pBS NL4-3 envFS eGFP PR-D25A

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载体基本信息

质粒名称: pBS NL4-3 envFS eGFP PR-D25A
出品公司: Addgene
目录编号: 101334
存储实验室: Jeremy Luban

载体骨架基本信息
载体名称: pBluescript
载体出品公司: --
空载体大小: --
完整质粒大小: --
载体修饰: --
载体类型: 慢病毒载体
筛选标记: --
载体抗性: 氨苄霉素
生长温度: 30 ℃
宿主菌: NEB Stable
拷贝数: 高拷贝

插入基因信息
插入基因名称: NL4-3 envFS
物种来源: --
插入大小: --
基因突变: Protease D25A
GenBank编号 --
启动子: --
标签/融合蛋白: --
克隆方法: 限制性内切酶
5’克隆位点: --
3’克隆位点: --
5’测序引物 --
3’测序引物 --

文献引用方法
材料和方法部分: pBS NL4-3 envFS eGFP PR-D25A was a gift from Jeremy Luban (Addgene plasmid # 101334 ; http://n2t.net/addgene:101334 ; RRID:Addgene_101334)
参考文献部分: Intron-containing RNA from the HIV-1 provirus activates type I interferon and inflammatory cytokines. McCauley SM, Kim K, Nowosielska A, Dauphin A, Yurkovetskiy L, Diehl WE, Luban J. Nat Commun. 2018 Dec 13;9(1):5305. doi: 10.1038/s41467-018-07753-2. 10.1038/s41467-018-07753-2 PubMed 30546110

