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pEF1α-IRES-ZsGreen1

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  • Ybscience
  • 中国/美国
  • 2025年07月16日
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    • 详细信息
    • 文献和实验
    • 技术资料
    • 保存条件：

      -20℃

    • 保质期：

      6个月

    • 英文名：

      pEF1α-IRES-ZsGreen1

    • 库存：

      大量

    • 供应商：

      钰博生物

    • 规格：

      1ug/5ug

    pEF1α-IRES-ZsGreen1

    载体基本信息

    出品公司: Ybscience
    载体名称: pEF1α-IRES-ZsGreen1
    质粒类型: 哺乳动物表达载体；双顺反子载体；荧光报告载体
    高拷贝/低拷贝: 高拷贝
    克隆方法: 多克隆位点，限制性内切酶
    启动子: EF1α
    载体大小: 6040 bp
    5' 测序引物及序列: EF1-F: TCAAGCCTCAGACAGTGGTTC
    3' 测序引物及序列: IRES-R
    载体标签: --
    载体抗性: 卡那霉素
    筛选标记: Neomycin、ZsGreen1
    克隆菌株: DH5alpha
    宿主细胞（系）: 大部分细胞类型，包括原代细胞、分化细胞、干细胞。
    备注: pEF1α-IRES-ZsGreen1载体是哺乳动物表达载体；
    目的基因与报告基因在EF1启动子驱动下转录为双顺反子；
    二者之间的IRES元件，使得目的基因 与报告基因独立翻译。
    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

    pEF1α-IRES-ZsGreen1载体图谱



    pEF1α-IRES-ZsGreen1 多克隆位点
     

    载体简介

     

    载体介绍
pEF1α-IRES-ZsGreen1 is designed to simultaneously and constitutively express a protein of interest and ZsGreen1 in mammalian cells. ZsGreen1 is a human-codon-optimized variant of the reef coral Zoanthus sp. green fluorescent protein (ZsGreen) that has been engineered for brighter fluorescence (1–3). The excitation and emission maxima of native ZsGreen1 are 493 nm and 505 nm, respectively. Simultaneous expression of a protein of interest and ZsGreen1 is made possible by the presence of an encephalomyocarditis virus internal ribosome entry site (IRES2; 4, 5) positioned between the multiple cloning site (MCS) and the ZsGreen1 gene. The IRES allows a protein of interest and ZsGreen1 to be translated from a single bicistronic mRNA. Stable, constitutive expression of the bicistronic transcript is driven by the EF1α promoter (PEF1α), which continues to be constitutively active even after vector integration into the host cell genome (6).
The vector backbone also contains an SV40 origin for replication in mammalian cells expressing the SV40 large T antigen, a pUC origin of replication for propagation in E. coli, and an f1 origin for single-stranded DNA production. This vector also has a neomycin-resistance cassette (Neor) that allows G418 selection of stably transfected eukaryotic cells (7).

This cassette consists of the SV40 early promoter (PSV40 e), a Tn5 kanamycin/neomycin resistance gene, and herpes simplex virus thymidine kinase (HSV TK) polyadenylation signals. A bacterial promoter upstream of the cassette drives expression of the kanamycin resistance gene in E. coli.

载体特征
PEF1α (human elongation factor 1 alpha promoter): 12–1346
MCS (multiple cloning site): 1348–1422
IRES2 (internal ribosome entry site): 1423–2007
ZsGreen1 (human-codon-optimized): 1999–2703
SV40 early polyA signal: 2859–2909
f1 origin of replication: 2956–3411 (complementary)
SV40 origin of replication: 3752–3887
PSV40e (SV40 early promoter and enhancer sequences): 3585–3853
Kanr/Neor (kanamycin/neomycin resistance gene): 3936–4730
HSV TK polyA signals: 4966–4984
pUC origin of replication: 5315–5958

