc_feature    4112..4128
                     /label="pBABE_5_primer"
                     /translation="MGQTVTTPLSLTLGHWKDVERIAHNQSVDVKKRRWVTFCSAEWP
                     TFNVGWPRDGTFNRDLITQVKIKVFSPGPHGHPDQVPYIVTWEALAFDPPPWVKPFVH
                     PKPPPPLPPSAPSLPLEPPRSTPPRSSLYPALTPSLGAKPKPQVLSDSGGPLIDLLTE
                     DPPPYRDPRPPPSDRDGNGGEATPAGEAPDPSPMASRLRGRREPPVADSTTSQAFPLR
                     AGGNGQLQYWPFSSSDLYNWKNNNPSFSEDPGKLTALIESVLITHQPTWDDCQQLLGT
                     LLTGEEKQRVLLEARKAVRGDDGRPTQLPNEVDAAFPLERPDWDYTTQAGRNHLVHYR
                     QLLLAGLQNAGRSPTNLAKVKGITQGPNESPSAFLERLKEAYRRYTPYDPEDPGQETN
                     VSMSFIWQSAPDIGRKLERLEDLKNKTLGDLVREAEKIFNKRETPEEREERIRRETEE
                     KEERRRTEDEQKEKERDRRRHREMSKLLATVVSGQKQDRQGGERRRSQLDRDQCAYCK
                     EKGHWAKDCPKKPRGPRGPRPQTSLLTLDD*"
     CDS             5669..8941
                     /label="ORF frame 2"
                     /translation="MGPMGQPLQVLTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAW
                     AETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQ
                     SPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVL
                     DLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL
                     ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQ
                     VKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAP
                     LYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVL
                     TQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHA
                     VEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLD
                     ILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAG
                     TSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKN
                     KDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLL
                     IENSSPYTSEHFHYTVTDIKDLTKLGAIYDKTKKYWVYQGKPVMPDQFTFELLDFLHQ
                     LTHLSFSKMKALLERSHSPYYMLNRDRTLKNITETCKACAQVNASKSAVKQGTRVRGH
                     RPGTHWEIDFTEIKPGLYGYKYLLVFIDTFSGWIEAFPTKKETAKVVTKKLLEEIFPR
                     FGMPQVLGTDNGPAFVSKVSQTVADLLGIDWKLHCAYRPQSSGQVERMNRTIKETLTK
                     LTLATGSRDWVLLLPLALYRARNTPGPHGLTPYEILYGAPPPLVNFPDPDMTRVTNSP
                     SLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWK
                     GPYPVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLTWRVQRSQNPLKIRLTRE
                     AP*"
     misc_feature    8816..8832
                     /label="pBMN_5_primer"
                     /translation="MGPMGQPLQVLTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAW
                     AETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQ
                     SPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVL
                     DLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL
                     ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQ
                     VKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAP
                     LYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVL
                     TQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHA
                     VEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLD
                     ILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAG
                     TSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKN
                     KDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLL
                     IENSSPYTSEHFHYTVTDIKDLTKLGAIYDKTKKYWVYQGKPVMPDQFTFELLDFLHQ
                     LTHLSFSKMKALLERSHSPYYMLNRDRTLKNITETCKACAQVNASKSAVKQGTRVRGH
                     RPGTHWEIDFTEIKPGLYGYKYLLVFIDTFSGWIEAFPTKKETAKVVTKKLLEEIFPR
                     FGMPQVLGTDNGPAFVSKVSQTVADLLGIDWKLHCAYRPQSSGQVERMNRTIKETLTK
                     LTLATGSRDWVLLLPLALYRARNTPGPHGLTPYEILYGAPPPLVNFPDPDMTRVTNSP
                     SLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWK
                     GPYPVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLTWRVQRSQNPLKIRLTRE
                     AP*"
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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