LOCUS       sequence-266286-       15124 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 15124)
  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 15124)
  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..15124
                     /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             8055..8684
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             9118..10185
                     /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"
     rep_origin      10251..10445
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     primer_bind     complement(10681..10703)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    10789..10929
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     10844..10863
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(10944..10961)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11115..11703)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11195..11214)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(11790..12584)
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     misc_feature    13009..13033
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     polyA_signal    13111..13285
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     promoter        complement(14375..15052)
                     /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 tcttgctcta gagctaagac agctcgtcga
     1561 cagctggcaa 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 ataacggata tttaaaaggg cgtgaaaagg tttatccgtt cgtccatttg tatgtgcatg
     6841 ccaaccacag ggttcccctc gggatcaaag tactttgatc caacccctcc gctgctatag
     6901 tgcagtcggc ttctgacgtt cagtgcagcc gtcttctgaa aacgacatgt cgcacaagtc
     6961 ctaagttacg cgacaggctg ccgccctgcc cttttcctgg cgttttcttg tcgcgtgttt
     7021 tagtcgcata aagtagaata cttgcgacta gaaccggaga cattacgcca tgaacaagag
     7081 cgccgccgct ggcctgctgg gctatgcccg cgtcagcacc gacgaccagg acttgaccaa
     7141 ccaacgggcc gaactgcacg cggccggctg caccaagctg ttttccgaga agatcaccgg
     7201 caccaggcgc gaccgcccgg agctggccag gatgcttgac cacctacgcc ctggcgacgt
     7261 tgtgacagtg accaggctag accgcctggc ccgcagcacc cgcgacctac tggacattgc
     7321 cgagcgcatc caggaggccg gcgcgggcct gcgtagcctg gcagagccgt gggccgacac
     7381 caccacgccg gccggccgca tggtgttgac cgtgttcgcc ggcattgccg agttcgagcg
     7441 ttccctaatc atcgaccgca cccggagcgg gcgcgaggcc gccaaggccc gaggcgtgaa
     7501 gtttggcccc cgccctaccc tcaccccggc acagatcgcg cacgcccgcg agctgatcga
     7561 ccaggaaggc cgcaccgtga aagaggcggc tgcactgctt ggcgtgcatc gctcgaccct
     7621 gtaccgcgca cttgagcgca gcgaggaagt gacgcccacc gaggccaggc ggcgcggtgc
     7681 cttccgtgag gacgcattga ccgaggccga cgccctggcg gccgccgaga atgaacgcca
     7741 agaggaacaa gcatgaaacc gcaccaggac ggccaggacg aaccgttttt cattaccgaa
     7801 gagatcgagg cggagatgat cgcggccggg tacgtgttcg agccgcccgc gcacggctca
     7861 accgtgcggc tgcatgaaat cctggccggt ttgtctgatg ccaagctggc ggcctggccg
     7921 gccagcttgg ccgctgaaga aaccgagcgc cgccgtctaa aaaggtgatg tgtatttgag
     7981 taaaacagct tgcgtcatgc ggtcgctgcg tatatgatgc gatgagtaaa taaacaaata
     8041 cgcaagggga acgcatgaag gttatcgctg tacttaacca gaaaggcggg tcaggcaaga
     8101 cgaccatcgc aacccatcta gcccgcgccc tgcaactcgc cggggccgat gttctgttag
     8161 tcgattccga tccccagggc agtgcccgcg attgggcggc cgtgcgggaa gatcaaccgc
     8221 taaccgttgt cggcatcgac cgcccgacga ttgaccgcga cgtgaaggcc atcggccggc
     8281 gcgacttcgt agtgatcgac ggagcgcccc aggcggcgga cttggctgtg tccgcgatca
     8341 aggcagccga cttcgtgctg attccggtgc agccaagccc ttacgacata tgggccaccg
     8401 ccgacctggt ggagctggtt aagcagcgca ttgaggtcac ggatggaagg ctacaagcgg
     8461 cctttgtcgt gtcgcgggcg atcaaaggca cgcgcatcgg cggtgaggtt gccgaggcgc
     8521 tggccgggta cgagctgccc attcttgagt cccgtatcac gcagcgcgtg agctacccag
     8581 gcactgccgc cgccggcaca accgttcttg aatcagaacc cgagggcgac gctgcccgcg
     8641 aggtccaggc gctggccgct gaaattaaat caaaactcat ttgagttaat gaggtaaaga
     8701 gaaaatgagc aaaagcacaa acacgctaag tgccggccgt ccgagcgcac gcagcagcaa
