LOCUS       sequence-272934-       15677 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Dual-mini35S-enhancer driven Cas9-Csy4 system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 15677)
  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 15677)
  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: pGEL051
FEATURES             Location/Qualifiers
     source          1..15677
                     /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        2129..2174
                     /label=minimal CaMV 35S promoter
                     /note="minimal 35S promoter from cauliflower mosaic virus"
     CDS             2205..2225
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2820..2876
                     /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             2877..6980
                     /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             2877..6980
                     /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             6993..7013
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      7024..7272
                     /label=HSP terminator
                     /note="efficient transcription terminator from the 
                     Arabidopsis thaliana heat shock protein 18.2 gene (Nagaya 
                     et al., 2010)"
     misc_feature    7293..7317
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             8618..9247
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             9676..10749
                     /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      10815..11009
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     primer_bind     complement(11245..11267)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    11343..11483
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     11398..11417
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(11498..11515)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11669..12257)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11749..11768)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(12344..13138)
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     misc_feature    13563..13587
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     polyA_signal    13665..13839
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     promoter        complement(14929..15605)
                     /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 tggtcttggg aacaagcgaa
     1561 agatcgaaac tccgacagac gtaatcttaa ttcgctgaga ggcgtgtgag aattgccttt
     1621 ttccttgaat ataaaatgca ctcccataat gtaatcaaac aaaagatata aatagaataa
     1681 gaaaaataac aactcagcgt aacggagtcc gattaacgga gtgagaaatc atctggtttc
     1741 tcgagagttc taagccaatg ataatcgaag gaaacgaacg gtgttgtgtg tgtgtggtta
     1801 agaaaagaac aaactcatac gagacacgtg caccgaagct ttttcccacg tgccaaagag
     1861 tttcgtcttc gttcacacag aaccaagatg ggtggtaacg aaggtaggac cctaaagcac
     1921 cagcctacaa cacatagatg aaatagtata tgcgtttaca tggatttgat gaattgttgt
     1981 gtttagtata taagtatatt gtttagaatt atcaaaaagt atactttata aaaagagtgg
     2041 tggtaacata tttagtttct gcaccaatat aaaaagaaat atggtgatgt ctaaagaaaa
     2101 aagagagaaa acttagccac catctagagc aagacccttc ctctatataa ggaagttcat
     2161 ttcatttgga gaggcctgca ggtagatcgc tcgtcgacat ggctccaaag aagaagagaa
     2221 aggttggaat ccacggagtt ccagctgcta tggatcatta tcttgatatt agacttagac
