pHDE-35s-Cas9-mCherry-UBQ植物编辑质

基本信息

启动子:	CaMV 35S promoter
复制子:	pUC ori，pVS1 oriV
终止子:	3A terminator
质粒分类:	植物系列质粒；植物编辑质粒；植物Cas9质粒
质粒大小:	16084bp
质粒标签:	C-SV40 NLS
原核抗性:	壮观霉素Spe
筛选标记:	潮霉素Hyg，红色荧光蛋白mCherry
克隆菌株:	DH5a等大肠杆菌
培养条件:	37℃，有氧，LB
表达宿主:	拟南芥等植物
诱导方式:	无需诱导，组成型表达
5'测序引物:	35S：GACGCACAATCCCACTATCC
3'测序引物:	3A-R：CCATTTGCATTTTGATGTCC
备注:	同时表达Cas9和sgRNA

质粒简介

          pHDE-35S-Cas9-mCherry-UBQ质粒是一个质粒可以在植物中表达Cas9酶和mCherry红色荧光蛋白，可视化基因编辑操作

质粒图谱

质粒序列

LOCUS       Exported               16084 bp ds-DNA     circular SYN 25-AUG-2017
DEFINITION  synthetic circular DNA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 16084)
  TITLE     Direct Submission
  JOURNAL   Exported Aug 25, 2017 from Plasmid
FEATURES             Location/Qualifiers
     source          1..16084
                     /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"
     CDS             271..1197
                     /codon_start=1
                     /label=mCherry
                     /translation="MMSGIVLFVTCHSFPTHNPDNNVRASLSLHTHSCMHAFLHVIAMQ
                     ISFLTYKYKPTLHYTLHSNQNKKTYTNSKTVPTMDNMAIIKEFMRFKVHMEGSVNGHEF
                     EIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLS
                     FPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEA
                     SSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSH
                     NEDYTIVEQYERAEGRHSTGGMDELYK"
     terminator      1203..1455
                     /label=MAS terminator
                     /note="mannopine synthase terminator"
     promoter        2568..2783
                     /label=NOS promoter
                     /note="nopaline synthase promoter"
     CDS             2835..3860
                     /codon_start=1
                     /label=Hyg
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDIGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPGSW
                     R"
     terminator      3898..4150
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     promoter        4283..4628
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     CDS             4633..8736
                     /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             8749..8769
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T 
                     antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_feature    8780..9249
                     /label=3A terminator
     misc_feature    9519..9543
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     CDS             10071..10862
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=Spe
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MGEAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      11106..11694
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    11880..12020
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      12364..12558
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     CDS             complement(12624..13691)
                     /codon_start=1
