pBUN6I11

价格:1200元

联系方式:I47-825O-882O

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载体基本信息

质粒名称: pBUN6I11 
出品公司: 爱迪基因
目录编号: 50580
存储实验室: Qi-Jun Chen

载体骨架基本信息
载体名称: pGreen-like binary vector
载体出品公司: Mullineaux Lab, see http://www.pgreen.ac.uk/
载体抗性: 卡那霉素和大观霉素
空载体大小: --
完整质粒大小: --
载体修饰: --
载体类型: CRISPR,植物表达
筛选标记: --
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: dCas9-KRAB,Cas9
物种来源: Synthetic
插入大小: --
基因突变: dCas9在DNA断裂中有缺陷;构建了玉米密码子优化的dCas9-KRAB(kruppel-associated-box)载体,用于靶向敲除感兴趣基因。
GenBank编号 --
启动子 Ubi1p
标签/融合蛋白:
3xFLAG(插入件上的N端子)
NLS(NLS N)
NLS(插入件上的C端子)
HA(插入件上的C端子)
克隆方法: --
5' 克隆位点: --
3' 克隆位点: --
5' 测序引物 hSpCas9-R1, CGCTCGTGCTTCTTATCCTC
3' 测序引物 --

文献引用方法
材料和方法部分: pBUN6I11 was a gift from Qi-Jun Chen (Addgene plasmid # 50580 ; http://n2t.net/addgene:50580 ; RRID:Addgene_50580)
参考文献部分: A CRISPR/Cas9 toolkit for multiplex genome editing in plants. Xing HL, Dong L, Wang ZP, Zhang HY, Han CY, Liu B, Wang XC, Chen QJ. BMC Plant Biol. 2014 Nov 29;14(1):327. 10.1186/s12870-014-0327-y PubMed 25432517

载体质粒图谱和多克隆位点信息

pBUN6I11质粒图

载体简介

载体序列

LOCUS       pBUN6I11               13880 bp ds-DNA     circular SYN 06-AUG-2018
DEFINITION  CRISPR/Cas based plant genome editing and gene regulation; expresses
            dCas9-KRAB, gRNA scaffold for insertion of target sequence (OsU3 
            promoter), Bar resistance.
ACCESSION   .
VERSION     .
KEYWORDS    pBUN6I11
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13880)
  AUTHORS   Xing HL, Dong L, Wang ZP, Zhang HY, Han CY, Liu B, Wang XC, Chen QJ
  TITLE     A CRISPR/Cas9 toolkit for multiplex genome editing in plants.
  JOURNAL   BMC Plant Biol. 2014 Nov 29;14(1):327.
  PUBMED    25432517
REFERENCE   2  (bases 1 to 13880)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 6, 2018 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..13880
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_feature    1..25
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     promoter        164..543
                     /label=OsU3 promoter
                     /note="Oryza sativa (rice) snRNA U3 promoter"
     CDS             823..1614
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MGEAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     misc_RNA        1766..1841
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2151..4141
                     /label=Ubi promoter
                     /note="maize polyubiquitin gene promoter"
     CDS             4187..4252
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             4259..4279
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4304..8404
                     /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="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     RBS             8317..8325
                     /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             8405..8452
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             8471..8491
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8507..8527
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8564..8749
                     /codon_start=1
                     /gene="ZNF10"
                     /gene_synonym="KOX1"
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEP"
     CDS             8756..8776
