pRetroQ-AcGFP1-N1

pRetroQ-AcGFP1-N1

载体基本信息

出品公司:	Ybscience
载体名称:	pRetroQ-AcGFP1-N1
质粒类型:	逆病毒载体；荧光报告载体
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 IE
载体大小:	7606 bp
5' 测序引物及序列:	--
3' 测序引物及序列:	--
载体标签:	AcGFP1(C-端）
载体抗性:	氨苄青霉素
筛选标记:	嘌呤霉素（Puromycin）
克隆菌株:	DH5α
宿主细胞（系）:	常规细胞系（293、CV-1、CHO等）
备注:	pRetroQ-AcGFP1-N1载体是自我失活型逆病毒载体，表达C端AcGFP1融合蛋白； pRetroQ-AcGFP1-N1载体能够高效递送目的基因到靶细胞中，尤其适用于原代细胞和其它难转染的细胞系。
稳定性:	稳表达
组成型/诱导型:	组成型
病毒/非病毒:	逆转录病毒

 

载体质粒图谱和多克隆位点信息

载体简介

 

pRetroQ-AcGFP1-N1载体描述

pRetroQ-AcGFP1-N1 is a high-titer, self-inactivating retroviral vector that facilitates efficient delivery and expression of AcGFP1, as well as N terminal fusions of AcGFP1, to target cells. AcGFP1 is the green fluorescent protein from Aequorea coerulescens (AcGFP1; Excitation maximum = 475 nm; emission maximum = 505 nm).
The coding sequence of the AcGFP1 gene contains silent base changes, which correspond to human codon-usage preferences (1). The MCS in pRetroQAcGFP1-N1 lies between the immediate early promoter of CMV (PCMV IE) and the AcGFP1 coding sequences. Genes cloned into the MCS are expressed as fusions to the N-terminus of AcGFP1 when they are in the same reading frame as AcGFP1 and there are no intervening stop codons.
The RetroQ retroviral vector backbone incorporates several unique features. This vector contains a puromycin resistance cassette (Puror) driven by the PGK promoter (PPGK) for selection of positively-infected cells (2).The hybrid 5’ long terminal repeat (LTR) consists of the CMV type I enhancer and the murine sarcoma virus (MSV) promoter. This vector demonstrates high levels of transcription in HEK 293-based packaging cell lines due, in part, to the presence of adenoviral E1A (3–6) in these cells. The self-inactivating feature of the vector is provided by a deletion in the 3’ LTR enhancer region (U3). During reverse transcription of the retroviral RNA, the inactivated 3’ LTR is copied and replaces the 5’ LTR CMV enhancer sequences. This can reduce the phenomenon known as promoter interference (7) and allow more efficient expression.

Additionally, the viral genomic transcript contains the necessary viral RNA processing elements, including the LTRs, packaging signal (ψ+), and tRNA primer binding site. pRetroQ-AcGFP1-N1 contains a bacterial origin of replication, an E. coli Ampr gene for propagation and selection in bacteria, and an SV40 origin for replication in mammalian cells expressing the SV40 large T antigen.

pRetroQ-AcGFP1-N1 is designed to efficiently deliver and express fusions to the N terminus of AcGFP1 into primary cells or cells that are difficult to transfect. Fusions to the N terminus of AcGFP1 retain the fluorescent properties of the native protein, allowing the in vivo localization of the fusion protein. The target gene should be cloned into pRetroQ AcGFP1-N1 so that it is in frame with the AcGFP1 coding sequences, with no intervening in-frame stop codons. The inserted gene should include the initiating ATG codon. The recombinant AcGFP1 vector can be infected or transfected into mammalian cells. If required, stable transformants can be selected using puromycin. pRetroQ-AcGFP1-N1 can also be used simply to express AcGFP1 in a cell line of interest (e.g., as an infection marker).
Once pRetroQ-AcGFP1-N1 is transfected into a packaging cell line (such as the RetroPack PT67 Cell line, AmphoPack-293, EcoPack2-293, Pantropic Expression System, or Retro-X Universal Packaging System , RNA from the vector is packaged into non-infectious, replication-incompetent retroviral particles, since pRetroQ-AcGFP1-N1 lacks the structural genes (gag, pol, and env) necessary for particle formation and replication. These genes, however, are stably integrated as part of the packaging cell genome. Once a high-titer supernatant is produced, these retroviral particles can infect target cells and transmit the gene of interest but cannot replicate within these cells due to the absence of viral structural genes. The separate introduction and integration of the structural genes into the packaging cell line minimizes the chances of producing replication-competent virus due to recombination events during cell proliferation.