载体图谱质粒图谱和多克隆位点信息

pBS NL4-3 envFS eGFP PR-D25A质粒图谱

载体简介

载体序列

LOCUS       pBS NL4-3 envFS eGFP PR-D25A             12625 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  pBS NL4-3 envFS eGFP with mutation that disrupts Protease catalytic 
            activity.
ACCESSION   .
VERSION     .
KEYWORDS    pBS NL4-3 envFS eGFP PR-D25A
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12625)
  AUTHORS   McCauley SM, Kim K, Nowosielska A, Dauphin A, Yurkovetskiy L, Diehl 
            WE, Luban J
  TITLE     Intron-containing RNA from the HIV-1 provirus activates type I 
            interferon and inflammatory cytokines.
  JOURNAL   Nat Commun. 2018 Dec 13;9(1):5305. doi: 10.1038/s41467-018-07753-2.
  PUBMED    30546110
REFERENCE   2  (bases 1 to 12625)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..12625
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     LTR             377..634
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     CDS             790..2292
                     /codon_start=1
                     /gene="gag"
                     /product="gag protein from human immunodeficiency virus 1"
                     /label=HIV-1 gag
                     /translation="MGARASVLSGGELDKWEKIRLRPGGKKQYKLKHIVWASRELERFA
                     VNPGLLETSEGCRQILGQLQPSLQTGSEELRSLYNTIAVLYCVHQRIDVKDTKEALDKI
                     EEEQNKSKKKAQQAAADTGNNSQVSQNYPIVQNLQGQMVHQAISPRTLNAWVKVVEEKA
                     FSPEVIPMFSALSEGATPQDLNTMLNTVGGHQAAMQMLKETINEEAAEWDRLHPVHAGP
                     IAPGQMREPRGSDIAGTTSTLQEQIGWMTHNPPIPVGEIYKRWIILGLNKIVRMYSPTS
                     ILDIRQGPKEPFRDYVDRFYKTLRAEQASQEVKNWMTETLLVQNANPDCKTILKALGPG
                     ATLEEMMTACQGVGGPGHKARVLAEAMSQVTNPATIMIQKGNFRNQRKTVKCFNCGKEG
                     HIAKNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSHKGRPGNFLQSRPEPTAP
                     PEESFRFGEETTTPSQKQEPIDKELYPLASLRSLFGSDPSSQ"
     CDS             2085..5096
                     /codon_start=1
                     /gene="pol"
                     /product="pol protein from human immunodeficiency virus 1"
                     /label=HIV-1 pol
                     /translation="FFREDLAFPQGKAREFSSEQTRANSPTRRELQVWGRDNNSLSEAG
                     ADRQGTVSFSFPQITLWQRPLVTIKIGGQLKEALLATGADDTVLEEMNLPGRWKPKMIG
                     GIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNFPISPIETV
                     PVKLKPGMDGPKVKQWPLTEEKIKALVEICTEMEKEGKISKIGPENPYNTPVFAIKKKD
                     STKWRKLVDFRELNKRTQDFWEVQLGIPHPAGLKQKKSVTVLDVGDAYFSVPLDKDFRK
                     YTAFTIPSINNETPGIRYQYNVLPQGWKGSPAIFQCSMTKILEPFRKQNPDIVIYQYMD
                     DLYVGSDLEIGQHRTKIEELRQHLLRWGFTTPDKKHQKEPPFLWMGYELHPDKWTVQPI
                     VLPEKDSWTVNDIQKLVGKLNWASQIYAGIKVRQLCKLLRGTKALTEVVPLTEEAELEL
                     AENREILKEPVHGVYYDPSKDLIAEIQKQGQGQWTYQIYQEPFKNLKTGKYARMKGAHT
                     NDVKQLTEAVQKIATESIVIWGKTPKFKLPIQKETWEAWWTEYWQATWIPEWEFVNTPP
                     LVKLWYQLEKEPIIGAETFYVDGAANRETKLGKAGYVTDRGRQKVVPLTDTTNQKTELQ
                     AIHLALQDSGLEVNIVTDSQYALGIIQAQPDKSESELVSQIIEQLIKKEKVYLAWVPAH
                     KGIGGNEQVDKLVSAGIRKVLFLDGIDKAQEEHEKYHSNWRAMASDFNLPPVVAKEIVA
                     SCDKCQLKGEAMHGQVDCSPGIWQLDCTHLEGKVILVAVHVASGYIEAEVIPAETGQET
                     AYFLLKLAGRWPVKTVHTDNGSNFTSTTVKAACWWAGIKQEFGIPYNPQSQGVIESMNK
                     ELKKIIGQVRDQAEHLKTAVQMAVFIHNFKRKGGIGGYSAGERIVDIIATDIQTKELQK
                     QITKIQNFRVYYRDSRDPVWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKAKIIRDYGKQ
                     MAGDDCVASRQDED"
     misc_feature    4781..4898
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     misc_feature    7761..7994
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             8179..8223
                     /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"
     CDS             8517..8543
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     CDS             8810..9526
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             8810..9523
