lentiCRISPRv2 neo

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lentiCRISPRv2neo载体基本信息

质粒名称: lentiCRISPRv2 neo 
出品公司: 爱迪基因
目录编号: 98292
存储实验室: Brett Stringer

载体骨架基本信息
载体名称: lentiCRISPRv2
载体出品公司: Feng Zhang (Addgene #52961)
载体抗性: 氨苄青霉素
空载体大小: 15072 bp
完整质粒大小: --
载体修饰: --
载体类型: 慢病毒,CRISPR
筛选标记: 新霉素G418
高拷贝/低拷贝: 高拷贝
生长条件: 37 ℃

插入基因信息
插入基因名称: P2a-neo
物种来源: --
插入大小: --
基因突变: --
GenBank编号 --
启动子 EF-1a
标签/融合蛋白: --
克隆方法: 限制性内切酶
5' 克隆位点: BamHI (未破坏)
3' 克隆位点: MluI (未破坏)
5' 测序引物 CCAAAGAGGTGCTGGACG
3' 测序引物 WPRE-R (CATAGCGTAAAAGGAGCAACA)

文献引用方法
材料和方法部分: lentiCRISPRv2 neo was a gift from Brett Stringer (Addgene plasmid # 98292 ; http://n2t.net/addgene:98292 ; RRID:Addgene_98292)
参考文献部分: A reference collection of patient-derived cell line and xenograft models of proneural, classical and mesenchymal glioblastoma. Stringer BW, Day BW, D'Souza RCJ, Jamieson PR, Ensbey KS, Bruce ZC, Lim YC, Goasdoue K, Offenhauser C, Akgul S, Allan S, Robertson T, Lucas P, Tollesson G, Campbell S, Winter C, Do H, Dobrovic A, Inglis PL, Jeffree RL, Johns TG, Boyd AW. Sci Rep. 2019 Mar 20;9(1):4902. doi: 10.1038/s41598-019-41277-z. 10.1038/s41598-019-41277-z PubMed 30894629