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8783..8809
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     terminator      8857..9109
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     primer_bind     complement(9118..9135)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(9118..9134)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9127..9149)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        9334..10011
                     /label=CaMV 35S promoter (enhanced)
                     /note="cauliflower mosaic virus 35S promoter with a 
                     duplicated enhancer region"
     CDS             10056..10607
                     /codon_start=1
                     /gene="Streptomyces hygroscopicus bar"
                     /product="phosphinothricin acetyltransferase"
                     /label=BlpR
                     /note="confers resistance to bialophos or phosphinothricin"
                     /translation="MSPERRPADIRRATEADMPAVCTIVNHYIETSTVNFRTEPQEPQE
                     WTDDLVRLRERYPWLVAEVDGEVAGIAYAGPWKARNAYDWTAESTVYVSPRHQRTGLGS
                     TLYTHLLKSLEAQGFKSVVAVIGLPNDPSVRMHEALGYAPRGMLRAAGFKHGNWHDVGF
                     WQLDFSLPVPPRPVLPVTEI"
     polyA_signal    10614..10788
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     misc_feature    10938..10962
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     primer_bind     complement(11210..11233)
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     terminator      11241..11268
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      11360..11446
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      11615..12050
                     /label=pSa ori
                     /note="origin of replication from bacterial plasmid pSa"
     rep_origin      complement(12165..12753)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(12245..12264)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(12843..13652)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVSLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     13566..13585
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     13644..13663
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 gtttacccgc caatatatcc tgtcaaacac tgatagttta aactgaaggc gggaaacgac
       61 aatctgatcc aagctcaagc taagctcacg tgacggaatt aagcttagta attcatccag
      121 gtcaccaagt tctaggattt tcagaactgc aacttatttt atcaaggaat ctttaaacat
      181 acgaacagat cacttaaagt tcttctgaag caacttaaag ttatcaggca tgcatggatc
      241 ttggaggaat cagatgtgca gtcagggacc atagcacaag acaggcgtct tctactggtg
      301 ctaccagcaa atgctggaag ccgggaacac tgggtacgtt ggaaaccacg tgatgtgaag
      361 aagtaagata aactgtagga gaaaagcatt tcgtagtggg ccatgaagcc tttcaggaca
      421 tgtattgcag tatgggccgg cccattacgc aattggacga caacaaagac tagtattagt
      481 accacctcgg ctatccacat agatcaaagc tgatttaaaa gagttgtgca gatgatccgt
      541 ggcgtgagac caacccagtg gacataagcc tgttcggttc gtaagctgta atgcaagtag
      601 cgtatgcgct cacgcaactg gtccagaacc ttgaccgaac gcagcggtgg taacggcgca
      661 gtggcggttt tcatggcttg ttatgactgt ttttttgggg tacagtctat gcctcgggca
      721 tccaagcagc aagcgcgtta cgccgtgggt cgatgtttga tgttatggag cagcaacgat
      781 gttacgcagc agggcagtcg ccctaaaaca aagttaaaca tcatggggga agcggtgatc
