LOCUS       sequence-272935-       15115 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Dual-mini35S driven Cas9-Csy4 system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 15115)
  AUTHORS   Ren Q, Zhong Z, Wang Y, You Q, Li Q, Yuan M, He Y, Qi C, Tang X, 
            Zheng X, Zhang T, Qi Y, Zhang Y
  TITLE     Bidirectional Promoter-Based CRISPR-Cas9 Systems for Plant Genome 
            Editing.
  JOURNAL   Front Plant Sci. 2019 Sep 20;10:1173. doi: 10.3389/fpls.2019.01173. 
            eCollection 2019.
  PUBMED    31616455
REFERENCE   2  (bases 1 to 15115)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Front Plant
            Sci."; date: "2019-09-20"; volume: "10:1173. doi"; pages: " 
            10.3389/fpls.2019.01173. eCollection 2019"
COMMENT     Sequence Label: pGEL050
FEATURES             Location/Qualifiers
     source          1..15115
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(16..723)
                     /label=OCS terminator
                     /note="octopine synthase terminator"
     protein_bind    complement(730..749)
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     misc_RNA        complement(750..825)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             1152..1457
                     /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="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     1336..1355
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(1465..1484)
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     promoter        complement(1490..1536)
                     /label=minimal CaMV 35S promoter
                     /note="minimal 35S promoter from cauliflower mosaic virus"
     promoter        1567..1612
                     /label=minimal CaMV 35S promoter
                     /note="minimal 35S promoter from cauliflower mosaic virus"
     CDS             1643..1663
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2258..2314
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             2315..6418
                     /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             2315..6418
                     /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             6431..6451
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      6462..6710
                     /label=HSP terminator
                     /note="efficient transcription terminator from the 
                     Arabidopsis thaliana heat shock protein 18.2 gene (Nagaya 
                     et al., 2010)"
     misc_feature    6731..6755
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             8056..8685
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             9114..10187
                     /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      10253..10447
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     primer_bind     complement(10683..10705)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    10781..10921
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     10836..10855
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(10936..10953)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11107..11695)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11187..11206)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(11782..12576)
