pYLCRISPR/Cas9pUbi-N

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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: pYLCRISPR/Cas9pUbi-N 
出品公司: 爱迪基因
目录编号: 106331
存储实验室: Yao-Guang Liu

载体骨架基本信息
载体名称: pCAMBIA1300
载体出品公司: CAMBIA
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: 植物表达,CRISPR
筛选标记: --
高拷贝/低拷贝: --
生长条件: 37 ℃

插入基因信息
插入基因名称: Cas9
物种来源: Synthetic
插入大小: --
基因突变: 植物密码子优化后,5'区(400bp)GC含量较高(62.5%),总GC含量为54.2%
GenBank编号 MG719602
启动子 ZmUbi
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: pYLCRISPR/Cas9pUbi-N was a gift from Yao-Guang Liu (Addgene plasmid # 106331 ; http://n2t.net/addgene:106331 ; RRID:Addgene_106331)
参考文献部分: A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, Wang B, Yang Z, Li H, Lin Y, Xie Y, Shen R, Chen S, Wang Z, Chen Y, Guo J, Chen L, Zhao X, Dong Z, Liu YG. Mol Plant. 2015 Apr 24. pii: S1674-2052(15)00204-X. doi: 10.1016/j.molp.2015.04.007. 10.1016/j.molp.2015.04.007 PubMed 25917172

载体图谱质粒图谱和多克隆位点信息

pYLCRISPRCas9pUbi-N质粒图谱

载体简介

载体序列

LOCUS       pYLCRISPR/Cas9pUbi-N             17355 bp ds-DNA     circular SYN 06-AUG-2018
DEFINITION  Expression of Cas9 in plants, ZmUbi promoter, Kanamycin selection.
ACCESSION   .
VERSION     .
KEYWORDS    pYLCRISPR/Cas9pUbi-N
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 17355)
  AUTHORS   Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, Wang B, Yang Z, Li H, 
            Lin Y, Xie Y, Shen R, Chen S, Wang Z, Chen Y, Guo J, Chen L, Zhao X,
            Dong Z, Liu YG
  TITLE     A robust CRISPR/Cas9 system for convenient, high-efficiency 
            multiplex genome editing in monocot and dicot plants.
  JOURNAL   Mol Plant. 2015 Apr 24. pii: S1674-2052(15)00204-X. doi: 
            10.1016/j.molp.2015.04.007.
  PUBMED    25917172
REFERENCE   2  (bases 1 to 17355)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 6, 2018 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..17355
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     protein_bind    94..115
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        130..160
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    168..184
                     /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)."
     primer_bind     173..195
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     192..208
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     192..208
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             2316..2336
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2361..6461
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     RBS             3547..3555
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             6462..6509
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     terminator      6522..6774
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    6892..6913
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        6928..6958
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6966..6982
                     /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        7024..7042
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     7024..7041
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             7116..7418
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMPSVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     7297..7316