Propagation in E. coli
 Suitable host strains: DH5α, Fusion Blue, and other general-purpose strains.
 Selectable marker: plasmid confers resistance to ampicillin (100 μg/ml) in E. coli hosts.
 E. coli replication origin: ColE1
 Copy number: low

 

载体序列

LOCUS       pRetroQ-AcGFP1-N1	7606 bp 	DNA	SYN
DEFINITION  pRetroQ-AcGFP1-N1
ACCESSION   
KEYWORDS    
SOURCE      
  ORGANISM  other sequences; artificial sequences; vectors.
FEATURES             Location/Qualifiers
     source          1..7606
                     /organism="pRetroQ-AcGFP1-N1"
                     /mol_type="other DNA"
     promoter        2..506
                     /label="CMV_immearly_promoter"
     misc_feature    6..688
                     /label="5_LTR"
     misc_feature    9..296
                     /label="CAG_enhancer"
     misc_feature    463..483
                     /label="CMV_fwd_primer"
     misc_feature    758..1567
                     /label="psi_plus_pack"
     misc_feature    1154..1561
                     /label="gag"
     misc_feature    1497..1519
                     /label="MSCV_primer"
     misc_feature    1497..1519
                     /label="pLXSN_5_primer"
     misc_feature    1535..1551
                     /label="pBABE_5_primer"
     promoter        1591..2143
                     /label="CMV_immearly_promoter"
     misc_feature    1646..1933
                     /label="CAG_enhancer"
     misc_feature    2100..2120
                     /label="CMV_fwd_primer"
     promoter        2101..2170
                     /label="CMV_promoter"
     CDS             2254..2973
                     /label="ORF frame 1"
                     /translation="MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKLT
                     LKFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFE
                     DDGNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKN
                     GIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDH
                     MIYFGFVTAAAITHGMDELYK*"
     gene            2257..2970
                     /label="AcGFP1"
                     /gene="AcGFP1"
                     /translation="MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKLT
                     LKFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFE
                     DDGNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKN
                     GIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDH
                     MIYFGFVTAAAITHGMDELYK*"
     misc_feature    complement(3036..3059)
                     /label="MSCV_rev_primer"
                     /translation="MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKLT
                     LKFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFE
                     DDGNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKN
                     GIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDH
                     MIYFGFVTAAAITHGMDELYK*"
     CDS             3281..4120
                     /label="ORF frame 2"
                     /translation="MEAGRPLGQRPIAALLLRFLGSEAGKGWVRGRAQGRAQGRGGRP
                     KVLRRPGILHASKAHVCRAVLLFLISGPFDLQPKLTMTEYKPTVRLATRDDVPRAVRT
                     LAAAFADYPATRHTVDPDRHIERVTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPE
                     SVEAGAVFAEIGPRMAELSGSRLAAQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKG
                     LGSAVVLPGVEAAERAGVPAFLETSAPRNLPFYERLGFTVTADVEVPEGPRTWCMTRK
                     PGA*"
     misc_feature    3433..3452
                     /label="mPGK_F_primer"
                     /translation="MEAGRPLGQRPIAALLLRFLGSEAGKGWVRGRAQGRAQGRGGRP
                     KVLRRPGILHASKAHVCRAVLLFLISGPFDLQPKLTMTEYKPTVRLATRDDVPRAVRT
                     LAAAFADYPATRHTVDPDRHIERVTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPE
                     SVEAGAVFAEIGPRMAELSGSRLAAQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKG
                     LGSAVVLPGVEAAERAGVPAFLETSAPRNLPFYERLGFTVTADVEVPEGPRTWCMTRK
                     PGA*"
     CDS             complement(3446..4171)
                     /label="ORF frame 1"
                     /translation="MGSCAPFGRALRVVGRASGTGLAGHAPGARSFGHLDVGGDGEAE
                     PLVEGEVAGRGGLQEGGHPGALGRLHSGEHDGAAQTLALVVGRDADGGQEPRGLLGPV
                     RRQEAFHLLLRGQPGTAQLGHARADLGEHRPRFDALRRGPDRHRGAVVRDPHLADVEP
                     DAREEEFLQLGDPLDVAVRIDGVARGGVVGERGGEGAYGPGDVVAGGEAHRGLVLGHG
                     KLGLQVERPGDEEEENSAADVRF*"
     gene            3521..4120
                     /label="puro (variant)"
                     /gene="puro (variant)"
                     /translation="MGSCAPFGRALRVVGRASGTGLAGHAPGARSFGHLDVGGDGEAE
                     PLVEGEVAGRGGLQEGGHPGALGRLHSGEHDGAAQTLALVVGRDADGGQEPRGLLGPV
                     RRQEAFHLLLRGQPGTAQLGHARADLGEHRPRFDALRRGPDRHRGAVVRDPHLADVEP
                     DAREEEFLQLGDPLDVAVRIDGVARGGVVGERGGEGAYGPGDVVAGGEAHRGLVLGHG
                     KLGLQVERPGDEEEENSAADVRF*"
     misc_feature    complement(4840..4862)
                     /label="pGEX_3_primer"
                     /translation="MGSCAPFGRALRVVGRASGTGLAGHAPGARSFGHLDVGGDGEAE
                     PLVEGEVAGRGGLQEGGHPGALGRLHSGEHDGAAQTLALVVGRDADGGQEPRGLLGPV
                     RRQEAFHLLLRGQPGTAQLGHARADLGEHRPRFDALRRGPDRHRGAVVRDPHLADVEP
                     DAREEEFLQLGDPLDVAVRIDGVARGGVVGERGGEGAYGPGDVVAGGEAHRGLVLGHG