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     misc_feature    13001..13025
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     polyA_signal    13103..13277
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     promoter        complement(14367..15043)
                     /label=CaMV 35S promoter (enhanced)
                     /note="cauliflower mosaic virus 35S promoter with a 
                     duplicated enhancer region"
ORIGIN
        1 ggcgcgccgc cggtcctgct gagcctcgac atgttgtcgc aaaattcgcc ctggacccgc
       61 ccaacgattt gtcgtcactg tcaaggtttg acctgcactt catttggggc ccacatacac
      121 caaaaaaatg ctgcataatt ctcggggcag caagtcggtt acccggccgc cgtgctggac
      181 cgggttgaat ggtgcccgta actttcggta gagcggacgg ccaatactca acttcaagga
      241 atctcaccca tgcgcgccgg cggggaaccg gagttccctt cagtgagcgt tattagttcg
      301 ccgctcggtg tgtcgtagat actagcccct ggggcacttt tgaaatttga ataagattta
      361 tgtaatcagt cttttaggtt tgaccggttc tgccgctttt tttaaaattg gatttgtaat
      421 aataaaacgc aattgtttgt tattgtggcg ctctatcata gatgtcgcta taaacctatt
      481 cagcacaata tattgttttc attttaatat tgtacatata agtagtaggg tacaatcagt
      541 aaattgaacg gagaatatta ttcataaaaa tacgatagta acgggtgata tattcattag
      601 aatgaaccga aaccggcggt aaggatctga gctacacatg ctcaggtttt ttacaacgtg
      661 cacaacagaa ttgaaagcaa atatcatgcg atcataggcg tctcgcatat ctcattaaag
      721 cagggtaccc tgcctatacg gcagtgaacg caccgactcg gtgccacttt ttcaagttga
      781 taacggacta gccttatttt aacttgctat ttctagctct aaaactgaga ccaagccaga
      841 taacagtatg cgtatttgcg cgctgatttt tgcggtataa gaatatatac tgatatgtat
      901 acccgaagta tgtcaaaaag aggtatgcta tgaagcagcg tattacagtg acagttgaca
      961 gcgacagcta tcagttgctc aaggcatata tgatgtcaat atctccggtc tggtaagcac
     1021 aaccatgcag aatgaagccc gtcgtctgcg tgccgaacgc tggaaagcgg aaaatcagga
     1081 agggatggct gaggtcgccc ggtttattga aatgaacggc tcttttgctg acgagaacag
     1141 gggctggtga aatgcagttt aaggtttaca cctataaaag agagagccgt tatcgtctgt
     1201 ttgtggatgt acagagtgat attattgaca cgcccgggcg acggatggtg atccccctgg
     1261 ccagtgcacg tctgctgtca gataaagtct cccgtgaact ttacccggtg gtgcatatcg
     1321 gggatgaaag ctggcgcatg atgaccaccg atatggccag tgtgccggtt tccgttatcg
     1381 gggaagaagt ggctgatctc agccaccgcg aaaatgacat caaaaacgcc attaacctga
     1441 tgttctgggg aatataaggt ctccctgcct atacggcagt gaacactagt cctctccaaa
     1501 tgaaatgaac ttccttatat agaggaaggg tcttgccagc tgtcgacgag ctgtcttagc
     1561 tctagagcaa gacccttcct ctatataagg aagttcattt catttggaga ggcctgcagg
     1621 tagatcgctc gtcgacatgg ctccaaagaa gaagagaaag gttggaatcc acggagttcc
     1681 agctgctatg gatcattatc ttgatattag acttagacct gatccagaat ttccaccagc
     1741 tcaacttatg tctgttcttt ttggaaaact tcatcaagct cttgttgctc aaggaggaga
     1801 tagaattgga gtttcttttc ctgatcttga tgaatcaaga tcaagacttg gagaaagact
     1861 tagaattcat gcttctgctg atgatcttag agctttgctt gctagacctt ggcttgaagg
     1921 acttagagat catcttcaat ttggagaacc agctgttgtt ccacatccaa ctccttatag
     1981 acaagtttca agagttcaag ctaaatctaa tccagaaaga cttagaagaa gacttatgag
     2041 aagacatgat ctttctgaag aagaagctag aaaaagaatt cctgatactg ttgctagagc
     2101 tttggatttg ccttttgtta cacttagatc acaatctact ggacaacatt ttagactttt