     2281 ctgatccaga atttccacca gctcaactta tgtctgttct ttttggaaaa cttcatcaag
     2341 ctcttgttgc tcaaggagga gatagaattg gagtttcttt tcctgatctt gatgaatcaa
     2401 gatcaagact tggagaaaga cttagaattc atgcttctgc tgatgatctt agagctttgc
     2461 ttgctagacc ttggcttgaa ggacttagag atcatcttca atttggagaa ccagctgttg
     2521 ttccacatcc aactccttat agacaagttt caagagttca agctaaatct aatccagaaa
     2581 gacttagaag aagacttatg agaagacatg atctttctga agaagaagct agaaaaagaa
     2641 ttcctgatac tgttgctaga gctttggatt tgccttttgt tacacttaga tcacaatcta
     2701 ctggacaaca ttttagactt tttattagac atggaccact tcaagttact gctgaagaag
     2761 gaggatttac ttgttatgga ctttctaagg gaggttttgt tccttggttt ggatctggag
     2821 ctactaattt ttctcttctt aagcaagctg gagatgttga agaaaatcct ggacccatgg
     2881 ataagaagta ctctatcgga ctcgatatcg gaactaactc tgtgggatgg gctgtgatca
     2941 ccgatgagta caaggtgcca tctaagaagt tcaaggttct cggaaacacc gataggcact
     3001 ctatcaagaa aaaccttatc ggtgctctcc tcttcgattc tggtgaaact gctgaggcta
     3061 ccagactcaa gagaaccgct agaagaaggt acaccagaag aaagaacagg atctgctacc
     3121 tccaagagat cttctctaac gagatggcta aagtggatga ttcattcttc cacaggctcg
     3181 aagagtcatt cctcgtggaa gaagataaga agcacgagag gcaccctatc ttcggaaaca
     3241 tcgttgatga ggtggcatac cacgagaagt accctactat ctaccacctc agaaagaagc
     3301 tcgttgattc tactgataag gctgatctca ggctcatcta cctcgctctc gctcacatga
     3361 tcaagttcag aggacacttc ctcatcgagg gtgatctcaa ccctgataac tctgatgtgg
     3421 ataagttgtt catccagctc gtgcagacct acaaccagct tttcgaagag aaccctatca
     3481 acgcttcagg tgtggatgct aaggctatcc tctctgctag gctctctaag tcaagaaggc
     3541 ttgagaacct cattgctcag ctccctggtg agaagaagaa cggacttttc ggaaacttga
     3601 tcgctctctc tctcggactc acccctaact tcaagtctaa cttcgatctc gctgaggatg
     3661 caaagctcca gctctcaaag gatacctacg atgatgatct cgataacctc ctcgctcaga
     3721 tcggagatca gtacgctgat ttgttcctcg ctgctaagaa cctctctgat gctatcctcc
     3781 tcagtgatat cctcagagtg aacaccgaga tcaccaaggc tccactctca gcttctatga
     3841 tcaagagata cgatgagcac caccaggatc tcacacttct caaggctctt gttagacagc
     3901 agctcccaga gaagtacaaa gagattttct tcgatcagtc taagaacgga tacgctggtt
     3961 acatcgatgg tggtgcatct caagaagagt tctacaagtt catcaagcct atcctcgaga
     4021 agatggatgg aaccgaggaa ctcctcgtga agctcaatag agaggatctt ctcagaaagc
     4081 agaggacctt cgataacgga tctatccctc atcagatcca cctcggagag ttgcacgcta
     4141 tccttagaag gcaagaggat ttctacccat tcctcaagga taacagggaa aagattgaga
     4201 agattctcac cttcagaatc ccttactacg tgggacctct cgctagagga aactcaagat
     4261 tcgcttggat gaccagaaag tctgaggaaa ccatcacccc ttggaacttc gaagaggtgg
     4321 tggataaggg tgctagtgct cagtctttca tcgagaggat gaccaacttc gataagaacc
     4381 ttccaaacga gaaggtgctc cctaagcact ctttgctcta cgagtacttc accgtgtaca
     4441 acgagttgac caaggttaag tacgtgaccg agggaatgag gaagcctgct tttttgtcag
     4501 gtgagcaaaa gaaggctatc gttgatctct tgttcaagac caacagaaag gtgaccgtga
     4561 agcagctcaa agaggattac ttcaagaaaa tcgagtgctt cgattcagtt gagatttctg
     4621 gtgttgagga taggttcaac gcatctctcg gaacctacca cgatctcctc aagatcatta
     4681 aggataagga tttcttggat aacgaggaaa acgaggatat cttggaggat atcgttctta
     4741 ccctcaccct ctttgaagat agagagatga ttgaagaaag gctcaagacc tacgctcatc
     4801 tcttcgatga taaggtgatg aagcagttga agagaagaag atacactggt tggggaaggc
     4861 tctcaagaaa gctcattaac ggaatcaggg ataagcagtc tggaaagaca atccttgatt