                     KLGLQVERPGDEEEENSAADVRF*"
     misc_feature    complement(4998..5018)
                     /label="pBABE_3_primer"
                     /translation="MGSCAPFGRALRVVGRASGTGLAGHAPGARSFGHLDVGGDGEAE
                     PLVEGEVAGRGGLQEGGHPGALGRLHSGEHDGAAQTLALVVGRDADGGQEPRGLLGPV
                     RRQEAFHLLLRGQPGTAQLGHARADLGEHRPRFDALRRGPDRHRGAVVRDPHLADVEP
                     DAREEEFLQLGDPLDVAVRIDGVARGGVVGERGGEGAYGPGDVVAGGEAHRGLVLGHG
                     KLGLQVERPGDEEEENSAADVRF*"
     misc_feature    complement(5004..5219)
                     /label="SV40_enhancer"
                     /translation="MGSCAPFGRALRVVGRASGTGLAGHAPGARSFGHLDVGGDGEAE
                     PLVEGEVAGRGGLQEGGHPGALGRLHSGEHDGAAQTLALVVGRDADGGQEPRGLLGPV
                     RRQEAFHLLLRGQPGTAQLGHARADLGEHRPRFDALRRGPDRHRGAVVRDPHLADVEP
                     DAREEEFLQLGDPLDVAVRIDGVARGGVVGERGGEGAYGPGDVVAGGEAHRGLVLGHG
                     KLGLQVERPGDEEEENSAADVRF*"
     promoter        5016..5284
                     /label="SV40_promoter"
                     /translation="MGSCAPFGRALRVVGRASGTGLAGHAPGARSFGHLDVGGDGEAE
                     PLVEGEVAGRGGLQEGGHPGALGRLHSGEHDGAAQTLALVVGRDADGGQEPRGLLGPV
                     RRQEAFHLLLRGQPGTAQLGHARADLGEHRPRFDALRRGPDRHRGAVVRDPHLADVEP
                     DAREEEFLQLGDPLDVAVRIDGVARGGVVGERGGEGAYGPGDVVAGGEAHRGLVLGHG
                     KLGLQVERPGDEEEENSAADVRF*"
     rep_origin      5183..5260
                     /label="SV40_origin"
                     /translation="MGSCAPFGRALRVVGRASGTGLAGHAPGARSFGHLDVGGDGEAE
                     PLVEGEVAGRGGLQEGGHPGALGRLHSGEHDGAAQTLALVVGRDADGGQEPRGLLGPV
                     RRQEAFHLLLRGQPGTAQLGHARADLGEHRPRFDALRRGPDRHRGAVVRDPHLADVEP
                     DAREEEFLQLGDPLDVAVRIDGVARGGVVGERGGEGAYGPGDVVAGGEAHRGLVLGHG
                     KLGLQVERPGDEEEENSAADVRF*"
     misc_feature    5245..5264
                     /label="SV40pro_F_primer"
                     /translation="MGSCAPFGRALRVVGRASGTGLAGHAPGARSFGHLDVGGDGEAE
                     PLVEGEVAGRGGLQEGGHPGALGRLHSGEHDGAAQTLALVVGRDADGGQEPRGLLGPV
                     RRQEAFHLLLRGQPGTAQLGHARADLGEHRPRFDALRRGPDRHRGAVVRDPHLADVEP
                     DAREEEFLQLGDPLDVAVRIDGVARGGVVGERGGEGAYGPGDVVAGGEAHRGLVLGHG
                     KLGLQVERPGDEEEENSAADVRF*"
     gene            complement(6384..7244)
                     /label="Ampicillin"
                     /gene="Ampicillin"
                     /translation="MGSCAPFGRALRVVGRASGTGLAGHAPGARSFGHLDVGGDGEAE
                     PLVEGEVAGRGGLQEGGHPGALGRLHSGEHDGAAQTLALVVGRDADGGQEPRGLLGPV
                     RRQEAFHLLLRGQPGTAQLGHARADLGEHRPRFDALRRGPDRHRGAVVRDPHLADVEP
                     DAREEEFLQLGDPLDVAVRIDGVARGGVVGERGGEGAYGPGDVVAGGEAHRGLVLGHG