     2161 tattagacat ggaccacttc aagttactgc tgaagaagga ggatttactt gttatggact
     2221 ttctaaggga ggttttgttc cttggtttgg atctggagct actaattttt ctcttcttaa
     2281 gcaagctgga gatgttgaag aaaatcctgg acccatggat aagaagtact ctatcggact
     2341 cgatatcgga actaactctg tgggatgggc tgtgatcacc gatgagtaca aggtgccatc
     2401 taagaagttc aaggttctcg gaaacaccga taggcactct atcaagaaaa accttatcgg
     2461 tgctctcctc ttcgattctg gtgaaactgc tgaggctacc agactcaaga gaaccgctag
     2521 aagaaggtac accagaagaa agaacaggat ctgctacctc caagagatct tctctaacga
     2581 gatggctaaa gtggatgatt cattcttcca caggctcgaa gagtcattcc tcgtggaaga
     2641 agataagaag cacgagaggc accctatctt cggaaacatc gttgatgagg tggcatacca
     2701 cgagaagtac cctactatct accacctcag aaagaagctc gttgattcta ctgataaggc
     2761 tgatctcagg ctcatctacc tcgctctcgc tcacatgatc aagttcagag gacacttcct
     2821 catcgagggt gatctcaacc ctgataactc tgatgtggat aagttgttca tccagctcgt
     2881 gcagacctac aaccagcttt tcgaagagaa ccctatcaac gcttcaggtg tggatgctaa
     2941 ggctatcctc tctgctaggc tctctaagtc aagaaggctt gagaacctca ttgctcagct
     3001 ccctggtgag aagaagaacg gacttttcgg aaacttgatc gctctctctc tcggactcac
     3061 ccctaacttc aagtctaact tcgatctcgc tgaggatgca aagctccagc tctcaaagga
     3121 tacctacgat gatgatctcg ataacctcct cgctcagatc ggagatcagt acgctgattt
     3181 gttcctcgct gctaagaacc tctctgatgc tatcctcctc agtgatatcc tcagagtgaa
     3241 caccgagatc accaaggctc cactctcagc ttctatgatc aagagatacg atgagcacca
     3301 ccaggatctc acacttctca aggctcttgt tagacagcag ctcccagaga agtacaaaga
     3361 gattttcttc gatcagtcta agaacggata cgctggttac atcgatggtg gtgcatctca
     3421 agaagagttc tacaagttca tcaagcctat cctcgagaag atggatggaa ccgaggaact
     3481 cctcgtgaag ctcaatagag aggatcttct cagaaagcag aggaccttcg ataacggatc
     3541 tatccctcat cagatccacc tcggagagtt gcacgctatc cttagaaggc aagaggattt
     3601 ctacccattc ctcaaggata acagggaaaa gattgagaag attctcacct tcagaatccc
     3661 ttactacgtg ggacctctcg ctagaggaaa ctcaagattc gcttggatga ccagaaagtc
     3721 tgaggaaacc atcacccctt ggaacttcga agaggtggtg gataagggtg ctagtgctca
     3781 gtctttcatc gagaggatga ccaacttcga taagaacctt ccaaacgaga aggtgctccc
     3841 taagcactct ttgctctacg agtacttcac cgtgtacaac gagttgacca aggttaagta
     3901 cgtgaccgag ggaatgagga agcctgcttt tttgtcaggt gagcaaaaga aggctatcgt
     3961 tgatctcttg ttcaagacca acagaaaggt gaccgtgaag cagctcaaag aggattactt
     4021 caagaaaatc gagtgcttcg attcagttga gatttctggt gttgaggata ggttcaacgc
     4081 atctctcgga acctaccacg atctcctcaa gatcattaag gataaggatt tcttggataa
     4141 cgaggaaaac gaggatatct tggaggatat cgttcttacc ctcaccctct ttgaagatag
     4201 agagatgatt gaagaaaggc tcaagaccta cgctcatctc ttcgatgata aggtgatgaa
     4261 gcagttgaag agaagaagat acactggttg gggaaggctc tcaagaaagc tcattaacgg
     4321 aatcagggat aagcagtctg gaaagacaat ccttgatttc ctcaagtctg atggattcgc
     4381 taacagaaac ttcatgcagc tcatccacga tgattctctc acctttaaag aggatatcca
     4441 gaaggctcag gtttcaggac agggtgatag tctccatgag catatcgcta acctcgctgg
     4501 atctcctgca atcaagaagg gaatcctcca gactgtgaag gttgtggatg agttggtgaa
     4561 ggtgatggga aggcataagc ctgagaacat cgtgatcgaa atggctagag agaaccagac
     4621 cactcagaag ggacagaaga actctaggga aaggatgaag aggatcgagg aaggtatcaa
     4681 agagcttgga tctcagatcc tcaaagagca ccctgttgag aacactcagc tccagaatga