     4921 tcctcaagtc tgatggattc gctaacagaa acttcatgca gctcatccac gatgattctc
     4981 tcacctttaa agaggatatc cagaaggctc aggtttcagg acagggtgat agtctccatg
     5041 agcatatcgc taacctcgct ggatctcctg caatcaagaa gggaatcctc cagactgtga
     5101 aggttgtgga tgagttggtg aaggtgatgg gaaggcataa gcctgagaac atcgtgatcg
     5161 aaatggctag agagaaccag accactcaga agggacagaa gaactctagg gaaaggatga
     5221 agaggatcga ggaaggtatc aaagagcttg gatctcagat cctcaaagag caccctgttg
     5281 agaacactca gctccagaat gagaagctct acctctacta cctccagaac ggaagggata
     5341 tgtatgtgga tcaagagttg gatatcaaca ggctctctga ttacgatgtt gatcatatcg
     5401 tgccacagtc attcttgaag gatgattcta tcgataacaa ggtgctcacc aggtctgata
     5461 agaacagggg taagagtgat aacgtgccaa gtgaagaggt tgtgaagaaa atgaagaact
     5521 attggaggca gctcctcaac gctaagctca tcactcagag aaagttcgat aacttgacta
     5581 aggctgagag gggaggactc tctgaattgg ataaggcagg attcatcaag aggcagcttg
     5641 tggaaaccag gcagatcact aagcacgttg cacagatcct cgattctagg atgaacacca
     5701 agtacgatga gaacgataag ttgatcaggg aagtgaaggt tatcaccctc aagtcaaagc
     5761 tcgtgtctga tttcagaaag gatttccaat tctacaaggt gagggaaatc aacaactacc
     5821 accacgctca cgatgcttac cttaacgctg ttgttggaac cgctctcatc aagaagtatc
     5881 ctaagctcga gtcagagttc gtgtacggtg attacaaggt gtacgatgtg aggaagatga
     5941 tcgctaagtc tgagcaagag atcggaaagg ctaccgctaa gtatttcttc tactctaaca
     6001 tcatgaattt cttcaagacc gagattaccc tcgctaacgg tgagatcaga aagaggccac
     6061 tcatcgagac aaacggtgaa acaggtgaga tcgtgtggga taagggaagg gatttcgcta
     6121 ccgttagaaa ggtgctctct atgccacagg tgaacatcgt taagaaaacc gaggtgcaga
     6181 ccggtggatt ctctaaagag tctatcctcc ctaagaggaa ctctgataag ctcattgcta
     6241 ggaagaagga ttgggaccct aagaaatacg gtggtttcga ttctcctacc gtggcttact
     6301 ctgttctcgt tgtggctaag gttgagaagg gaaagagtaa gaagctcaag tctgttaagg
     6361 aacttctcgg aatcactatc atggaaaggt catctttcga gaagaaccca atcgatttcc
     6421 tcgaggctaa gggatacaaa gaggttaaga aggatctcat catcaagctc ccaaagtact
     6481 cactcttcga actcgagaac ggtagaaaga ggatgctcgc ttctgctggt gagcttcaaa
     6541 agggaaacga gcttgctctc ccatctaagt acgttaactt tctttacctc gcttctcact
     6601 acgagaagtt gaagggatct ccagaagata acgagcagaa gcaacttttc gttgagcagc
     6661 acaagcacta cttggatgag atcatcgagc agatctctga gttctctaaa agggtgatcc
     6721 tcgctgatgc aaacctcgat aaggtgttgt ctgcttacaa caagcacaga gataagccta
     6781 tcagggaaca ggcagagaac atcatccatc tcttcaccct taccaacctc ggtgctcctg
     6841 ctgctttcaa gtacttcgat acaaccatcg ataggaagag atacacctct accaaagaag
     6901 tgctcgatgc taccctcatc catcagtcta tcactggact ctacgagact aggatcgatc
     6961 tctcacagct cggtggtgat tcaagggctg atcctaagaa gaagaggaag gtttgaggat
     7021 ccatatgaag atgaagatga aatatttggt gtgtcaaata aaaagcttgt gtgcttaagt
     7081 ttgtgttttt ttcttggctt gttgtgttat gaatttgtgg ctttttctaa tattaaatga
     7141 atgtaagatc acattataat gaataaacaa atgtttctat aatccattgt gaatgttttg
     7201 ttggatctct tctgcagcat ataactactg tatgtgctat ggtatggact atggaatatg
     7261 attaaagata aggagctcaa actatcagtg tttgacagga tatattggcg ggtaaaccta
     7321 agagaaaaga gcgtttatta gaataatcgg atatttaaaa gggcgtgaaa aggtttatcc
     7381 gttcgtccat ttgtatgtgc atgccaacca cagggttccc ctcgggatca aagtactttg
     7441 atccaacccc tccgctgcta tagtgcagtc ggcttctgac gttcagtgca gccgtcttct
     7501 gaaaacgaca tgtcgcacaa gtcctaagtt acgcgacagg ctgccgccct gcccttttcc