                     KLGLQVERPGDEEEENSAADVRF*"
     CDS             complement(6384..7244)
                     /label="ORF frame 3"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVE
                     YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
                     DRWEPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
                     LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
                     EIGASLIKHW*"
     promoter        complement(7286..7314)
                     /label="AmpR_promoter"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVE
                     YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
                     DRWEPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
                     LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
                     EIGASLIKHW*"
     promoter        7536..506
                     /label="CMV_immearly_promoter"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVE
                     YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
                     DRWEPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
                     LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
                     EIGASLIKHW*"
     misc_feature    7600..688
                     /label="5_LTR"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVE
                     YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
                     DRWEPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
                     LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
                     EIGASLIKHW*"
ORIGIN
    1 TCCGCGTTAC ATAACTTACG GTAAATGGCC CGCCTGGCTG ACCGCCCAAC GACCCCCGCC
   61 CATTGACGTC AATAATGACG TATGTTCCCA TAGTAACGCC AATAGGGACT TTCCATTGAC
  121 GTCAATGGGT GGAGTATTTA CGGTAAACTG CCCACTTGGC AGTACATCAA GTGTATCATA
  181 TGCCAAGTAC GCCCCCTATT GACGTCAATG ACGGTAAATG GCCCGCCTGG CATTATGCCC
  241 AGTACATGAC CTTATGGGAC TTTCCTACTT GGCAGTACAT CTACGTATTA GTCATCGCTA
  301 TTACCATGGT GATGCGGTTT TGGCAGTACA TCAATGGGCG TGGATAGCGG TTTGACTCAC
  361 GGGGATTTCC AAGTCTCCAC CCCATTGACG TCAATGGGAG TTTGTTTTGG CACCAAAATC
  421 AACGGGACTT TCCAAAATGT CGTAACAACT CCGCCCCATT GACGCAAATG GGCGGTAGGC
  481 GTGTACGGTG GGAGGTCTAT ATAAGCAGAG CTCAATAAAA GAGCCCACAA CCCCTCACTC
  541 GGCGCGCCAG TCTTCCGATA GACTGCGTCG CCCGGGTACC CGTATTCCCA ATAAAGCCTC
  601 TTGCTGTTTG CATCCGAATC GTGGTCTCGC TGTTCCTTGG GAGGGTCTCC TCTGAGTGAT
  661 TGACTACCCA CGACGGGGGT CTTTCATTTG GGGGCTCGTC CGGGATTTGG AGACCCCTGC
  721 CCAGGGACCA CCGACCCACC ACCGGGAGGT AAGCTGGCCA GCAACTTATC TGTGTCTGTC
  781 CGATTGTCTA GTGTCTATGT TTGATGTTAT GCGCCTGCGT CTGTACTAGT TAGCTAACTA