补充信息
pEF1α-IRES2-ZsGreen1 can be used to quickly identify cells expressing a gene of interest by screening for ZsGreen1 fluorescence. Genes inserted into the MCS must contain a start codon (ATG) and a stop codon. pEF1α-IRES2-ZsGreen1 can be introduced into mammalian cells using any standard transfection method. Cells expressing ZsGreen1 can be detected by flow cytometry or fluorescence microscopy 24 hours after transfection. However, in some cases, up to 48 hours may be required for detection. If required, stable transformants can be selected using G418 (7).
Propagation in E. coli
Suitable host strains: DH5α and other general purpose strains. Single-stranded DNA production requires a host containing an F plasmid such as JM101 or XL1-Blue.
Selectable marker: plasmid confers resistance to kanamycin (50 μg/ml) in E. coli hosts.
E. coli replication origin: pUC
Copy number: high
Excitation and Emission Maxima of ZsGreen1
Excitation: 493 nm
Emission: 505 nm

     

    载体序列

    LOCUS       pEF1α-IRES-ZsGreen1	6040 bp 	DNA	SYN
DEFINITION  pEF1α-IRES-ZsGreen1
ACCESSION   
KEYWORDS    
SOURCE      
  ORGANISM  other sequences; artificial sequences; vectors.
FEATURES             Location/Qualifiers
     source          1..6040
                     /organism="pEF1α-IRES-ZsGreen1"
                     /mol_type="other DNA"
     promoter        159..1346
                     /label="EF1a_promoter"
     CDS             complement(439..1086)
                     /label="ORF frame 2"
                     /translation="MKRRLRTERPFSFVWVTHPPALPSAASSILSSLQQGREAAIFPL
                     TQLVPTGPALPPRAGRYTAARGQAPEQAGQLETTPVRFSVAALAGPASPNMCAGTHGP
                     RRRPRPQKPKYQCADLGPHLQDYLARKKASQQVIKNFKWLETYRKQRDRREGATRFAR
                     GGPSAQARPQLKHEAKGLLKRKASNSPTHFQPEARDQESRTAARGVEVIQGTQGP*"
     misc_feature    1294..1314
                     /label="EF1a_fwd_primer"
                     /translation="MKRRLRTERPFSFVWVTHPPALPSAASSILSSLQQGREAAIFPL
                     TQLVPTGPALPPRAGRYTAARGQAPEQAGQLETTPVRFSVAALAGPASPNMCAGTHGP
                     RRRPRPQKPKYQCADLGPHLQDYLARKKASQQVIKNFKWLETYRKQRDRREGATRFAR
                     GGPSAQARPQLKHEAKGLLKRKASNSPTHFQPEARDQESRTAARGVEVIQGTQGP*"
     misc_feature    1423..2007
                     /label="IRES"