lentiCRISPRv2neo载体图谱质粒图谱和多克隆位点信息

lentiCRISPRv2 neo质粒图谱

lentiCRISPRv2neo载体简介

lentiCRISPRv2neo载体序列

LOCUS       lentiCRISPRv2 neo               15072 bp ds-DNA     circular SYN 01-FEB-2019
DEFINITION  Variant of lentiCRISPRv2 that confers G418 resistance.
ACCESSION   .
VERSION     .
KEYWORDS    lentiCRISPRv2 neo
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 15072)
  AUTHORS   Brett Stringer
  TITLE     Brett Stringer Lab Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 15072)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Feb 1, 2019 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..15072
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        242..621
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        623..821
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     776..796
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             839..1019
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1066..1191
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1684..1917
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    2444..2561
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2612..2852
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     2612..2632
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2783..2802
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4742..4817
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        4879..5090
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      5109..5118
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             5115..9218
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             9219..9266
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             9267..9290
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             9300..9356
                     /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             9357..10148
                     /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="IEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRPV
                     LFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLSS
                     HLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQG
                     LAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIAL
                     ATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(9408..9427)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     10018..10037
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     misc_feature    10164..10752
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(10217..10237)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(10635..10646)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             10824..11057
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(11083..11100)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    11089..11313
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      11359..11787
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11446..11465)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     11656..11677
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(11796..11816)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        11801..12130
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      11981..12116
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     12043..12062
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        12178..12225
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             12244..12618
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    12748..12869
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(12785..12804)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     12839..12858
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(12918..12934)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12918..12934)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(12931..12953)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    12942..12958
                     /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(12966..12996)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    13011..13032
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(13149..13166)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(13320..13908)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(13400..13419)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(14079..14939)
                     /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     14702..14721
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(14940..15044)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgcgcgt
      241 tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc
      301 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
      361 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg
      421 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
      481 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc
      541 tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta
      601 ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag
      661 cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt
      721 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa
      781 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gcgcgttttg cctgtactgg
      841 gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact
      901 gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg
      961 tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag
     1021 tggcgcccga acagggactt gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg
     1081 actcggcttg ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt gagtacgcca
     1141 aaaattttga ctagcggagg ctagaaggag agagatgggt gcgagagcgt cagtattaag
     1201 cgggggagaa ttagatcgcg atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa
     1261 tataaattaa aacatatagt atgggcaagc agggagctag aacgattcgc agttaatcct
     1321 ggcctgttag aaacatcaga aggctgtaga caaatactgg gacagctaca accatccctt
     1381 cagacaggat cagaagaact tagatcatta tataatacag tagcaaccct ctattgtgtg
     1441 catcaaagga tagagataaa agacaccaag gaagctttag acaagataga ggaagagcaa
     1501 aacaaaagta agaccaccgc acagcaagcg gccgctgatc ttcagacctg gaggaggaga
     1561 tatgagggac aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt
     1621 aggagtagca cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg
     1681 aataggagct ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc
     1741 aatgacgctg acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa
     1801 tttgctgagg gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa
     1861 gcagctccag gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg
     1921 gatttggggt tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg
     1981 gagtaataaa tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga
     2041 aattaacaat tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga
     2101 aaagaatgaa caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa
     2161 cataacaaat tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg
     2221 tttaagaata gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc
     2281 attatcgttt cagacccacc tcccaacccc gaggggaccc gacaggcccg aaggaataga
     2341 agaagaaggt ggagagagag acagagacag atccattcga ttagtgaacg gatcggcact
     2401 gcgtgcgcca attctgcaga caaatggcag tattcatcca caattttaaa agaaaagggg
     2461 ggattggggg gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa
     2521 ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca
     2581 gcagagatcc agtttggtta attaaggtac cgagggccta tttcccatga ttccttcata
     2641 tttgcatata cgatacaagg ctgttagaga gataattaga attaatttga ctgtaaacac
     2701 aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt
     2761 tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga
     2821 tttcttggct ttatatatct tgtggaaagg acgaaacacc ggagacggtt gtaaatgagc
     2881 acacaaaata cacatgctaa aatattatat tctatgacct ttataaaatc aaccaaaatc
     2941 ttctttttaa taactttagt atcaataatt agaattttta tgttcctttt tgcaaacttt
     3001 taataaaaat gagcaaaata aaaaaacgct agttttagta actcgcgttg ttttcttcac
     3061 ctttaataat agctactcca ccacttgttc ctaagcggtc agctcctgct tcaatcattt
     3121 tttgagcatc ttcaaatgtt ctaactccac cagctgcttt aactaaagca ttgtctttaa
     3181 caactgactt cattagttta acatcttcaa atgttgcacc tgattttgaa aatcctgttg
     3241 atgttttaac aaattctaat ccagcttcaa cagctatttc acaagctttc atgatttctt
     3301 cttttgttaa taaacaattt tccataatac atttaacaac atgtgatcca gctgcttttt
     3361 ttacagcttt catgtcttct aaaactaatt cataattttt gtcttttaat gcaccaatat
     3421 ttaataccat atcaatttct gttgcaccat ctttaattgc ttcagaaact tcgaatgctt
     3481 ttgtagctgt tgtgcatgca cctagaggaa aacctacaac atttgttatt cctacatttg
     3541 tgccttttaa taattcttta caatagcttg ttcaatatga attaacacaa actgttgcaa
     3601 aatcaaattc aattgcttca tcacataatt gtttaatttc agctttcgta gcatcttgtt
     3661 ttaataatgt gtgatctata tatttgttta gtttcatttt ttctcctata tattcatttt
     3721 taattttaat tctttaataa tttcgtctac tttaacttta gcgttttgaa cagattcacc
     3781 aacacctata aaataaattt ttagtttagg ttcagttcca cttgggcgaa cagcaaatca
     3841 tgacttatct tctaaataaa attttagtaa gtcttgtcct ggcatattat acattccatc
     3901 gatgtagtct tcaacattaa caactttaag tccagcaatt tgagttaagg gtgttgctct
     3961 caatgatttc attaatggtt caatttttaa tttcttttct tctggtttaa aattcaagtt
     4021 taaagtgaaa gtgtaatatg cacccatttc tttaaataaa tcttctaaat agtctactaa
     4081 tgttttattt tgttttttat aaaatcaagc agcctctgct attaatatag aagcttgtat
     4141 tccatcttta tctctagctg agtcatcaat tacatatcca taactttctt cataagcaaa
     4201 aacaaaattt aatccgttat cttcttcttt agcaatttct ctacccattc atttaaatcc
     4261 agttaaagtt tttacaatat taactccata tttttcatga gcgattctat cacccaaatc
     4321 acttgttaca aaacttgaat atagagccgg attttttgga atgctattta agcgttttag
     4381 atttgataat tttcaatcaa ttaaaattgg tcctgtttga tttccatcta atcttacaaa
     4441 atgaccatca tgttttattg ccattccaaa tctgtcagca tctgggtcat tcataataat
     4501 aatatctgca tcatgtttaa taccatattc aagcggtatt tttcatgcag gatcaaattc
     4561 tggatttgga tttacaacat ttttaaatgt ttcatcttca aatgcatgct cttcaacctc
     4621 aataacgtta tatcctgatt cacgtaatat ttttggggta aatttagttc ctgttccatt
     4681 aactgcgcta aaaataattt ttaaatcttt tttagcttct tgctcttttt tgtacgtctc
     4741 tgttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag
     4801 tggcaccgag tcggtgcttt tttgaattcg ctagctaggt cttgaaagga gtgggaattg
     4861 gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg
     4921 gaggggtcgg caattgatcc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg
     4981 atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag
     5041 tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gaccggttct
     5101 agagcgctgc caccatggac aagaagtaca gcatcggcct ggacatcggc accaactctg
     5161 tgggctgggc cgtgatcacc gacgagtaca aggtgcccag caagaaattc aaggtgctgg
     5221 gcaacaccga ccggcacagc atcaagaaga acctgatcgg agccctgctg ttcgacagcg
     5281 gcgaaacagc cgaggccacc cggctgaaga gaaccgccag aagaagatac accagacgga
     5341 agaaccggat ctgctatctg caagagatct tcagcaacga gatggccaag gtggacgaca
     5401 gcttcttcca cagactggaa gagtccttcc tggtggaaga ggataagaag cacgagcggc
     5461 accccatctt cggcaacatc gtggacgagg tggcctacca cgagaagtac cccaccatct