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(7628..7661)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     primer_bind     complement(7691..7708)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7691..7707)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7700..7722)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     misc_feature    7910..7934
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             9235..9864
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             10293..11366
                     /codon_start=1
                     /product="replication protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 RepA
                     /translation="MSGRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESW
                     QAAADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLS
                     KRDRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKP
                     GRVFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGE
                     ALISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYR
                     LARRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPI
                     LVMRYRNLIEGEASAGS"
     rep_origin      11432..11626
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     primer_bind     complement(11862..11884)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    11970..12110
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     12025..12044
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(12125..12142)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(12296..12884)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(12376..12395)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(12971..13765)
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     misc_feature    15389..15413
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     polyA_signal    15491..15665
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     CDS             complement(15722..16519)
                     /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="MGIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR
                     PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLL
                     SSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEH
                     QGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDI
                     ALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(15833..15852)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     16443..16462
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(16582..17258)
                     /label=CaMV 35S promoter (enhanced)
                     /note="cauliflower mosaic virus 35S promoter with a 
                     duplicated enhancer region"
     primer_bind     complement(16625..16645)
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
ORIGIN
        1 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
       61 cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc
      121 accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata
      181 acaatttcac acaggaaaca gctatgacat gattacgaat tcgagctcgg taccgctcgc
      241 taccttaaga gaggatatcc ctccatccta taatgtaggc tataggaact agggcaaggc
      301 cggccatgcg gccgcaagct gggtgcagcg tgacccggtc gtgcccctct ctagagataa
      361 tgagcattgc atgtctaagt tataaaaaat taccacatat tttttttgtc acacttgttt
      421 gaagtgcagt ttatctatct ttatacatat atttaaactt tactctacga ataatataat
      481 ctatagtact acaataatat cagtgtttta gagaatcata taaatgaaca gttagacatg
      541 gtctaaagga caattgagta ttttgacaac aggactctac agttttatct ttttagtgtg
