pPlatTET-gRNA2

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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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pPlatTETgRNA2载体基本信息

质粒名称: pPlatTET-gRNA2 
出品公司: 爱迪基因
目录编号: 82559
存储实验室: Izuho Hatada

载体骨架基本信息
载体名称: pCAG
载体出品公司: --
载体抗性: 卡那霉素
空载体大小: --
完整质粒大小: --
载体修饰: --
载体类型: 哺乳细胞表达
筛选标记: 新霉素G418
高拷贝/低拷贝: --
生长条件: 37 ℃

插入基因信息
插入基因名称: dCas9-5xPlat2AflD-P2A-scFvGCN4sfGFPTET1CD
物种来源: --
插入大小: --
基因突变: --
GenBank编号 --
启动子 --
标签/融合蛋白: --
克隆方法: --
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: pPlatTET-gRNA2 was a gift from Izuho Hatada (Addgene plasmid # 82559 ; http://n2t.net/addgene:82559 ; RRID:Addgene_82559)
参考文献部分: Targeted DNA demethylation in vivo using dCas9-peptide repeat and scFv-TET1 catalytic domain fusions. Morita S, Noguchi H, Horii T, Nakabayashi K, Kimura M, Okamura K, Sakai A, Nakashima H, Hata K, Nakashima K, Hatada I. Nat Biotechnol. 2016 Aug 29. doi: 10.1038/nbt.3658. 10.1038/nbt.3658 PubMed 27571369

pPlatTETgRNA2载体图谱质粒图谱和多克隆位点信息

pPlatTET-gRNA2质粒图谱

pPlatTETgRNA2载体简介

pPlatTETgRNA2载体序列

LOCUS       pPlatTET-gRNA2             14290 bp ds-DNA     circular SYN 06-AUG-2018
DEFINITION  All in one vector which contains Cas9 peptide array (linker length: 
            22aa), antibody-sfGFP-TET1CD, and gRNA expression system..
ACCESSION   .
VERSION     .
KEYWORDS    pPlatTET-gRNA2
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14290)
  AUTHORS   Morita S, Noguchi H, Horii T, Nakabayashi K, Kimura M, Okamura K, 
            Sakai A, Nakashima H, Hata K, Nakashima K, Hatada I
  TITLE     Targeted DNA demethylation in vivo using dCas9-peptide repeat and 
            scFv-TET1 catalytic domain fusions.
  JOURNAL   Nat Biotechnol. 2016 Aug 29. doi: 10.1038/nbt.3658.
  PUBMED    27571369
REFERENCE   2  (bases 1 to 14290)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 6, 2018 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..14290
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          582..1598
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1522..1544
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1606..1625
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             1674..1694
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      1743..1752
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1749..5852
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             5856..5882
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             5901..5921
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5928..5948
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6012..6068
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             6135..6191
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             6258..6314
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             6381..6437
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             6504..6560
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             6648..6704
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             7452..7478
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             7569..8279
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGT
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKAN
                     FKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(7596..7624)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     8219..8239
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     polyA_signal    10606..10687
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10643..10662)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10697..10716
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     10804..10824