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(8852..8873)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(9113..9132)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     9460..9481
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     LTR             9716..10349
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(10425..11013)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10505..10524)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(11184..12044)
                     /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     11807..11826
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12045..12149)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      12176..12604
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(12263..12282)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     12473..12494
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 tggaagggct aatttggtcc caaaaaagac aagagatcct tgatctgtgg atctaccaca
       61 cacaaggcta cttccctgat tggcagaact acacaccagg gccagggatc agatatccac
      121 tgacctttgg atggtgcttc aagttagtac cagttgaacc agagcaagta gaagaggcca
      181 atgaaggaga gaacaacagc ttgttacacc ctatgagcca gcatgggatg gaggacccgg
      241 agggagaagt attagtgtgg aagtttgaca gcctcctagc atttcgtcac atggcccgag
      301 agctgcatcc ggagtactac aaagactgct gacatcgagc tttctacaag ggactttccg
      361 ctggggactt tccagggagg tgtggcctgg gcgggactgg ggagtggcga gccctcagat
      421 gctacatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga
      481 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
      541 tgagtgctca aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
      601 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
      661 cgaaagtaaa gccagaggag atctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
      721 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga
      781 aggagagaga tgggtgcgag agcgtcggta ttaagcgggg gagaattaga taaatgggaa
      841 aaaattcggt taaggccagg gggaaagaaa caatataaac taaaacatat agtatgggca
      901 agcagggagc tagaacgatt cgcagttaat cctggccttt tagagacatc agaaggctgt
      961 agacaaatac tgggacagct acaaccatcc cttcagacag gatcagaaga acttagatca
     1021 ttatataata caatagcagt cctctattgt gtgcatcaaa ggatagatgt aaaagacacc
     1081 aaggaagcct tagataagat agaggaagag caaaacaaaa gtaagaaaaa ggcacagcaa
     1141 gcagcagctg acacaggaaa caacagccag gtcagccaaa attaccctat agtgcagaac
     1201 ctccaggggc aaatggtaca tcaggccata tcacctagaa ctttaaatgc atgggtaaaa
     1261 gtagtagaag agaaggcttt cagcccagaa gtaataccca tgttttcagc attatcagaa
     1321 ggagccaccc cacaagattt aaataccatg ctaaacacag tggggggaca tcaagcagcc
     1381 atgcaaatgt taaaagagac catcaatgag gaagctgcag aatgggatag attgcatcca
     1441 gtgcatgcag ggcctattgc accaggccag atgagagaac caaggggaag tgacatagca
     1501 ggaactacta gtacccttca ggaacaaata ggatggatga cacataatcc acctatccca
     1561 gtaggagaaa tctataaaag atggataatc ctgggattaa ataaaatagt aagaatgtat
     1621 agccctacca gcattctgga cataagacaa ggaccaaagg aaccctttag agactatgta
     1681 gaccgattct ataaaactct aagagccgag caagcttcac aagaggtaaa aaattggatg
     1741 acagaaacct tgttggtcca aaatgcgaac ccagattgta agactatttt aaaagcattg
     1801 ggaccaggag cgacactaga agaaatgatg acagcatgtc agggagtggg gggacccggc
     1861 cataaagcaa gagttttggc tgaagcaatg agccaagtaa caaatccagc taccataatg
     1921 atacagaaag gcaattttag gaaccaaaga aagactgtta agtgtttcaa ttgtggcaaa
     1981 gaagggcaca tagccaaaaa ttgcagggcc cctaggaaaa agggctgttg gaaatgtgga
     2041 aaggaaggac accaaatgaa agattgtact gagagacagg ctaatttttt agggaagatc
     2101 tggccttccc acaagggaag gccagggaat tttcttcaga gcagaccaga gccaacagcc
     2161 ccaccagaag agagcttcag gtttggggaa gagacaacaa ctccctctca gaagcaggag
     2221 ccgatagaca aggaactgta tcctttagct tccctcagat cactctttgg cagcgacccc
     2281 tcgtcacaat aaagataggg gggcaattaa aggaagctct attagctaca ggagcagatg
     2341 atacagtatt agaagaaatg aatttgccag gaagatggaa accaaaaatg atagggggaa
     2401 ttggaggttt tatcaaagta agacagtatg atcagatact catagaaatc tgcggacata
     2461 aagctatagg tacagtatta gtaggaccta cacctgtcaa cataattgga agaaatctgt
     2521 tgactcagat tggctgcact ttaaattttc ccattagtcc tattgagact gtaccagtaa