      601 catgtgttct cctttttttt tgcaaatagc ttcacctata taatacttca tccattttat
      661 tagtacatcc atttagggtt tagggttaat ggtttttata gactaatttt tttagtacat
      721 ctattttatt ctattttagc ctctaaatta agaaaactaa aactctattt tagttttttt
      781 atttaataat ttagatataa aatagaataa aataaagtga ctaaaaatta aacaaatacc
      841 ctttaagaaa ttaaaaaaac taaggaaaca tttttcttgt ttcgagtaga taatgccagc
      901 ctgttaaacg ccgtcgacga gtctaacgga caccaaccag cgaaccagca gcgtcgcgtc
      961 gggccaagcg aagcagacgg cacggcatct ctgtcgctgc ctctggaccc ctctcgagag
     1021 ttccgctcca ccgttggact tgctccgctg tcggcatcca gaaattgcgt ggcggagcgg
     1081 cagacgtgag ccggcacggc aggcggcctc ctcctcctct cacggcaccg gcagctacgg
     1141 gggattcctt tcccaccgct ccttcgcttt cccttcctcg cccgccgtaa taaatagaca
     1201 ccccctccac accctctttc cccaacctcg tgttgttcgg agcgcacaca cacacaacca
     1261 gatctccccc aaatccaccc gtcggcacct ccgcttcaag gtacgccgct cgtcctnccc
     1321 cccccccctc tctaccttct ctagatcggc gttccggtcc atggttaggg cccggtagtt
     1381 ctacttctgt tcatgtttgt gttagatccg tgtttgtgtt agatccgtgc tgctagcgtt
     1441 cgtacacgga tgcgacctgt acgtcagaca cgttctgatt gctaacttgc cagtgtttct
     1501 ctttggggaa tcctgggatg gctctagccg ttccgcagac gggatcgatt tcatgatttt
     1561 ttttgtttcg ttgcataggg tttggtttgc ccttttcctt tatttcaata tatgccgtgc
     1621 acttgtttgt cgggtcatct tttcatgctt ttttttgtct tggttgtgat gatgtggtct
     1681 ggttgggcgg tcgttctaga tcggagtaga attctgtttc aaactacctg gtggatttat
     1741 taattttgga tctgtatgtg tgtgccatac atattcatag ttacgaattg aagatgatgg
     1801 atggaaatat cgatctagga taggtataca tgttgatgcg ggttttactg atgcatatac
     1861 agagatgctt tttgttcgct tggttgtgat gatgtggtgt ggttgggcgg tcgttcattc
     1921 gttctagatc ggagtagaat actgtttcaa actacctggt gtatttatta attttggaac
     1981 tgtatgtgtg tgtcatacat cttcatagtt acgagtttaa gatggatgga aatatcgatc
     2041 taggataggt atacatgttg atgtgggttt tactgatgca tatacatgat ggcatatgca
     2101 gcatctattc atatgctcta accttgagta cctatctatt ataataaaca agtatgtttt
     2161 ataattattt tgatcttgat atacttggat gatggcatat gcagcagcta tatgtggatt
     2221 tttttagccc tgccttcata cgctatttat ttgcttggta ctgtttcttt tgtcgatgct
     2281 caccctgttg tttggtgtta cttctgcaga tggctcctaa gaagaagcgg aaggttggta
     2341 ttcacggggt gcctgcggct gacaagaagt actccatcgg cctcgacatc ggcaccaaca
     2401 gcgtcggctg ggcggtgatc accgacgagt acaaggtccc gtccaagaag ttcaaggtcc
     2461 tgggcaacac cgaccgccac tccatcaaga agaacctcat cggcgccctc ctcttcgact
     2521 ccggcgagac ggcggaggcg acccgcctca agcgcaccgc ccgccgccgc tacacccgcc
     2581 gcaagaaccg catctgctac ctccaggaga tcttctccaa cgagatggcg aaggtcgacg
     2641 actccttctt ccaccgcctc gaggagtcct tcctcgtgga ggaggacaag aagcacgagc
     2701 gccaccccat cttcggcaac atcgtcgacg aggtcgccta ccacgagaag taccccacta
     2761 tctaccacct tcgtaagaag cttgttgact ctactgataa ggctgatctt cgtctcatct
     2821 accttgctct cgctcacatg atcaagttcc gtggtcactt ccttatcgag ggtgacctta
     2881 accctgataa ctccgacgtg gacaagctct tcatccagct cgtccagacc tacaaccagc
     2941 tcttcgagga gaaccctatc aacgcttccg gtgtcgacgc taaggcgatc ctttccgcta
     3001 ggctctccaa gtccaggcgt ctcgagaacc tcatcgccca gctccctggt gagaagaaga
     3061 acggtctttt cggtaacctc atcgctctct ccctcggtct gacccctaac ttcaagtcca
     3121 acttcgacct cgctgaggac gctaagcttc agctctccaa ggatacctac gacgatgatc
     3181 tcgacaacct cctcgctcag attggagatc agtacgctga tctcttcctt gctgctaaga
     3241 acctctccga tgctatcctc ctttcggata tccttagggt taacactgag atcactaagg
     3301 ctcctctttc tgcttccatg atcaagcgct acgacgagca ccaccaggac ctcaccctcc
     3361 tcaaggctct tgttcgtcag cagctccccg agaagtacaa ggagatcttc ttcgaccagt