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     10975..10994
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(11101..11125)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     rep_origin      complement(11146..11601)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11283..11304)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     11495..11514
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        11628..11732
                     /gene="bla"
                     /label=AmpR promoter
     promoter        11734..12091
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     primer_bind     complement(11757..11777)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      11942..12077
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     12004..12023
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             12126..12920
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(12180..12199)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     12790..12809
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(13108..13127)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    13152..13199
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      13528..14116
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     14017..14036
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catggtcgag gtgagcccca cgttctgctt cactctcccc atctcccccc cctccccacc
      361 cccaattttg tatttattta ttttttaatt attttgtgca gcgatggggg cggggggggg
      421 gggggggcgc gcccggcggg ggggggcggg gcgaggggcg gggcggggcg aggcggagag
      481 gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt tccttttatg gcgaggcggc
      541 ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc gggagtcgct gcgcgctgcc
      601 ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc gccccggctc tgactgaccg
      661 cgttactccc acaggtgagc gggcgggacg gcccttctcc tccgggctgt aattagcgct
      721 tggtttaatg acggcttgtt tcttttctgt ggctgcgtga aagccttgag gggctccggg
      781 agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg cgtgtgtgtg tgcgtgggga
      841 gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg ctgcgggcgc ggcgcggggc
      901 tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg gggcggtgcc ccgcggtgcg
      961 gggggggctg cgaggggaac aaaggctgcg tgcggggtgt gtgcgtgggg gggtgagcag
     1021 ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca cccccctccc cgagttgctg
     1081 agcacggccc ggcttcgggt gcggggctcc gtacggggcg tggcgcgggg ctcgccgtgc
     1141 cgggcggggg gtggcggcag gtgggggtgc cgggcggggc ggggccgcct cgggccgggg
     1201 agggctcggg ggaggggcgc ggcggccccc ggagcgccgg cggctgtcga ggcgcggcga
     1261 gccgcagcca ttgcctttta tggtaatcgt gcgagagggc gcagggactt cctttgtccc
     1321 aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac cccctctagc gggcgcgggg
     1381 cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga gggccttcgt gcgtcgccgc
     1441 gccgccgtcc ccttctccct ctccagcctc ggggctgtcc gcggggggac ggctgccttc
     1501 gggggggacg gggcagggcg gggttcggct tctggcgtgt gaccggcggc tctagagcct
     1561 ctgctaacca tgttcatgcc ttcttctttt tcctacagct cctgggcaac gtgctggtta
     1621 ttgtgctgtc tcatcatttt ggcaaagaat tcccccgcga tcgcgccacc atgcccaaga
     1681 agaagcgcaa ggtgggacgc gtctgcagga tatcaagctt gcggtaccgc gggcccggga
     1741 tcgccaccat ggacaagaag tacagcatcg gcctggccat cggcaccaac tctgtgggct
     1801 gggccgtgat caccgacgag tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca
     1861 ccgaccggca cagcatcaag aagaacctga tcggcgccct gctgttcgac agcggagaaa
     1921 cagccgaggc cacccggctg aagagaaccg ccagaagaag atacaccaga cggaagaacc
     1981 ggatctgcta tctgcaagag atcttcagca acgagatggc caaggtggac gacagcttct
     2041 tccacagact ggaagagtcc ttcctggtgg aagaggataa gaagcacgag cggcacccca