     4741 gaagctctac ctctactacc tccagaacgg aagggatatg tatgtggatc aagagttgga
     4801 tatcaacagg ctctctgatt acgatgttga tcatatcgtg ccacagtcat tcttgaagga
     4861 tgattctatc gataacaagg tgctcaccag gtctgataag aacaggggta agagtgataa
     4921 cgtgccaagt gaagaggttg tgaagaaaat gaagaactat tggaggcagc tcctcaacgc
     4981 taagctcatc actcagagaa agttcgataa cttgactaag gctgagaggg gaggactctc
     5041 tgaattggat aaggcaggat tcatcaagag gcagcttgtg gaaaccaggc agatcactaa
     5101 gcacgttgca cagatcctcg attctaggat gaacaccaag tacgatgaga acgataagtt
     5161 gatcagggaa gtgaaggtta tcaccctcaa gtcaaagctc gtgtctgatt tcagaaagga
     5221 tttccaattc tacaaggtga gggaaatcaa caactaccac cacgctcacg atgcttacct
     5281 taacgctgtt gttggaaccg ctctcatcaa gaagtatcct aagctcgagt cagagttcgt
     5341 gtacggtgat tacaaggtgt acgatgtgag gaagatgatc gctaagtctg agcaagagat
     5401 cggaaaggct accgctaagt atttcttcta ctctaacatc atgaatttct tcaagaccga
     5461 gattaccctc gctaacggtg agatcagaaa gaggccactc atcgagacaa acggtgaaac
     5521 aggtgagatc gtgtgggata agggaaggga tttcgctacc gttagaaagg tgctctctat
     5581 gccacaggtg aacatcgtta agaaaaccga ggtgcagacc ggtggattct ctaaagagtc
     5641 tatcctccct aagaggaact ctgataagct cattgctagg aagaaggatt gggaccctaa
     5701 gaaatacggt ggtttcgatt ctcctaccgt ggcttactct gttctcgttg tggctaaggt
     5761 tgagaaggga aagagtaaga agctcaagtc tgttaaggaa cttctcggaa tcactatcat
     5821 ggaaaggtca tctttcgaga agaacccaat cgatttcctc gaggctaagg gatacaaaga
     5881 ggttaagaag gatctcatca tcaagctccc aaagtactca ctcttcgaac tcgagaacgg
     5941 tagaaagagg atgctcgctt ctgctggtga gcttcaaaag ggaaacgagc ttgctctccc
     6001 atctaagtac gttaactttc tttacctcgc ttctcactac gagaagttga agggatctcc
     6061 agaagataac gagcagaagc aacttttcgt tgagcagcac aagcactact tggatgagat
     6121 catcgagcag atctctgagt tctctaaaag ggtgatcctc gctgatgcaa acctcgataa
     6181 ggtgttgtct gcttacaaca agcacagaga taagcctatc agggaacagg cagagaacat
     6241 catccatctc ttcaccctta ccaacctcgg tgctcctgct gctttcaagt acttcgatac
     6301 aaccatcgat aggaagagat acacctctac caaagaagtg ctcgatgcta ccctcatcca
     6361 tcagtctatc actggactct acgagactag gatcgatctc tcacagctcg gtggtgattc
     6421 aagggctgat cctaagaaga agaggaaggt ttgaggatcc atatgaagat gaagatgaaa
     6481 tatttggtgt gtcaaataaa aagcttgtgt gcttaagttt gtgttttttt cttggcttgt
     6541 tgtgttatga atttgtggct ttttctaata ttaaatgaat gtaagatcac attataatga
     6601 ataaacaaat gtttctataa tccattgtga atgttttgtt ggatctcttc tgcagcatat
     6661 aactactgta tgtgctatgg tatggactat ggaatatgat taaagataag gagctcaaac
     6721 tatcagtgtt tgacaggata tattggcggg taaacctaag agaaaagagc gtttattaga
     6781 ataatcggat atttaaaagg gcgtgaaaag gtttatccgt tcgtccattt gtatgtgcat
     6841 gccaaccaca gggttcccct cgggatcaaa gtactttgat ccaacccctc cgctgctata
     6901 gtgcagtcgg cttctgacgt tcagtgcagc cgtcttctga aaacgacatg tcgcacaagt
     6961 cctaagttac gcgacaggct gccgccctgc ccttttcctg gcgttttctt gtcgcgtgtt
     7021 ttagtcgcat aaagtagaat acttgcgact agaaccggag acattacgcc atgaacaaga
     7081 gcgccgccgc tggcctgctg ggctatgccc gcgtcagcac cgacgaccag gacttgacca
     7141 accaacgggc cgaactgcac gcggccggct gcaccaagct gttttccgag aagatcaccg
     7201 gcaccaggcg cgaccgcccg gagctggcca ggatgcttga ccacctacgc cctggcgacg