                     /translation="MKRRLRTERPFSFVWVTHPPALPSAASSILSSLQQGREAAIFPL
                     TQLVPTGPALPPRAGRYTAARGQAPEQAGQLETTPVRFSVAALAGPASPNMCAGTHGP
                     RRRPRPQKPKYQCADLGPHLQDYLARKKASQQVIKNFKWLETYRKQRDRREGATRFAR
                     GGPSAQARPQLKHEAKGLLKRKASNSPTHFQPEARDQESRTAARGVEVIQGTQGP*"
     CDS             1999..2706
                     /label="ORF frame 1"
                     /translation="MATTMAQSKHGLTKEMTMKYRMEGCVDGHKFVITGEGIGYPFKG
                     KQAINLCVVEGGPLPFAEDILSAAFMYGNRVFTEYPQDIVDYFKNSCPAGYTWDRSFL
                     FEDGAVCICNADITVSVEENCMYHESKFYGVNFPADGPVMKKMTDNWEPSCEKIIPVP
                     KQGILKGDVSMYLLLKDGGRLRCQFDTVYKAKSVPRKMPDWHFIQHKLTREDRSDAKN
                     QKWHLTEHAIASGSALP*"
     gene            2014..2703
                     /label="Zs_Green"
                     /gene="Zs_Green"
                     /translation="MATTMAQSKHGLTKEMTMKYRMEGCVDGHKFVITGEGIGYPFKG
                     KQAINLCVVEGGPLPFAEDILSAAFMYGNRVFTEYPQDIVDYFKNSCPAGYTWDRSFL
                     FEDGAVCICNADITVSVEENCMYHESKFYGVNFPADGPVMKKMTDNWEPSCEKIIPVP
                     KQGILKGDVSMYLLLKDGGRLRCQFDTVYKAKSVPRKMPDWHFIQHKLTREDRSDAKN
                     QKWHLTEHAIASGSALP*"
     misc_feature    2919..2938
                     /label="EBV_rev_primer"
                     /translation="MATTMAQSKHGLTKEMTMKYRMEGCVDGHKFVITGEGIGYPFKG
                     KQAINLCVVEGGPLPFAEDILSAAFMYGNRVFTEYPQDIVDYFKNSCPAGYTWDRSFL
                     FEDGAVCICNADITVSVEENCMYHESKFYGVNFPADGPVMKKMTDNWEPSCEKIIPVP
                     KQGILKGDVSMYLLLKDGGRLRCQFDTVYKAKSVPRKMPDWHFIQHKLTREDRSDAKN
                     QKWHLTEHAIASGSALP*"
     rep_origin      complement(3088..3394)
                     /label="f1_origin"
                     /translation="MATTMAQSKHGLTKEMTMKYRMEGCVDGHKFVITGEGIGYPFKG
                     KQAINLCVVEGGPLPFAEDILSAAFMYGNRVFTEYPQDIVDYFKNSCPAGYTWDRSFL
                     FEDGAVCICNADITVSVEENCMYHESKFYGVNFPADGPVMKKMTDNWEPSCEKIIPVP
                     KQGILKGDVSMYLLLKDGGRLRCQFDTVYKAKSVPRKMPDWHFIQHKLTREDRSDAKN
                     QKWHLTEHAIASGSALP*"
     promoter        3473..3501
                     /label="AmpR_promoter"
                     /translation="MATTMAQSKHGLTKEMTMKYRMEGCVDGHKFVITGEGIGYPFKG
                     KQAINLCVVEGGPLPFAEDILSAAFMYGNRVFTEYPQDIVDYFKNSCPAGYTWDRSFL