     8761 ggctgcaacg ttggccagcc tggcagacac gccagccatg aagcgggtca actttcagtt
     8821 gccggcggag gatcacacca agctgaagat gtacgcggta cgccaaggca agaccattac
     8881 cgagctgcta tctgaataca tcgcgcagct accagagtaa atgagcaaat gaataaatga
     8941 gtagatgaat tttagcggct aaaggaggcg gcatggaaaa tcaagaacaa ccaggcaccg
     9001 acgccgtgga atgccccatg tgtggaggaa cgggcggttg gccaggcgta agcggctggg
     9061 ttgtctgccg gccctgcaat ggcactggaa cccccaagcc cgaggaatcg gcgtgacggt
     9121 cgcaaaccat ccggcccggt acaaatcggc gcggcgctgg gtgatgacct ggtggagaag
     9181 ttgaaggccg cgcaggccgc ccagcggcaa cgcatcgagg cagaagcacg ccccggtgaa
     9241 tcgtggcaag cggccgctga tcgaatccgc aaagaatccc ggcaaccgcc ggcagccggt
     9301 gcgccgtcga ttaggaagcc gcccaagggc gacgagcaac cagatttttt cgttccgatg
     9361 ctctatgacg tgggcacccg cgatagtcgc agcatcatgg acgtggccgt tttccgtctg
     9421 tcgaagcgtg accgacgagc tggcgaggtg atccgctacg agcttccaga cgggcacgta
     9481 gaggtttccg cagggccggc cggcatggcc agtgtgtggg attacgacct ggtactgatg
     9541 gcggtttccc atctaaccga atccatgaac cgataccggg aagggaaggg agacaagccc
     9601 ggccgcgtgt tccgtccaca cgttgcggac gtactcaagt tctgccggcg agccgatggc
     9661 ggaaagcaga aagacgacct ggtagaaacc tgcattcggt taaacaccac gcacgttgcc
     9721 atgcagcgta cgaagaaggc caagaacggc cgcctggtga cggtatccga gggtgaagcc
     9781 ttgattagcc gctacaagat cgtaaagagc gaaaccgggc ggccggagta catcgagatc
     9841 gagctagctg attggatgta ccgcgagatc acagaaggca agaacccgga cgtgctgacg
     9901 gttcaccccg attacttttt gatcgatccc ggcatcggcc gttttctcta ccgcctggca
     9961 cgccgcgccg caggcaaggc agaagccaga tggttgttca agacgatcta cgaacgcagt
    10021 ggcagcgccg gagagttcaa gaagttctgt ttcaccgtgc gcaagctgat cgggtcaaat
    10081 gacctgccgg agtacgattt gaaggaggag gcggggcagg ctggcccgat cctagtcatg
    10141 cgctaccgca acctgatcga gggcgaagca tccgccggtt cctaatgtac ggagcagatg
    10201 ctagggcaaa ttgccctagc aggggaaaaa ggtcgaaaag gcctctttcc tgtggatagc
    10261 acgtacattg ggaacccaaa gccgtacatt gggaaccgga acccgtacat tgggaaccca
    10321 aagccgtaca ttgggaaccg gtcacacatg taagtgactg atataaaaga gaaaaaaggc
    10381 gatttttccg cctaaaactc tttaaaactt attaaaactc ttaaaacccg cctggcctgt
    10441 gcataactgt ctggccagcg cacagccgaa gagctgcaaa aagcgcctac ccttcggtcg
    10501 ctgcgctccc tacgccccgc cgcttcgcgt cggcctatcg cggccgctgg ccgctcaaaa
    10561 atggctggcc tacggccagg caatctacca gggcgcggac aagccgcgcc gtcgccactc
    10621 gaccgccggc gcccacatca aggcaccctg cctcgcgcgt ttcggtgatg acggtgaaaa
    10681 cctctgacac atgcagctcc cggaaacggt cacagcttgt ctgtaagcgg atgccgggag
    10741 cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg cagccatgac
    10801 ccagtcacgt agcgatagcg gagtgtatac tggcttaact atgcggcatc agagcagatt
    10861 gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac
    10921 cgcatcaggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
    10981 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
    11041 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
    11101 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
    11161 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
    11221 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
    11281 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
    11341 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
    11401 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
    11461 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
    11521 ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg
    11581 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
    11641 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
    11701 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
    11761 taagggattt tggtcatgca ttctaggtac taaaacaatt catccagtaa aatataatat
    11821 tttattttct cccaatcagg cttgatcccc agtaagtcaa aaaatagctc gacatactgt
    11881 tcttccccga tatcctccct gatcgaccgg acgcagaagg caatgtcata ccacttgtcc
    11941 gccctgccgc ttctcccaag atcaataaag ccacttactt tgccatcttt cacaaagatg
