LOCUS       sequence-266287-       15686 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 15686)
  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 15686)
  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..15686
                     /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             8617..9246
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             9680..10747
                     /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      10813..11007
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     primer_bind     complement(11243..11265)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    11351..11491
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     11406..11425
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(11506..11523)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11677..12265)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11757..11776)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(12352..13146)
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     misc_feature    13571..13595
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     polyA_signal    13673..13847
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     promoter        complement(14937..15614)
                     /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 gaataacgga tatttaaaag ggcgtgaaaa ggtttatccg
     7381 ttcgtccatt tgtatgtgca tgccaaccac agggttcccc tcgggatcaa agtactttga
     7441 tccaacccct ccgctgctat agtgcagtcg gcttctgacg ttcagtgcag ccgtcttctg
     7501 aaaacgacat gtcgcacaag tcctaagtta cgcgacaggc tgccgccctg cccttttcct
     7561 ggcgttttct tgtcgcgtgt tttagtcgca taaagtagaa tacttgcgac tagaaccgga
     7621 gacattacgc catgaacaag agcgccgccg ctggcctgct gggctatgcc cgcgtcagca
     7681 ccgacgacca ggacttgacc aaccaacggg ccgaactgca cgcggccggc tgcaccaagc
     7741 tgttttccga gaagatcacc ggcaccaggc gcgaccgccc ggagctggcc aggatgcttg
     7801 accacctacg ccctggcgac gttgtgacag tgaccaggct agaccgcctg gcccgcagca
     7861 cccgcgacct actggacatt gccgagcgca tccaggaggc cggcgcgggc ctgcgtagcc
     7921 tggcagagcc gtgggccgac accaccacgc cggccggccg catggtgttg accgtgttcg
     7981 ccggcattgc cgagttcgag cgttccctaa tcatcgaccg cacccggagc gggcgcgagg
     8041 ccgccaaggc ccgaggcgtg aagtttggcc cccgccctac cctcaccccg gcacagatcg
     8101 cgcacgcccg cgagctgatc gaccaggaag gccgcaccgt gaaagaggcg gctgcactgc
     8161 ttggcgtgca tcgctcgacc ctgtaccgcg cacttgagcg cagcgaggaa gtgacgccca
     8221 ccgaggccag gcggcgcggt gccttccgtg aggacgcatt gaccgaggcc gacgccctgg
     8281 cggccgccga gaatgaacgc caagaggaac aagcatgaaa ccgcaccagg acggccagga
     8341 cgaaccgttt ttcattaccg aagagatcga ggcggagatg atcgcggccg ggtacgtgtt
     8401 cgagccgccc gcgcacggct caaccgtgcg gctgcatgaa atcctggccg gtttgtctga
     8461 tgccaagctg gcggcctggc cggccagctt ggccgctgaa gaaaccgagc gccgccgtct
     8521 aaaaaggtga tgtgtatttg agtaaaacag cttgcgtcat gcggtcgctg cgtatatgat
     8581 gcgatgagta aataaacaaa tacgcaaggg gaacgcatga aggttatcgc tgtacttaac
     8641 cagaaaggcg ggtcaggcaa gacgaccatc gcaacccatc tagcccgcgc cctgcaactc
     8701 gccggggccg atgttctgtt agtcgattcc gatccccagg gcagtgcccg cgattgggcg
     8761 gccgtgcggg aagatcaacc gctaaccgtt gtcggcatcg accgcccgac gattgaccgc
     8821 gacgtgaagg ccatcggccg gcgcgacttc gtagtgatcg acggagcgcc ccaggcggcg
     8881 gacttggctg tgtccgcgat caaggcagcc gacttcgtgc tgattccggt gcagccaagc
     8941 ccttacgaca tatgggccac cgccgacctg gtggagctgg ttaagcagcg cattgaggtc
     9001 acggatggaa ggctacaagc ggcctttgtc gtgtcgcggg cgatcaaagg cacgcgcatc
     9061 ggcggtgagg ttgccgaggc gctggccggg tacgagctgc ccattcttga gtcccgtatc
     9121 acgcagcgcg tgagctaccc aggcactgcc gccgccggca caaccgttct tgaatcagaa
     9181 cccgagggcg acgctgcccg cgaggtccag gcgctggccg ctgaaattaa atcaaaactc
     9241 atttgagtta atgaggtaaa gagaaaatga gcaaaagcac aaacacgcta agtgccggcc
     9301 gtccgagcgc acgcagcagc aaggctgcaa cgttggccag cctggcagac acgccagcca