                     FEDGAVCICNADITVSVEENCMYHESKFYGVNFPADGPVMKKMTDNWEPSCEKIIPVP
                     KQGILKGDVSMYLLLKDGGRLRCQFDTVYKAKSVPRKMPDWHFIQHKLTREDRSDAKN
                     QKWHLTEHAIASGSALP*"
     misc_feature    complement(3567..3587)
                     /label="pBABE_3_primer"
                     /translation="MATTMAQSKHGLTKEMTMKYRMEGCVDGHKFVITGEGIGYPFKG
                     KQAINLCVVEGGPLPFAEDILSAAFMYGNRVFTEYPQDIVDYFKNSCPAGYTWDRSFL
                     FEDGAVCICNADITVSVEENCMYHESKFYGVNFPADGPVMKKMTDNWEPSCEKIIPVP
                     KQGILKGDVSMYLLLKDGGRLRCQFDTVYKAKSVPRKMPDWHFIQHKLTREDRSDAKN
                     QKWHLTEHAIASGSALP*"
     misc_feature    complement(3573..3788)
                     /label="SV40_enhancer"
                     /translation="MATTMAQSKHGLTKEMTMKYRMEGCVDGHKFVITGEGIGYPFKG
                     KQAINLCVVEGGPLPFAEDILSAAFMYGNRVFTEYPQDIVDYFKNSCPAGYTWDRSFL
                     FEDGAVCICNADITVSVEENCMYHESKFYGVNFPADGPVMKKMTDNWEPSCEKIIPVP
                     KQGILKGDVSMYLLLKDGGRLRCQFDTVYKAKSVPRKMPDWHFIQHKLTREDRSDAKN
                     QKWHLTEHAIASGSALP*"
     promoter        3585..3853
                     /label="SV40_promoter"
                     /translation="MATTMAQSKHGLTKEMTMKYRMEGCVDGHKFVITGEGIGYPFKG
                     KQAINLCVVEGGPLPFAEDILSAAFMYGNRVFTEYPQDIVDYFKNSCPAGYTWDRSFL
                     FEDGAVCICNADITVSVEENCMYHESKFYGVNFPADGPVMKKMTDNWEPSCEKIIPVP
                     KQGILKGDVSMYLLLKDGGRLRCQFDTVYKAKSVPRKMPDWHFIQHKLTREDRSDAKN
                     QKWHLTEHAIASGSALP*"
     rep_origin      3752..3829
                     /label="SV40_origin"
                     /translation="MATTMAQSKHGLTKEMTMKYRMEGCVDGHKFVITGEGIGYPFKG
                     KQAINLCVVEGGPLPFAEDILSAAFMYGNRVFTEYPQDIVDYFKNSCPAGYTWDRSFL
                     FEDGAVCICNADITVSVEENCMYHESKFYGVNFPADGPVMKKMTDNWEPSCEKIIPVP
                     KQGILKGDVSMYLLLKDGGRLRCQFDTVYKAKSVPRKMPDWHFIQHKLTREDRSDAKN
                     QKWHLTEHAIASGSALP*"
     misc_feature    3814..3833
                     /label="SV40pro_F_primer"
                     /translation="MATTMAQSKHGLTKEMTMKYRMEGCVDGHKFVITGEGIGYPFKG
                     KQAINLCVVEGGPLPFAEDILSAAFMYGNRVFTEYPQDIVDYFKNSCPAGYTWDRSFL
                     FEDGAVCICNADITVSVEENCMYHESKFYGVNFPADGPVMKKMTDNWEPSCEKIIPVP