  841 GCTCTGTATC TGGCGGACCC GTGGTGGAAC TGACGAGTTC TGAACACCCG GCCGCAACCC
  901 TGGGAGACGT CCCAGGGACT TTGGGGGCCG TTTTTGTGGC CCGACCTGAG GAAGGGAGTC
  961 GATGTGGAAT CCGACCCCGT CAGGATATGT GGTTCTGGTA GGAGACGAGA ACCTAAAACA
 1021 GTTCCCGCCT CCGTCTGAAT TTTTGCTTTC GGTTTGGAAC CGAAGCCGCG CGTCTTGTCT
 1081 GCTGCAGCGC TGCAGCATCG TTCTGTGTTG TCTCTGTCTG ACTGTGTTTC TGTATTTGTC
 1141 TGAAAATTAG GGCCAGACTG TTACCACTCC CTTAAGTTTG ACCTTAGGTC ACTGGAAAGA
 1201 TGTCGAGCGG ATCGCTCACA ACCAGTCGGT AGATGTCAAG AAGAGACGTT GGGTTACCTT
 1261 CTGCTCTGCA GAATGGCCAA CCTTTAACGT CGGATGGCCG CGAGACGGCA CCTTTAACCG
 1321 AGACCTCATC ACCCAGGTTA AGATCAAGGT CTTTTCACCT GGCCCGCATG GACACCCAGA
 1381 CCAGGTCCCC TACATCGTGA CCTGGGAAGC CTTGGCTTTT GACCCCCCTC CCTGGGTCAA
 1441 GCCCTTTGTA CACCCTAAGC CTCCGCCTCC TCTTCCTCCA TCCGCCCCGT CTCTCCCCCT
 1501 TGAACCTCCT CGTTCGACCC CGCCTCGATC CTCCCTTTAT CCAGCCCTCA CTCCTTCTCT
 1561 AGGCGCCGGA ATTGAAGATC ATAGTTATTA ATAGTAATCA ATTACGGGGT CATTAGTTCA
 1621 TAGCCCATAT ATGGAGTTCC GCGTTACATA ACTTACGGTA AATGGCCCGC CTGGCTGACC
 1681 GCCCAACGAC CCCCGCCCAT TGACGTCAAT AATGACGTAT GTTCCCATAG TAACGCCAAT
 1741 AGGGACTTTC CATTGACGTC AATGGGTGGA GTATTTACGG TAAACTGCCC ACTTGGCAGT
 1801 ACATCAAGTG TATCATATGC CAAGTACGCC CCCTATTGAC GTCAATGACG GTAAATGGCC
 1861 CGCCTGGCAT TATGCCCAGT ACATGACCTT ATGGGACTTT CCTACTTGGC AGTACATCTA
 1921 CGTATTAGTC ATCGCTATTA CCATGGTGAT GCGGTTTTGG CAGTACATCA ATGGGCGTGG
 1981 ATAGCGGTTT GACTCACGGG GATTTCCAAG TCTCCACCCC ATTGACGTCA ATGGGAGTTT
 2041 GTTTTGGCAC CAAAATCAAC GGGACTTTCC AAAATGTCGT AACAACTCCG CCCCATTGAC
 2101 GCAAATGGGC GGTAGGCGTG TACGGTGGGA GGTCTATATA AGCAGAGCTG GTTTAGTGAA
 2161 CCGTCAGATC CGCTAGCGCT ACCGGACTCA GATCTCGAGC TCAAGCTTCG AATTCTGCAG
 2221 TCGACGGTAC CGCGGGCCCG GGATCCACCG GTCATGGTGA GCAAGGGCGC CGAGCTGTTC
 2281 ACCGGCATCG TGCCCATCCT GATCGAGCTG AATGGCGATG TGAATGGCCA CAAGTTCAGC
 2341 GTGAGCGGCG AGGGCGAGGG CGATGCCACC TACGGCAAGC TGACCCTGAA GTTCATCTGC
 2401 ACCACCGGCA AGCTGCCTGT GCCCTGGCCC ACCCTGGTGA CCACCCTGAG CTACGGCGTG
 2461 CAGTGCTTCT CACGCTACCC CGATCACATG AAGCAGCACG ACTTCTTCAA GAGCGCCATG
 2521 CCTGAGGGCT ACATCCAGGA GCGCACCATC TTCTTCGAGG ATGACGGCAA CTACAAGTCG
 2581 CGCGCCGAGG TGAAGTTCGA GGGCGATACC CTGGTGAATC GCATCGAGCT GACCGGCACC
 2641 GATTTCAAGG AGGATGGCAA CATCCTGGGC AATAAGATGG AGTACAACTA CAACGCCCAC
 2701 AATGTGTACA TCATGACCGA CAAGGCCAAG AATGGCATCA AGGTGAACTT CAAGATCCGC
 2761 CACAACATCG AGGATGGCAG CGTGCAGCTG GCCGACCACT ACCAGCAGAA TACCCCCATC
 2821 GGCGATGGCC CTGTGCTGCT GCCCGATAAC CACTACCTGT CCACCCAGAG CGCCCTGTCC
 2881 AAGGACCCCA ACGAGAAGCG CGATCACATG ATCTACTTCG GCTTCGTGAC CGCCGCCGCC
 2941 ATCACCCACG GCATGGATGA GCTGTACAAG TGAGCGGCCG CGACTCTAGA TAATTCTACC
 3001 GGGTAGGGGA GGCGCTTTTC CCAAGGCAGT CTGGAGCATG CGCTTTAGCA GCCCCGCTGG
 3061 GCACTTGGCG CTACACAAGT GGCCTCTGGC CTCGCACACA TTCCACATCC ACCGGTAGGC