     2101 tcttcggcaa catcgtggac gaggtggcct accacgagaa gtaccccacc atctaccacc
     2161 tgagaaagaa actggtggac agcaccgaca aggccgacct gcggctgatc tatctggccc
     2221 tggcccacat gatcaagttc cggggccact tcctgatcga gggcgacctg aaccccgaca
     2281 acagcgacgt ggacaagctg ttcatccagc tggtgcagac ctacaaccag ctgttcgagg
     2341 aaaaccccat caacgccagc ggcgtggacg ccaaggccat cctgtctgcc agactgagca
     2401 agagcagacg gctggaaaat ctgatcgccc agctgcccgg cgagaagaag aatggcctgt
     2461 tcggcaacct gattgccctg agcctgggcc tgacccccaa cttcaagagc aacttcgacc
     2521 tggccgagga tgccaaactg cagctgagca aggacaccta cgacgacgac ctggacaacc
     2581 tgctggccca gatcggcgac cagtacgccg acctgtttct ggccgccaag aacctgtccg
     2641 acgccatcct gctgagcgac atcctgagag tgaacaccga gatcaccaag gcccccctga
     2701 gcgcctctat gatcaagaga tacgacgagc accaccagga cctgaccctg ctgaaagctc
     2761 tcgtgcggca gcagctgcct gagaagtaca aagagatttt cttcgaccag agcaagaacg
     2821 gctacgccgg ctacatcgat ggcggagcca gccaggaaga gttctacaag ttcatcaagc
     2881 ccatcctgga aaagatggac ggcaccgagg aactgctcgt gaagctgaac agagaggacc
     2941 tgctgcggaa gcagcggacc ttcgacaacg gcagcatccc ccaccagatc cacctgggag
     3001 agctgcacgc cattctgcgg cggcaggaag atttttaccc attcctgaag gacaaccggg
     3061 aaaagatcga gaagatcctg accttccgca tcccctacta cgtgggccct ctggccaggg
     3121 gaaacagcag attcgcctgg atgaccagaa agagcgagga aaccatcacc ccctggaact
     3181 tcgaggaagt ggtggacaag ggcgccagcg cccagagctt catcgagcgg atgaccaact
     3241 tcgataagaa cctgcccaac gagaaggtgc tgcccaagca cagcctgctg tacgagtact
     3301 tcaccgtgta caacgagctg accaaagtga aatacgtgac cgagggaatg agaaagcccg
     3361 ccttcctgag cggcgagcag aaaaaagcca tcgtggacct gctgttcaag accaaccgga
     3421 aagtgaccgt gaagcagctg aaagaggact acttcaagaa aatcgagtgc ttcgactccg
     3481 tggaaatctc cggcgtggaa gatcggttca acgcctccct gggcacatac cacgatctgc
     3541 tgaaaattat caaggacaag gacttcctgg acaatgagga aaacgaggac attctggaag
     3601 atatcgtgct gaccctgaca ctgtttgagg acagagagat gatcgaggaa cggctgaaaa
     3661 cctatgccca cctgttcgac gacaaagtga tgaagcagct gaagcggcgg agatacaccg
     3721 gctggggcag gctgagccgg aagctgatca acggcatccg ggacaagcag tccggcaaga
     3781 caatcctgga tttcctgaag tccgacggct tcgccaacag aaacttcatg cagctgatcc
     3841 acgacgacag cctgaccttt aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg
     3901 atagcctgca cgagcacatt gccaatctgg ccggcagccc cgccattaag aagggcatcc
     3961 tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat gggccggcac aagcccgaga
     4021 acatcgtgat cgaaatggcc agagagaacc agaccaccca gaagggacag aagaacagcc
     4081 gcgagagaat gaagcggatc gaagagggca tcaaagagct gggcagccag atcctgaaag
     4141 aacaccccgt ggaaaacacc cagctgcaga acgagaagct gtacctgtac tacctgcaga
     4201 atgggcggga tatgtacgtg gaccaggaac tggacatcaa ccggctgtcc gactacgatg
     4261 tggacgctat cgtgcctcag agctttctga aggacgactc catcgataac aaagtgctga
     4321 ctcggagcga caagaaccgg ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga
     4381 agatgaagaa ctactggcgc cagctgctga atgccaagct gattacccag aggaagttcg
     4441 acaatctgac caaggccgag agaggcggcc tgagcgaact ggataaggcc ggcttcatca
     4501 agagacagct ggtggaaacc cggcagatca caaagcacgt ggcacagatc ctggactccc
     4561 ggatgaacac taagtacgac gagaacgaca aactgatccg ggaagtgaaa gtgatcaccc
     4621 tgaagtccaa gctggtgtcc gatttccgga aggatttcca gttttacaaa gtgcgcgaga
     4681 tcaacaacta ccaccacgcc cacgacgcct acctgaacgc cgtcgtggga accgccctga
     4741 tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg
     4801 tgcggaagat gatcgccaag agcgagcagg aaatcggcaa ggctaccgcc aagtacttct
     4861 tctacagcaa catcatgaac tttttcaaga ccgagattac cctggccaac ggcgagatcc
     4921 ggaagcggcc tctgatcgag acaaacggcg aaacaggcga gatcgtgtgg gataagggcc
     4981 gggactttgc caccgtgcgg aaagtgctgt ctatgcccca agtgaatatc gtgaaaaaga
