LOCUS       sequence-309990-       17230 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  OsBiP1 promoter driven Cas9-csy4 system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 17230)
  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 17230)
  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: pGEL053
FEATURES             Location/Qualifiers
     source          1..17230
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(158..865)
                     /label=OCS terminator
                     /note="octopine synthase terminator"
     protein_bind    complement(872..891)
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     misc_RNA        complement(892..967)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             1294..1599
                     /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     1478..1497
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(1607..1626)
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     CDS             3900..3920
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4515..4571
                     /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             4572..8675
                     /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             4572..8675
                     /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             8688..8708
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      8719..8967
                     /label=HSP terminator
                     /note="efficient transcription terminator from the 
                     Arabidopsis thaliana heat shock protein 18.2 gene (Nagaya 
                     et al., 2010)"
     misc_feature    8988..9012
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             10313..10942
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             11371..12444
                     /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      12510..12704
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     primer_bind     complement(12940..12962)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    13038..13178
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     13093..13112
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(13193..13210)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(13364..13952)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(13444..13463)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(14039..14833)
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     misc_feature    15258..15282
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     polyA_signal    15360..15534
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     promoter        complement(join(16624..17230,1..70))
                     /label=CaMV 35S promoter (enhanced)
                     /note="cauliflower mosaic virus 35S promoter with a 
                     duplicated enhancer region"
     primer_bind     complement(16667..16687)
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
ORIGIN
        1 gaagtgacag atagctgggc aatggaatcc gaggaggttt cccgatatta ccctttgttg