     7561 tggcgttttc ttgtcgcgtg ttttagtcgc ataaagtaga atacttgcga ctagaaccgg
     7621 agacattacg ccatgaacaa gagcgccgcc gctggcctgc tgggctatgc ccgcgtcagc
     7681 accgacgacc aggacttgac caaccaacgg gccgaactgc acgcggccgg ctgcaccaag
     7741 ctgttttccg agaagatcac cggcaccagg cgcgaccgcc cggagctggc caggatgctt
     7801 gaccacctac gccctggcga cgttgtgaca gtgaccaggc tagaccgcct ggcccgcagc
     7861 acccgcgacc tactggacat tgccgagcgc atccaggagg ccggcgcggg cctgcgtagc
     7921 ctggcagagc cgtgggccga caccaccacg ccggccggcc gcatggtgtt gaccgtgttc
     7981 gccggcattg ccgagttcga gcgttcccta atcatcgacc gcacccggag cgggcgcgag
     8041 gccgccaagg cccgaggcgt gaagtttggc ccccgcccta ccctcacccc ggcacagatc
     8101 gcgcacgccc gcgagctgat cgaccaggaa ggccgcaccg tgaaagaggc ggctgcactg
     8161 cttggcgtgc atcgctcgac cctgtaccgc gcacttgagc gcagcgagga agtgacgccc
     8221 accgaggcca ggcggcgcgg tgccttccgt gaggacgcat tgaccgaggc cgacgccctg
     8281 gcggccgccg agaatgaacg ccaagaggaa caagcatgaa accgcaccag gacggccagg
     8341 acgaaccgtt tttcattacc gaagagatcg aggcggagat gatcgcggcc gggtacgtgt
     8401 tcgagccgcc cgcgcacggc tcaaccgtgc ggctgcatga aatcctggcc ggtttgtctg
     8461 atgccaagct ggcggcctgg ccggccagct tggccgctga agaaaccgag cgccgccgtc
     8521 taaaaaggtg atgtgtattt gagtaaaaca gcttgcgtca tgcggtcgct gcgtatatga
     8581 tgcgatgagt aaataaacaa atacgcaagg ggaacgcatg aaggttatcg ctgtacttaa
     8641 ccagaaaggc gggtcaggca agacgaccat cgcaacccat ctagcccgcg ccctgcaact
     8701 cgccggggcc gatgttctgt tagtcgattc cgatccccag ggcagtgccc gcgattgggc
     8761 ggccgtgcgg gaagatcaac cgctaaccgt tgtcggcatc gaccgcccga cgattgaccg
     8821 cgacgtgaag gccatcggcc ggcgcgactt cgtagtgatc gacggagcgc cccaggcggc
     8881 ggacttggct gtgtccgcga tcaaggcagc cgacttcgtg ctgattccgg tgcagccaag
     8941 cccttacgac atatgggcca ccgccgacct ggtggagctg gttaagcagc gcattgaggt
     9001 cacggatgga aggctacaag cggcctttgt cgtgtcgcgg gcgatcaaag gcacgcgcat
     9061 cggcggtgag gttgccgagg cgctggccgg gtacgagctg cccattcttg agtcccgtat
     9121 cacgcagcgc gtgagctacc caggcactgc cgccgccggc acaaccgttc ttgaatcaga
     9181 acccgagggc gacgctgccc gcgaggtcca ggcgctggcc gctgaaatta aatcaaaact
     9241 catttgagtt aatgaggtaa agagaaaatg agcaaaagca caaacacgct aagtgccggc
     9301 cgtccgagcg cacgcagcag caaggctgca acgttggcca gcctggcaga cacgccagcc
     9361 atgaagcggg tcaactttca gttgccggcg gaggatcaca ccaagctgaa gatgtacgcg
     9421 gtacgccaag gcaagaccat taccgagctg ctatctgaat acatcgcgca gctaccagag
     9481 taaatgagca aatgaataaa tgagtagatg aattttagcg gctaaaggag gcggcatgga
     9541 aaatcaagaa caaccaggca ccgacgccgt ggaatgcccc atgtgtggag gaacgggcgg
     9601 ttggccaggc gtaagcggct gggttgcctg ccggccctgc aatggcactg gaacccccaa
     9661 gcccgaggaa tcggcgtgag cggtcgcaaa ccatccggcc cggtacaaat cggcgcggcg
     9721 ctgggtgatg acctggtgga gaagttgaag gccgcgcagg ccgcccagcg gcaacgcatc
     9781 gaggcagaag cacgccccgg tgaatcgtgg caagcggccg ctgatcgaat ccgcaaagaa
     9841 tcccggcaac cgccggcagc cggtgcgccg tcgattagga agccgcccaa gggcgacgag
     9901 caaccagatt ttttcgttcc gatgctctat gacgtgggca cccgcgatag tcgcagcatc
     9961 atggacgtgg ccgttttccg tctgtcgaag cgtgaccgac gagctggcga ggtgatccgc
    10021 tacgagcttc cagacgggca cgtagaggtt tccgcagggc cggccggcat ggccagtgtg
    10081 tgggattacg acctggtact gatggcggtt tcccatctaa ccgaatccat gaaccgatac
    10141 cgggaaggga agggagacaa gcccggccgc gtgttccgtc cacacgttgc ggacgtactc
    10201 aagttctgcc ggcgagccga tggcggaaag cagaaagacg acctggtaga aacctgcatt