                     KQGILKGDVSMYLLLKDGGRLRCQFDTVYKAKSVPRKMPDWHFIQHKLTREDRSDAKN
                     QKWHLTEHAIASGSALP*"
     CDS             3936..4730
                     /label="ORF frame 3"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR
                     PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL
                     LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY
                     QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*"
     gene            3939..4727
                     /label="NeoR/KanR"
                     /gene="NeoR/KanR"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR
                     PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL
                     LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY
                     QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*"
     CDS             complement(4245..4781)
                     /label="ORF frame 3"
                     /translation="MAGWASLGRSFRTPESRSEELVKKAIEGDALRIGSGDTVKHEEA
                     VSPFAAKLFSNITGSQRYVLIAVRHTQPATVDESRKAAIFHHDIRQAGIAMGHDEILA
                     VGHARLEPGEQFGWREPLMLFVQIILIDKTGFHPSTCSLDAMFRLVVEWAGSRIKRMQ
                     PPHCISHDGYFLGRSKVR*"
     terminator      4905..5174
                     /label="TK_PA_terminator"
                     /translation="MAGWASLGRSFRTPESRSEELVKKAIEGDALRIGSGDTVKHEEA
                     VSPFAAKLFSNITGSQRYVLIAVRHTQPATVDESRKAAIFHHDIRQAGIAMGHDEILA
                     VGHARLEPGEQFGWREPLMLFVQIILIDKTGFHPSTCSLDAMFRLVVEWAGSRIKRMQ
                     PPHCISHDGYFLGRSKVR*"
     rep_origin      5322..5941
                     /label="pBR322_origin"
                     /translation="MAGWASLGRSFRTPESRSEELVKKAIEGDALRIGSGDTVKHEEA
                     VSPFAAKLFSNITGSQRYVLIAVRHTQPATVDESRKAAIFHHDIRQAGIAMGHDEILA
                     VGHARLEPGEQFGWREPLMLFVQIILIDKTGFHPSTCSLDAMFRLVVEWAGSRIKRMQ
                     PPHCISHDGYFLGRSKVR*"
ORIGIN
    1 TAGTTATTAA TGAGTAATTC ATACAAAAGG ACTCGCCCCT GCCTTGGGGA ATCCCAGGGA
   61 CCGTCGTTAA ACTCCCACTA ACGTAGAACC CAGAGATCGC TGCGTTCCCG CCCCCTCACC
  121 CGCCCGCTCT CGTCATCACT GAGGTGGAGA AGAGCATGCG TGAGGCTCCG GTGCCCGTCA
  181 GTGGGCAGAG CGCACATCGC CCACAGTCCC CGAGAAGTTG GGGGGAGGGG TCGGCAATTG
  241 AACCGGTGCC TAGAGAAGGT GGCGCGGGGT AAACTGGGAA AGTGATGTCG TGTACTGGCT