       61 aaaagtctca atagcccttt ggtcttctga gactgtatct ttgatattct tggagtagac
      121 gagagtgtcg tgctccacca tgggcgcgcc gccggtcctg ctgagcctcg acatgttgtc
      181 gcaaaattcg ccctggaccc gcccaacgat ttgtcgtcac tgtcaaggtt tgacctgcac
      241 ttcatttggg gcccacatac accaaaaaaa tgctgcataa ttctcggggc agcaagtcgg
      301 ttacccggcc gccgtgctgg accgggttga atggtgcccg taactttcgg tagagcggac
      361 ggccaatact caacttcaag gaatctcacc catgcgcgcc ggcggggaac cggagttccc
      421 ttcagtgagc gttattagtt cgccgctcgg tgtgtcgtag atactagccc ctggggcact
      481 tttgaaattt gaataagatt tatgtaatca gtcttttagg tttgaccggt tctgccgctt
      541 tttttaaaat tggatttgta ataataaaac gcaattgttt gttattgtgg cgctctatca
      601 tagatgtcgc tataaaccta ttcagcacaa tatattgttt tcattttaat attgtacata
      661 taagtagtag ggtacaatca gtaaattgaa cggagaatat tattcataaa aatacgatag
      721 taacgggtga tatattcatt agaatgaacc gaaaccggcg gtaaggatct gagctacaca
      781 tgctcaggtt ttttacaacg tgcacaacag aattgaaagc aaatatcatg cgatcatagg
      841 cgtctcgcat atctcattaa agcagggtac cctgcctata cggcagtgaa cgcaccgact
      901 cggtgccact ttttcaagtt gataacggac tagccttatt ttaacttgct atttctagct
      961 ctaaaactga gaccaagcca gataacagta tgcgtatttg cgcgctgatt tttgcggtat
     1021 aagaatatat actgatatgt atacccgaag tatgtcaaaa agaggtatgc tatgaagcag
     1081 cgtattacag tgacagttga cagcgacagc tatcagttgc tcaaggcata tatgatgtca
     1141 atatctccgg tctggtaagc acaaccatgc agaatgaagc ccgtcgtctg cgtgccgaac
     1201 gctggaaagc ggaaaatcag gaagggatgg ctgaggtcgc ccggtttatt gaaatgaacg
     1261 gctcttttgc tgacgagaac aggggctggt gaaatgcagt ttaaggttta cacctataaa
     1321 agagagagcc gttatcgtct gtttgtggat gtacagagtg atattattga cacgcccggg
     1381 cgacggatgg tgatccccct ggccagtgca cgtctgctgt cagataaagt ctcccgtgaa
     1441 ctttacccgg tggtgcatat cggggatgaa agctggcgca tgatgaccac cgatatggcc
     1501 agtgtgccgg tttccgttat cggggaagaa gtggctgatc tcagccaccg cgaaaatgac
     1561 atcaaaaacg ccattaacct gatgttctgg ggaatataag gtctccctgc ctatacggca
     1621 gtgaacacta gtagctgcaa acataacaaa tatacctcag aatcaataaa ctaggctaaa
     1681 agggaactga agagaatcaa cagactgtag tctctgagca gtaactacac aaaaacatat
     1741 gatagaacaa tctggtaaaa acatatgcag tatgttctac accacaaatt tattacatac
     1801 aacaattaca tctatcagcg tgaagaacgc atgtcaaata gtttctttca atttttcata
     1861 caccaaaaca aaattcatgg gctaaaaccc caagacttta aattgcataa atactgacaa
     1921 gtggcaacaa aagctgattt taagggtatg taaacctacc agtaaggtgg acgttgacag
     1981 ggcaaacaaa ttagaatttt gcactgatac taaaatgggc ttccaaaccc taataggcac
     2041 tagatcagac tgcacacaca tacaactact aatgggaaaa ctaggtcagc atatacattc
     2101 aacgccgtaa attggcatag aaaacttcat catcacagat gaaaccaagc agcttgtatt
     2161 tcttatgtgg ttataaaggg gagcacttgc cattgctgtt acaagacata catttatgta
     2221 gattaataat gattttccat ggaatccaca agcatttgta agcgaaaacg aagcaaacat
     2281 tacaaaattc ccaagtatca ctaaagcaga atagcagatg caagtaacat ggcacctatt
     2341 agtaacctaa gccgtagcaa acttcacact ccacgtctcc agaacaaata aaagctctgc
     2401 aacgacctaa agttatgcga aaagtgcaaa atccccaaca agcgccactt cgtcatgtga
     2461 accgaaccag ccaaaatctc aaacctcccc tccccttctc gattctcata acgcaataga