     3421 ccaagaacgg ctacgccggt tacattgacg gtggagctag ccaggaggag ttctacaagt
     3481 tcatcaagcc aatccttgag aagatggatg gtactgagga gcttctcgtt aagcttaacc
     3541 gtgaggacct ccttaggaag cagaggactt tcgataacgg ctctatccct caccagatcc
     3601 accttggtga gcttcacgcc atccttcgta ggcaggagga cttctaccct ttcctcaagg
     3661 acaaccgtga gaagatcgag aagatcctta ctttccgtat tccttactac gttggtcctc
     3721 ttgctcgtgg taactcccgt ttcgcttgga tgactaggaa gtccgaggag actatcaccc
     3781 cttggaactt cgaggaggtt gttgacaagg gtgcttccgc ccagtccttc atcgagcgca
     3841 tgaccaactt cgacaagaac ctccccaacg agaaggtcct ccccaagcac tccctcctct
     3901 acgagtactt cacggtctac aacgagctca ccaaggtcaa gtacgtcacc gagggtatgc
     3961 gcaagcctgc cttcctctcc ggcgagcaga agaaggctat cgttgacctc ctcttcaaga
     4021 ccaaccgcaa ggtcaccgtc aagcagctca aggaggacta cttcaagaag atcgagtgct
     4081 tcgactccgt cgagatcagc ggcgttgagg accgtttcaa cgcttctctc ggtacctacc
     4141 acgatctcct caagatcatc aaggacaagg acttcctcga caacgaggag aacgaggaca
     4201 tcctcgagga catcgtcctc actcttactc tcttcgagga tagggagatg atcgaggaga
     4261 ggctcaagac ttacgctcat ctcttcgatg acaaggttat gaagcagctc aagcgtcgcc
     4321 gttacaccgg ttggggtagg ctctcccgca agctcatcaa cggtatcagg gataagcaga
     4381 gcggcaagac tatcctcgac ttcctcaagt ctgatggttt cgctaacagg aacttcatgc
     4441 agctcatcca cgatgactct cttaccttca aggaggatat tcagaaggct caggtgtccg
     4501 gtcagggcga ctctctccac gagcacattg ctaaccttgc tggttcccct gctatcaaga
     4561 agggcatcct tcagactgtt aaggttgtcg atgagcttgt caaggttatg ggtcgtcaca
     4621 agcctgagaa catcgtcatc gagatggctc gtgagaacca gactacccag aagggtcaga
     4681 agaactcgag ggagcgcatg aagaggattg aggagggtat caaggagctt ggttctcaga
     4741 tccttaagga gcaccctgtc gagaacaccc agctccagaa cgagaagctc tacctctact
     4801 acctccagaa cggtagggat atgtacgttg accaggagct cgacatcaac aggctttctg
     4861 actacgacgt cgaccacatt gttcctcagt ctttccttaa ggatgactcc atcgacaaca
     4921 aggtcctcac gaggtccgac aagaacaggg gtaagtcgga caacgtccct tccgaggagg
     4981 ttgtcaagaa gatgaagaac tactggaggc agcttctcaa cgctaagctc attacccaga
     5041 ggaagttcga caacctcacg aaggctgaga ggggtggcct ttccgagctt gacaaggctg
     5101 gtttcatcaa gaggcagctt gttgagacga ggcagattac caagcacgtt gctcagatcc
     5161 tcgattctag gatgaacacc aagtacgacg agaacgacaa gctcatccgc gaggtcaagg
     5221 tgatcaccct caagtccaag ctcgtctccg acttccgcaa ggacttccag ttctacaagg
     5281 tccgcgagat caacaactac caccacgctc acgatgctta ccttaacgct gtcgttggta
     5341 ccgctcttat caagaagtac cctaagcttg agtccgagtt cgtctacggt gactacaagg
     5401 tctacgacgt tcgtaagatg atcgccaagt ccgagcagga gatcggcaag gccaccgcca
     5461 agtacttctt ctactccaac atcatgaact tcttcaagac cgagatcacc ctcgccaacg
     5521 gcgagatccg caagcgccct cttatcgaga cgaacggtga gactggtgag atcgtttggg
     5581 acaagggtcg cgacttcgct actgttcgca aggtcctttc tatgcctcag gttaacatcg
     5641 tcaagaagac cgaggtccag accggtggct tctccaagga gtctatcctt ccaaagagaa
     5701 actcggacaa gctcatcgct aggaagaagg attgggaccc taagaagtac ggtggtttcg
     5761 actcccctac tgtcgcctac tccgtcctcg tggtcgccaa ggtggagaag ggtaagtcga
     5821 agaagctcaa gtccgtcaag gagctcctcg gcatcaccat catggagcgc tcctccttcg
     5881 agaagaaccc gatcgacttc ctcgaggcca agggctacaa ggaggtcaag aaggacctca
     5941 tcatcaagct ccccaagtac tctcttttcg agctcgagaa cggtcgtaag aggatgctgg
     6001 cttccgctgg tgagctccag aagggtaacg agcttgctct tccttccaag tacgtgaact
     6061 tcctctacct cgcctcccac tacgagaagc tcaagggttc ccctgaggat aacgagcaga
     6121 agcagctctt cgtggagcag cacaagcact acctcgacga gatcatcgag cagatctccg