     7261 ttgtgacagt gaccaggcta gaccgcctgg cccgcagcac ccgcgaccta ctggacattg
     7321 ccgagcgcat ccaggaggcc ggcgcgggcc tgcgtagcct ggcagagccg tgggccgaca
     7381 ccaccacgcc ggccggccgc atggtgttga ccgtgttcgc cggcattgcc gagttcgagc
     7441 gttccctaat catcgaccgc acccggagcg ggcgcgaggc cgccaaggcc cgaggcgtga
     7501 agtttggccc ccgccctacc ctcaccccgg cacagatcgc gcacgcccgc gagctgatcg
     7561 accaggaagg ccgcaccgtg aaagaggcgg ctgcactgct tggcgtgcat cgctcgaccc
     7621 tgtaccgcgc acttgagcgc agcgaggaag tgacgcccac cgaggccagg cggcgcggtg
     7681 ccttccgtga ggacgcattg accgaggccg acgccctggc ggccgccgag aatgaacgcc
     7741 aagaggaaca agcatgaaac cgcaccagga cggccaggac gaaccgtttt tcattaccga
     7801 agagatcgag gcggagatga tcgcggccgg gtacgtgttc gagccgcccg cgcacggctc
     7861 aaccgtgcgg ctgcatgaaa tcctggccgg tttgtctgat gccaagctgg cggcctggcc
     7921 ggccagcttg gccgctgaag aaaccgagcg ccgccgtcta aaaaggtgat gtgtatttga
     7981 gtaaaacagc ttgcgtcatg cggtcgctgc gtatatgatg cgatgagtaa ataaacaaat
     8041 acgcaagggg aacgcatgaa ggttatcgct gtacttaacc agaaaggcgg gtcaggcaag
     8101 acgaccatcg caacccatct agcccgcgcc ctgcaactcg ccggggccga tgttctgtta
     8161 gtcgattccg atccccaggg cagtgcccgc gattgggcgg ccgtgcggga agatcaaccg
     8221 ctaaccgttg tcggcatcga ccgcccgacg attgaccgcg acgtgaaggc catcggccgg
     8281 cgcgacttcg tagtgatcga cggagcgccc caggcggcgg acttggctgt gtccgcgatc
     8341 aaggcagccg acttcgtgct gattccggtg cagccaagcc cttacgacat atgggccacc
     8401 gccgacctgg tggagctggt taagcagcgc attgaggtca cggatggaag gctacaagcg
     8461 gcctttgtcg tgtcgcgggc gatcaaaggc acgcgcatcg gcggtgaggt tgccgaggcg
     8521 ctggccgggt acgagctgcc cattcttgag tcccgtatca cgcagcgcgt gagctaccca
     8581 ggcactgccg ccgccggcac aaccgttctt gaatcagaac ccgagggcga cgctgcccgc
     8641 gaggtccagg cgctggccgc tgaaattaaa tcaaaactca tttgagttaa tgaggtaaag
     8701 agaaaatgag caaaagcaca aacacgctaa gtgccggccg tccgagcgca cgcagcagca
     8761 aggctgcaac gttggccagc ctggcagaca cgccagccat gaagcgggtc aactttcagt
     8821 tgccggcgga ggatcacacc aagctgaaga tgtacgcggt acgccaaggc aagaccatta
     8881 ccgagctgct atctgaatac atcgcgcagc taccagagta aatgagcaaa tgaataaatg
     8941 agtagatgaa ttttagcggc taaaggaggc ggcatggaaa atcaagaaca accaggcacc
     9001 gacgccgtgg aatgccccat gtgtggagga acgggcggtt ggccaggcgt aagcggctgg
     9061 gttgcctgcc ggccctgcaa tggcactgga acccccaagc ccgaggaatc ggcgtgagcg
     9121 gtcgcaaacc atccggcccg gtacaaatcg gcgcggcgct gggtgatgac ctggtggaga
     9181 agttgaaggc cgcgcaggcc gcccagcggc aacgcatcga ggcagaagca cgccccggtg
     9241 aatcgtggca agcggccgct gatcgaatcc gcaaagaatc ccggcaaccg ccggcagccg
     9301 gtgcgccgtc gattaggaag ccgcccaagg gcgacgagca accagatttt ttcgttccga
     9361 tgctctatga cgtgggcacc cgcgatagtc gcagcatcat ggacgtggcc gttttccgtc
     9421 tgtcgaagcg tgaccgacga gctggcgagg tgatccgcta cgagcttcca gacgggcacg
     9481 tagaggtttc cgcagggccg gccggcatgg ccagtgtgtg ggattacgac ctggtactga
     9541 tggcggtttc ccatctaacc gaatccatga accgataccg ggaagggaag ggagacaagc
     9601 ccggccgcgt gttccgtcca cacgttgcgg acgtactcaa gttctgccgg cgagccgatg
     9661 gcggaaagca gaaagacgac ctggtagaaa cctgcattcg gttaaacacc acgcacgttg
     9721 ccatgcagcg tacgaagaag gccaagaacg gccgcctggt gacggtatcc gagggtgaag
     9781 ccttgattag ccgctacaag atcgtaaaga gcgaaaccgg gcggccggag tacatcgaga