     2521 agtatagaga agggattgct tcacgggacg cacctttgat cccgagggac gtctggccgg
     2581 atccgaccgc ggtgagcgcg tgctcgatct gcaccagctt ccccgacggg ctgcaaacga
     2641 aatcgccaca aaaaccacag atgacgaaac cgaaaccgca ccagatcacg cccgatctcc
     2701 gggcgaggga ggcgcaaggc agcacatacc tgaaggtggt gagggagaag gagtactggc
     2761 tgtctcccat ggccgccgac gcgcaggcct acctgtgagc tgctgctctt cgtgtctcgc
     2821 ggcggaagag gaggcgaccg agcgagcgat tgcggcgaga cggcgtggga agggagaggg
     2881 aggcagagca caactcgcca aggaagaaga ggagagagag aaagagactg gatggatgga
     2941 ttgctccgca agaaggtggt gggtcttggg ccggaaatct ggcccatctg ttattgggtc
     3001 agcccatgac tcacccacct ccccgtcacg tcgtcttcct cctcataact tcttccccac
     3061 gggccaacgg ctgcgcgtct tcccgttcgt cctcctgtcc ggtccgagtc cctctgacga
     3121 cgcgactttc cccacgccgg gatcgagctc cggggagggg agctgcgagc tggtacgtac
     3181 gcttgatttc ttcccttgcc accgcgatat tccgatgtat ccgtgatgtg tcgttcatgg
     3241 gtagatcggc tagggatttg gcggcgttga ggaaagcgat ggctgccggg tggggggaat
     3301 tgttgtatta gactctcgaa gctgatatct ggtgtggatt tgcggtggaa gccatggtcg
     3361 gaggcccagt gccttccggg agttgttggg ttatcgggag cccggagggc agtgcggctc
     3421 gccgactgct gccatgatcc ccgtcggcaa aatctccaga ttgggggaga tggggggtag
     3481 gaaccatttg gattagtagt gtatacacgg ggtttcgttt attatattta gagcgctcta
     3541 catgtagaca gcacacagca gctttaacgt atggatggtg tatcgcaggg aatgtatctc
     3601 gaagacattg gattaggcta ttattacatt tagagaacca gattgggttt gcgataacta
     3661 gaaattgagt ctgttatgaa tttattgatg gagcggacag ggaattacga ttactatctg
     3721 tttagctttt cagttgtgaa tgatgattat ttcaatttag ctaatcaagg ttcctcaatc
     3781 aaaccgttct aactttttct atttatgctc ttggttacat tttgacagaa acactttgtc
     3841 atatcagtca acagtagaga ggagacgagc ctgcaggtag atcgctcgtc gacatggctc
     3901 caaagaagaa gagaaaggtt ggaatccacg gagttccagc tgctatggat cattatcttg
     3961 atattagact tagacctgat ccagaatttc caccagctca acttatgtct gttctttttg
     4021 gaaaacttca tcaagctctt gttgctcaag gaggagatag aattggagtt tcttttcctg
     4081 atcttgatga atcaagatca agacttggag aaagacttag aattcatgct tctgctgatg
     4141 atcttagagc tttgcttgct agaccttggc ttgaaggact tagagatcat cttcaatttg
     4201 gagaaccagc tgttgttcca catccaactc cttatagaca agtttcaaga gttcaagcta
     4261 aatctaatcc agaaagactt agaagaagac ttatgagaag acatgatctt tctgaagaag
     4321 aagctagaaa aagaattcct gatactgttg ctagagcttt ggatttgcct tttgttacac
     4381 ttagatcaca atctactgga caacatttta gactttttat tagacatgga ccacttcaag
     4441 ttactgctga agaaggagga tttacttgtt atggactttc taagggaggt tttgttcctt
     4501 ggtttggatc tggagctact aatttttctc ttcttaagca agctggagat gttgaagaaa
     4561 atcctggacc catggataag aagtactcta tcggactcga tatcggaact aactctgtgg
     4621 gatgggctgt gatcaccgat gagtacaagg tgccatctaa gaagttcaag gttctcggaa
     4681 acaccgatag gcactctatc aagaaaaacc ttatcggtgc tctcctcttc gattctggtg
     4741 aaactgctga ggctaccaga ctcaagagaa ccgctagaag aaggtacacc agaagaaaga
     4801 acaggatctg ctacctccaa gagatcttct ctaacgagat ggctaaagtg gatgattcat
     4861 tcttccacag gctcgaagag tcattcctcg tggaagaaga taagaagcac gagaggcacc
     4921 ctatcttcgg aaacatcgtt gatgaggtgg cataccacga gaagtaccct actatctacc