     6181 agttctccaa gcgcgtcatc ctcgctgacg ctaacctcga caaggtcctc tccgcctaca
     6241 acaagcaccg cgacaagccc atccgcgagc aggccgagaa catcatccac ctcttcacgc
     6301 tcacgaacct cggcgcccct gctgctttca agtacttcga caccaccatc gacaggaagc
     6361 gttacacgtc caccaaggag gttctcgacg ctactctcat ccaccagtcc atcaccggtc
     6421 tttacgagac tcgtatcgac ctttcccagc ttggtggtga taagcgtcct gctgccacca
     6481 aaaaggccgg acaggctaag aaaaagaagt aggatcctcc cgatcgttca aacatttggc
     6541 aataaagttt cttaagattg aatcctgttg ccggtcttgc gatgattatc atataatttc
     6601 tgttgaatta cgttaagcat gtaataatta acatgtaatg catgacgtta tttatgaggt
     6661 gggtttttat gattagagtc ccgcaattat acatttaata cgcgatagaa aacaaaatat
     6721 agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc tatgttacta gatcgggagc
     6781 accggtaagg cgcgccgtag tgctcgagag acctctgaag tggccgattc attaatgcag
     6841 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
     6901 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
     6961 tggaattgtg agcggataac aatttcacac aagaaacagc tatgaccatg attacgccaa
     7021 gctatttagg tgacactata gaatactcaa gctatgcatc aagctcaatg ggtctagtct
     7081 gtagataccc atcacactgg cgaccgctcg aacatcagtt taaggtttac acctataaaa
     7141 gagagagccg ttatcgtctg tttgtggatg tacagagtga tattattgac acgccggggc
     7201 gacggatggt gatccccctg gccagtgcac gtctgctgtc agataaagtc tcccgtgaac
     7261 tttacccggt ggtgcatatc ggggatgaaa gctggcgcat gatgaccacc gatatgccca
     7321 gtgtgcctgt ctccgttatc ggggaagaag tggctgatct cagccaccgc gaaaatgaca
     7381 tcaaaaacgc cattaacctg atgttctggg gaatataaat gtcaggcctg aatggcgaat
     7441 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgag cgtgggtctc gcggtatcat
     7501 tggcgcgcct ctcgagctag cggccgcatg catcgatctc ctacatcgta taaattagcc
     7561 tatacgaagt tattgcatct atgtcgggtg cggagaaaga ggtaatgaaa tggcagtatt
     7621 agatctgata acttcgtata atgtatgcta tacgaagtta tgactgcaag tcgacaccca
     7681 taagcttggc actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc
     7741 aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc
     7801 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatgctag agcagcttga
     7861 gcttggatca gattgtcgtt tcccgccttc agtttaaact atcagtgttt gacaggatat
     7921 attggcgggt aaacctaaga gaaaagagcg tttattagaa taatcggata tttaaaaggg
     7981 cgtgaaaagg tttatccgtt cgtccatttg tatgtgcatg ccaaccacag ggttcccctc
     8041 gggatcaaag tactttgatc caacccctcc gctgctatag tgcagtcggc ttctgacgtt
     8101 cagtgcagcc gtcttctgaa aacgacatgt cgcacaagtc ctaagttacg cgacaggctg
     8161 ccgccctgcc cttttcctgg cgttttcttg tcgcgtgttt tagtcgcata aagtagaata
     8221 cttgcgacta gaaccggaga cattacgcca tgaacaagag cgccgccgct ggcctgctgg
     8281 gctatgcccg cgtcagcacc gacgaccagg acttgaccaa ccaacgggcc gaactgcacg
     8341 cggccggctg caccaagctg ttttccgaga agatcaccgg caccaggcgc gaccgcccgg
     8401 agctggccag gatgcttgac cacctacgcc ctggcgacgt tgtgacagtg accaggctag
     8461 accgcctggc ccgcagcacc cgcgacctac tggacattgc cgagcgcatc caggaggccg
     8521 gcgcgggcct gcgtagcctg gcagagccgt gggccgacac caccacgccg gccggccgca
     8581 tggtgttgac cgtgttcgcc ggcattgccg agttcgagcg ttccctaatc atcgaccgca
     8641 cccggagcgg gcgcgaggcc gccaaggccc gaggcgtgaa gtttggcccc cgccctaccc
     8701 tcaccccggc acagatcgcg cacgcccgcg agctgatcga ccaggaaggc cgcaccgtga
     8761 aagaggcggc tgcactgctt ggcgtgcatc gctcgaccct gtaccgcgca cttgagcgca
     8821 gcgaggaagt gacgcccacc gaggccaggc ggcgcggtgc cttccgtgag gacgcattga
     8881 ccgaggccga cgccctggcg gccgccgaga atgaacgcca agaggaacaa gcatgaaacc
