pLVX-EF1alpha-SARS-CoV-2-M-2xStrep-IRES-Puro

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相关技术服务:质粒构建    基因合成    质粒大提

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载体基本信息

质粒名称: pLVX-EF1alpha-SARS-CoV-2-M-2xStrep-IRES-Puro
出品公司: Addgene
目录编号: 141386
存储实验室: Nevan Krogan

载体骨架基本信息
载体名称: pLVX-EF1alpha-IRES-Puro
载体出品公司: Takara Bio
空载体大小: 8825 bp
完整质粒大小: 8825 bp
载体类型: 哺乳细胞表达, Lentiviral
筛选标记: Puromycin
载体抗性: 氨苄霉素
生长温度: 370℃
宿主菌: NEB Stable
拷贝数: 未知

插入基因信息
插入基因名称: SARS-CoV-2-M
物种来源: SARS-CoV-2
基因突变: human codon optimized
启动子: EF1alpha
标签/融合蛋白: 2xStrep (C terminal on insert)
克隆方法: 未知

文献引用方法
材料和方法部分:
pLVX-EF1alpha-SARS-CoV-2-M-2xStrep-IRES-Puro was a gift from Nevan Krogan (Addgene plasmid # 141386 ; http://n2t.net/addgene:141386 ; RRID:Addgene_141386)
For viral preps, please replace (Addgene plasmid # 141386) in the above sentence with: (Addgene viral prep # 141386-LV)
参考文献部分: A SARS-CoV-2 protein interaction map reveals targets for drug repurposing. Gordon DE, Jang GM, Bouhaddou M, Xu J, Obernier K, White KM, O'Meara MJ, Rezelj VV, Guo JZ, Swaney DL, Tummino TA, Huttenhain R, Kaake RM, Richards AL, Tutuncuoglu B, Foussard H, Batra J, Haas K, Modak M, Kim M, Haas P, Polacco BJ, Braberg H, Fabius JM, Eckhardt M, Soucheray M, Bennett MJ, Cakir M, McGregor MJ, Li Q, Meyer B, Roesch F, Vallet T, Mac Kain A, Miorin L, Moreno E, Naing ZZC, Zhou Y, Peng S, Shi Y, Zhang Z, Shen W, Kirby IT, Melnyk JE, Chorba JS, Lou K, Dai SA, Barrio-Hernandez I, Memon D, Hernandez-Armenta C, Lyu J, Mathy CJP, Perica T, Pilla KB, Ganesan SJ, Saltzberg DJ, Rakesh R, Liu X, Rosenthal SB, Calviello L, Venkataramanan S, Liboy-Lugo J, Lin Y, Huang XP, Liu Y, Wankowicz SA, Bohn M, Safari M, Ugur FS, Koh C, Savar NS, Tran QD, Shengjuler D, Fletcher SJ, O'Neal MC, Cai Y, Chang JCJ, Broadhurst DJ, Klippsten S, Sharp PP, Wenzell NA, Kuzuoglu-Ozturk D, Wang HY, Trenker R, Young JM, Cavero DA, Hiatt J, Roth TL, Rathore U, Subramanian A, Noack J, Hubert M, Stroud RM, Frankel AD, Rosenberg OS, Verba KA, Agard DA, Ott M, Emerman M, Jura N, von Zastrow M, Verdin E, Ashworth A, Schwartz O, d'Enfert C, Mukherjee S, Jacobson M, Malik HS, Fujimori DG, Ideker T, Craik CS, Floor SN, Fraser JS, Gross JD, Sali A, Roth BL, Ruggero D, Taunton J, Kortemme T, Beltrao P, Vignuzzi M, Garcia-Sastre A, Shokat KM, Shoichet BK, Krogan NJ. Nature. 2020 Jul;583(7816):459-468. doi: 10.1038/s41586-020-2286-9. Epub 2020 Apr 30. 10.1038/s41586-020-2286-9 PubMed 32353859