     4981 acctcagaaa gaagctcgtt gattctactg ataaggctga tctcaggctc atctacctcg
     5041 ctctcgctca catgatcaag ttcagaggac acttcctcat cgagggtgat ctcaaccctg
     5101 ataactctga tgtggataag ttgttcatcc agctcgtgca gacctacaac cagcttttcg
     5161 aagagaaccc tatcaacgct tcaggtgtgg atgctaaggc tatcctctct gctaggctct
     5221 ctaagtcaag aaggcttgag aacctcattg ctcagctccc tggtgagaag aagaacggac
     5281 ttttcggaaa cttgatcgct ctctctctcg gactcacccc taacttcaag tctaacttcg
     5341 atctcgctga ggatgcaaag ctccagctct caaaggatac ctacgatgat gatctcgata
     5401 acctcctcgc tcagatcgga gatcagtacg ctgatttgtt cctcgctgct aagaacctct
     5461 ctgatgctat cctcctcagt gatatcctca gagtgaacac cgagatcacc aaggctccac
     5521 tctcagcttc tatgatcaag agatacgatg agcaccacca ggatctcaca cttctcaagg
     5581 ctcttgttag acagcagctc ccagagaagt acaaagagat tttcttcgat cagtctaaga
     5641 acggatacgc tggttacatc gatggtggtg catctcaaga agagttctac aagttcatca
     5701 agcctatcct cgagaagatg gatggaaccg aggaactcct cgtgaagctc aatagagagg
     5761 atcttctcag aaagcagagg accttcgata acggatctat ccctcatcag atccacctcg
     5821 gagagttgca cgctatcctt agaaggcaag aggatttcta cccattcctc aaggataaca
     5881 gggaaaagat tgagaagatt ctcaccttca gaatccctta ctacgtggga cctctcgcta
     5941 gaggaaactc aagattcgct tggatgacca gaaagtctga ggaaaccatc accccttgga
     6001 acttcgaaga ggtggtggat aagggtgcta gtgctcagtc tttcatcgag aggatgacca
     6061 acttcgataa gaaccttcca aacgagaagg tgctccctaa gcactctttg ctctacgagt
     6121 acttcaccgt gtacaacgag ttgaccaagg ttaagtacgt gaccgaggga atgaggaagc
     6181 ctgctttttt gtcaggtgag caaaagaagg ctatcgttga tctcttgttc aagaccaaca
     6241 gaaaggtgac cgtgaagcag ctcaaagagg attacttcaa gaaaatcgag tgcttcgatt
     6301 cagttgagat ttctggtgtt gaggataggt tcaacgcatc tctcggaacc taccacgatc
     6361 tcctcaagat cattaaggat aaggatttct tggataacga ggaaaacgag gatatcttgg
     6421 aggatatcgt tcttaccctc accctctttg aagatagaga gatgattgaa gaaaggctca
     6481 agacctacgc tcatctcttc gatgataagg tgatgaagca gttgaagaga agaagataca
     6541 ctggttgggg aaggctctca agaaagctca ttaacggaat cagggataag cagtctggaa
     6601 agacaatcct tgatttcctc aagtctgatg gattcgctaa cagaaacttc atgcagctca
     6661 tccacgatga ttctctcacc tttaaagagg atatccagaa ggctcaggtt tcaggacagg
     6721 gtgatagtct ccatgagcat atcgctaacc tcgctggatc tcctgcaatc aagaagggaa
     6781 tcctccagac tgtgaaggtt gtggatgagt tggtgaaggt gatgggaagg cataagcctg
     6841 agaacatcgt gatcgaaatg gctagagaga accagaccac tcagaaggga cagaagaact
     6901 ctagggaaag gatgaagagg atcgaggaag gtatcaaaga gcttggatct cagatcctca
     6961 aagagcaccc tgttgagaac actcagctcc agaatgagaa gctctacctc tactacctcc
     7021 agaacggaag ggatatgtat gtggatcaag agttggatat caacaggctc tctgattacg
     7081 atgttgatca tatcgtgcca cagtcattct tgaaggatga ttctatcgat aacaaggtgc
     7141 tcaccaggtc tgataagaac aggggtaaga gtgataacgt gccaagtgaa gaggttgtga
     7201 agaaaatgaa gaactattgg aggcagctcc tcaacgctaa gctcatcact cagagaaagt
     7261 tcgataactt gactaaggct gagaggggag gactctctga attggataag gcaggattca
     7321 tcaagaggca gcttgtggaa accaggcaga tcactaagca cgttgcacag atcctcgatt
     7381 ctaggatgaa caccaagtac gatgagaacg ataagttgat cagggaagtg aaggttatca