     5521 accacctgag aaagaaactg gtggacagca ccgacaaggc cgacctgcgg ctgatctatc
     5581 tggccctggc ccacatgatc aagttccggg gccacttcct gatcgagggc gacctgaacc
     5641 ccgacaacag cgacgtggac aagctgttca tccagctggt gcagacctac aaccagctgt
     5701 tcgaggaaaa ccccatcaac gccagcggcg tggacgccaa ggccatcctg tctgccagac
     5761 tgagcaagag cagacggctg gaaaatctga tcgcccagct gcccggcgag aagaagaatg
     5821 gcctgttcgg aaacctgatt gccctgagcc tgggcctgac ccccaacttc aagagcaact
     5881 tcgacctggc cgaggatgcc aaactgcagc tgagcaagga cacctacgac gacgacctgg
     5941 acaacctgct ggcccagatc ggcgaccagt acgccgacct gtttctggcc gccaagaacc
     6001 tgtccgacgc catcctgctg agcgacatcc tgagagtgaa caccgagatc accaaggccc
     6061 ccctgagcgc ctctatgatc aagagatacg acgagcacca ccaggacctg accctgctga
     6121 aagctctcgt gcggcagcag ctgcctgaga agtacaaaga gattttcttc gaccagagca
     6181 agaacggcta cgccggctac attgacggcg gagccagcca ggaagagttc tacaagttca
     6241 tcaagcccat cctggaaaag atggacggca ccgaggaact gctcgtgaag ctgaacagag
     6301 aggacctgct gcggaagcag cggaccttcg acaacggcag catcccccac cagatccacc
     6361 tgggagagct gcacgccatt ctgcggcggc aggaagattt ttacccattc ctgaaggaca
     6421 accgggaaaa gatcgagaag atcctgacct tccgcatccc ctactacgtg ggccctctgg
     6481 ccaggggaaa cagcagattc gcctggatga ccagaaagag cgaggaaacc atcaccccct
     6541 ggaacttcga ggaagtggtg gacaagggcg cttccgccca gagcttcatc gagcggatga
     6601 ccaacttcga taagaacctg cccaacgaga aggtgctgcc caagcacagc ctgctgtacg
     6661 agtacttcac cgtgtataac gagctgacca aagtgaaata cgtgaccgag ggaatgagaa
     6721 agcccgcctt cctgagcggc gagcagaaaa aggccatcgt ggacctgctg ttcaagacca
     6781 accggaaagt gaccgtgaag cagctgaaag aggactactt caagaaaatc gagtgcttcg
     6841 actccgtgga aatctccggc gtggaagatc ggttcaacgc ctccctgggc acataccacg
     6901 atctgctgaa aattatcaag gacaaggact tcctggacaa tgaggaaaac gaggacattc
     6961 tggaagatat cgtgctgacc ctgacactgt ttgaggacag agagatgatc gaggaacggc
     7021 tgaaaaccta tgcccacctg ttcgacgaca aagtgatgaa gcagctgaag cggcggagat
     7081 acaccggctg gggcaggctg agccggaagc tgatcaacgg catccgggac aagcagtccg
     7141 gcaagacaat cctggatttc ctgaagtccg acggcttcgc caacagaaac ttcatgcagc
     7201 tgatccacga cgacagcctg acctttaaag aggacatcca gaaagcccag gtgtccggcc
     7261 agggcgatag cctgcacgag cacattgcca atctggccgg cagccccgcc attaagaagg
     7321 gcatcctgca gacagtgaag gtggtggacg agctcgtgaa agtgatgggc cggcacaagc
     7381 ccgagaacat cgtgatcgaa atggccagag agaaccagac cacccagaag ggacagaaga
     7441 acagccgcga gagaatgaag cggatcgaag agggcatcaa agagctgggc agccagatcc
     7501 tgaaagaaca ccccgtggaa aacacccagc tgcagaacga gaagctgtac ctgtactacc
     7561 tgcagaatgg gcgggatatg tacgtggacc aggaactgga catcaaccgg ctgtccgact
     7621 acgatgtgga ccatatcgtg cctcagagct ttctgaagga cgactccatc gacaacaagg
     7681 tgctgaccag aagcgacaag aaccggggca agagcgacaa cgtgccctcc gaagaggtcg
     7741 tgaagaagat gaagaactac tggcggcagc tgctgaacgc caagctgatt acccagagaa
     7801 agttcgacaa tctgaccaag gccgagagag gcggcctgag cgaactggat aaggccggct
     7861 tcatcaagag acagctggtg gaaacccggc agatcacaaa gcacgtggca cagatcctgg
     7921 actcccggat gaacactaag tacgacgaga atgacaagct gatccgggaa gtgaaagtga
     7981 tcaccctgaa gtccaagctg gtgtccgatt tccggaagga tttccagttt tacaaagtgc
     8041 gcgagatcaa caactaccac cacgcccacg acgcctacct gaacgccgtc gtgggaaccg
     8101 ccctgatcaa aaagtaccct aagctggaaa gcgagttcgt gtacggcgac tacaaggtgt
     8161 acgacgtgcg gaagatgatc gccaagagcg agcaggaaat cggcaaggct accgccaagt
     8221 acttcttcta cagcaacatc atgaactttt tcaagaccga gattaccctg gccaacggcg
     8281 agatccggaa gcggcctctg atcgagacaa acggcgaaac cggggagatc gtgtgggata
     8341 agggccggga ttttgccacc gtgcggaaag tgctgagcat gccccaagtg aatatcgtga
     8401 aaaagaccga ggtgcagaca ggcggcttca gcaaagagtc tatcctgccc aagaggaaca
     8461 gcgataagct gatcgccaga aagaaggact gggaccctaa gaagtacggc ggcttcgaca
     8521 gccccaccgt ggcctattct gtgctggtgg tggccaaagt ggaaaagggc aagtccaaga
     8581 aactgaagag tgtgaaagag ctgctgggga tcaccatcat ggaaagaagc agcttcgaga
     8641 agaatcccat cgactttctg gaagccaagg gctacaaaga agtgaaaaag gacctgatca
     8701 tcaagctgcc taagtactcc ctgttcgagc tggaaaacgg ccggaagaga atgctggcct
     8761 ctgccggcga actgcagaag ggaaacgaac tggccctgcc ctccaaatat gtgaacttcc
     8821 tgtacctggc cagccactat gagaagctga agggctcccc cgaggataat gagcagaaac
     8881 agctgtttgt ggaacagcac aagcactacc tggacgagat catcgagcag atcagcgagt
     8941 tctccaagag agtgatcctg gccgacgcta atctggacaa agtgctgtcc gcctacaaca
     9001 agcaccggga taagcccatc agagagcagg ccgagaatat catccacctg tttaccctga
     9061 ccaatctggg agcccctgcc gccttcaagt actttgacac caccatcgac cggaagaggt
     9121 acaccagcac caaagaggtg ctggacgcca ccctgatcca ccagagcatc accggcctgt
     9181 acgagacacg gatcgacctg tctcagctgg gaggcgacaa gcgacctgcc gccacaaaga
     9241 aggctggaca ggctaagaag aagaaagatt acaaagacga tgacgataag ggatccggcg
     9301 caacaaactt ctctctgctg aaacaagccg gagatgtcga agagaatcct ggaccgattg
     9361 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
     9421 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
     9481 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg
     9541 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg
     9601 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
     9661 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
     9721 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
     9781 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
     9841 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg
     9901 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
     9961 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
    10021 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