     9361 tgaagcgggt caactttcag ttgccggcgg aggatcacac caagctgaag atgtacgcgg
     9421 tacgccaagg caagaccatt accgagctgc tatctgaata catcgcgcag ctaccagagt
     9481 aaatgagcaa atgaataaat gagtagatga attttagcgg ctaaaggagg cggcatggaa
     9541 aatcaagaac aaccaggcac cgacgccgtg gaatgcccca tgtgtggagg aacgggcggt
     9601 tggccaggcg taagcggctg ggttgtctgc cggccctgca atggcactgg aacccccaag
     9661 cccgaggaat cggcgtgacg gtcgcaaacc atccggcccg gtacaaatcg gcgcggcgct
     9721 gggtgatgac ctggtggaga agttgaaggc cgcgcaggcc gcccagcggc aacgcatcga
     9781 ggcagaagca cgccccggtg aatcgtggca agcggccgct gatcgaatcc gcaaagaatc
     9841 ccggcaaccg ccggcagccg gtgcgccgtc gattaggaag ccgcccaagg gcgacgagca
     9901 accagatttt ttcgttccga tgctctatga cgtgggcacc cgcgatagtc gcagcatcat
     9961 ggacgtggcc gttttccgtc tgtcgaagcg tgaccgacga gctggcgagg tgatccgcta
    10021 cgagcttcca gacgggcacg tagaggtttc cgcagggccg gccggcatgg ccagtgtgtg
    10081 ggattacgac ctggtactga tggcggtttc ccatctaacc gaatccatga accgataccg
    10141 ggaagggaag ggagacaagc ccggccgcgt gttccgtcca cacgttgcgg acgtactcaa
    10201 gttctgccgg cgagccgatg gcggaaagca gaaagacgac ctggtagaaa cctgcattcg
    10261 gttaaacacc acgcacgttg ccatgcagcg tacgaagaag gccaagaacg gccgcctggt
    10321 gacggtatcc gagggtgaag ccttgattag ccgctacaag atcgtaaaga gcgaaaccgg
    10381 gcggccggag tacatcgaga tcgagctagc tgattggatg taccgcgaga tcacagaagg
    10441 caagaacccg gacgtgctga cggttcaccc cgattacttt ttgatcgatc ccggcatcgg
    10501 ccgttttctc taccgcctgg cacgccgcgc cgcaggcaag gcagaagcca gatggttgtt
    10561 caagacgatc tacgaacgca gtggcagcgc cggagagttc aagaagttct gtttcaccgt
    10621 gcgcaagctg atcgggtcaa atgacctgcc ggagtacgat ttgaaggagg aggcggggca
    10681 ggctggcccg atcctagtca tgcgctaccg caacctgatc gagggcgaag catccgccgg
    10741 ttcctaatgt acggagcaga tgctagggca aattgcccta gcaggggaaa aaggtcgaaa
    10801 aggcctcttt cctgtggata gcacgtacat tgggaaccca aagccgtaca ttgggaaccg
    10861 gaacccgtac attgggaacc caaagccgta cattgggaac cggtcacaca tgtaagtgac
    10921 tgatataaaa gagaaaaaag gcgatttttc cgcctaaaac tctttaaaac ttattaaaac
    10981 tcttaaaacc cgcctggcct gtgcataact gtctggccag cgcacagccg aagagctgca
    11041 aaaagcgcct acccttcggt cgctgcgctc cctacgcccc gccgcttcgc gtcggcctat
    11101 cgcggccgct ggccgctcaa aaatggctgg cctacggcca ggcaatctac cagggcgcgg
    11161 acaagccgcg ccgtcgccac tcgaccgccg gcgcccacat caaggcaccc tgcctcgcgc
    11221 gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggaaacg gtcacagctt
    11281 gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg
    11341 ggtgtcgggg cgcagccatg acccagtcac gtagcgatag cggagtgtat actggcttaa
    11401 ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca
    11461 cagatgcgta aggagaaaat accgcatcag gcgctcttcc gcttcctcgc tcactgactc
    11521 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
    11581 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
    11641 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
    11701 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
    11761 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
    11821 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
    11881 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
    11941 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
    12001 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
    12061 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac
    12121 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
    12181 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
    12241 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
    12301 tcagtggaac gaaaactcac gttaagggat tttggtcatg cattctaggt actaaaacaa
    12361 ttcatccagt aaaatataat attttatttt ctcccaatca ggcttgatcc ccagtaagtc
    12421 aaaaaatagc tcgacatact gttcttcccc gatatcctcc ctgatcgacc ggacgcagaa
    12481 ggcaatgtca taccacttgt ccgccctgcc gcttctccca agatcaataa agccacttac
    12541 tttgccatct ttcacaaaga tgttgctgtc tcccaggtcg ccgtgggaaa agacaagttc