     7441 ccctcaagtc aaagctcgtg tctgatttca gaaaggattt ccaattctac aaggtgaggg
     7501 aaatcaacaa ctaccaccac gctcacgatg cttaccttaa cgctgttgtt ggaaccgctc
     7561 tcatcaagaa gtatcctaag ctcgagtcag agttcgtgta cggtgattac aaggtgtacg
     7621 atgtgaggaa gatgatcgct aagtctgagc aagagatcgg aaaggctacc gctaagtatt
     7681 tcttctactc taacatcatg aatttcttca agaccgagat taccctcgct aacggtgaga
     7741 tcagaaagag gccactcatc gagacaaacg gtgaaacagg tgagatcgtg tgggataagg
     7801 gaagggattt cgctaccgtt agaaaggtgc tctctatgcc acaggtgaac atcgttaaga
     7861 aaaccgaggt gcagaccggt ggattctcta aagagtctat cctccctaag aggaactctg
     7921 ataagctcat tgctaggaag aaggattggg accctaagaa atacggtggt ttcgattctc
     7981 ctaccgtggc ttactctgtt ctcgttgtgg ctaaggttga gaagggaaag agtaagaagc
     8041 tcaagtctgt taaggaactt ctcggaatca ctatcatgga aaggtcatct ttcgagaaga
     8101 acccaatcga tttcctcgag gctaagggat acaaagaggt taagaaggat ctcatcatca
     8161 agctcccaaa gtactcactc ttcgaactcg agaacggtag aaagaggatg ctcgcttctg
     8221 ctggtgagct tcaaaaggga aacgagcttg ctctcccatc taagtacgtt aactttcttt
     8281 acctcgcttc tcactacgag aagttgaagg gatctccaga agataacgag cagaagcaac
     8341 ttttcgttga gcagcacaag cactacttgg atgagatcat cgagcagatc tctgagttct
     8401 ctaaaagggt gatcctcgct gatgcaaacc tcgataaggt gttgtctgct tacaacaagc
     8461 acagagataa gcctatcagg gaacaggcag agaacatcat ccatctcttc acccttacca
     8521 acctcggtgc tcctgctgct ttcaagtact tcgatacaac catcgatagg aagagataca
     8581 cctctaccaa agaagtgctc gatgctaccc tcatccatca gtctatcact ggactctacg
     8641 agactaggat cgatctctca cagctcggtg gtgattcaag ggctgatcct aagaagaaga
     8701 ggaaggtttg aggatccata tgaagatgaa gatgaaatat ttggtgtgtc aaataaaaag
     8761 cttgtgtgct taagtttgtg tttttttctt ggcttgttgt gttatgaatt tgtggctttt
     8821 tctaatatta aatgaatgta agatcacatt ataatgaata aacaaatgtt tctataatcc
     8881 attgtgaatg ttttgttgga tctcttctgc agcatataac tactgtatgt gctatggtat
     8941 ggactatgga atatgattaa agataaggag ctcaaactat cagtgtttga caggatatat
     9001 tggcgggtaa acctaagaga aaagagcgtt tattagaata atcggatatt taaaagggcg
     9061 tgaaaaggtt tatccgttcg tccatttgta tgtgcatgcc aaccacaggg ttcccctcgg
     9121 gatcaaagta ctttgatcca acccctccgc tgctatagtg cagtcggctt ctgacgttca
     9181 gtgcagccgt cttctgaaaa cgacatgtcg cacaagtcct aagttacgcg acaggctgcc
     9241 gccctgccct tttcctggcg ttttcttgtc gcgtgtttta gtcgcataaa gtagaatact
     9301 tgcgactaga accggagaca ttacgccatg aacaagagcg ccgccgctgg cctgctgggc
     9361 tatgcccgcg tcagcaccga cgaccaggac ttgaccaacc aacgggccga actgcacgcg
     9421 gccggctgca ccaagctgtt ttccgagaag atcaccggca ccaggcgcga ccgcccggag
     9481 ctggccagga tgcttgacca cctacgccct ggcgacgttg tgacagtgac caggctagac
     9541 cgcctggccc gcagcacccg cgacctactg gacattgccg agcgcatcca ggaggccggc
     9601 gcgggcctgc gtagcctggc agagccgtgg gccgacacca ccacgccggc cggccgcatg
     9661 gtgttgaccg tgttcgccgg cattgccgag ttcgagcgtt ccctaatcat cgaccgcacc
     9721 cggagcgggc gcgaggccgc caaggcccga ggcgtgaagt ttggcccccg ccctaccctc
     9781 accccggcac agatcgcgca cgcccgcgag ctgatcgacc aggaaggccg caccgtgaaa