     2581 aattaaagcc aggaatggat ggcccaaaag ttaaacaatg gccattgaca gaagaaaaaa
     2641 taaaagcatt agtagaaatt tgtacagaaa tggaaaagga aggaaaaatt tcaaaaattg
     2701 ggcctgaaaa tccatacaat actccagtat ttgccataaa gaaaaaagac agtactaaat
     2761 ggagaaaatt agtagatttc agagaactta ataagagaac tcaagatttc tgggaagttc
     2821 aattaggaat accacatcct gcagggttaa aacagaaaaa atcagtaaca gtactggatg
     2881 tgggcgatgc atatttttca gttcccttag ataaagactt caggaagtat actgcattta
     2941 ccatacctag tataaacaat gagacaccag ggattagata tcagtacaat gtgcttccac
     3001 agggatggaa aggatcacca gcaatattcc agtgtagcat gacaaaaatc ttagagcctt
     3061 ttagaaaaca aaatccagac atagtcatct atcaatacat ggatgatttg tatgtaggat
     3121 ctgacttaga aatagggcag catagaacaa aaatagagga actgagacaa catctgttga
     3181 ggtggggatt taccacacca gacaaaaaac atcagaaaga acctccattc ctttggatgg
     3241 gttatgaact ccatcctgat aaatggacag tacagcctat agtgctgcca gaaaaggaca
     3301 gctggactgt caatgacata cagaaattag tgggaaaatt gaattgggca agtcagattt
     3361 atgcagggat taaagtaagg caattatgta aacttcttag gggaaccaaa gcactaacag
     3421 aagtagtacc actaacagaa gaagcagagc tagaactggc agaaaacagg gagattctaa
     3481 aagaaccggt acatggagtg tattatgacc catcaaaaga cttaatagca gaaatacaga
     3541 agcaggggca aggccaatgg acatatcaaa tttatcaaga gccatttaaa aatctgaaaa
     3601 caggaaagta tgcaagaatg aagggtgccc acactaatga tgtgaaacaa ttaacagagg
     3661 cagtacaaaa aatagccaca gaaagcatag taatatgggg aaagactcct aaatttaaat
     3721 tacccataca aaaggaaaca tgggaagcat ggtggacaga gtattggcaa gccacctgga
     3781 ttcctgagtg ggagtttgtc aatacccctc ccttagtgaa gttatggtac cagttagaga
     3841 aagaacccat aataggagca gaaactttct atgtagatgg ggcagccaat agggaaacta
     3901 aattaggaaa agcaggatat gtaactgaca gaggaagaca aaaagttgtc cccctaacgg
     3961 acacaacaaa tcagaagact gagttacaag caattcatct agctttgcag gattcgggat
     4021 tagaagtaaa catagtgaca gactcacaat atgcattggg aatcattcaa gcacaaccag
     4081 ataagagtga atcagagtta gtcagtcaaa taatagagca gttaataaaa aaggaaaaag
     4141 tctacctggc atgggtacca gcacacaaag gaattggagg aaatgaacaa gtagataaat
     4201 tggtcagtgc tggaatcagg aaagtactat ttttagatgg aatagataag gcccaagaag
     4261 aacatgagaa atatcacagt aattggagag caatggctag tgattttaac ctaccacctg
     4321 tagtagcaaa agaaatagta gccagctgtg ataaatgtca gctaaaaggg gaagccatgc
     4381 atggacaagt agactgtagc ccaggaatat ggcagctaga ttgtacacat ttagaaggaa
     4441 aagttatctt ggtagcagtt catgtagcca gtggatatat agaagcagaa gtaattccag
     4501 cagagacagg gcaagaaaca gcatacttcc tcttaaaatt agcaggaaga tggccagtaa
     4561 aaacagtaca tacagacaat ggcagcaatt tcaccagtac tacagttaag gccgcctgtt
     4621 ggtgggcggg gatcaagcag gaatttggca ttccctacaa tccccaaagt caaggagtaa
     4681 tagaatctat gaataaagaa ttaaagaaaa ttataggaca ggtaagagat caggctgaac
     4741 atcttaagac agcagtacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga
     4801 ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta
     4861 aagaattaca aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca
     4921 gagatccagt ttggaaagga ccagcaaagc tcctctggaa aggtgaaggg gcagtagtaa
     4981 tacaagataa tagtgacata aaagtagtgc caagaagaaa agcaaagatc atcagggatt
     5041 atggaaaaca gatggcaggt gatgattgtg tggcaagtag acaggatgag gattaacaca
     5101 tggaaaagat tagtaaaaca ccatatgtat atttcaagga aagctaagga ctggttttat
     5161 agacatcact atgaaagtac taatccaaaa ataagttcag aagtacacat cccactaggg
     5221 gatgctaaat tagtaataac aacatattgg ggtctgcata caggagaaag agactggcat
     5281 ttgggtcagg gagtctccat agaatggagg aaaaagagat atagcacaca agtagaccct
     5341 gacctagcag accaactaat tcatctgcac tattttgatt gtttttcaga atctgctata
     5401 agaaatacca tattaggacg tatagttagt cctaggtgtg aatatcaagc aggacataac
     5461 aaggtaggat ctctacagta cttggcacta gcagcattaa taaaaccaaa acagataaag
     5521 ccacctttgc ctagtgttag gaaactgaca gaggacagat ggaacaagcc ccagaagacc
     5581 aagggccaca gagggagcca tacaatgaat ggacactaga gcttttagag gaacttaaga
     5641 gtgaagctgt tagacatttt cctaggatat ggctccataa cttaggacaa catatctatg
     5701 aaacttacgg ggatacttgg gcaggagtgg aagccataat aagaattctg caacaactgc