      841 gccgaagtat cgactcaact atcagaggta gttggcgtca tcgagcgcca tctcgaaccg
      901 acgttgctgg ccgtacattt gtacggctcc gcagtggatg gcggcctgaa gccacacagt
      961 gatattgatt tgctggttac ggtgaccgta aggcttgatg aaacaacgcg gcgagctttg
     1021 atcaacgacc ttttggaaac ttcggcttcc cctggagaga gcgagattct ccgcgctgta
     1081 gaagtcacca ttgttgtgca cgacgacatc attccgtggc gttatccagc taagcgcgaa
     1141 ctgcaatttg gagaatggca gcgcaatgac attcttgcag gtatcttcga gccagccacg
     1201 atcgacattg atctggctat cttgctgaca aaagcaagag aacatagcgt tgccttggta
     1261 ggtccagcgg cggaggaact ctttgatccg gttcctgaac aggatctatt tgaggcgcta
     1321 aatgaaacct taacgctatg gaactcgccg cccgactggg ctggcgatga gcgaaatgta
     1381 gtgcttacgt tgtcccgcat ttggtacagc gcagtaaccg gcaaaatcgc gccgaaggat
     1441 gtcgctgccg actgggcaat ggagcgcctg ccggcccagt atcagcccgt catacttgaa
     1501 gctagacagg cttatcttgg acaagaagaa gatcgcttgg cctcgcgcgc agatcagttg
     1561 gaagaatttg tccactacgt gaaaggcgag atcaccaagg tagtcggcaa ataatgtcta
     1621 gctagaaatt cgttcaagcc gacgccgctt cgcggcgcgg cttaactcaa gcgttagatg
     1681 cactaagcac ataattgctc acagccaaac tatcaggtca agtctgcttt tattattttt
     1741 aagcgtgcat aataagccgg tctcggtttt agagctagaa atagcaagtt aaaataaggc
     1801 tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg cttttttttt tcgttttgca
     1861 ttgagttttc tccgtcgcat gtttgcagtt ttattttccg ttttgcattg aaatttctcc
     1921 gtctcatgtt tgcagcgtgt tcaaaaagta cgcagctgta tttcacttat ttacggcgcc
     1981 acattttcat gccgtttgtg ccaactatcc cgagctagtg aatacagctt ggcttcacac
     2041 aacactggtg acccgctgac ctgctcgtac ctcgtaccgt cgtacggcac agcatttgga
     2101 attaaagggt gtgatcgata ctgcttgctg ctaagcttga tatcgaattc ctgcagtgca
     2161 gcgtgacccg gtcgtgcccc tctctagaga taatgagcat tgcatgtcta agttataaaa
     2221 aattaccaca tatttttttt gtcacacttg tttgaagtgc agtttatcta tctttataca
     2281 tatatttaaa ctttactcta cgaataatat aatctatagt actacaataa tatcagtgtt
     2341 ttagagaatc atataaatga acagttagac atggtctaaa ggacaattga gtattttgac
     2401 aacaggactc tacagtttta tctttttagt gtgcatgtgt tctccttttt ttttgcaaat
     2461 agcttcacct atataatact tcatccattt tattagtaca tccatttagg gtttagggtt
     2521 aatggttttt atagactaat ttttttagta catctatttt attctatttt agcctctaaa
     2581 ttaagaaaac taaaactcta ttttagtttt tttatttaat aatttagata taaaatagaa
     2641 taaaataaag tgactaaaaa ttaaacaaat accctttaag aaattaaaaa aactaaggaa
     2701 acatttttct tgtttcgagt agataatgcc agcctgttaa acgccgtcga cgagtctaac
     2761 ggacaccaac cagcgaacca gcagcgtcgc gtcgggccaa gcgaagcaga cggcacggca
     2821 tctctgtcgc tgcctctgga cccctctcga gagttccgct ccaccgttgg acttgctccg
     2881 ctgtcggcat ccagaaatgc gtggcggagc ggcagacgtg agccggcacg gcaggcggcc
     2941 tcctcctcct ctcacggcac ggcagctacg ggggattcct ttcccaccgc tccttcgctt
     3001 tcccttcctc gcccgccgta ataaatagac accccctcca caccctcttt ccccaacctc
     3061 gtgttgttcg gagcgcacac acacacaacc agatctcccc caaatccacc cgtcggcacc
     3121 tccgcttcaa ggtacgccgc tcgtcctccc cccccccccc tctctacctt ctctagatcg
     3181 gcgttccggt ccatggttag ggcccggtag ttctacttct gttcatgttt gtgttagatc
     3241 cgtgtttgtg ttagatccgt gctgctagcg ttcgtacacg gatgcgacct gtacgtcaga
     3301 cacgttctga ttgctaactt gccagtgttt ctctttgggg aatcctggga tggctctagc
     3361 cgttccgcag acgggatcga tttcatgatt ttttttgttt cgttgcatag ggtttggttt