     9841 gaggcggctg cactgcttgg cgtgcatcgc tcgaccctgt accgcgcact tgagcgcagc
     9901 gaggaagtga cgcccaccga ggccaggcgg cgcggtgcct tccgtgagga cgcattgacc
     9961 gaggccgacg ccctggcggc cgccgagaat gaacgccaag aggaacaagc atgaaaccgc
    10021 accaggacgg ccaggacgaa ccgtttttca ttaccgaaga gatcgaggcg gagatgatcg
    10081 cggccgggta cgtgttcgag ccgcccgcgc acggctcaac cgtgcggctg catgaaatcc
    10141 tggccggttt gtctgatgcc aagctggcgg cctggccggc cagcttggcc gctgaagaaa
    10201 ccgagcgccg ccgtctaaaa aggtgatgtg tatttgagta aaacagcttg cgtcatgcgg
    10261 tcgctgcgta tatgatgcga tgagtaaata aacaaatacg caaggggaac gcatgaaggt
    10321 tatcgctgta cttaaccaga aaggcgggtc aggcaagacg accatcgcaa cccatctagc
    10381 ccgcgccctg caactcgccg gggccgatgt tctgttagtc gattccgatc cccagggcag
    10441 tgcccgcgat tgggcggccg tgcgggaaga tcaaccgcta accgttgtcg gcatcgaccg
    10501 cccgacgatt gaccgcgacg tgaaggccat cggccggcgc gacttcgtag tgatcgacgg
    10561 agcgccccag gcggcggact tggctgtgtc cgcgatcaag gcagccgact tcgtgctgat
    10621 tccggtgcag ccaagccctt acgacatatg ggccaccgcc gacctggtgg agctggttaa
    10681 gcagcgcatt gaggtcacgg atggaaggct acaagcggcc tttgtcgtgt cgcgggcgat
    10741 caaaggcacg cgcatcggcg gtgaggttgc cgaggcgctg gccgggtacg agctgcccat
    10801 tcttgagtcc cgtatcacgc agcgcgtgag ctacccaggc actgccgccg ccggcacaac
    10861 cgttcttgaa tcagaacccg agggcgacgc tgcccgcgag gtccaggcgc tggccgctga
    10921 aattaaatca aaactcattt gagttaatga ggtaaagaga aaatgagcaa aagcacaaac
    10981 acgctaagtg ccggccgtcc gagcgcacgc agcagcaagg ctgcaacgtt ggccagcctg
    11041 gcagacacgc cagccatgaa gcgggtcaac tttcagttgc cggcggagga tcacaccaag
    11101 ctgaagatgt acgcggtacg ccaaggcaag accattaccg agctgctatc tgaatacatc
    11161 gcgcagctac cagagtaaat gagcaaatga ataaatgagt agatgaattt tagcggctaa
    11221 aggaggcggc atggaaaatc aagaacaacc aggcaccgac gccgtggaat gccccatgtg
    11281 tggaggaacg ggcggttggc caggcgtaag cggctgggtt gcctgccggc cctgcaatgg
    11341 cactggaacc cccaagcccg aggaatcggc gtgagcggtc gcaaaccatc cggcccggta
    11401 caaatcggcg cggcgctggg tgatgacctg gtggagaagt tgaaggccgc gcaggccgcc
    11461 cagcggcaac gcatcgaggc agaagcacgc cccggtgaat cgtggcaagc ggccgctgat
    11521 cgaatccgca aagaatcccg gcaaccgccg gcagccggtg cgccgtcgat taggaagccg
    11581 cccaagggcg acgagcaacc agattttttc gttccgatgc tctatgacgt gggcacccgc
    11641 gatagtcgca gcatcatgga cgtggccgtt ttccgtctgt cgaagcgtga ccgacgagct
    11701 ggcgaggtga tccgctacga gcttccagac gggcacgtag aggtttccgc agggccggcc
    11761 ggcatggcca gtgtgtggga ttacgacctg gtactgatgg cggtttccca tctaaccgaa
    11821 tccatgaacc gataccggga agggaaggga gacaagcccg gccgcgtgtt ccgtccacac
    11881 gttgcggacg tactcaagtt ctgccggcga gccgatggcg gaaagcagaa agacgacctg
    11941 gtagaaacct gcattcggtt aaacaccacg cacgttgcca tgcagcgtac gaagaaggcc
    12001 aagaacggcc gcctggtgac ggtatccgag ggtgaagcct tgattagccg ctacaagatc
    12061 gtaaagagcg aaaccgggcg gccggagtac atcgagatcg agctagctga ttggatgtac
    12121 cgcgagatca cagaaggcaa gaacccggac gtgctgacgg ttcaccccga ttactttttg
    12181 atcgatcccg gcatcggccg ttttctctac cgcctggcac gccgcgccgc aggcaaggca
    12241 gaagccagat ggttgttcaa gacgatctac gaacgcagtg gcagcgccgg agagttcaag