    12601 ctcttcgggc ttttccgtct ttaaaaaatc atacagctcg cgcggatctt taaatggagt
    12661 gtcttcttcc cagttttcgc aatccacatc ggccagatcg ttattcagta agtaatccaa
    12721 ttcggctaag cggctgtcta agctattcgt atagggacaa tccgatatgt cgatggagtg
    12781 aaagagcctg atgcactccg catacagctc gataatcttt tcagggcttt gttcatcttc
    12841 atactcttcc gagcaaagga cgccatcggc ctcactcatg agcagattgc tccagccatc
    12901 atgccgttca aagtgcagga cctttggaac aggcagcttt ccttccagcc atagcatcat
    12961 gtccttttcc cgttccacat cataggtggt ccctttatac cggctgtccg tcatttttaa
    13021 atataggttt tcattttctc ccaccagctt atatacctta gcaggagaca ttccttccgt
    13081 atcttttacg cagcggtatt tttcgatcag ttttttcaat tccggtgata ttctcatttt
    13141 agccatttat tatttccttc ctcttttcta cagtatttaa agatacccca agaagctaat
    13201 tataacaaga cgaactccaa ttcactgttc cttgcattct aaaaccttaa ataccagaaa
    13261 acagcttttt caaagttgtt ttcaaagttg gcgtataaca tagtatcgac ggagccgatt
    13321 ttgaaaccgc ggtgatcaca ggcagcaacg ctctgtcatc gttacaatca acatgctacc
    13381 ctccgcgaga tcatccgtgt ttcaaacccg gcagcttagt tgccgttctt ccgaatagca
    13441 tcggtaacat gagcaaagtc tgccgcctta caacggctct cccgctgacg ccgtcccgga
    13501 ctgatgggct gcctgtatcg agtggtgatt ttgtgccgag ctgccggtcg gggagctgtt
    13561 ggctggctgg tggcaggata tattgtggtg taaacaaatt gacgcttaga caacttaata
    13621 acacattgcg gacgttttta atgtactgaa ttaacgccga attaattcgg gggatctgga
    13681 ttttagtact ggattttggt tttaggaatt agaaatttta ttgatagaag tattttacaa
    13741 atacaaatac atactaaggg tttcttatat gctcaacaca tgagcgaaac cctataggaa
    13801 ccctaattcc cttatctggg aactactcac acattattat ggagaaaata gagcttgtcg
    13861 atcgttaatt aatcacctcc aagatcccgg acgagtgctg gggcgtcggt ttccactatc
    13921 ggcgagtact tctacacagc catcggtcca gacggccgcg cttctgcggg cgatttgtgt
    13981 acgcccgaca gtcccggctc cggatcggac gattgcgtcg catcgaccct gcgcccaagc
    14041 tgcatcatcg aaattgccgt caaccaagct ctgatagagt tggtcaagac caatgcggag
    14101 catatacgcc cggagccgcg gcgatcctgc aagctccgga tgcctccgct cgaagtagcg
    14161 cgtctgctgc tccatacaag ccaaccacgg cctccagaag aagatgttgg cgacctcgta
    14221 ttgggaatcc ccgaacatcg cctcgctcca gtcaatgacc gctgttatgc ggccattgtc
    14281 cgtcaggaca ttgttggagc cgaaatccgc gtgcacgagg tgccggactt cggggcagtc
    14341 ctcggcccaa agcatcagct catcgagagc ctgcgcgacg gacgcactga cggtgtcgtc
    14401 catcacagtt tgccagtgat acacatgggg atcagcaatc gcgcatatga aatcacgcca
    14461 tgtagtgtat tgaccgattc cttgcggtcc gaatgggccg aacccgctcg tctggctaag
    14521 atcggccgca gcgatcgcat ccatggcctc cgcgaccggc tgcagaacag cgggcagttc
    14581 ggtttcaggc aggtcttgca acgtgacacc ctgtgcacgg cgggagatgc aataggtcag
    14641 gctctcgctg aattccccaa tgtcaagcac ttccggaatc gggagcgcgg ccgatgcaaa
    14701 gtgccgataa acataacgat ctttgtagaa accatcggcg cagctattta cccgcaggac
    14761 atatccacgc cctcctacat cgaagctgaa agcacgagat tcttcgccct ccgagagctg
    14821 catcaggtcg gacacgctgt cgaacttttc gatcagaaac ttctcgacag acgtcgcggt
    14881 gagttcaggc tttttcatcc taggagagat agatttgtag agagagactg gtgatttcag
    14941 cgtgtcctct ccaaatgaaa tgaacttcct tatatagagg aaggtcttgc gaaggatagt
    15001 gggattgtgc gtcatccctt acgtcagtgg agatatcaca tcaatccact tgctttgaag
    15061 acgtggttgg aacgtcttct ttttccacga tgctcctcgt gggtgggggt ccatctttgg
    15121 gaccactgtc ggcagaggca tcttgaacga tagcctttcc tttatcgcaa tgatggcatt
    15181 tgtaggtgcc accttccttt tctactgtcc ttttgatgaa gtgacagata gctgggcaat
    15241 ggaatccgag gaggtttccc gatattaccc tttgttgaaa agtctcaata gccctttggt
    15301 cttctgagac tgtatctttg atattcttgg agtagacgag agtgtcgtgc tccaccatgt
    15361 tatcacatca atccacttgc tttgaagacg tggttggaac gtcttctttt tccacgatgc
    15421 tcctcgtggg tgggggtcca tctttgggac cactgtcggc agaggcatct tgaacgatag
    15481 cctttccttt atcgcaatga tggcatttgt aggtgccacc ttccttttct actgtccttt
    15541 tgatgaagtg acagatagct gggcaatgga atccgaggag gtttcccgat attacccttt
    15601 gttgaaaagt ctcaatagcc ctttggtctt ctgagactgt atctttgata ttcttggagt
    15661 agacgagagt gtcgtgctcc accatg
//