                     /product="replication protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 RepA
                     /translation="GRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESWQA
                     AADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLSKR
                     DRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKPGR
                     VFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGEAL
                     ISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYRLA
                     RRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPILV
                     MRYRNLIEGEASAGS"
     CDS             complement(14125..14754)
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
ORIGIN
        1 gtttacccgc caatatatcc tgtcaaacac tgatagttta aactagtgaa gtgaagcttg
       61 gtctcaacta tgagacctaa gctggcacaa ctatatttcc aacatcacta gctaccatca
      121 aaagattgac ttctcatctt actcgattga aaccaaatta acatagggtt tttatttaaa
      181 taaaagttta accttctttt taaaaaattg ttcatagtgt catgtcagaa caagagctac
      241 aaatcacaca tagcatgcat aagcggagct atgatgagtg gtattgtttt gttcgtcact
      301 tgtcactctt ttccaacaca taatcccgac aacaacgtaa gagcatctct ctctctccac
      361 acacactcat gcatgcatgc attcttacac gtgattgcca tgcaaatctc ctttctcacc
      421 tataaataca aaccaaccct tcactacact cttcactcaa accaaaacaa gaaaacatac
      481 acaaatagca aaacggtacc aacaatggat aacatggcca tcatcaagga gttcatgcgc
      541 ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt tcgagatcga gggcgagggc
      601 gagggccgcc cctacgaggg cacccagacc gccaagctga aggtgaccaa gggtggcccc
      661 ctgcccttcg cctgggacat cctgtcccct cagttcatgt acggctccaa ggcctacgtg
      721 aagcaccccg ccgacatccc cgactacttg aagctgtcct tccccgaggg cttcaagtgg
      781 gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg tgacccagga ctcctccctg
      841 caggacggcg agttcatcta caaggtgaag ctgcgcggca ccaacttccc ctccgacggc
      901 cccgtaatgc agaagaagac catgggctgg gaggcctcct ccgagcggat gtaccccgag
      961 gacggcgccc tgaagggcga gatcaagcag aggctgaagc tgaaggacgg cggccactac
     1021 gacgctgagg tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc cggcgcctac
     1081 aacgtcaaca tcaagttgga catcacctcc cacaacgagg actacaccat cgtggaacag
     1141 tacgaacgcg ccgagggccg ccactccacc ggcggcatgg acgagctgta caagtgagag
     1201 ctcttggact cccatgttgg caaaggcaac caaacaaaca atgaatgatc cgctcctgca
     1261 tatggggcgg tttgagtatt tcaactgcca tttgggctga attgaagaca tgctcctgtc
     1321 agaaattccg tgatcttact caatattcag taatctcggc caatatccta aatgtgcgtg
     1381 gctttatctg tctttgtatt gtttcatcaa ttcatgtaac gtttgctttt cttatgaatt
     1441 ttcaaataaa ttatcgtatt gagaccgaat tctcgtcgac gagtcagtaa taaacggcgt
     1501 caaagtggtt gcagccggca cacacgagtc gtgtttatca actcaaagca caaatacttt
     1561 tcctcaacct aaaaataagg caattagcca aaaacaactt tgcgtgtaaa caacgctcaa
     1621 tacacgtgtc attttattat tagctattgc ttcaccgcct tagctttctc gtgacctagt
     1681 cgtcctcgtc ttttcttctt cttcttctat aaaacaatac ccaaagagct cttcttcttc