     8941 gcaccaggac ggccaggacg aaccgttttt cattaccgaa gagatcgagg cggagatgat
     9001 cgcggccggg tacgtgttcg agccgcccgc gcacgtctca accgtgcggc tgcatgaaat
     9061 cctggccggt ttgtctgatg ccaagctggc ggcctggccg gccagcttgg ccgctgaaga
     9121 aaccgagcgc cgccgtctaa aaaggtgatg tgtatttgag taaaacagct tgcgtcatgc
     9181 ggtcgctgcg tatatgatgc gatgagtaaa taaacaaata cgcaagggga acgcatgaag
     9241 gttatcgctg tacttaacca gaaaggcggg tcaggcaaga cgaccatcgc aacccatcta
     9301 gcccgcgccc tgcaactcgc cggggccgat gttctgttag tcgattccga tccccagggc
     9361 agtgcccgcg attgggcggc cgtgcgggaa gatcaaccgc taaccgttgt cggcatcgac
     9421 cgcccgacga ttgaccgcga cgtgaaggcc atcggccggc gcgacttcgt agtgatcgac
     9481 ggagcgcccc aggcggcgga cttggctgtg tccgcgatca aggcagccga cttcgtgctg
     9541 attccggtgc agccaagccc ttacgacata tgggccaccg ccgacctggt ggagctggtt
     9601 aagcagcgca ttgaggtcac ggatggaagg ctacaagcgg cctttgtcgt gtcgcgggcg
     9661 atcaaaggca cgcgcatcgg cggtgaggtt gccgaggcgc tggccgggta cgagctgccc
     9721 attcttgagt cccgtatcac gcagcgcgtg agctacccag gcactgccgc cgccggcaca
     9781 accgttcttg aatcagaacc cgagggcgac gctgcccgcg aggtccaggc gctggccgct
     9841 gaaattaaat caaaactcat ttgagttaat gaggtaaaga gaaaatgagc aaaagcacaa
     9901 acacgctaag tgccggccgt ccgagcgcac gcagcagcaa ggctgcaacg ttggccagcc
     9961 tggcagacac gccagccatg aagcgggtca actttcagtt gccggcggag gatcacacca
    10021 agctgaagat gtacgcggta cgccaaggca agaccattac cgagctgcta tctgaataca
    10081 tcgcgcagct accagagtaa atgagcaaat gaataaatga gtagatgaat tttagcggct
    10141 aaaggaggcg gcatggaaaa tcaagaacaa ccaggcaccg acgccgtgga atgccccatg
    10201 tgtggaggaa cgggcggttg gccaggcgta agcggctggg ttgcctgccg gccctgcaat
    10261 ggcactggaa cccccaagcc cgaggaatcg gcgtgagcgg tcgcaaacca tccggcccgg
    10321 tacaaatcgg cgcggcgctg ggtgatgacc tggtggagaa gttgaaggcc gcgcaggccg
    10381 cccagcggca acgcatcgag gcagaagcac gccccggtga atcgtggcaa gcggccgctg
    10441 atcgaatccg caaagaatcc cggcaaccgc cggcagccgg tgcgccgtcg attaggaagc
    10501 cgcccaaggg cgacgagcaa ccagattttt tcgttccgat gctctatgac gtgggcaccc
    10561 gcgatagtcg cagcatcatg gacgtggccg ttttccgtct gtcgaagcgt gaccgacgag
    10621 ctggcgaggt gatccgctac gagcttccag acgggcacgt agaggtttcc gcagggccgg
    10681 ccggcatggc cagtgtgtgg gattacgacc tggtactgat ggcggtttcc catctaaccg
    10741 aatccatgaa ccgataccgg gaagggaagg gagacaagcc cggccgcgtg ttccgtccac
    10801 acgttgcgga cgtactcaag ttctgccggc gagccgatgg cggaaagcag aaagacgacc
    10861 tggtagaaac ctgcattcgg ttaaacacca cgcacgttgc catgcagcgt acgaagaagg
    10921 ccaagaacgg ccgcctggtg acggtatccg agggtgaagc cttgattagc cgctacaaga
    10981 tcgtaaagag cgaaaccggg cggccggagt acatcgagat cgagctagct gattggatgt
    11041 accgcgagat cacagaaggc aagaacccgg acgtgctgac ggttcacccc gattactttt
    11101 tgatcgatcc cggcatcggc cgttttctct accgcctggc acgccgcgcc gcaggcaagg
    11161 cagaagccag atggttgttc aagacgatct acgaacgcag tggcagcgcc ggagagttca
    11221 agaagttctg tttcaccgtg cgcaagctga tcgggtcaaa tgacctgccg gagtacgatt
    11281 tgaaggagga ggcggggcag gctggcccga tcctagtcat gcgctaccgc aacctgatcg
    11341 agggcgaagc atccgccggt tcctaatgta cggagcagat gctagggcaa attgccctag
    11401 caggggaaaa aggtcgaaaa gttctctttc ctgtggatag cacgtacatt gggaacccaa
    11461 agccgtacat tgggaaccgg aacccgtaca ttgggaaccc aaagccgtac attgggaacc
    11521 ggtcacacat gtaagtgact gatataaaag agaaaaaagg cgatttttcc gcctaaaact
    11581 ctttaaaact tattaaaact cttaaaaccc gcctggcctg tgcataactg tctggccagc
    11641 gcacagccga agagctgcaa aaagcgccta cccttcggtc gctgcgctcc ctacgccccg