    10261 cggttaaaca ccacgcacgt tgccatgcag cgtacgaaga aggccaagaa cggccgcctg
    10321 gtgacggtat ccgagggtga agccttgatt agccgctaca agatcgtaaa gagcgaaacc
    10381 gggcggccgg agtacatcga gatcgagcta gctgattgga tgtaccgcga gatcacagaa
    10441 ggcaagaacc cggacgtgct gacggttcac cccgattact ttttgatcga tcccggcatc
    10501 ggccgttttc tctaccgcct ggcacgccgc gccgcaggca aggcagaagc cagatggttg
    10561 ttcaagacga tctacgaacg cagtggcagc gccggagagt tcaagaagtt ctgtttcacc
    10621 gtgcgcaagc tgatcgggtc aaatgacctg ccggagtacg atttgaagga ggaggcgggg
    10681 caggctggcc cgatcctagt catgcgctac cgcaacctga tcgagggcga agcatccgcc
    10741 ggttcctaat gtacggagca gatgctaggg caaattgccc tagcagggga aaaaggtcga
    10801 aaaggcctct ttcctgtgga tagcacgtac attgggaacc caaagccgta cattgggaac
    10861 cggaacccgt acattgggaa cccaaagccg tacattggga accggtcaca catgtaagtg
    10921 actgatataa aagagaaaaa aggcgatttt tccgcctaaa actctttaaa acttattaaa
    10981 actcttaaaa cccgcctggc ctgtgcataa ctgtctggcc agcgcacagc cgaagagctg
    11041 caaaaagcgc ctacccttcg gtcgctgcgc tccctacgcc ccgccgcttc gcgtcggcct
    11101 atcgcggccg ctggccgctc aaaaatggct ggcctacggc caggcaatct accagggcgc
    11161 ggacaagccg cgccgtcgcc actcgaccgc cggcgcccac atcaaggcac cctgcctcgc
    11221 gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaaa cggtcacagc
    11281 ttgtctgtaa gcggatgccg ggagcagaca agcgcgtcag cgggtgttgg cgggtgtcgg
    11341 ggcgcagcca tgacccagtc acgtagcgat agcggagtgt atactggctt aactatgcgg
    11401 catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg
    11461 taaggagaaa ataccgcatc aggcgctctt ccgcttcctc gctcactgac tcgctgcgct
    11521 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
    11581 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
    11641 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
    11701 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
    11761 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
    11821 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
    11881 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
    11941 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
    12001 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
    12061 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg
    12121 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
    12181 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
    12241 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
    12301 acgaaaactc acgttaaggg attttggtca tgcattctag gtactaaaac aattcatcca
    12361 gtaaaatata atattttatt ttctcccaat caggcttgat ccccagtaag tcaaaaaata
    12421 gctcgacata ctgttcttcc ccgatatcct ccctgatcga ccggacgcag aaggcaatgt
    12481 cataccactt gtccgccctg ccgcttctcc caagatcaat aaagccactt actttgccat
    12541 ctttcacaaa gatgttgctg tctcccaggt cgccgtggga aaagacaagt tcctcttcgg
    12601 gcttttccgt ctttaaaaaa tcatacagct cgcgcggatc tttaaatgga gtgtcttctt
    12661 cccagttttc gcaatccaca tcggccagat cgttattcag taagtaatcc aattcggcta
    12721 agcggctgtc taagctattc gtatagggac aatccgatat gtcgatggag tgaaagagcc
    12781 tgatgcactc cgcatacagc tcgataatct tttcagggct ttgttcatct tcatactctt
    12841 ccgagcaaag gacgccatcg gcctcactca tgagcagatt gctccagcca tcatgccgtt
    12901 caaagtgcag gacctttgga acaggcagct ttccttccag ccatagcatc atgtcctttt
    12961 cccgttccac atcataggtg gtccctttat accggctgtc cgtcattttt aaatataggt