  301 CCGCCTTTTT CCCGAGGGTG GGGGAGAACC GTATATAAGT GCAGTAGTCG CCGTGAACGT
  361 TCTTTTTCGC AACGGGTTTG CCGCCAGAAC ACAGGTAAGT GCCGTGTGTG GTTCCCGCGG
  421 GCCTGGCCTC TTTACGGGTT ATGGCCCTTG CGTGCCTTGA ATTACTTCCA CGCCCCTGGC
  481 TGCAGTACGT GATTCTTGAT CCCGAGCTTC GGGTTGGAAG TGGGTGGGAG AGTTCGAGGC
  541 CTTGCGCTTA AGGAGCCCCT TCGCCTCGTG CTTGAGTTGA GGCCTGGCTT GGGCGCTGGG
  601 GCCGCCGCGT GCGAATCTGG TGGCACCTTC GCGCCTGTCT CGCTGCTTTC GATAAGTCTC
  661 TAGCCATTTA AAATTTTTGA TGACCTGCTG CGACGCTTTT TTTCTGGCAA GATAGTCTTG
  721 TAAATGCGGG CCAAGATCTG CACACTGGTA TTTCGGTTTT TGGGGCCGCG GGCGGCGACG
  781 GGGCCCGTGC GTCCCAGCGC ACATGTTCGG CGAGGCGGGG CCTGCGAGCG CGGCCACCGA
  841 GAATCGGACG GGGGTAGTCT CAAGCTGGCC GGCCTGCTCT GGTGCCTGGC CTCGCGCCGC
  901 CGTGTATCGC CCCGCCCTGG GCGGCAAGGC TGGCCCGGTC GGCACCAGTT GCGTGAGCGG
  961 AAAGATGGCC GCTTCCCGGC CCTGCTGCAG GGAGCTCAAA ATGGAGGACG CGGCGCTCGG
 1021 GAGAGCGGGC GGGTGAGTCA CCCACACAAA GGAAAAGGGC CTTTCCGTCC TCAGCCGTCG
 1081 CTTCATGTGA CTCCACGGAG TACCGGGCGC CGTCCAGGCA CCTCGATTAG TTCTCGAGCT
 1141 TTTGGAGTAC GTCGTCTTTA GGTTGGGGGG AGGGGTTTTA TGCGATGGAG TTTCCCCACA
 1201 CTGAGTGGGT GGAGACTGAA GTTAGGCCAG CTTGGCACTT GATGTAATTC TCCTTGGAAT
 1261 TTGCCCTTTT TGAGTTTGGA TCTTGGTTCA TTCTCAAGCC TCAGACAGTG GTTCAAAGTT
 1321 TTTTTCTTCC ATTTCAGGTG TCGTGACGCT AGCGCTACCG GACTCAGATC TCGAGCTCAA
 1381 GCTTCGAATT CTGCAGTCGA CGGTACCGCG GGCCCGGGAT CCGCCCCTCT CCCTCCCCCC
 1441 CCCCTAACGT TACTGGCCGA AGCCGCTTGG AATAAGGCCG GTGTGCGTTT GTCTATATGT
 1501 TATTTTCCAC CATATTGCCG TCTTTTGGCA ATGTGAGGGC CCGGAAACCT GGCCCTGTCT
 1561 TCTTGACGAG CATTCCTAGG GGTCTTTCCC CTCTCGCCAA AGGAATGCAA GGTCTGTTGA
 1621 ATGTCGTGAA GGAAGCAGTT CCTCTGGAAG CTTCTTGAAG ACAAACAACG TCTGTAGCGA
 1681 CCCTTTGCAG GCAGCGGAAC CCCCCACCTG GCGACAGGTG CCTCTGCGGC CAAAAGCCAC
 1741 GTGTATAAGA TACACCTGCA AAGGCGGCAC AACCCCAGTG CCACGTTGTG AGTTGGATAG
 1801 TTGTGGAAAG AGTCAAATGG CTCTCCTCAA GCGTATTCAA CAAGGGGCTG AAGGATGCCC
 1861 AGAAGGTACC CCATTGTATG GGATCTGATC TGGGGCCTCG GTACACATGC TTTACATGTG
 1921 TTTAGTCGAG GTTAAAAAAA CGTCTAGGCC CCCCGAACCA CGGGGACGTG GTTTTCCTTT
 1981 GAAAAACACG ATGATAATAT GGCCACAACC ATGGCCCAGT CCAAGCACGG CCTGACCAAG
 2041 GAGATGACCA TGAAGTACCG CATGGAGGGC TGCGTGGACG GCCACAAGTT CGTGATCACC
 2101 GGCGAGGGCA TCGGCTACCC CTTCAAGGGC AAGCAGGCCA TCAACCTGTG CGTGGTGGAG
 2161 GGCGGCCCCT TGCCCTTCGC CGAGGACATC TTGTCCGCCG CCTTCATGTA CGGCAACCGC