     5041 ccgaggtgca gacaggcggc ttcagcaaag agtctatcct gcccaagagg aacagcgaca
     5101 agctgatcgc cagaaagaag gactgggacc ctaagaagta cggcggcttc gacagcccca
     5161 ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa gggcaagtcc aagaaactga
     5221 agagtgtgaa agagctgctg gggatcacca tcatggaaag aagcagcttc gagaagaatc
     5281 ccatcgactt tctggaagcc aagggctaca aagaagtgaa aaaggacctg atcatcaagc
     5341 tgcctaagta ctccctgttc gagctggaaa acggccggaa gagaatgctg gcctctgccg
     5401 gcgaactgca gaagggaaac gaactggccc tgccctccaa atatgtgaac ttcctgtacc
     5461 tggccagcca ctatgagaag ctgaagggct cccccgagga taatgagcag aaacagctgt
     5521 ttgtggaaca gcacaaacac tacctggacg agatcatcga gcagatcagc gagttctcca
     5581 agagagtgat cctggccgac gctaatctgg acaaggtgct gagcgcctac aacaagcaca
     5641 gagacaagcc tatcagagag caggccgaga atatcatcca cctgtttacc ctgaccaatc
     5701 tgggagcccc tgccgccttc aagtactttg acaccaccat cgaccggaag aggtacacca
     5761 gcaccaaaga ggtgctggac gccaccctga tccaccagag catcaccggc ctgtacgaga
     5821 cacggatcga cctgtctcag ctgggaggcg acgcctatcc ctatgacgtg cccgattatg
     5881 ccagcctggg cagcggctcc cccaagaaaa aacgcaaggt ggaagatcct aagaaaaagc
     5941 ggaaagtgga cggcattggt agtgggagca acggcagcag cggatccaac ggtccgactg
     6001 acgccgcgga agaggagctt ctgagcaaaa actatcacct cgaaaacgag gttgcgcgac
     6061 tgaagaaagg aagcgggtcc ggtggaagtg gctccggatc tggaggttct ggcagcggag
     6121 gtagcggcag tggcgaagag ctccttagta agaactatca tctggaaaat gaggtagcgc
     6181 gcttaaagaa agggtcggga agtggcggca gcggaagtgg gagtggaggg agcggttctg
     6241 gcggttccgg cagtggagag gagttgctgt ctaagaacta ccacttagaa aacgaagtcg
     6301 cacggctaaa aaaaggttcc ggctccggcg gctccggttc tggaagcggg ggctcgggat
     6361 caggtggatc tggatcagga gaggaattgc tttccaaaaa ctaccacctt gagaatgagg
     6421 tggccaggtt aaagaagggg agcggctcgg ggggtagtgg atcggggtcg ggcgggtcag
     6481 gaagcggtgg tagcggatct ggggaggagc tgctctcgaa gaattaccat ttggagaacg
     6541 aagtggcgag actaaagaag ggaagcggta gtggtggttc agggtctggt tcaggtggca
     6601 gtgggtctgg gggctcaggg tccgggacgg ccggcctcgg aagcggagct actaacttca
     6661 gcctgctgaa gcaggctgga gacgtggagg agaaccctgg acctagtacc atgggccccg
     6721 acatcgtgat gacccagagc cccagcagcc tgagcgccag cgtgggcgac cgcgtgacca
     6781 tcacctgccg cagcagcacc ggcgccgtga ccaccagcaa ctacgccagc tgggtgcagg
     6841 agaagcccgg caagctgttc aagggcctga tcggcggcac caacaaccgc gcccccggcg
     6901 tgcccagccg cttcagcggc agcctgatcg gcgacaaggc caccctgacc atcagcagcc
     6961 tgcagcccga ggacttcgcc acctacttct gcgccctgtg gtacagcaac cactgggtgt
     7021 tcggccaggg caccaaggtg gagctgaagc gcggcggcgg cggcagcggc ggcggcggca
     7081 gcggcggcgg cggcagcagc ggcggcggca gcgaggtgaa gctgctggag agcggcggcg
     7141 gcctggtgca gcccggcggc agcctgaagc tgagctgcgc cgtgagcggc ttcagcctga
     7201 ccgactacgg cgtgaactgg gtgcgccagg cccccggccg cggcctggag tggatcggcg
     7261 tgatctgggg cgacggcatc accgactaca acagcgccct gaaggaccgc ttcatcatca
     7321 gcaaggacaa cggcaagaac accgtgtacc tgcagatgag caaggtgcgc agcgacgaca
     7381 ccgccctgta ctactgcgtg accggcctgt tcgactactg gggccagggc accctggtga
     7441 ccgtgagcag ctacccatac gatgttccag attacgctgg tggaggcgga ggttctgggg
     7501 gaggaggtag tggcggtggt ggttcaggag gcggcggaag cttggatcca ggtggaggtg
     7561 gaagcggtag caaaggagaa gaacttttca ctggagttgt cccaattctt gttgaattag
     7621 atggtgatgt taatgggcac aaattttctg tccgtggaga gggtgaaggt gatgctacaa
     7681 acggaaaact cacccttaaa tttatttgca ctactggaaa actacctgtt ccgtggccaa
     7741 cacttgtcac tactctgacc tatggtgttc aatgcttttc ccgttatccg gatcacatga
     7801 aacggcatga ctttttcaag agtgccatgc ccgaaggtta tgtacaggaa cgcactatat
     7861 ctttcaaaga tgacgggacc tacaagacgc gtgctgaagt caagtttgaa ggtgataccc
     7921 ttgttaatcg tatcgagtta aagggtattg attttaaaga agatggaaac attcttggac
     7981 acaaactcga gtacaacttt aactcacaca atgtatacat cacggcagac aaacaaaaga