 3121 GCCAACCGGC TCCGTTCTTT GGTGGCCCCT TCGCGCCACC TTCTACTCCT CCCCTAGTCA
 3181 GGAAGTTCCC CCCCGCCCCG CAGCTCGCGT CGTGCAGGAC GTGACAAATG GAAGTAGCAC
 3241 GTCTCACTAG TCTCGTGCAG ATGGACAGCA CCGCTGAGCA ATGGAAGCGG GTAGGCCTTT
 3301 GGGGCAGCGG CCAATAGCAG CTTTGCTCCT TCGCTTTCTG GGCTCAGAGG CTGGGAAGGG
 3361 GTGGGTCCGG GGGCGGGCTC AGGGGCGGGC TCAGGGGCGG GGCGGGCGCC CGAAGGTCCT
 3421 CCGGAGGCCC GGCATTCTGC ACGCTTCAAA AGCGCACGTC TGCCGCGCTG TTCTCCTCTT
 3481 CCTCATCTCC GGGCCTTTCG ACCTGCAGCC CAAGCTTACC ATGACCGAGT ACAAGCCCAC
 3541 GGTGCGCCTC GCCACCCGCG ACGACGTCCC CAGGGCCGTA CGCACCCTCG CCGCCGCGTT
 3601 CGCCGACTAC CCCGCCACGC GCCACACCGT CGATCCGGAC CGCCACATCG AGCGGGTCAC
 3661 CGAGCTGCAA GAACTCTTCC TCACGCGCGT CGGGCTCGAC ATCGGCAAGG TGTGGGTCGC
 3721 GGACGACGGC GCCGCGGTGG CGGTCTGGAC CACGCCGGAG AGCGTCGAAG CGGGGGCGGT
 3781 GTTCGCCGAG ATCGGCCCGC GCATGGCCGA GTTGAGCGGT TCCCGGCTGG CCGCGCAGCA
 3841 ACAGATGGAA GGCCTCCTGG CGCCGCACCG GCCCAAGGAG CCCGCGTGGT TCCTGGCCAC
 3901 CGTCGGCGTC TCGCCCGACC ACCAGGGCAA GGGTCTGGGC AGCGCCGTCG TGCTCCCCGG
 3961 AGTGGAGGCG GCCGAGCGCG CCGGGGTGCC CGCCTTCCTG GAGACCTCCG CGCCCCGCAA
 4021 CCTCCCCTTC TACGAGCGGC TCGGCTTCAC CGTCACCGCC GACGTCGAGG TGCCCGAAGG
 4081 ACCGCGCACC TGGTGCATGA CCCGCAAGCC CGGTGCCTGA CGCCCGCCCC ACGACCCGCA
 4141 GCGCCCGACC GAAAGGAGCG CACGACCCCA TGCATCGGCG TCTCGATCGA GATATCAGTG
 4201 GTCCAGGCTC TAGTTTTGAC TCAACAATAT CACCAGCTGA AGCCTATAGA GTACGAGCCA
 4261 TAGATAAAAT AAAAGATTTT ATTTAGTCTC CAGAAAAAGG GGGGAATGAA AGACCCCACC
 4321 TGTAGGTTTG GCAAGCTACT AGCTTAAGTA ACGCCATTTT GCAAGGCATG GAAAAATACA
 4381 TAACTGAGAA TAGAGAAGTT CAGATCAAGG TCAGGAACAG ATGGAACAGG GTCGATCGAC
 4441 CCTAGAGAAC CATCAGATGT TTCCAGGGTG CCCCAAGGAC CTGAAATGAC CCTGTGCCTT
 4501 ATTTGAACTA ACCAATCAGT TCGCTTCTCG CTTCTGTTCG CGCGCTTCTG CTCCCCGAGC
 4561 TCAATAAAAG AGCCCACAAC CCCTCACTCG GGGCGCCAGT CCTCCGATTG ACTGAGTCGC
 4621 CCGGGTACCC GTGTATCCAA TAAACCCTCT TGCAGTTGCA TCCGACTTGT GGTCTCGCTG
 4681 TTCCTTGGGA GGGTCTCCTC TGAGTGATTG ACTACCCGTC AGCGGGGGTC TTTCATTTGG
 4741 GGGCTCGTCC GGGATCGGGA GACCCCTGCC CAGGGACCAC CGACCCACCA CCGGGAGGTA
 4801 AGCTGGCTGC CTCGCGCGTT TCGGTGATGA CGGTGAAAAC CTCTGACACA TGCAGCTCCC
 4861 GGAGACGGTC ACAGCTTGTC TGTAAGCGGA TGCCGGGAGC AGACAAGCCC GTCAGGGCGC
 4921 GTCAGCGGGT GTTGGCGGGT GTCGGGGCGC AGCCATGACC CAGTCACGTA GCGATAGCGG
 4981 AGTGTAGATC CGGCTGTGGA ATGTGTGTCA GTTAGGGTGT GGAAAGTCCC CAGGCTCCCC
 5041 AGCAGGCAGA AGTATGCAAA GCATGCATCT CAATTAGTCA GCAACCAGGT GTGGAAAGTC
 5101 CCCAGGCTCC CCAGCAGGCA GAAGTATGCA AAGCATGCAT CTCAATTAGT CAGCAACCAT
 5161 AGTCCCGCCC CTAACTCCGC CCATCCCGCC CCTAACTCCG CCCAGTTCCG CCCATTCTCC
 5221 GCCCCATGGC TGACTAATTT TTTTTATTTA TGCAGAGGCC GAGGCCGCCT CGGCCTCTGA
 5281 GCTATTCCAG AAGTAGTGAG GAGGCTTTTT TGGAGGCCTA GGCTTTTGCA AAAAGCTTAC