 2221 GTGTTCACCG AGTACCCCCA GGACATCGTC GACTACTTCA AGAACTCCTG CCCCGCCGGC
 2281 TACACCTGGG ACCGCTCCTT CCTGTTCGAG GACGGCGCCG TGTGCATCTG CAACGCCGAC
 2341 ATCACCGTGA GCGTGGAGGA GAACTGCATG TACCACGAGT CCAAGTTCTA CGGCGTGAAC
 2401 TTCCCCGCCG ACGGCCCCGT GATGAAGAAG ATGACCGACA ACTGGGAGCC CTCCTGCGAG
 2461 AAGATCATCC CCGTGCCCAA GCAGGGCATC TTGAAGGGCG ACGTGAGCAT GTACCTGCTG
 2521 CTGAAGGACG GTGGCCGCTT GCGCTGCCAG TTCGACACCG TGTACAAGGC CAAGTCCGTG
 2581 CCCCGCAAGA TGCCCGACTG GCACTTCATC CAGCACAAGC TGACCCGCGA GGACCGCAGC
 2641 GACGCCAAGA ACCAGAAGTG GCACCTGACC GAGCACGCCA TCGCCTCCGG CTCCGCCTTG
 2701 CCCTGAGCGG CCGCGACTCT AGATCATAAT CAGCCATACC ACATTTGTAG AGGTTTTACT
 2761 TGCTTTAAAA AACCTCCCAC ACCTCCCCCT GAACCTGAAA CATAAAATGA ATGCAATTGT
 2821 TGTTGTTAAC TTGTTTATTG CAGCTTATAA TGGTTACAAA TAAAGCAATA GCATCACAAA
 2881 TTTCACAAAT AAAGCATTTT TTTCACTGCA TTCTAGTTGT GGTTTGTCCA AACTCATCAA
 2941 TGTATCTTAA GGCGTAAATT GTAAGCGTTA ATATTTTGTT AAAATTCGCG TTAAATTTTT
 3001 GTTAAATCAG CTCATTTTTT AACCAATAGG CCGAAATCGG CAAAATCCCT TATAAATCAA
 3061 AAGAATAGAC CGAGATAGGG TTGAGTGTTG TTCCAGTTTG GAACAAGAGT CCACTATTAA
 3121 AGAACGTGGA CTCCAACGTC AAAGGGCGAA AAACCGTCTA TCAGGGCGAT GGCCCACTAC
 3181 GTGAACCATC ACCCTAATCA AGTTTTTTGG GGTCGAGGTG CCGTAAAGCA CTAAATCGGA
 3241 ACCCTAAAGG GAGCCCCCGA TTTAGAGCTT GACGGGGAAA GCCGGCGAAC GTGGCGAGAA
 3301 AGGAAGGGAA GAAAGCGAAA GGAGCGGGCG CTAGGGCGCT GGCAAGTGTA GCGGTCACGC
 3361 TGCGCGTAAC CACCACACCC GCCGCGCTTA ATGCGCCGCT ACAGGGCGCG TCAGGTGGCA
 3421 CTTTTCGGGG AAATGTGCGC GGAACCCCTA TTTGTTTATT TTTCTAAATA CATTCAAATA
 3481 TGTATCCGCT CATGAGACAA TAACCCTGAT AAATGCTTCA ATAATATTGA AAAAGGAAGA
 3541 GTCCTGAGGC GGAAAGAACC AGCTGTGGAA TGTGTGTCAG TTAGGGTGTG GAAAGTCCCC
 3601 AGGCTCCCCA GCAGGCAGAA GTATGCAAAG CATGCATCTC AATTAGTCAG CAACCAGGTG
 3661 TGGAAAGTCC CCAGGCTCCC CAGCAGGCAG AAGTATGCAA AGCATGCATC TCAATTAGTC
 3721 AGCAACCATA GTCCCGCCCC TAACTCCGCC CATCCCGCCC CTAACTCCGC CCAGTTCCGC
 3781 CCATTCTCCG CCCCATGGCT GACTAATTTT TTTTATTTAT GCAGAGGCCG AGGCCGCCTC
 3841 GGCCTCTGAG CTATTCCAGA AGTAGTGAGG AGGCTTTTTT GGAGGCCTAG GCTTTTGCAA
 3901 AGATCGATCA AGAGACAGGA TGAGGATCGT TTCGCATGAT TGAACAAGAT GGATTGCACG
 3961 CAGGTTCTCC GGCCGCTTGG GTGGAGAGGC TATTCGGCTA TGACTGGGCA CAACAGACAA
 4021 TCGGCTGCTC TGATGCCGCC GTGTTCCGGC TGTCAGCGCA GGGGCGCCCG GTTCTTTTTG
 4081 TCAAGACCGA CCTGTCCGGT GCCCTGAATG AACTGCAAGA CGAGGCAGCG CGGCTATCGT