     1741 acaattcaga tttcaatttc tcaaaatctt aaaaactttc tctcaattct ctctaccgtg
     1801 atcaaggtaa atttctgtgt tccttattct ctcaaaatct tcgattttgt tttcgttcga
     1861 tcccaatttc gtatatgttc tttggtttag attctgttaa tcttagatcg aagacgattt
     1921 tctgggtttg atcgttagat atcatcttaa ttctcgatta gggtttcata gatatcatcc
     1981 gatttgttca aataatttga gttttgtcga ataattactc ttcgatttgt gatttctatc
     2041 tagatctggt gttagtttct agtttgtgcg atcgaatttg tcgattaatc tgagtttttc
     2101 tgattaacag ctcgcaattg acctaactga gtaagctagc ttgttcgagt attatggcat
     2161 tgggaaaact gtttttcttg taccatttgt tgtgcttgta atttactgtg ttttttattc
     2221 ggttttcgct atcgaactgt gaaatggaaa tggatggaga agagttaatg aatgatatgg
     2281 tccttttgtt cattctcaaa ttaatattat ttgttttttc tcttatttgt tgtgtgttga
     2341 atttgaaatt ataagagata tgcaaacatt ttgttttgag taaaaatgtg tcaaatcgtg
     2401 gcctctaatg accgaagtta atatgaggag taaaacacta gatccccaaa caagcttgaa
     2461 actgaaggcg ggaaacgaca atctgatcat gagcggagaa ttaagggagt cacgttatga
     2521 cccccgccga tgacgcggga caagccgttt tacgtttgga actgacagaa ccgcaacgtt
     2581 gaaggagcca ctcagccgcg ggtttctgga gtttaatgag ctaagcacat acgtcagaaa
     2641 ccattattgc gcgttcaaaa gtcgcctaag gtcactatca gctagcaaat atttcttgtc
     2701 aaaaatgctc cactgacgtt ccataaattc ccctcggtat ccaattagag tctcatattc
     2761 actctcaatc caaataatct gcaccggatc cgctagagga tctcgacctg caagatcccg
     2821 gggggcaatg agatatgaaa aagcctgaac tcaccgcgac gtctgtcgag aagtttctga
     2881 tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc ggagggcgaa gaatctcgtg
     2941 ctttcagctt cgatatagga gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg
     3001 gtttctacaa agatcgttat gtttatcggc actttgcatc ggccgcgctc ccgattccgg
     3061 aagtgcttga cattggggaa ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac
     3121 agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg
     3181 cggaggccat ggatgcgatc gctgcggccg atcttagcca gacgagcggg ttcggcccat
     3241 tcggaccgca aggaatcggt caatacacta catggcgtga tttcatatgc gcgattgctg
     3301 atccccatgt gtatcactgg caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc
     3361 aggctctcga tgagctgatg ctttgggccg aggactgccc cgaagtccgg cacctcgtgc
     3421 acgcggattt cggctccaac aatgtcctga cggacaatgg ccgcataaca gcggtcattg
     3481 actggagcga ggcgatgttc ggggattccc aatacgaggt cgccaacatc ttcttctgga
     3541 ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt cgagcggagg catccggagc
     3601 ttgcaggatc gccgcggctc cgggcgtata tgctccgcat tggtcttgac caactctatc
     3661 agagcttggt tgacggcaat ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa
     3721 tcgtccgatc cggagccggg actgtcgggc gtacacaaat cgcccgcaga agcgcggccg
     3781 tctggaccga tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc cccagcactc
     3841 gtccgggatc ttggaggtga tgtaacatga tcacaagctg atcccccgaa tttccccgat
     3901 cgttcaaaca tttggcaata aagtttctta agattgaatc ctgttgccgg tcttgcgatg
     3961 attatcatat aatttctgtt gaattacgtt aagcatgtaa taattaacat gtaatgcatg
     4021 acgttattta tgagatgggt ttttatgatt agagtcccgc aattatacat ttaatacgcg