     3421 gcccttttcc tttatttcaa tatatgccgt gcacttgttt gtcgggtcat cttttcatgc
     3481 ttttttttgt cttggttgtg atgatgtggt ctggttgggc ggtcgttcta gatcggagta
     3541 gaattctgtt tcaaactacc tggtggattt attaattttg gatctgtatg tgtgtgccat
     3601 acatattcat agttacgaat tgaagatgat ggatggaaat atcgatctag gataggtata
     3661 catgttgatg cgggttttac tgatgcatat acagagatgc tttttgttcg cttggttgtg
     3721 atgatgtggt gtggttgggc ggtcgttcat tcgttctaga tcggagtaga atactgtttc
     3781 aaactacctg gtgtatttat taattttgga actgtatgtg tgtgtcatac atcttcatag
     3841 ttacgagttt aagatggatg gaaatatcga tctaggatag gtatacatgt tgatgtgggt
     3901 tttactgatg catatacatg atggcatatg cagcatctat tcatatgctc taaccttgag
     3961 tacctatcta ttataataaa caagtatgtt ttataattat tttgatcttg atatacttgg
     4021 atgatggcat atgcagcagc tatatgtgga tttttttagc cctgccttca tacgctattt
     4081 atttgcttgg tactgtttct tttgtcgatg ctcaccctgt tgtttggtgt tacttctgca
     4141 gcccggggga tccccaatac ttgtatggcc gcggccgctc tagatggatt acaaggacca
     4201 cgacggggat tacaaggacc acgacattga ttacaaggat gatgatgaca agatggctcc
     4261 gaagaagaag aggaaggttg gcatccacgg ggtgccagct gctgacaaga agtactcgat
     4321 cggcctcgcc attgggacta actctgttgg ctgggccgtg atcaccgacg agtacaaggt
     4381 gccctcaaag aagttcaagg tcctgggcaa caccgatcgg cattccatca agaagaatct
     4441 cattggcgct ctcctgttcg acagcggcga gacggctgag gctacgcggc tcaagcgcac
     4501 cgcccgcagg cggtacacgc gcaggaagaa tcgcatctgc tacctgcagg agattttctc
     4561 caacgagatg gcgaaggttg acgattcttt cttccacagg ctggaggagt cattcctcgt
     4621 ggaggaggat aagaagcacg agcggcatcc aatcttcggc aacattgtcg acgaggttgc
     4681 ctaccacgag aagtacccta cgatctacca tctgcggaag aagctcgtgg actccacaga
     4741 taaggcggac ctccgcctga tctacctcgc tctggcccac atgattaagt tcaggggcca
     4801 tttcctgatc gagggggatc tcaacccgga caatagcgat gttgacaagc tgttcatcca
     4861 gctcgtgcag acgtacaacc agctcttcga ggagaacccc attaatgcgt caggcgtcga
     4921 cgcgaaggct atcctgtccg ctaggctctc gaagtctcgg cgcctcgaga acctgatcgc
     4981 ccagctgccg ggcgagaaga agaacggcct gttcgggaat ctcattgcgc tcagcctggg
     5041 gctcacgccc aacttcaagt cgaatttcga tctcgctgag gacgccaagc tgcagctctc
     5101 caaggacaca tacgacgatg acctggataa cctcctggcc cagatcggcg atcagtacgc
     5161 ggacctgttc ctcgctgcca agaatctgtc ggacgccatc ctcctgtctg atattctcag
     5221 ggtgaacacc gagattacga aggctccgct ctcagcctcc atgatcaagc gctacgacga
     5281 gcaccatcag gatctgaccc tcctgaaggc gctggtcagg cagcagctcc ccgagaagta
     5341 caaggagatc ttcttcgatc agtcgaagaa cggctacgct gggtacattg acggcggggc
     5401 ctctcaggag gagttctaca agttcatcaa gccgattctg gagaagatgg acggcacgga
     5461 ggagctgctg gtgaagctca atcgcgagga cctcctgagg aagcagcgga cattcgataa
     5521 cggcagcatc ccacaccaga ttcatctcgg ggagctgcac gctatcctga ggaggcagga
     5581 ggacttctac cctttcctca aggataaccg cgagaagatc gagaagattc tgactttcag
     5641 gatcccgtac tacgtcggcc cactcgctag gggcaactcc cgcttcgctt ggatgacccg
     5701 caagtcagag gagacgatca cgccgtggaa cttcgaggag gtggtcgaca agggcgctag
     5761 cgctcagtcg ttcatcgaga ggatgacgaa tttcgacaag aacctgccaa atgagaaggt
     5821 gctccctaag cactcgctcc tgtacgagta cttcacagtc tacaacgagc tgactaaggt
     5881 gaagtatgtg accgagggca tgaggaagcc ggctttcctg tctggggagc agaagaaggc
     5941 catcgtggac ctcctgttca agaccaaccg gaaggtcacg gttaagcagc tcaaggagga