载体图谱质粒图谱和多克隆位点信息

pLVX-EF1alpha-SARS-CoV-2-M-2xStrep-IRES-Puro载体图谱

载体简介

载体序列

LOCUS      pLVX-EF1alpha-SARS-CoV-2-M-2xStrep-IRES-Puro               9579 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Lentiviral expression of SARS-CoV-2 membrane (M) protein; transient 
            expression and generate lentivirus.
ACCESSION   .
VERSION     .
KEYWORDS    pLVX-EF1alpha-SARS-CoV-2-M-2xStrep-IRES-Puro
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9579)
  AUTHORS   Gordon DE, et al.
  TITLE     A SARS-CoV-2-Human Protein-Protein Interaction Map Reveals Drug 
            Targets and Potential Drug Repurposing
  JOURNAL   BioRxiv March 2020
REFERENCE   2  (bases 1 to 9579)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.biofeng.com
FEATURES             Location/Qualifiers
     source          1..9579
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     LTR             1..634
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    681..806
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1303..1536
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1721..1765
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    2028..2143
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1 (lacking the first T)"
     promoter        2338..3519
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2568..3510
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3467..3487
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      3544..3553
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             4234..4257
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             4294..4317
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     misc_feature    4329..4902
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4496..4513)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4723..4742
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4935..5534
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4935..4954)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5431..5451
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    5548..6136
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5601..5621)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6019..6030)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             6343..6976
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(7104..7120)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7104..7120)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7117..7139)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7128..7144
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(7152..7182)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7197..7218
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7335..7352)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7506..8094)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7586..7605)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(8265..9125)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     8888..8907
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9126..9230)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    9278..9412
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(9315..9334)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     9369..9388
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca
       61 cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac
      121 tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca
      181 ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg
      241 agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag
      301 agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg
      361 ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat
      421 cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga
      481 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
      541 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
      601 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
      661 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
      721 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga
      781 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg
      841 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg
      901 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct
      961 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat
     1021 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca
     1081 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc
     1141 aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag
     1201 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc
     1261 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg
     1321 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc
     1381 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc
     1441 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct
     1501 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa
     1561 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca
     1621 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt
     1681 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt
     1741 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta
     1801 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt
     1861 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct
     1921 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga
     1981 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccttt aaaagaaaag
     2041 gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac
     2101 aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg
     2161 acagcagaga tccagtttat cgatgagtaa ttcatacaaa aggactcgcc cctgccttgg
     2221 ggaatcccag ggaccgtcgt taaactccca ctaacgtaga acccagagat cgctgcgttc
     2281 ccgccccctc acccgcccgc tctcgtcatc actgaggtgg agaagagcat gcgtgaggct
     2341 ccggtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag ttggggggag
     2401 gggtcggcaa ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg
     2461 tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata agtgcagtag
     2521 tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacaggta agtgccgtgt
     2581 gtggttcccg cgggcctggc ctctttacgg gttatggccc ttgcgtgcct tgaattactt
     2641 ccacgcccct ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg
     2701 gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg
     2761 cttgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct
     2821 ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg
     2881 caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc
     2941 gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga
     3001 gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct
     3061 ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca
     3121 gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg
     3181 acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg
     3241 tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat
     3301 tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg
     3361 gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa
     3421 ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca
     3481 gtggttcaaa gtttttttct tccatttcag gtgtcgtgag gatctatttc cggtgaattc
     3541 gccgccacca tggccgactc aaatgggacc attactgtag aggagctgaa aaaactgttg
     3601 gagcaatgga accttgtgat tggatttctc ttcttgacat ggatttgtct cctgcaattc
     3661 gcatacgcga atcgcaaccg attcctctat attataaaac ttatattcct ttggctcctg
     3721 tggcctgtga cccttgcctg cttcgtactc gcggcggtgt atcgaataaa ctggataact
     3781 ggcggcatag caatagctat ggcatgcctg gtaggtttga tgtggctctc ctattttata
     3841 gcgtcattca gactttttgc acgcacgcga agcatgtggt catttaaccc tgaaacaaat
     3901 atcttgctta atgtccccct gcatggcaca attttgaccc gaccgctcct tgaatcagaa
     3961 ttggtgattg gagccgtgat cctgaggggc cacttgagga tcgcaggaca tcatcttggt
     4021 cgctgtgata ttaaggattt gccaaaagaa ataactgtag ctaccagtcg aactttgagc
     4081 tattacaagc ttggagcgtc ccaaagggtc gctggcgata gtggatttgc agcctattca
     4141 cgctacagga tagggaacta taaactgaat acagaccact catcatcttc tgataacatt
     4201 gctttgctcg tccagctcga aggcggcggg ggatggagcc atccacaatt cgagaaaggc
     4261 ggtggttcag gaggaggtag cgggggtgga tcatggtcac atccgcagtt tgaaaagtaa
     4321 ggatcccgcc cctctccctc ccccccccct aacgttactg gccgaagccg cttggaataa
     4381 ggccggtgtg cgtttgtcta tatgttattt tccaccatat tgccgtcttt tggcaatgtg
     4441 agggcccgga aacctggccc tgtcttcttg acgagcattc ctaggggtct ttcccctctc
     4501 gccaaaggaa tgcaaggtct gttgaatgtc gtgaaggaag cagttcctct ggaagcttct
     4561 tgaagacaaa caacgtctgt agcgaccctt tgcaggcagc ggaacccccc acctggcgac
     4621 aggtgcctct gcggccaaaa gccacgtgta taagatacac ctgcaaaggc ggcacaaccc
     4681 cagtgccacg ttgtgagttg gatagttgtg gaaagagtca aatggctctc ctcaagcgta
     4741 ttcaacaagg ggctgaagga tgcccagaag gtaccccatt gtatgggatc tgatctgggg
     4801 cctcggtgca catgctttac atgtgtttag tcgaggttaa aaaaacgtct aggccccccg
     4861 aaccacgggg acgtggtttt cctttgaaaa acacgatgat aagcttgcca caacccacaa
     4921 ggagacgacc ttccatgacc gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg
     4981 tcccccgggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca
     5041 ccgtcgaccc ggaccgccac atcgagcggg tcaccgagct gcaagaactc ttcctcacgc
     5101 gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct
     5161 ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg
     5221 ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc
     5281 accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg
     5341 gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg
     5401 tgcccgcctt cctggagacc tccgcgcccc gcaacctccc cttctacgag cggctcggct
     5461 tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc atgacccgca
     5521 agcccggtgc ctagacgcgt ctggaacaat caacctctgg attacaaaat ttgtgaaaga
     5581 ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg
     5641 cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc
     5701 tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc
     5761 actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt
     5821 tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt
     5881 gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg
     5941 aagctgacgt cctttccatg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg
     6001 tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg
     6061 ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt
     6121 tgggccgcct ccccgcctgg aattaattct gcagtcgaga cctagaaaaa catggagcaa
     6181 tcacaagtag caatacagca gctaccaatg ctgattgtgc ctggctagaa gcacaagagg
     6241 aggaggaggt gggttttcca gtcacacctc aggtaccttt aagaccaatg acttacaagg
     6301 cagctgtaga tcttagccac tttttaaaag aaaagagggg actggaaggg ctaattcact
     6361 cccaacgaag acaagatatc cttgatctgt ggatctacca cacacaaggc tacttccctg
     6421 attagcagaa ctacacacca gggccagggg tcagatatcc actgaccttt ggatggtgct
     6481 acaagctagt accagttgag ccagataagg tagaagaggc caataaagga gagaacacca
     6541 gcttgttaca ccctgtgagc ctgcatggga tggatgaccc ggagagagaa gtgttagagt
     6601 ggaggtttga cagccgccta gcatttcatc acgtggcccg agagctgcat ccggagtact
     6661 tcaagaactg ctgatatcga gcttgctaca agggactttc cgctggggac tttccaggga
     6721 ggcgtggcct gggcgggact ggggagtggc gagccctcag atcctgcata taagcagctg
     6781 ctttttgcct gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc
     6841 taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg
     6901 tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg
     6961 tggaaaatct ctagcagtag tagttcatgt catcttatta ttcagtattt ataacttgca
     7021 aagaaatgaa tatcagagag tgagaggcct tgacattgct agcgtttacc gtcgacctct
     7081 agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     7141 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     7201 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     7261 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     7321 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     7381 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     7441 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     7501 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     7561 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     7621 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     7681 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     7741 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     7801 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     7861 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     7921 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     7981 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     8041 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     8101 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     8161 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     8221 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     8281 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     8341 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     8401 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     8461 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     8521 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     8581 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     8641 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     8701 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     8761 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     8821 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     8881 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     8941 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     9001 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     9061 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     9121 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     9181 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     9241 cgaaaagtgc cacctgacgt cgacggatcg ggagatcaac ttgtttattg cagcttataa
     9301 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     9361 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgga tcaactggat
     9421 aactcaagct aaccaaaatc atcccaaact tcccacccca taccctatta ccactgccaa
     9481 ttacctgtgg tttcatttac tctaaacctg tgattcctct gaattatttt cattttaaag
     9541 aaattgtatt tgttaaatat gtactacaaa cttagtagt
//

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