     9841 tcgagctagc tgattggatg taccgcgaga tcacagaagg caagaacccg gacgtgctga
     9901 cggttcaccc cgattacttt ttgatcgatc ccggcatcgg ccgttttctc taccgcctgg
     9961 cacgccgcgc cgcaggcaag gcagaagcca gatggttgtt caagacgatc tacgaacgca
    10021 gtggcagcgc cggagagttc aagaagttct gtttcaccgt gcgcaagctg atcgggtcaa
    10081 atgacctgcc ggagtacgat ttgaaggagg aggcggggca ggctggcccg atcctagtca
    10141 tgcgctaccg caacctgatc gagggcgaag catccgccgg ttcctaatgt acggagcaga
    10201 tgctagggca aattgcccta gcaggggaaa aaggtcgaaa aggcctcttt cctgtggata
    10261 gcacgtacat tgggaaccca aagccgtaca ttgggaaccg gaacccgtac attgggaacc
    10321 caaagccgta cattgggaac cggtcacaca tgtaagtgac tgatataaaa gagaaaaaag
    10381 gcgatttttc cgcctaaaac tctttaaaac ttattaaaac tcttaaaacc cgcctggcct
    10441 gtgcataact gtctggccag cgcacagccg aagagctgca aaaagcgcct acccttcggt
    10501 cgctgcgctc cctacgcccc gccgcttcgc gtcggcctat cgcggccgct ggccgctcaa
    10561 aaatggctgg cctacggcca ggcaatctac cagggcgcgg acaagccgcg ccgtcgccac
    10621 tcgaccgccg gcgcccacat caaggcaccc tgcctcgcgc gtttcggtga tgacggtgaa
    10681 aacctctgac acatgcagct cccggaaacg gtcacagctt gtctgtaagc ggatgccggg
    10741 agcagacaag cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac
    10801 gtagcgatag cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag
    10861 agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag
    10921 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
    10981 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
    11041 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
    11101 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
    11161 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
    11221 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
    11281 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
    11341 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
    11401 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
    11461 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
    11521 gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc
    11581 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
    11641 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
    11701 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
    11761 tttggtcatg cattctaggt actaaaacaa ttcatccagt aaaatataat attttatttt
    11821 ctcccaatca ggcttgatcc ccagtaagtc aaaaaatagc tcgacatact gttcttcccc
    11881 gatatcctcc ctgatcgacc ggacgcagaa ggcaatgtca taccacttgt ccgccctgcc
    11941 gcttctccca agatcaataa agccacttac tttgccatct ttcacaaaga tgttgctgtc
    12001 tcccaggtcg ccgtgggaaa agacaagttc ctcttcgggc ttttccgtct ttaaaaaatc
    12061 atacagctcg cgcggatctt taaatggagt gtcttcttcc cagttttcgc aatccacatc
    12121 ggccagatcg ttattcagta agtaatccaa ttcggctaag cggctgtcta agctattcgt
    12181 atagggacaa tccgatatgt cgatggagtg aaagagcctg atgcactccg catacagctc
    12241 gataatcttt tcagggcttt gttcatcttc atactcttcc gagcaaagga cgccatcggc
    12301 ctcactcatg agcagattgc tccagccatc atgccgttca aagtgcagga cctttggaac
    12361 aggcagcttt ccttccagcc atagcatcat gtccttttcc cgttccacat cataggtggt
    12421 ccctttatac cggctgtccg tcatttttaa atataggttt tcattttctc ccaccagctt
    12481 atatacctta gcaggagaca ttccttccgt atcttttacg cagcggtatt tttcgatcag