     6001 ctacttcaag aagattgagt gcttcgattc ggtcgagatc tctggcgttg aggaccgctt
     6061 caacgcctcc ctggggacct accacgatct cctgaagatc attaaggata aggacttcct
     6121 ggacaacgag gagaatgagg atatcctcga ggacattgtg ctgacactca ctctgttcga
     6181 ggaccgggag atgatcgagg agcgcctgaa gacttacgcc catctcttcg atgacaaggt
     6241 catgaagcag ctcaagagga ggaggtacac cggctggggg aggctgagca ggaagctcat
     6301 caacggcatt cgggacaagc agtccgggaa gacgatcctc gacttcctga agagcgatgg
     6361 cttcgcgaac cgcaatttca tgcagctgat tcacgatgac agcctcacat tcaaggagga
     6421 tatccagaag gctcaggtga gcggccaggg ggactcgctg cacgagcata tcgcgaacct
     6481 cgctggctcg ccagctatca agaaggggat tctgcagacc gtgaaggttg tggacgagct
     6541 ggtgaaggtc atgggcaggc acaagcctga gaacatcgtc attgagatgg cccgggagaa
     6601 tcagaccacg cagaagggcc agaagaactc acgcgagagg atgaagagga tcgaggaggg
     6661 cattaaggag ctggggtccc agatcctcaa ggagcacccg gtggagaaca cgcagctgca
     6721 gaatgagaag ctctacctgt actacctcca gaatggccgc gatatgtatg tggaccagga
     6781 gctggatatt aacaggctca gcgattacga cgtcgatgcc atcgttccac agtcattcct
     6841 gaaggatgac tccattgaca acaaggtcct caccaggtcg gacaagaacc ggggcaagtc
     6901 tgataatgtt ccttcagagg aggtcgttaa gaagatgaag aactactggc gccagctcct
     6961 gaatgccaag ctgatcacgc agcggaagtt cgataacctc acaaaggctg agaggggcgg
     7021 gctctctgag ctggacaagg cgggcttcat caagaggcag ctggtcgaga cacggcagat
     7081 cactaagcac gttgcgcaga ttctcgactc acggatgaac actaagtacg atgagaatga
     7141 caagctgatc cgcgaggtga aggtcatcac cctgaagtca aagctcgtct ccgacttcag
     7201 gaaggatttc cagttctaca aggttcggga gatcaacaat taccaccatg cccatgacgc
     7261 gtacctgaac gcggtggtcg gcacagctct gatcaagaag tacccaaagc tcgagagcga
     7321 gttcgtgtac ggggactaca aggtttacga tgtgaggaag atgatcgcca agtcggagca
     7381 ggagattggc aaggctaccg ccaagtactt cttctactct aacattatga atttcttcaa
     7441 gacagagatc actctggcca atggcgagat ccggaagcgc cccctcatcg agacgaacgg
     7501 cgagacgggg gagatcgtgt gggacaaggg cagggatttc gcgaccgtca ggaaggttct
     7561 ctccatgcca caagtgaata tcgtcaagaa gacagaggtc cagactggcg ggttctctaa
     7621 ggagtcaatt ctgcctaagc ggaacagcga caagctcatc gcccgcaaga aggactggga
     7681 tccgaagaag tacggcgggt tcgacagccc cactgtggcc tactcggtcc tggttgtggc
     7741 gaaggttgag aagggcaagt ccaagaagct caagagcgtg aaggagctgc tggggatcac
     7801 gattatggag cgctccagct tcgagaagaa cccgatcgat ttcctggagg cgaagggcta
     7861 caaggaggtg aagaaggacc tgatcattaa gctccccaag tactcactct tcgagctgga
     7921 gaacggcagg aagcggatgc tggcttccgc tggcgagctg cagaagggga acgagctggc
     7981 tctgccgtcc aagtatgtga acttcctcta cctggcctcc cactacgaga agctcaaggg
     8041 cagccccgag gacaacgagc agaagcagct gttcgtcgag cagcacaagc attacctcga
     8101 cgagatcatt gagcagattt ccgagttctc caagcgcgtg atcctggccg acgcgaatct
     8161 ggataaggtc ctctccgcgt acaacaagca ccgcgacaag ccaatcaggg agcaggctga
     8221 gaatatcatt catctcttca ccctgacgaa cctcggcgcc cctgctgctt tcaagtactt
     8281 cgacacaact atcgatcgca agaggtacac aagcactaag gaggtcctgg acgcgaccct
     8341 catccaccag tcgattaccg gcctctacga gacgcgcatc gacctgtctc agctcggggg
     8401 cgacaagcgg ccagcggcga cgaagaaggc ggggcaggcg aagaagaaga agtccgggtc
     8461 ctcggggtcg ccaaagaaga agcggaaggt cggctcaagc gggtcaccaa agaagaagcg
     8521 gaaggtcggc tcgatggacg ctaagtccct caccgcttgg agccgcaccc tggtgacgtt