    13021 tttcattttc tcccaccagc ttatatacct tagcaggaga cattccttcc gtatctttta
    13081 cgcagcggta tttttcgatc agttttttca attccggtga tattctcatt ttagccattt
    13141 attatttcct tcctcttttc tacagtattt aaagataccc caagaagcta attataacaa
    13201 gacgaactcc aattcactgt tccttgcatt ctaaaacctt aaataccaga aaacagcttt
    13261 ttcaaagttg ttttcaaagt tggcgtataa catagtatcg acggagccga ttttgaaacc
    13321 gcggtgatca caggcagcaa cgctctgtca tcgttacaat caacatgcta ccctccgcga
    13381 gatcatccgt gtttcaaacc cggcagctta gttgccgttc ttccgaatag catcggtaac
    13441 atgagcaaag tctgccgcct tacaacggct ctcccgctga cgccgtcccg gactgatggg
    13501 ctgcctgtat cgagtggtga ttttgtgccg agctgccggt cggggagctg ttggctggct
    13561 ggtggcagga tatattgtgg tgtaaacaaa ttgacgctta gacaacttaa taacacattg
    13621 cggacgtttt taatgtactg aattaacgcc gaattaattc gggggatctg gattttagta
    13681 ctggattttg gttttaggaa ttagaaattt tattgataga agtattttac aaatacaaat
    13741 acatactaag ggtttcttat atgctcaaca catgagcgaa accctatagg aaccctaatt
    13801 cccttatctg ggaactactc acacattatt atggagaaaa tagagcttgt cgatcgttaa
    13861 ttaatcacct ccaagatccc ggacgagtgc tggggcgtcg gtttccacta tcggcgagta
    13921 cttctacaca gccatcggtc cagacggccg cgcttctgcg ggcgatttgt gtacgcccga
    13981 cagtcccggc tccggatcgg acgattgcgt cgcatcgacc ctgcgcccaa gctgcatcat
    14041 cgaaattgcc gtcaaccaag ctctgataga gttggtcaag accaatgcgg agcatatacg
    14101 cccggagccg cggcgatcct gcaagctccg gatgcctccg ctcgaagtag cgcgtctgct
    14161 gctccataca agccaaccac ggcctccaga agaagatgtt ggcgacctcg tattgggaat
    14221 ccccgaacat cgcctcgctc cagtcaatga ccgctgttat gcggccattg tccgtcagga
    14281 cattgttgga gccgaaatcc gcgtgcacga ggtgccggac ttcggggcag tcctcggccc
    14341 aaagcatcag ctcatcgaga gcctgcgcga cggacgcact gacggtgtcg tccatcacag
    14401 tttgccagtg atacacatgg ggatcagcaa tcgcgcatat gaaatcacgc catgtagtgt
    14461 attgaccgat tccttgcggt ccgaatgggc cgaacccgct cgtctggcta agatcggccg
    14521 cagcgatcgc atccatggcc tccgcgaccg gctgcagaac agcgggcagt tcggtttcag
    14581 gcaggtcttg caacgtgaca ccctgtgcac ggcgggagat gcaataggtc aggctctcgc
    14641 tgaattcccc aatgtcaagc acttccggaa tcgggagcgc ggccgatgca aagtgccgat
    14701 aaacataacg atctttgtag aaaccatcgg cgcagctatt tacccgcagg acatatccac
    14761 gccctcctac atcgaagctg aaagcacgag attcttcgcc ctccgagagc tgcatcaggt
    14821 cggacacgct gtcgaacttt tcgatcagaa acttctcgac agacgtcgcg gtgagttcag
    14881 gctttttcat cctaggagag atagatttgt agagagagac tggtgatttc agcgtgtcct
    14941 ctccaaatga aatgaacttc cttatataga ggaagggtct tgcgaaggat agtgggattg
    15001 tgcgtcatcc cttacgtcag tggagatatc acatcaatcc acttgctttg aagacgtggt
    15061 tggaacgtct tctttttcca cgatgctcct cgtgggtggg ggtccatctt tgggaccact
    15121 gtcggcagag gcatcttgaa cgatagcctt tcctttatcg caatgatggc atttgtaggt
    15181 gccaccttcc ttttctactg tccttttgat gaagtgacag atagctgggc aatggaatcc
    15241 gaggaggttt cccgatatta ccctttgttg aaaagtctca atagcccttt ggtcttctga
    15301 gactgtatct ttgatattct tggagtagac gagagtgtcg tgctccacca tgttcacatc
    15361 aatccacttg ctttgaagac gtggttggaa cgtcttcttt ttccacgatg ctcctcgtgg
    15421 gtgggggtcc atctttggga ccactgtcgg cagaggcatc ttgaacgata gcctttcctt
    15481 tatcgcaatg atggcatttg taggtgccac cttccttttc tactgtcctt ttgatgaagt
    15541 gacagatagc tgggcaatgg aatccgagga ggtttcccga tattaccctt tgttgaaaag
    15601 tctcaatagc cctttggtct tctgagactg tatctttgat attcttggag tagacgagag
    15661 tgtcgtgctc caccatg
//