    12541 ttttttcaat tccggtgata ttctcatttt agccatttat tatttccttc ctcttttcta
    12601 cagtatttaa agatacccca agaagctaat tataacaaga cgaactccaa ttcactgttc
    12661 cttgcattct aaaaccttaa ataccagaaa acagcttttt caaagttgtt ttcaaagttg
    12721 gcgtataaca tagtatcgac ggagccgatt ttgaaaccgc ggtgatcaca ggcagcaacg
    12781 ctctgtcatc gttacaatca acatgctacc ctccgcgaga tcatccgtgt ttcaaacccg
    12841 gcagcttagt tgccgttctt ccgaatagca tcggtaacat gagcaaagtc tgccgcctta
    12901 caacggctct cccgctgacg ccgtcccgga ctgatgggct gcctgtatcg agtggtgatt
    12961 ttgtgccgag ctgccggtcg gggagctgtt ggctggctgg tggcaggata tattgtggtg
    13021 taaacaaatt gacgcttaga caacttaata acacattgcg gacgttttta atgtactgaa
    13081 ttaacgccga attaattcgg gggatctgga ttttagtact ggattttggt tttaggaatt
    13141 agaaatttta ttgatagaag tattttacaa atacaaatac atactaaggg tttcttatat
    13201 gctcaacaca tgagcgaaac cctataggaa ccctaattcc cttatctggg aactactcac
    13261 acattattat ggagaaaata gagcttgtcg atcgttaatt aatcacctcc aagatcccgg
    13321 acgagtgctg gggcgtcggt ttccactatc ggcgagtact tctacacagc catcggtcca
    13381 gacggccgcg cttctgcggg cgatttgtgt acgcccgaca gtcccggctc cggatcggac
    13441 gattgcgtcg catcgaccct gcgcccaagc tgcatcatcg aaattgccgt caaccaagct
    13501 ctgatagagt tggtcaagac caatgcggag catatacgcc cggagccgcg gcgatcctgc
    13561 aagctccgga tgcctccgct cgaagtagcg cgtctgctgc tccatacaag ccaaccacgg
    13621 cctccagaag aagatgttgg cgacctcgta ttgggaatcc ccgaacatcg cctcgctcca
    13681 gtcaatgacc gctgttatgc ggccattgtc cgtcaggaca ttgttggagc cgaaatccgc
    13741 gtgcacgagg tgccggactt cggggcagtc ctcggcccaa agcatcagct catcgagagc
    13801 ctgcgcgacg gacgcactga cggtgtcgtc catcacagtt tgccagtgat acacatgggg
    13861 atcagcaatc gcgcatatga aatcacgcca tgtagtgtat tgaccgattc cttgcggtcc
    13921 gaatgggccg aacccgctcg tctggctaag atcggccgca gcgatcgcat ccatggcctc
    13981 cgcgaccggc tgcagaacag cgggcagttc ggtttcaggc aggtcttgca acgtgacacc
    14041 ctgtgcacgg cgggagatgc aataggtcag gctctcgctg aattccccaa tgtcaagcac
    14101 ttccggaatc gggagcgcgg ccgatgcaaa gtgccgataa acataacgat ctttgtagaa
    14161 accatcggcg cagctattta cccgcaggac atatccacgc cctcctacat cgaagctgaa
    14221 agcacgagat tcttcgccct ccgagagctg catcaggtcg gacacgctgt cgaacttttc
    14281 gatcagaaac ttctcgacag acgtcgcggt gagttcaggc tttttcatcc taggagagat
    14341 agatttgtag agagagactg gtgatttcag cgtgtcctct ccaaatgaaa tgaacttcct
    14401 tatatagagg aagggtcttg cgaaggatag tgggattgtg cgtcatccct tacgtcagtg
    14461 gagatatcac atcaatccac ttgctttgaa gacgtggttg gaacgtcttc tttttccacg
    14521 atgctcctcg tgggtggggg tccatctttg ggaccactgt cggcagaggc atcttgaacg
    14581 atagcctttc ctttatcgca atgatggcat ttgtaggtgc caccttcctt ttctactgtc
    14641 cttttgatga agtgacagat agctgggcaa tggaatccga ggaggtttcc cgatattacc
    14701 ctttgttgaa aagtctcaat agccctttgg tcttctgaga ctgtatcttt gatattcttg
    14761 gagtagacga gagtgtcgtg ctccaccatg ttcacatcaa tccacttgct ttgaagacgt
    14821 ggttggaacg tcttcttttt ccacgatgct cctcgtgggt gggggtccat ctttgggacc
    14881 actgtcggca gaggcatctt gaacgatagc ctttccttta tcgcaatgat ggcatttgta
    14941 ggtgccacct tccttttcta ctgtcctttt gatgaagtga cagatagctg ggcaatggaa
    15001 tccgaggagg tttcccgata ttaccctttg ttgaaaagtc tcaatagccc tttggtcttc
    15061 tgagactgta tctttgatat tcttggagta gacgagagtg tcgtgctcca ccatg
//