 5341 TGGCTTAACT ATGCGGCATC AGAGCAGATT GTACTGAGAG TGCACCATAT GCGGTGTGAA
 5401 ATACCGCACA GATGCGTAAG GAGAAAATAC CGCATCAGGC GCTCTTCCGC TTCCTCGCTC
 5461 ACTGACTCGC TGCGCTCGGT CGTTCGGCTG CGGCGAGCGG TATCAGCTCA CTCAAAGGCG
 5521 GTAATACGGT TATCCACAGA ATCAGGGGAT AACGCAGGAA AGAACATGTG AGCAAAAGGC
 5581 CAGCAAAAGG CCAGGAACCG TAAAAAGGCC GCGTTGCTGG CGTTTTTCCA TAGGCTCCGC
 5641 CCCCCTGACG AGCATCACAA AAATCGACGC TCAAGTCAGA GGTGGCGAAA CCCGACAGGA
 5701 CTATAAAGAT ACCAGGCGTT TCCCCCTGGA AGCTCCCTCG TGCGCTCTCC TGTTCCGACC
 5761 CTGCCGCTTA CCGGATACCT GTCCGCCTTT CTCCCTTCGG GAAGCGTGGC GCTTTCTCAT
 5821 AGCTCACGCT GTAGGTATCT CAGTTCGGTG TAGGTCGTTC GCTCCAAGCT GGGCTGTGTG
 5881 CACGAACCCC CCGTTCAGCC CGACCGCTGC GCCTTATCCG GTAACTATCG TCTTGAGTCC
 5941 AACCCGGTAA GACACGACTT ATCGCCACTG GCAGCAGCCA CTGGTAACAG GATTAGCAGA
 6001 GCGAGGTATG TAGGCGGTGC TACAGAGTTC TTGAAGTGGT GGCCTAACTA CGGCTACACT
 6061 AGAAGGACAG TATTTGGTAT CTGCGCTCTG CTGAAGCCAG TTACCTTCGG AAAAAGAGTT
 6121 GGTAGCTCTT GATCCGGCAA ACAAACCACC GCTGGTAGCG GTGGTTTTTT TGTTTGCAAG
 6181 CAGCAGATTA CGCGCAGAAA AAAAGGATCT CAAGAAGATC CTTTGATCTT TTCTACGGGG
 6241 TCTGACGCTC AGTGGAACGA AAACTCACGT TAAGGGATTT TGGTCATGAG ATTATCAAAA
 6301 AGGATCTTCA CCTAGATCCT TTTAAATTAA AAATGAAGTT TTAAATCAAT CTAAAGTATA
 6361 TATGAGTAAA CTTGGTCTGA CAGTTACCAA TGCTTAATCA GTGAGGCACC TATCTCAGCG
 6421 ATCTGTCTAT TTCGTTCATC CATAGTTGCC TGACTCCCCG TCGTGTAGAT AACTACGATA
 6481 CGGGAGGGCT TACCATCTGG CCCCAGTGCT GCAATGATAC CGCGAGACCC ACGCTCACCG
 6541 GCTCCAGATT TATCAGCAAT AAACCAGCCA GCCGGAAGGG CCGAGCGCAG AAGTGGTCCT
 6601 GCAACTTTAT CCGCCTCCAT CCAGTCTATT AATTGTTGCC GGGAAGCTAG AGTAAGTAGT
 6661 TCGCCAGTTA ATAGTTTGCG CAACGTTGTT GCCATTGCTG CAGGCATCGT GGTGTCACGC
 6721 TCGTCGTTTG GTATGGCTTC ATTCAGCTCC GGTTCCCAAC GATCAAGGCG AGTTACATGA
 6781 TCCCCCATGT TGTGCAAAAA AGCGGTTAGC TCCTTCGGTC CTCCGATCGT TGTCAGAAGT
 6841 AAGTTGGCCG CAGTGTTATC ACTCATGGTT ATGGCAGCAC TGCATAATTC TCTTACTGTC
 6901 ATGCCATCCG TAAGATGCTT TTCTGTGACT GGTGAGTACT CAACCAAGTC ATTCTGAGAA
 6961 TAGTGTATGC GGCGACCGAG TTGCTCTTGC CCGGCGTCAA CACGGGATAA TACCGCGCCA
 7021 CATAGCAGAA CTTTAAAAGT GCTCATCATT GGAAAACGTT CTTCGGGGCG AAAACTCTCA
 7081 AGGATCTTAC CGCTGTTGAG ATCCAGTTCG ATGTAACCCA CTCGTGCACC CAACTGATCT
 7141 TCAGCATCTT TTACTTTCAC CAGCGTTTCT GGGTGAGCAA AAACAGGAAG GCAAAATGCC
 7201 GCAAAAAAGG GAATAAGGGC GACACGGAAA TGTTGAATAC TCATACTCTT CCTTTTTCAA
 7261 TATTATTGAA GCATTTATCA GGGTTATTGT CTCATGAGCG GATACATATT TGAATGTATT
 7321 TAGAAAAATA AACAAATAGG GGTTCCGCGC ACATTTCCCC GAAAAGTGCC ACCTGACGTC
 7381 TAAGAAACCA TTATTATCAT GACATTAACC TATAAAAATA GGCGTATCAC GAGGCCCTTT
 7441 CGTCTTCAAG AATTAGCTTG GCCATTGCAT ACGTTGTATC CATATCATAA TATGTACATT
 7501 TATATTGGCT CATGTCCAAC ATTACCGCCA TGTTGACATT GATTATTGAC TAGTTATTAA
 7561 TAGTAATCAA TTACGGGGTC ATTAGTTCAT AGCCCATATA TGGAGT