    11701 ccgcttcgcg tcggcctatc gcggccgctg gccgctcaaa aatggctggc ctacggccag
    11761 gcaatctacc agggcgcgga caagccgcgc cgtcgccact cgaccgccgg cgcccacatc
    11821 aaggcaccct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc
    11881 ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc
    11941 gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc
    12001 ggagtgtata ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata
    12061 tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg
    12121 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
    12181 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
    12241 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
    12301 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
    12361 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
    12421 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
    12481 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
    12541 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
    12601 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
    12661 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
    12721 acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
    12781 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
    12841 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
    12901 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatgc
    12961 attctaggta ctaaaacaat tcatccagta aaatataata ttttattttc tcccaatcag
    13021 gcttgatccc cagtaagtca aaaaatagct cgacatactg ttcttccccg atatcctccc
    13081 tgatcgaccg gacgcagaag gcaatgtcat accacttgtc cgccctgccg cttctcccaa
    13141 gatcaataaa gccacttact ttgccatctt tcacaaagat gttgctgtct cccaggtcgc
    13201 cgtgggaaaa gacaagttcc tcttcgggct tttccgtctt taaaaaatca tacagctcgc
    13261 gcggatcttt aaatggagtg tcttcttccc agttttcgca atccacatcg gccagatcgt
    13321 tattcagtaa gtaatccaat tcggctaagc ggctgtctaa gctattcgta tagggacaat
    13381 ccgatatgtc gatggagtga aagagcctga tgcactccgc atacagctcg ataatctttt
    13441 cagggctttg ttcatcttca tactcttccg agcaaaggac gccatcggcc tcactcatga
    13501 gcagattgct ccagccatca tgccgttcaa agtgcaggac ctttggaaca ggcagctttc
    13561 cttccagcca tagcatcatg tccttttccc gttccacatc ataggtggtc cctttatacc
    13621 ggctgtccgt catttttaaa tataggtttt cattttctcc caccagctta tataccttag
    13681 caggagacat tccttccgta tcttttacgc agcggtattt ttcgatcagt tttttcaatt
    13741 ccggtgatat tctcatttta gccatttatt atttccttcc tcttttctac agtatttaaa
    13801 gataccccaa gaagctaagg aaggtgcgaa caagtccctg atatgagatc atgtttgtca
    13861 tctggagcca tagaacaggg ttcatcatga gtcatcaact taccttcgcc gacagtgaat
    13921 tcagcagtaa gcgccgtcag accagaaaag agattttctt gtcccgcatg gagcagattc
    13981 tgccatggca aaacatggtg gaagtcatcg agccgtttta ccccaaggct ggtaatggcc
    14041 ggcgacctta tccgctggaa accatgctac gcattcactg catgcagcat tggtacaacc
    14101 tgagcgatgg cgcgatggaa gatgctctgt acgaaatcgc ctccatgcgt ctgtttgccc
    14161 ggttatccct ggatagcgcc ttgccggacc gcaccaccat catgaatttc cgccacctgc
    14221 tggagcagca tcaactggcc cgccaattgt tcaagaccat caatcgctgg ctggccgaag
    14281 caggcgtcat gatgactcaa ggcaccttgg tcgatgccac catcattgag gcacccagct
    14341 cgaccaagaa caaagagcag caacgcgatc cggagatgca tcagaccaag aaaggcaatc
    14401 agtggcactt tggcatgaag gcccacattg gtgtcgatgc caagagtggc ctgacccaca
    14461 gcctggtcac caccgcggcc aacgagcatg acctcaatca gctgggtaat ctgctgcatg
    14521 gagaggagca atttgtctca gccgatgccg gctaccaagg ggcgccacag cgcgaggagc
    14581 tggccgaggt ggatgtggac tggctgatcg ccgagcgccc cggcaaggta agaaccttga