     8041 atggaatcaa agctaacttc aaaattcgcc acaacgttga agatggttcc gttcaactag
     8101 cagaccatta tcaacaaaat actccaattg gcgatggccc tgtcctttta ccagacaacc
     8161 attacctgtc gacacaatct gtcctttcga aagatcccaa cgaaaagcgt gaccacatgg
     8221 tccttcttga gtttgtaact gctgctggga ttacacatgg catggatgag ctctacaaag
     8281 gtggaggtcg gaccggtggc ggtggcggag gggctagcag atccgaactg cccacctgca
     8341 gctgtcttga tcgagttata caaaaagaca aaggcccata ttatacacac cttggggcag
     8401 gaccaagtgt tgctgctgtc agggaaatca tggagaatag gtatggtcaa aaaggaaacg
     8461 caataaggat agaaatagta gtgtacaccg gtaaagaagg gaaaagctct catgggtgtc
     8521 caattgctaa gtgggtttta agaagaagca gtgatgaaga aaaagttctt tgtttggtcc
     8581 ggcagcgtac aggccaccac tgtccaactg ctgtgatggt ggtgctcatc atggtgtggg
     8641 atggcatccc tcttccaatg gccgaccggc tatacacaga gctcacagag aatctaaagt
     8701 catacaatgg gcaccctacc gacagaagat gcaccctcaa tgaaaatcgt acctgtacat
     8761 gtcaaggaat tgatccagag acttgtggag cttcattctc ttttggctgt tcatggagta
     8821 tgtactttaa tggctgtaag tttggtagaa gcccaagccc cagaagattt agaattgatc
     8881 caagctctcc cttacatgaa aaaaaccttg aagataactt acagagtttg gctacacgat
     8941 tagctccaat ttataagcag tatgctccag tagcttacca aaatcaggtg gaatatgaaa
     9001 atgttgcccg agaatgtcgg cttggcagca aggaaggtcg tcccttctct ggggtcactg
     9061 cttgcctgga cttctgtgct catccccaca gggacattca caacatgaat aatggaagca
     9121 ctgtggtttg taccttaact cgagaagata accgctcttt gggtgttatt cctcaagatg
     9181 agcagctcca tgtgctacct ctttataagc tttcagacac agatgagttt ggctccaagg
     9241 aaggaatgga agccaagatc aaatctgggg ccatcgaggt cctggcaccc cgccgcaaaa
     9301 aaagaacgtg tttcactcag cctgttcccc gttctggaaa gaagagggct gcgatgatga
     9361 cagaggttct tgcacataag ataagggcag tggaaaagaa acctattccc cgaatcaagc
     9421 ggaagaataa ctcaacaaca acaaacaaca gtaagccttc gtcactgcca accttaggga
     9481 gtaacactga gaccgtgcaa cctgaagtaa aaagtgaaac cgaaccccat tttatcttaa
     9541 aaagttcaga caacactaaa acttattcgc tgatgccatc cgctcctcac ccagtgaaag
     9601 aggcatctcc aggcttctcc tggtccccga agactgcttc agccacacca gctccactga
     9661 agaatgacgc aacagcctca tgcgggtttt cagaaagaag cagcactccc cactgtacga
     9721 tgccttcggg aagactcagt ggtgccaatg cagctgctgc tgatggccct ggcatttcac
     9781 agcttggcga agtggctcct ctccccaccc tgtctgctcc tgtgatggag cccctcatta
     9841 attctgagcc ttccactggt gtgactgagc cgctaacgcc tcatcagcca aaccaccagc
     9901 cctccttcct cacctctcct caagaccttg cctcttctcc aatggaagaa gatgagcagc
     9961 attctgaagc agatgagcct ccatcagacg aacccctatc tgatgacccc ctgtcacctg
    10021 ctgaggagaa attgccccac attgatgagt attggtcaga cagtgagcac atctttttgg
    10081 atgcaaatat tggtggggtg gccatcgcac ctgctcacgg ctcggttttg attgagtgtg
    10141 cccggcgaga gctgcacgct accactcctg ttgagcaccc caaccgtaat catccaaccc
    10201 gcctctccct tgtcttttac cagcacaaaa acctaaataa gccccaacat ggttttgaac
    10261 taaacaagat taagtttgag gctaaagaag ctaagaataa gaaaatgaag gcctcagagc
    10321 aaaaagacca ggcagctaat gaaggtccag aacagtcctc tgaagtaaat gaattgaacc
    10381 aaattccttc tcataaagca ttaacattaa cccatgacaa tgttgtcacc gtgtcccctt
    10441 atgctctcac acacgttgcg gggccctata accattgggt ctgagcggcc gcgactctag
    10501 atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac
    10561 ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca
    10621 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
    10681 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttggcg cgcctgtaca
    10741 aaaaagcagg ctttaaagga accaattcag tcgactggat ccggtaccaa ggtcgggcag
    10801 gaagagggcc tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga
    10861 gagataatta gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt
    10921 agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat
    10981 catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttaagttaa
    11041 aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct ttttttctag
    11101 acccagcttt cttgtacaaa gttggcatta ggcgcgccaa ggcgtaaatt gtaagcgtta
    11161 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg
    11221 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg
    11281 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa
    11341 aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg
    11401 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt
    11461 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
    11521 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta
    11581 atgcgccgct acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
    11641 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
    11701 aaatgcttca ataatattga aaaaggaaga gtcctgaggc ggaaagaacc agctgtggaa
    11761 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
    11821 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag
    11881 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
    11941 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
    12001 ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg
    12061 aggctttttt ggaggcctag gcttttgcaa agatcgatca agagacagga tgaggatcgt
    12121 ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc
    12181 tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc
    12241 tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg
    12301 aactgcaaga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag
    12361 ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg
    12421 ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg
    12481 caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac
    12541 atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg
    12601 acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgagcatgc
    12661 ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg
    12721 aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc
    12781 aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc
    12841 gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc
    12901 ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc
    12961 caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg
    13021 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt
    13081 cttcgcccac cctaggggga ggctaactga aacacggaag gagacaatac cggaaggaac
    13141 ccgcgctatg acggcaataa aaagacagaa taaaacgcac ggtgttgggt cgtttgttca
    13201 taaacgcggg gttcggtccc agggctggca ctctgtcgat accccaccga gaccccattg
    13261 gggccaatac gcccgcgttt cttccttttc cccaccccac cccccaagtt cgggtgaagg
    13321 cccagggctc gcagccaacg tcggggcggc aggccctgcc atagcctcag gttactcata
    13381 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
    13441 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
    13501 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
    13561 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
    13621 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
    13681 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
    13741 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
    13801 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
    13861 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
    13921 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
    13981 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
    14041 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
    14101 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
    14161 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
    14221 cgccatgcat tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
    14281 tggagttccg
//

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