     5761 tgtttatcca tttcagaatt gggtgtcgac atagcagaat aggcgttact cgacagagga
     5821 gagcaagaaa tggagccagt agatcctaga ctagagccct ggaagcatcc aggaagtcag
     5881 cctaaaactg cttgtaccaa ttgctattgt aaaaagtgtt gctttcattg ccaagtttgt
     5941 ttcatgacaa aagccttagg catctcctat ggcaggaaga agcggagaca gcgacgaaga
     6001 gctcatcaga acagtcagac tcatcaagct tctctatcaa agcagtaagt agtacatgta
     6061 atgcaaccta taatagtagc aatagtagca ttagtagtag caataataat agcaatagtt
     6121 gtgtggtcca tagtaatcat agaatatagg aaaatattaa gacaaagaaa aatagacagg
     6181 ttaattgata gactaataga aagagcagaa gacagtggca atgagagtga aggagaagta
     6241 tcagcacttg tggagatggg ggtggaaatg gggcaccatg ctccttggga tattgatgat
     6301 ctgtagtgct acagaaaaat tgtgggtcac cgtctattat ggggtacctg tgtggaagga
     6361 agcaaccacc actctatttt gtgcatcaga tgctaaagca tatatgatac agaggtacat
     6421 aatgtttggg ccacacatgc ctgtgtaccc acagacccca acccacaaga agtagtattg
     6481 gtaaatgtga cagaaaattt taacatgtgg aaaaatgaca tggtagaaca gatgcatgag
     6541 gatataatca gtttatggga tcaaagccta aagccatgtg taaaattaac cccactctgt
     6601 gttagtttaa agtgcactga tttgaagaat gatactaata ccaatagtag tagcgggaga
     6661 atgataatgg agaaaggaga gataaaaaac tgctctttca atatcagcac aagcataaga
     6721 gataaggtgc agaaagaata tgcattcttt tataaacttg atatagtacc aatagataat
     6781 accagctata ggttgataag ttgtaacacc tcagtcatta cacaggcctg tccaaaggta
     6841 tcctttgagc caattcccat acattattgt gccccggctg gttttgcgat tctaaaatgt
     6901 aataataaga cgttcaatgg aacaggacca tgtacaaatg tcagcacagt acaatgtaca
     6961 catggaatca ggccagtagt atcaactcaa ctgctgttaa atggcagtct agcagaagaa
     7021 gatgtagtaa ttagatctgc caatttcaca gacaatgcta aaaccataat agtacagctg
     7081 aacacatctg tagaaattaa ttgtacaaga cccaacaaca atacaagaaa aagtatccgt
     7141 atccagaggg gaccagggag agcatttgtt acaataggaa aaataggaaa tatgagacaa
     7201 gcacattgta acattagtag agcaaaatgg aatgccactt taaaacagat agctagcaaa
     7261 ttaagagaac aatttggaaa taataaaaca ataatcttta agcaatcctc aggaggggac
     7321 ccagaaattg taacgcacag ttttaattgt ggaggggaat ttttctactg taattcaaca
     7381 caactgttta atagtacttg gtttaatagt acttggagta ctgaagggtc aaataacact
     7441 gaaggaagtg acacaatcac actcccatgc agaataaaac aatttataaa catgtggcag
     7501 gaagtaggaa aagcaatgta tgcccctccc atcagtggac aaattagatg ttcatcaaat
     7561 attactgggc tgctattaac aagagatggt ggtaataaca acaatgggtc cgagatcttc
     7621 agacctggag gaggcgatat gagggacaat tggagaagtg aattatataa atataaagta
     7681 gtaaaaattg aaccattagg agtagcaccc accaaggcaa agagaagagt ggtgcagaga
     7741 gaaaaaagag cagtgggaat aggagctttg ttccttgggt tcttgggagc agcaggaagc
     7801 actatgggcg cagcgtcaat gacgctgacg gtacaggcca gacaattatt gtctgatata
     7861 gtgcagcagc agaacaattt gctgagggct attgaggcgc aacagcatct gttgcaactc
     7921 acagtctggg gcatcaaaca gctccaggca agaatcctgg ctgtggaaag atacctaaag
     7981 gatcaacagc tcctggggat ttggggttgc tctggaaaac tcatttgcac cactgctgtg
     8041 ccttggaatg ctagttggag taataaatct ctggaacaga tttggaataa catgacctgg
     8101 atggagtggg acagagaaat taacaattac acaagcttaa tacactcctt aattgaagaa
     8161 tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg aattagataa atgggcaagt
     8221 ttgtggaatt ggtttaacat aacaaattgg ctgtggtata taaaattatt cataatgata
     8281 gtaggaggct tggtaggttt aagaatagtt tttgctgtac tttctatagt gaatagagtt
     8341 aggcagggat attcaccatt atcgtttcag acccacctcc caatcccgag gggacccgac
     8401 aggcccgaag gaatagaaga agaaggtgga gagagagaca gagacagatc cattcgatta
     8461 gtgaacggat ccttagcact tatctgggac gatctgcgga gcctgtgcct cttcagctac
     8521 caccgcttga gagacttact cttgattgta acgaggattg tggaacttct gggacgcagg
     8581 gggtgggaag ccctcaaata ttggtggaat ctcctacaat attggagtca ggagctaaag
     8641 aatagtgctg ttagcttgct caatgccaca gccatagcag tagctgaggg gacagatagg
     8701 gttatagaag tagtacaagg agcttgtaga gctattcgcc acatacctag aagaataaga
     8761 cagggcttgg aaaggatttt gctataagat gggtggcgcg gccgctctgg tgagcaaggg
     8821 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg
     8881 ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct
     8941 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct
     9001 gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt
     9061 caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg
     9121 caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
     9181 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
     9241 ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa
     9301 cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca
     9361 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca
     9421 gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
     9481 gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtaactcg agacctagaa
     9541 aaacatggag caatcacaag tagcaataca gcagctaaca atgctgcttg tgcctggcta
     9601 gaagcacaag aggaggaaga ggtgggtttt ccagtcacac ctcaggtacc tttaagacca
     9661 atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa
     9721 gggctaattc actcccaaag aagacaagat atccttgatc tgtggatcta ccacacacaa
     9781 ggctacttcc ctgattggca gaactacaca ccagggccag gggtcagata tccactgacc
     9841 tttggatggt gctacaagct agtaccagtt gagccagata aggtagaaga ggccaataaa
     9901 ggagagaaca ccagcttgtt acaccctgtg agcctgcatg gaatggatga ccctgagaga
     9961 gaagtgttag agtggaggtt tgacagccgc ctagcatttc atcacgtggc ccgagagctg
    10021 catccggagt acttcaagaa ctgctgacat cgagcttgct acaagggact ttccgctggg
    10081 gactttccag ggaggcgtgg cctgggcggg actggggagt ggcgagccct cagatgctgc
    10141 atataagcag ctgctttttg cctgtactgg gtctctctgg ttagaccaga tctgagcctg
    10201 ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt
    10261 gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc
    10321 cttttagtca gtgtggaaaa tctctagcag gccggcccaa ttgacatgtg agcaaaaggc
    10381 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
    10441 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
    10501 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
    10561 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
    10621 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
    10681 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
    10741 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
    10801 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
    10861 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
    10921 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
    10981 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
    11041 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
    11101 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
    11161 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
    11221 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
    11281 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
    11341 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
    11401 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
    11461 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
    11521 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
    11581 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
    11641 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
    11701 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
    11761 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
    11821 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
    11881 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
    11941 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
    12001 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
    12061 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
    12121 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgcg
    12181 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
    12241 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
    12301 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
    12361 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
    12421 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
    12481 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
    12541 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
    12601 aattttaaca aaatatttta attaa
//

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