    14641 aacagcatcc acgcaagaac aaaacggcca tcaacatcga atacatgaaa gccagcatcc
    14701 gggccagggt ggagcaccca tttcgcatca tcaagcgaca gttcggcttc gtgaaagcca
    14761 gatacaaggg gttgctgaaa aacgataacc aactggcgat gttattcacg ctggccaacc
    14821 tgtttcgggc ggaccaaatg atacgtcagt gggagagatc tcactaaaaa ctggggataa
    14881 cgccttaaat ggcgaagaaa cggtctaaat aggctgattc aaggcattta cgggagaaaa
    14941 aatcggctca aacatgaaga aatgaaatga ctgagtcagc cgagaagaat ttccccgctt
    15001 attcgcacct tccctaatta taacaagacg aactccaatt cactgttcct tgcattctaa
    15061 aaccttaaat accagaaaac agctttttca aagttgtttt caaagttggc gtataacata
    15121 gtatcgacgg agccgatttt gaaaccgcgg tgatcacagg cagcaacgct ctgtcatcgt
    15181 tacaatcaac atgctaccct ccgcgagatc atccgtgttt caaacccggc agcttagttg
    15241 ccgttcttcc gaatagcatc ggtaacatga gcaaagtctg ccgccttaca acggctctcc
    15301 cgctgacgcc gtcccggact gatgggctgc ctgtatcgag tggtgatttt gtgccgagct
    15361 gccggtcggg gagctgttgg ctggctggtg gcaggatata ttgtggtgta aacaaattga
    15421 cgcttagaca acttaataac acattgcgga cgtttttaat gtactgaatt aacgccgaat
    15481 taattcgggg gatctggatt ttagtactgg attttggttt taggaattag aaattttatt
    15541 gatagaagta ttttacaaat acaaatacat actaagggtt tcttatatgc tcaacacatg
    15601 agcgaaaccc tataggaacc ctaattccct tatctgggaa ctactcacac attattatgg
    15661 agaaactcga gcttgtcgat cgactctagc tagaggatcg atccgaaccc cagagtcccg
    15721 ctcagaagaa ctcgtcaaga aggcgataga aggcgatgcg ctgcgaatcg ggagcggcga
    15781 taccgtaaag cacgaggaag cggtcagccc attcgccgcc aagctcttca gcaatatcac
    15841 gggtagccaa cgctatgtcc tgatagcggt ccgccacacc cagccggcca cagtcgatga
    15901 atccagaaaa gcggccattt tccaccatga tattcggcaa gcaggcatcg ccatgtgtca
    15961 cgacgagatc ctcgccgtcg ggcatgcgcg ccttgagcct ggcgaacagt tcggctggcg
    16021 cgagcccctg atgctcttcg tccagatcat cctgatcgac aagaccggct tccatccgag
    16081 tacgtgctcg ctcgatgcga tgtttcgctt ggtggtcgaa tgggcaggta gccggatcaa
    16141 gcgtatgcag ccgccgcatt gcatcagcca tgatggatac tttctcggca ggagcaaggt
    16201 gagatgacag gagatcctgc cccggcactt cgcccaatag cagccagtcc cttcccgctt
    16261 cagtgacaac gtcgagcaca gctgcgcaag gaacgcccgt cgtggccagc cacgatagcc
    16321 gcgctgcctc gtcctggagt tcattcaggg caccggacag gtcggtcttg acaaaaagaa
    16381 ccgggcgccc ctgcgctgac agccgaaaca cggcggcatc agagcagccg attgtctgtt
    16441 gtgcccagtc atagccgaat agcctctcca cccaagcggc cggagaacct gcgtgcaatc
    16501 catcttgttc aatccccatg gtcgatcgac agatctgcga aagctcgaga gagatagatt
    16561 tgtagagaga gactggtgat ttcagcgtgt cctctccaaa tgaaatgaac ttccttatat
    16621 agaggaaggg tcttgcgaag gatagtggga ttgtgcgtca tcccttacgt cagtggagat
    16681 atcacatcaa tccacttgct ttgaagacgt ggttggaacg tcttcttttt ccacgatgct
    16741 cttcgtgggt gggggtccat ctttgggacc actgtcggca gaggcatctt gaacgatagc
    16801 ctttccttta tcgcaatgat ggcatttgta ggtgccacct tccttttcta ctgtcctttt
    16861 gatgaagtga cagatagctg ggcaatggaa tccgaggagg tttcccgata ttaccctttg
    16921 ttgaaaagtc tcaatagccc tttggtcttc tgagactgta tctttgatat tcttggagta
    16981 gacgagagtg tcgtgctcca ccatgttcac atcaatccac ttgctttgaa gacgtggttg
    17041 gaacgtcttc tttttccacg atgctcctcg tgggtggggg tccatctttg ggaccactgt
    17101 cggcagaggc atcttgaacg atagcctttc ctttatcgca atgatggcat ttgtaggtgc
    17161 caccttcctt ttctactgtc cttttgatga agtgacagat agctgggcaa tggaatccga
    17221 ggaggtttcc cgatattacc ctttgttgaa aagtctcaat agccctttgg tcttctgaga
    17281 ctgtatcttt gatattcttg gagtagacga gagtgtcgtg ctccaccatg ttggcaagct
    17341 gctctagcca atacg
//

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