    12001 ttgctgtctc ccaggtcgcc gtgggaaaag acaagttcct cttcgggctt ttccgtcttt
    12061 aaaaaatcat acagctcgcg cggatcttta aatggagtgt cttcttccca gttttcgcaa
    12121 tccacatcgg ccagatcgtt attcagtaag taatccaatt cggctaagcg gctgtctaag
    12181 ctattcgtat agggacaatc cgatatgtcg atggagtgaa agagcctgat gcactccgca
    12241 tacagctcga taatcttttc agggctttgt tcatcttcat actcttccga gcaaaggacg
    12301 ccatcggcct cactcatgag cagattgctc cagccatcat gccgttcaaa gtgcaggacc
    12361 tttggaacag gcagctttcc ttccagccat agcatcatgt ccttttcccg ttccacatca
    12421 taggtggtcc ctttataccg gctgtccgtc atttttaaat ataggttttc attttctccc
    12481 accagcttat ataccttagc aggagacatt ccttccgtat cttttacgca gcggtatttt
    12541 tcgatcagtt ttttcaattc cggtgatatt ctcattttag ccatttatta tttccttcct
    12601 cttttctaca gtatttaaag ataccccaag aagctaatta taacaagacg aactccaatt
    12661 cactgttcct tgcattctaa aaccttaaat accagaaaac agctttttca aagttgtttt
    12721 caaagttggc gtataacata gtatcgacgg agccgatttt gaaaccgcgg tgatcacagg
    12781 cagcaacgct ctgtcatcgt tacaatcaac atgctaccct ccgcgagatc atccgtgttt
    12841 caaacccggc agcttagttg ccgttcttcc gaatagcatc ggtaacatga gcaaagtctg
    12901 ccgccttaca acggctctcc cgctgacgcc gtcccggact gatgggctgc ctgtatcgag
    12961 tggtgatttt gtgccgagct gccggtcggg gagctgttgg ctggctggtg gcaggatata
    13021 ttgtggtgta aacaaattga cgcttagaca acttaataac acattgcgga cgtttttaat
    13081 gtactgaatt aacgccgaat taattcgggg gatctggatt ttagtactgg attttggttt
    13141 taggaattag aaattttatt gatagaagta ttttacaaat acaaatacat actaagggtt
    13201 tcttatatgc tcaacacatg agcgaaaccc tataggaacc ctaattccct tatctgggaa
    13261 ctactcacac attattatgg agaaaataga gcttgtcgat cgttaattaa tcacctccaa
    13321 gatcccggac gagtgctggg gcgtcggttt ccactatcgg cgagtacttc tacacagcca
    13381 tcggtccaga cggccgcgct tctgcgggcg atttgtgtac gcccgacagt cccggctccg
    13441 gatcggacga ttgcgtcgca tcgaccctgc gcccaagctg catcatcgaa attgccgtca
    13501 accaagctct gatagagttg gtcaagacca atgcggagca tatacgcccg gagccgcggc
    13561 gatcctgcaa gctccggatg cctccgctcg aagtagcgcg tctgctgctc catacaagcc
    13621 aaccacggcc tccagaagaa gatgttggcg acctcgtatt gggaatcccc gaacatcgcc
    13681 tcgctccagt caatgaccgc tgttatgcgg ccattgtccg tcaggacatt gttggagccg
    13741 aaatccgcgt gcacgaggtg ccggacttcg gggcagtcct cggcccaaag catcagctca
    13801 tcgagagcct gcgcgacgga cgcactgacg gtgtcgtcca tcacagtttg ccagtgatac
    13861 acatggggat cagcaatcgc gcatatgaaa tcacgccatg tagtgtattg accgattcct
    13921 tgcggtccga atgggccgaa cccgctcgtc tggctaagat cggccgcagc gatcgcatcc
    13981 atggcctccg cgaccggctg cagaacagcg ggcagttcgg tttcaggcag gtcttgcaac
    14041 gtgacaccct gtgcacggcg ggagatgcaa taggtcaggc tctcgctgaa ttccccaatg
    14101 tcaagcactt ccggaatcgg gagcgcggcc gatgcaaagt gccgataaac ataacgatct
    14161 ttgtagaaac catcggcgca gctatttacc cgcaggacat atccacgccc tcctacatcg
    14221 aagctgaaag cacgagattc ttcgccctcc gagagctgca tcaggtcgga cacgctgtcg
    14281 aacttttcga tcagaaactt ctcgacagac gtcgcggtga gttcaggctt tttcatccta
    14341 ggagagatag atttgtagag agagactggt gatttcagcg tgtcctctcc aaatgaaatg
    14401 aacttcctta tatagaggaa ggtcttgcga aggatagtgg gattgtgcgt catcccttac
    14461 gtcagtggag atatcacatc aatccacttg ctttgaagac gtggttggaa cgtcttcttt
    14521 ttccacgatg ctcctcgtgg gtgggggtcc atctttggga ccactgtcgg cagaggcatc
    14581 ttgaacgata gcctttcctt tatcgcaatg atggcatttg taggtgccac cttccttttc
    14641 tactgtcctt ttgatgaagt gacagatagc tgggcaatgg aatccgagga ggtttcccga
    14701 tattaccctt tgttgaaaag tctcaatagc cctttggtct tctgagactg tatctttgat
    14761 attcttggag tagacgagag tgtcgtgctc caccatgtta tcacatcaat ccacttgctt
    14821 tgaagacgtg gttggaacgt cttctttttc cacgatgctc ctcgtgggtg ggggtccatc
    14881 tttgggacca ctgtcggcag aggcatcttg aacgatagcc tttcctttat cgcaatgatg
    14941 gcatttgtag gtgccacctt ccttttctac tgtccttttg atgaagtgac agatagctgg
    15001 gcaatggaat ccgaggaggt ttcccgatat taccctttgt tgaaaagtct caatagccct
    15061 ttggtcttct gagactgtat ctttgatatt cttggagtag acgagagtgt cgtgctccac
    15121 catg
//