 4141 GGCTGGCCAC GACGGGCGTT CCTTGCGCAG CTGTGCTCGA CGTTGTCACT GAAGCGGGAA
 4201 GGGACTGGCT GCTATTGGGC GAAGTGCCGG GGCAGGATCT CCTGTCATCT CACCTTGCTC
 4261 CTGCCGAGAA AGTATCCATC ATGGCTGATG CAATGCGGCG GCTGCATACG CTTGATCCGG
 4321 CTACCTGCCC ATTCGACCAC CAAGCGAAAC ATCGCATCGA GCGAGCACGT ACTCGGATGG
 4381 AAGCCGGTCT TGTCGATCAG GATGATCTGG ACGAAGAGCA TCAGGGGCTC GCGCCAGCCG
 4441 AACTGTTCGC CAGGCTCAAG GCGAGCATGC CCGACGGCGA GGATCTCGTC GTGACCCATG
 4501 GCGATGCCTG CTTGCCGAAT ATCATGGTGG AAAATGGCCG CTTTTCTGGA TTCATCGACT
 4561 GTGGCCGGCT GGGTGTGGCG GACCGCTATC AGGACATAGC GTTGGCTACC CGTGATATTG
 4621 CTGAAGAGCT TGGCGGCGAA TGGGCTGACC GCTTCCTCGT GCTTTACGGT ATCGCCGCTC
 4681 CCGATTCGCA GCGCATCGCC TTCTATCGCC TTCTTGACGA GTTCTTCTGA GCGGGACTCT
 4741 GGGGTTCGAA ATGACCGACC AAGCGACGCC CAACCTGCCA TCACGAGATT TCGATTCCAC
 4801 CGCCGCCTTC TATGAAAGGT TGGGCTTCGG AATCGTTTTC CGGGACGCCG GCTGGATGAT
 4861 CCTCCAGCGC GGGGATCTCA TGCTGGAGTT CTTCGCCCAC CCTAGGGGGA GGCTAACTGA
 4921 AACACGGAAG GAGACAATAC CGGAAGGAAC CCGCGCTATG ACGGCAATAA AAAGACAGAA
 4981 TAAAACGCAC GGTGTTGGGT CGTTTGTTCA TAAACGCGGG GTTCGGTCCC AGGGCTGGCA
 5041 CTCTGTCGAT ACCCCACCGA GACCCCATTG GGGCCAATAC GCCCGCGTTT CTTCCTTTTC
 5101 CCCACCCCAC CCCCCAAGTT CGGGTGAAGG CCCAGGGCTC GCAGCCAACG TCGGGGCGGC
 5161 AGGCCCTGCC ATAGCCTCAG GTTACTCATA TATACTTTAG ATTGATTTAA AACTTCATTT
 5221 TTAATTTAAA AGGATCTAGG TGAAGATCCT TTTTGATAAT CTCATGACCA AAATCCCTTA
 5281 ACGTGAGTTT TCGTTCCACT GAGCGTCAGA CCCCGTAGAA AAGATCAAAG GATCTTCTTG
 5341 AGATCCTTTT TTTCTGCGCG TAATCTGCTG CTTGCAAACA AAAAAACCAC CGCTACCAGC
 5401 GGTGGTTTGT TTGCCGGATC AAGAGCTACC AACTCTTTTT CCGAAGGTAA CTGGCTTCAG
 5461 CAGAGCGCAG ATACCAAATA CTGTTCTTCT AGTGTAGCCG TAGTTAGGCC ACCACTTCAA
 5521 GAACTCTGTA GCACCGCCTA CATACCTCGC TCTGCTAATC CTGTTACCAG TGGCTGCTGC
 5581 CAGTGGCGAT AAGTCGTGTC TTACCGGGTT GGACTCAAGA CGATAGTTAC CGGATAAGGC
 5641 GCAGCGGTCG GGCTGAACGG GGGGTTCGTG CACACAGCCC AGCTTGGAGC GAACGACCTA
 5701 CACCGAACTG AGATACCTAC AGCGTGAGCT ATGAGAAAGC GCCACGCTTC CCGAAGGGAG
 5761 AAAGGCGGAC AGGTATCCGG TAAGCGGCAG GGTCGGAACA GGAGAGCGCA CGAGGGAGCT
 5821 TCCAGGGGGA AACGCCTGGT ATCTTTATAG TCCTGTCGGG TTTCGCCACC TCTGACTTGA
 5881 GCGTCGATTT TTGTGATGCT CGTCAGGGGG GCGGAGCCTA TGGAAAAACG CCAGCAACGC
 5941 GGCCTTTTTA CGGTTCCTGG CCTTTTGCTG GCCTTTTGCT CACATGTTCT TTCCTGCGTT
 6001 ATCCCCTGAT TCTGTGGATA ACCGTATTAC CGCCATGCAT

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