     4081 atagaaaaca aaatatagcg cgcaaactag gataaattat cgcgcgcggt gtcatctatg
     4141 ttactagatc gggaattgat cccccctcga cagcttgcat gccagcttgg gctgcaggtc
     4201 gacgcgtcaa catggtggag cacgacactc tcgtctactc caagaatatc aaagatacag
     4261 tctcagaaga ccaaagggct attgagactt ttcaacaaag ggtaatatcg ggaaacctcc
     4321 tcggattcca ttgcccagct atctgtcact tcatcaaaag gacagtagaa aaggaaggtg
     4381 gcacctacaa atgccatcat tgcgataaag gaaaggctat cgttcaagat gcctctgccg
     4441 acagtggtcc caaagatgga cccccaccca cgaggagcat cgtggaaaaa gaagacgttc
     4501 caaccacgtc ttcaaagcaa gtggattgat gtgatatctc cactgacgta agggatgacg
     4561 cacaatccca ctatccttcg caagaccctt cctctatata aggaagttca tttcatttgg
     4621 agaggacacg atatggacaa gaagtactcc attgggctcg atatcggcac aaacagcgtc
     4681 ggctgggccg tcattacgga cgagtacaag gtgccgagca aaaaattcaa agttctgggc
     4741 aataccgatc gccacagcat aaagaagaac ctcattggcg ccctcctgtt cgactccggg
     4801 gagacggccg aagccacgcg gctcaaaaga acagcacggc gcagatatac ccgcagaaag
     4861 aatcggatct gctacctgca ggagatcttt agtaatgaga tggctaaggt ggatgactct
     4921 ttcttccata ggctggagga gtcctttttg gtggaggagg ataaaaagca cgagcgccac
     4981 ccaatctttg gcaatatcgt ggacgaggtg gcgtaccatg aaaagtaccc aaccatatat
     5041 catctgagga agaagcttgt agacagtact gataaggctg acttgcggtt gatctatctc
     5101 gcgctggcgc atatgatcaa atttcgggga cacttcctca tcgaggggga cctgaaccca
     5161 gacaacagcg atgtcgacaa actctttatc caactggttc agacttacaa tcagcttttc
     5221 gaagagaacc cgatcaacgc atccggagtt gacgccaaag caatcctgag cgctaggctg
     5281 tccaaatccc ggcggctcga aaacctcatc gcacagctcc ctggggagaa gaagaacggc
     5341 ctgtttggta atcttatcgc cctgtcactc gggctgaccc ccaactttaa atctaacttc
     5401 gacctggccg aagatgccaa gcttcaactg agcaaagaca cctacgatga tgatctcgac
     5461 aatctgctgg cccagatcgg cgaccagtac gcagaccttt ttttggcggc aaagaacctg
     5521 tcagacgcca ttctgctgag tgatattctg cgagtgaaca cggagatcac caaagctccg
     5581 ctgagcgcta gtatgatcaa gcgctatgat gagcaccacc aagacttgac tttgctgaag
     5641 gcccttgtca gacagcaact gcctgagaag tacaaggaaa ttttcttcga tcagtctaaa
     5701 aatggctacg ccggatacat tgacggcgga gcaagccagg aggaatttta caaatttatt
     5761 aagcccatct tggaaaaaat ggacggcacc gaggagctgc tggtaaagct taacagagaa
     5821 gatctgttgc gcaaacagcg cactttcgac aatggaagca tcccccacca gattcacctg
     5881 ggcgaactgc acgctatcct caggcggcaa gaggatttct accccttttt gaaagataac
     5941 agggaaaaga ttgagaaaat cctcacattt cggataccct actatgtagg ccccctcgcc
     6001 cggggaaatt ccagattcgc gtggatgact cgcaaatcag aagagaccat cactccctgg
     6061 aacttcgagg aagtcgtgga taagggggcc tctgcccagt ccttcatcga aaggatgact
     6121 aactttgata aaaatctgcc taacgaaaag gtgcttccta aacactctct gctgtacgag
     6181 tacttcacag tttataacga gctcaccaag gtcaaatacg tcacagaagg gatgagaaag
     6241 ccagcattcc tgtctggaga gcagaagaaa gctatcgtgg acctcctctt caagacgaac
     6301 cggaaagtta ccgtgaaaca gctcaaagaa gactatttca aaaagattga atgtttcgac
     6361 tctgttgaaa tcagcggagt ggaggatcgc ttcaacgcat ccctgggaac gtatcacgat