     8581 caaggacgtg ttcgtcgatt tcacaaggga ggagtggaag ctcctggaca ctgctcagca
     8641 gatcgtttac cggaacgtga tgctggagaa ctacaagaac ctcgtgtccc tcggctacca
     8701 gctgacgaag ccggatgtta ttctccgcct ggagaagggc gaggagcccg ggtcccctaa
     8761 gaagaagcgg aaggtcggct cctacccata cgatgtccct gactacgcct catgagctca
     8821 ggcctccata caagtattgg ggatccgaat ttccccgatc gttcaaacat ttggcaataa
     8881 agtttcttaa gattgaatcc tgttgccggt cttgcgatga ttatcatata atttctgttg
     8941 aattacgtta agcatgtaat aattaacatg taatgcatga cgttatttat gagatgggtt
     9001 tttatgatta gagtcccgca attatacatt taatacgcga tagaaaacaa aatatagcgc
     9061 gcaaactagg ataaattatc gcgcgcggtg tcatctatgt tactagatcg ggaattcact
     9121 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
     9181 tgcagcacat ccccctttcg ccaggggtac cgagctctag aattcgaggc ggtttgcgta
     9241 ttggctagag cagcttgcca acatggtgga gcacgacact ctcgtctact ccaagaatat
     9301 caaagataca gtctcagaag accaaagggc tattgagact tttcaacaaa gggtaatatc
     9361 gggaaacctc ctcggattcc attgcccagc tatctgtcac ttcatcaaaa ggacagtaga
     9421 aaaggaaggt ggcacctaca aatgccatca ttgcgataaa ggaaaggcta tcgttcaaga
     9481 tgcctctgcc gacagtggtc ccaaagatgg acccccaccc acgaggagca tcgtggaaaa
     9541 agaagacgtt ccaaccacgt cttcaaagca agtggattga tgtgataaca tggtggagca
     9601 cgacactctc gtctactcca agaatatcaa agatacagtc tcagaagacc aaagggctat
     9661 tgagactttt caacaaaggg taatatcggg aaacctcctc ggattccatt gcccagctat
     9721 ctgtcacttc atcaaaagga cagtagaaaa ggaaggtggc acctacaaat gccatcattg
     9781 cgataaagga aaggctatcg ttcaagatgc ctctgccgac agtggtccca aagatggacc
     9841 cccacccacg aggagcatcg tggaaaaaga agacgttcca accacgtctt caaagcaagt
     9901 ggattgatgt gatatctcca ctgacgtaag ggatgacgca caatcccact atccttcgca
     9961 agaccttcct ctatataagg aagttcattt catttggaga ggacacgctg aaatcaccag
    10021 tctctctcta caaatctatc tctctcgagt ctaccatgag cccagaacga cgcccggccg
    10081 acatccgccg tgccaccgag gcggacatgc cggcggtctg caccatcgtc aaccactaca
    10141 tcgagacaag cacggtcaac ttccgtaccg agccgcagga accgcaggag tggacggacg
    10201 acctcgtccg tctgcgggag cgctatccct ggctcgtcgc cgaggtggac ggcgaggtcg
    10261 ccggcatcgc ctacgcgggc ccctggaagg cacgcaacgc ctacgactgg acggccgagt
    10321 cgaccgtgta cgtctccccc cgccaccagc ggacgggact gggctccacg ctctacaccc
    10381 acctgctgaa gtccctggag gcacagggct tcaagagcgt ggtcgctgtc atcgggctgc
    10441 ccaacgaccc gagcgtgcgc atgcacgagg cgctcggata tgccccccgc ggcatgctgc
    10501 gggcggccgg cttcaagcac gggaactggc atgacgtggg tttctggcag ctggacttca
    10561 gcctgccggt accgccccgt ccggtcctgc ccgtcaccga gatttgactc gagtttctcc
    10621 ataataatgt gtgagtagtt cccagataag ggaattaggg ttcctatagg gtttcgctca
    10681 tgtgttgagc atataagaaa cccttagtat gtatttgtat ttgtaaaata cttctatcaa
    10741 taaaatttct aattcctaaa accaaaatcc agtactaaaa tccagatccc ccgaattaat
    10801 tcggcgttaa ttcagtacat taaaaacgtc cgcaatgtgt tattaagttg tcactagtca
    10861 ggttaactca attcggcgtt aattcagtac attaaaaacg tccgcaatgt gttattaagt
    10921 tgtctaagcg tcaatttgtt tacaccacaa tatatcctgc caccagccag ccaacagctc
    10981 cccgaccggc agctcggcac aaaatcacca ctcgatacag gcagcccatc agtccgggac
    11041 ggcgtcagcg ggagagccgt tgtaaggcgg cagactttgc tcatgttacc gatgctattc
    11101 ggaagaacgg caactaagct gccgggtttg aaacacggat gatctcgcgg agggtagcat