    10081 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
    10141 tcttctgaac gcgttaagtc gacaatcaac ctctggatta caaaatttgt gaaagattga
    10201 ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt
    10261 tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt
    10321 tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg
    10381 tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg
    10441 ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc
    10501 gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat
    10561 catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct
    10621 tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg
    10681 ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg
    10741 ccgcctcccc gcgtcgactt taagaccaat gacttacaag gcagctgtag atcttagcca
    10801 ctttttaaaa gaaaaggggg gactggaagg gctaattcac tcccaacgaa gacaagatct
    10861 gctttttgct tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg
    10921 ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt
    10981 gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt
    11041 gtggaaaatc tctagcaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta
    11101 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
    11161 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
    11221 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
    11281 gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg
    11341 gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
    11401 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
    11461 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
    11521 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
    11581 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
    11641 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
    11701 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
    11761 tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg
    11821 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
    11881 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
    11941 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
    12001 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
    12061 ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
    12121 cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc agcacgtgtt
    12181 gacaattaat catcggcata gtatatcggc atagtataat acgacaaggt gaggaactaa
    12241 accatggcca agttgaccag tgccgttccg gtgctcaccg cgcgcgacgt cgccggagcg
    12301 gtcgagttct ggaccgaccg gctcgggttc tcccgggact tcgtggagga cgacttcgcc
    12361 ggtgtggtcc gggacgacgt gaccctgttc atcagcgcgg tccaggacca ggtggtgccg
    12421 gacaacaccc tggcctgggt gtgggtgcgc ggcctggacg agctgtacgc cgagtggtcg
    12481 gaggtcgtgt ccacgaactt ccgggacgcc tccgggccgg ccatgaccga gatcggcgag
    12541 cagccgtggg ggcgggagtt cgccctgcgc gacccggccg gcaactgcgt gcacttcgtg
    12601 gccgaggagc aggactgaca cgtgctacga gatttcgatt ccaccgccgc cttctatgaa
    12661 aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat
    12721 ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa
    12781 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
    12841 ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag
    12901 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
    12961 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
    13021 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
    13081 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
    13141 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
    13201 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
    13261 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
    13321 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
    13381 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
    13441 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
    13501 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
    13561 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
    13621 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
    13681 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
    13741 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
    13801 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
    13861 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
    13921 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
    13981 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
    14041 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
    14101 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
    14161 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
    14221 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
    14281 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
    14341 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
    14401 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
    14461 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
    14521 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
    14581 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
    14641 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
    14701 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
    14761 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
    14821 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
    14881 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
    14941 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
    15001 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
    15061 gtgccacctg ac
//

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