     6421 ctcctgaaaa tcattaaaga caaggacttc ctggacaatg aggagaacga ggacattctt
     6481 gaggacattg tcctcaccct tacgttgttt gaagataggg agatgattga agaacgcttg
     6541 aaaacttacg ctcatctctt cgacgacaaa gtcatgaaac agctcaagag gcgccgatat
     6601 acaggatggg ggcggctgtc aagaaaactg atcaatggga tccgagacaa gcagagtgga
     6661 aagacaatcc tggattttct taagtccgat ggatttgcca accggaactt catgcagttg
     6721 atccatgatg actctctcac ctttaaggag gacatccaga aagcacaagt ttctggccag
     6781 ggggacagtc ttcacgagca catcgctaat cttgcaggta gcccagctat caaaaaggga
     6841 atactgcaga ccgttaaggt cgtggatgaa ctcgtcaaag taatgggaag gcataagccc
     6901 gagaatatcg ttatcgagat ggcccgagag aaccaaacta cccagaaggg acagaagaac
     6961 agtagggaaa ggatgaagag gattgaagag ggtataaaag aactggggtc ccaaatcctt
     7021 aaggaacacc cagttgaaaa cacccagctt cagaatgaga agctctacct gtactacctg
     7081 cagaacggca gggacatgta cgtggatcag gaactggaca tcaatcggct ctccgactac
     7141 gacgtggatc atatcgtgcc ccagtctttt ctcaaagatg attctattga taataaagtg
     7201 ttgacaagat ccgataaaaa tagagggaag agtgataacg tcccctcaga agaagttgtc
     7261 aagaaaatga aaaattattg gcggcagctg ctgaacgcca aactgatcac acaacggaag
     7321 ttcgataatc tgactaaggc tgaacgaggt ggcctgtctg agttggataa agccggcttc
     7381 atcaaaaggc agcttgttga gacacgccag atcaccaagc acgtggccca aattctcgat
     7441 tcacgcatga acaccaagta cgatgaaaat gacaaactga ttcgagaggt gaaagttatt
     7501 actctgaagt ctaagctggt ctcagatttc agaaaggact ttcagtttta taaggtgaga
     7561 gagatcaaca attaccacca tgcgcatgat gcctacctga atgcagtggt aggcactgca
     7621 cttatcaaaa aatatcccaa gcttgaatct gaatttgttt acggagacta taaagtgtac
     7681 gatgttagga aaatgatcgc aaagtctgag caggaaatag gcaaggccac cgctaagtac
     7741 ttcttttaca gcaatattat gaattttttc aagaccgaga ttacactggc caatggagag
     7801 attcggaagc gaccacttat cgaaacaaac ggagaaacag gagaaatcgt gtgggacaag
     7861 ggtagggatt tcgcgacagt ccggaaggtc ctgtccatgc cgcaggtgaa catcgttaaa
     7921 aagaccgaag tacagaccgg aggcttctcc aaggaaagta tcctcccgaa aaggaacagc
     7981 gacaagctga tcgcacgcaa aaaagattgg gaccccaaga aatacggcgg attcgattct
     8041 cctacagtcg cttacagtgt actggttgtg gccaaagtgg agaaagggaa gtctaaaaaa
     8101 ctcaaaagcg tcaaggaact gctgggcatc acaatcatgg agcgatcaag cttcgaaaaa
     8161 aaccccatcg actttctcga ggcgaaagga tataaagagg tcaaaaaaga cctcatcatt
     8221 aagcttccca agtactctct ctttgagctt gaaaacggcc ggaaacgaat gctcgctagt
     8281 gcgggcgagc tgcagaaagg taacgagctg gcactgccct ctaaatacgt taatttcttg
     8341 tatctggcca gccactatga aaagctcaaa gggtctcccg aagataatga gcagaagcag
     8401 ctgttcgtgg aacaacacaa acactacctt gatgagatca tcgagcaaat aagcgaattc
     8461 tccaaaagag tgatcctcgc cgacgctaac ctcgataagg tgctttctgc ttacaataag
     8521 cacagggata agcccatcag ggagcaggca gaaaacatta tccacttgtt tactctgacc
     8581 aacttgggcg cgcctgcagc cttcaagtac ttcgacacca ccatagacag aaagcggtac
     8641 acctctacaa aggaggtcct ggacgccaca ctgattcatc agtcaattac ggggctctat
     8701 gaaacaagaa tcgacctctc tcagctcggt ggagacagca gggctgaccc caagaagaag
     8761 aggaaggtgt gatcgatcca ggcctcccag ctttcgtccg tatcatcggt ttcgacaacg