    11161 gttgattgta acgatgacag agcgttgctg cctgtgatca cttaagtaac taactaacag
    11221 gaagagtttg tagaaacgca aaaaggccat ccgtcaggat ggccttctgc ttagtttgat
    11281 gcctggcagt ttatggcggg cgtcctgccc gccaccctcc gggccgttgc ttcacaacgt
    11341 tcaaatccgc tcccggcgga tttgtcctac tcaggagagc gttcaccgac aaacaacaga
    11401 taaaacgaaa ggcccagtct tccgactgag cctttcgttt tatttgatgc ctggcagttc
    11461 cctactctcg cttagtagtt agacgtcccc gagatccatg ctagaccatg aatccagaag
    11521 cccgagaggt tgccgccttt cgggcttttt ctttttcaaa aaaaaaaatt tataaaacga
    11581 tctgttgcgg ccggccgccg ggttgtgggc aaaggcgctc gacggtgggc aaccgcttgc
    11641 ggttgtccac gggcggagcc ggtgcgcgta gcgcattgtc cacaagccaa gggcgaccaa
    11701 taattgatat atatattcat aattgaaaag ctaattgaac atactacttg ctgtaactac
    11761 ttgccggagc gaggggtgtt tgcaagctgt tgatctgaaa gggctattag cgttctcacg
    11821 tgcctttttg attagcgatt tcacgtgacc ttattagcga tttcacgtac tccgattagc
    11881 gatttcacgt accctgatta gcgatttcac gtggatagtt tttggagcgg gccggaaagc
    11941 cccgtgaatc aaggctttgc ggggcattag cggtttcacg tggataacta ccctctatcc
    12001 acaggcttcc ggggataaaa aagcccgctc gacggcgggc tgttggatgg ggatctagcg
    12061 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
    12121 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
    12181 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
    12241 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
    12301 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
    12361 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
    12421 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
    12481 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
    12541 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
    12601 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
    12661 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
    12721 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctaccggg
    12781 tctgacgctc agtggaacgg ggcccaatct gaataatgtt acaaccaatt aaccaattct
    12841 gattagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca
    12901 ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc
    12961 cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa
    13021 cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg
    13081 actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc
    13141 cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat
    13201 tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc
    13261 gaatgcaacc ggcgcaggga cactgccagc gcatcaacaa tattttcacc tgaatcagga
    13321 tattcttcta atacctggaa tgctgttttt ccggggatcg cagtggtgag taaccatgca
    13381 tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag
    13441 tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga
    13501 aacaactctg gcgcatcggg cttcccatac aagcgataga ttgtcgcacc tgattgcccg
    13561 acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc
    13621 ggcctcgacg tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa
    13681 gcagacagtt ttattgttca tgatgatata tttttatctt gtgcaatgta acatcagaga
    13741 ttttgagaca cgggccagag ctgcagtttg atcccgaggg gaaccctgtg gttgacatgc
    13801 acatacaaat ggacgaacgg ataaaccttt tcacgccctt ttaaatatcc gttattctaa
    13861 taaacgctct tttctcttag
//

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