     8821 ttcgtcaagt tcaatgcatc agtttcattg cccacacacc agaatcctac taagtttgag
     8881 tattatggca ttggaaaagc tgttttcttc tatcatttgt tctgcttgta atttactgtg
     8941 ttctttcagt ttttgttttc ggacatcaaa atgcaaatgg atggataaga gttaataaat
     9001 gatatggtcc ttttgttcat tctcaaatta ttattatctg ttgtttttac tttaatgggt
     9061 tgaatttaag taagaaagga actaacagtg tgatattaag gtgcaatgtt agacatataa
     9121 aacagtcttt cacctctctt tggttatgtc ttgaattggt ttgtttcttc acttatctgt
     9181 gtaatcaagt ttactatgag tctatgatca agtaattatg caatcaagtt aagtacagta
     9241 taggcttttt gtgtcgaggg ggtaccgagt cgaggaattc actggccgtc gttttacaac
     9301 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
     9361 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
     9421 gcctgaatgg cgggtaccga gctcgaattc aattcggcgt taattcagta cattaaaaac
     9481 gtccgcaatg tgttattaag ttgtctaagc gtcaatttgt ttacaccaca atatatcctg
     9541 ccaccagcca gccaacagct ccccgaccgg cagctcggca caaaatcacc actcgataca
     9601 ggcagcccat cagtccggga cggcgtcagc gggagagccg ttgtaaggcg gcagactttg
     9661 ctcatgttac cgatgctatt cggaagaacg gcaactaagc tgccgggttt gaaacacgga
     9721 tgatctcgcg gagggtagca tgttgattgt aacgatgaca gagcgttgct gcctgtgatc
     9781 aattcgggca cgaacccagt ggacataagc ctcgttcggt tcgtaagctg taatgcaagt
     9841 agcgtaactg ccgtcacgca actggtccag aaccttgacc gaacgcagcg gtggtaacgg
     9901 cgcagtggcg gttttcatgg cttcttgtta tgacatgttt ttttggggta cagtctatgc
     9961 ctcgggcatc caagcagcaa gcgcgttacg ccgtgggtcg atgtttgatg ttatggagca
    10021 gcaacgatgt tacgcagcag ggcagtcgcc ctaaaacaaa gttaaacatc atgggggaag
    10081 cggtgatcgc cgaagtatcg actcaactat cagaggtagt tggcgtcatc gagcgccatc
    10141 tcgaaccgac gttgctggcc gtacatttgt acggctccgc agtggatggc ggcctgaagc
    10201 cacacagtga tattgatttg ctggttacgg tgaccgtaag gcttgatgaa acaacgcggc
    10261 gagctttgat caacgacctt ttggaaactt cggcttcccc tggagagagc gagattctcc
    10321 gcgctgtaga agtcaccatt gttgtgcacg acgacatcat tccgtggcgt tatccagcta
    10381 agcgcgaact gcaatttgga gaatggcagc gcaatgacat tcttgcaggt atcttcgagc
    10441 cagccacgat cgacattgat ctggctatct tgctgacaaa agcaagagaa catagcgttg
    10501 ccttggtagg tccagcggcg gaggaactct ttgatccggt tcctgaacag gatctatttg
    10561 aggcgctaaa tgaaacctta acgctatgga actcgccgcc cgactgggct ggcgatgagc
    10621 gaaatgtagt gcttacgttg tcccgcattt ggtacagcgc agtaaccggc aaaatcgcgc
    10681 cgaaggatgt cgctgccgac tgggcaatgg agcgcctgcc ggcccagtat cagcccgtca
    10741 tacttgaagc tagacaggct tatcttggac aagaagaaga tcgcttggcc tcgcgcgcag
    10801 atcagttgga agaatttgtc cactacgtga aaggcgagat caccaaggta gtcggcaaat
    10861 aatgtctagc tagaaattcg ttcaagccga cgccgcttcg ccggcgttaa ctcaagcgat
    10921 tagatgcact aagcacataa ttgctcacag ccaaactatc aggtcaagtc tgcttttatt
    10981 atttttaagc gtgcataata agccctacac aaattgggag atatatcatg catgaccaaa
    11041 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
    11101 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
    11161 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
    11221 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
    11281 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
    11341 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
    11401 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
    11461 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
    11521 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
    11581 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
    11641 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
    11701 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
    11761 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
    11821 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
    11881 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact
    11941 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac
    12001 gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
    12061 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
    12121 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gggtgccttg atgtgggcgc
    12181 cggcggtcga gtggcgacgg cgcggcttgt ccgcgccctg gtagattgcc tggccgtagg
    12241 ccagccattt ttgagcggcc agcggccgcg ataggccgac gcgaagcggc ggggcgtagg
    12301 gagcgcagcg accgaagggt aggcgctttt tgcagctctt cggctgtgcg ctggccagac
    12361 agttatgcac aggccaggcg ggttttaaga gttttaataa gttttaaaga gttttaggcg
    12421 gaaaaatcgc cttttttctc ttttatatca gtcacttaca tgtgtgaccg gttcccaatg
    12481 tacggctttg ggttcccaat gtacgggttc cggttcccaa tgtacggctt tgggttccca
    12541 atgtacgtgc tatccacagg aaagagacct tttcgacctt tttcccctgc tagggcaatt
    12601 tgccctagca tctgctccgt acattaggaa ccggcggatg cttcgccctc gatcaggttg
    12661 cggtagcgca tgactaggat cgggccagcc tgccccgcct cctccttcaa atcgtactcc
    12721 ggcaggtcat ttgacccgat cagcttgcgc acggtgaaac agaacttctt gaactctccg
    12781 gcgctgccac tgcgttcgta gatcgtcttg aacaaccatc tggcttctgc cttgcctgcg
    12841 gcgcggcgtg ccaggcggta gagaaaacgg ccgatgccgg gatcgatcaa aaagtaatcg
    12901 gggtgaaccg tcagcacgtc cgggttcttg ccttctgtga tctcgcggta catccaatca
    12961 gctagctcga tctcgatgta ctccggccgc ccggtttcgc tctttacgat cttgtagcgg
    13021 ctaatcaagg cttcaccctc ggataccgtc accaggcggc cgttcttggc cttcttcgta
    13081 cgctgcatgg caacgtgcgt ggtgtttaac cgaatgcagg tttctaccag gtcgtctttc
    13141 tgctttccgc catcggctcg ccggcagaac ttgagtacgt ccgcaacgtg tggacggaac
    13201 acgcggccgg gcttgtctcc cttcccttcc cggtatcggt tcatggattc ggttagatgg
    13261 gaaaccgcca tcagtaccag gtcgtaatcc cacacactgg ccatgccggc cggccctgcg
    13321 gaaacctcta cgtgcccgtc tggaagctcg tagcggatca cctcgccagc tcgtcggtca
    13381 cgcttcgaca gacggaaaac ggccacgtcc atgatgctgc gactatcgcg ggtgcccacg
    13441 tcatagagca tcggaacgaa aaaatctggt tgctcgtcgc ccttgggcgg cttcctaatc
    13501 gacggcgcac cggctgccgg cggttgccgg gattctttgc ggattcgatc agcggccgct
    13561 tgccacgatt caccggggcg tgcttctgcc tcgatgcgtt gccgctgggc ggcctgcgcg
    13621 gccttcaact tctccaccag gtcatcaccc agcgccgcgc cgatttgtac cgggccggat
    13681 ggtttgcgac cgtcacgccg attcctcggg cttgggggtt ccagtgccat tgcagggccg
    13741 gcagacaacc cagccgctta cgcctggcca accgcccgtt cctccacaca tggggcattc
    13801 cacggcgtcg gtgcctggtt gttcttgatt ttccatgccg cctcctttag ccgctaaaat
    13861 tcatctactc atttattcat ttgctcattt actctggtag ctgcgcgatg tattcagata
    13921 gcagctcggt aatggtcttg ccttggcgta ccgcgtacat cttcagcttg gtgtgatcct
    13981 ccgccggcaa ctgaaagttg acccgcttca tggctggcgt gtctgccagg ctggccaacg
    14041 ttgcagcctt gctgctgcgt gcgctcggac ggccggcact tagcgtgttt gtgcttttgc
    14101 tcattttctc tttacctcat taactcaaat gagttttgat ttaatttcag cggccagcgc
    14161 ctggacctcg cgggcagcgt cgccctcggg ttctgattca agaacggttg tgccggcggc
    14221 ggcagtgcct gggtagctca cgcgctgcgt gatacgggac tcaagaatgg gcagctcgta
    14281 cccggccagc gcctcggcaa cctcaccgcc gatgcgcgtg cctttgatcg cccgcgacac
    14341 gacaaaggcc gcttgtagcc ttccatccgt gacctcaatg cgctgcttaa ccagctccac
    14401 caggtcggcg gtggcccata tgtcgtaagg gcttggctgc accggaatca gcacgaagtc
    14461 ggctgccttg atcgcggaca cagccaagtc cgccgcctgg ggcgctccgt cgatcactac
    14521 gaagtcgcgc cggccgatgg ccttcacgtc gcggtcaatc gtcgggcggt cgatgccgac
    14581 aacggttagc ggttgatctt cccgcacggc cgcccaatcg cgggcactgc cctggggatc
    14641 ggaatcgact aacagaacat cggccccggc gagttgcagg gcgcgggcta gatgggttgc
    14701 gatggtcgtc ttgcctgacc cgcctttctg gttaagtaca gcgataacct tcatgcgttc
    14761 cccttgcgta tttgtttatt tactcatcgc atcatatacg cagcgaccgc atgacgcaag
    14821 ctgttttact caaatacaca tcaccttttt agacggcggc gctcggtttc ttcagcggcc
    14881 aagctggccg gccaggccgc cagcttggca tcagacaaac cggccaggat ttcatgcagc
    14941 cgcacggttg agacgtgcgc gggcggctcg aacacgtacc cggccgcgat catctccgcc
    15001 tcgatctctt cggtaatgaa aaacggttcg tcctggccgt cctggtgcgg tttcatgctt
    15061 gttcctcttg gcgttcattc tcggcggccg ccagggcgtc ggcctcggtc aatgcgtcct
    15121 cacggaaggc accgcgccgc ctggcctcgg tgggcgtcac ttcctcgctg cgctcaagtg
    15181 cgcggtacag ggtcgagcga tgcacgccaa gcagtgcagc cgcctctttc acggtgcggc
    15241 cttcctggtc gatcagctcg cgggcgtgcg cgatctgtgc cggggtgagg gtagggcggg
    15301 ggccaaactt cacgcctcgg gccttggcgg cctcgcgccc gctccgggtg cggtcgatga
    15361 ttagggaacg ctcgaactcg gcaatgccgg cgaacacggt caacaccatg cggccggccg
    15421 gcgtggtggt gtcggcccac ggctctgcca ggctacgcag gcccgcgccg gcctcctgga
    15481 tgcgctcggc aatgtccagt aggtcgcggg tgctgcgggc caggcggtct agcctggtca
    15541 ctgtcacaac gtcgccaggg cgtaggtggt caagcatcct ggccagctcc gggcggtcgc
    15601 gcctggtgcc ggtgatcttc tcggaaaaca gcttggtgca gccggccgcg tgcagttcgg
    15661 cccgttggtt ggtcaagtcc tggtcgtcgg tgctgacgcg ggcatagccc agcaggccag
    15721 cggcggcgct cttgttcatg gcgtaatgtc tccggttcta gtcgcaagta ttctacttta
    15781 tgcgactaaa acacgcgaca agaaaacgcc aggaaaaggg cagggcggca gcctgtcgcg
    15841 taacttagga cttgtgcgac atgtcgtttt cagaagacgg ctgcactgaa cgtcagaagc
    15901 cgactgcact atagcagcgg aggggttgga tcaaagtact ttaaagtact ttaaagtact
    15961 ttaaagtact ttgatcccga ggggaaccct gtggttggca tgcacataca aatggacgaa
    16021 cggataaacc ttttcacgcc cttttaaata tccgattatt ctaataaacg ctcttttctc
    16081 ttag