    12301 aagttctgtt tcaccgtgcg caagctgatc gggtcaaatg acctgccgga gtacgatttg
    12361 aaggaggagg cggggcaggc tggcccgatc ctagtcatgc gctaccgcaa cctgatcgag
    12421 ggcgaagcat ccgccggttc ctaatgtacg gagcagatgc tagggcaaat tgccctagca
    12481 ggggaaaaag gtcgaaaagg cctctttcct gtggatagca cgtacattgg gaacccaaag
    12541 ccgtacattg ggaaccggaa cccgtacatt gggaacccaa agccgtacat tgggaaccgg
    12601 tcacacatgt aagtgactga tataaaagag aaaaaaggcg atttttccgc ctaaaactct
    12661 ttaaaactta ttaaaactct taaaacccgc ctggcctgtg cataactgtc tggccagcgc
    12721 acagccgaag agctgcaaaa agcgcctacc cttcggtcgc tgcgctccct acgccccgcc
    12781 gcttcgcgtc ggcctatcgc ggccgctggc cgctcaaaaa tggctggcct acggccaggc
    12841 aatctaccag ggcgcggaca agccgcgccg tcgccactcg accgccggcg cccacatcaa
    12901 ggcaccctgc ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc
    12961 ggaaacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagcgc gtcagcgggt
    13021 gttggcgggt gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtatact
    13081 ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg cggtgtgaaa
    13141 taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct tcctcgctca
    13201 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
    13261 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
    13321 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
    13381 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
    13441 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
    13501 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
    13561 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
    13621 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
    13681 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
    13741 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
    13801 gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
    13861 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
    13921 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
    13981 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgcat tctaggtact
    14041 aaaacaattc atccagtaaa atataatatt ttattttctc ccaatcaggc ttgatcccca
    14101 gtaagtcaaa aaatagctcg acatactgtt cttccccgat atcctccctg atcgaccgga
    14161 cgcagaaggc aatgtcatac cacttgtccg ccctgccgct tctcccaaga tcaataaagc
    14221 cacttacttt gccatctttc acaaagatgt tgctgtctcc caggtcgccg tgggaaaaga
    14281 caagttcctc ttcgggcttt tccgtcttta aaaaatcata cagctcgcgc ggatctttaa
    14341 atggagtgtc ttcttcccag ttttcgcaat ccacatcggc cagatcgtta ttcagtaagt
    14401 aatccaattc ggctaagcgg ctgtctaagc tattcgtata gggacaatcc gatatgtcga
    14461 tggagtgaaa gagcctgatg cactccgcat acagctcgat aatcttttca gggctttgtt
    14521 catcttcata ctcttccgag caaaggacgc catcggcctc actcatgagc agattgctcc
    14581 agccatcatg ccgttcaaag tgcaggacct ttggaacagg cagctttcct tccagccata
    14641 gcatcatgtc cttttcccgt tccacatcat aggtggtccc tttataccgg ctgtccgtca
    14701 tttttaaata taggttttca ttttctccca ccagcttata taccttagca ggagacattc
    14761 cttccgtatc ttttacgcag cggtattttt cgatcagttt tttcaattcc ggtgatattc
    14821 tcattttagc catttattat ttccttcctc ttttctacag tatttaaaga taccccaaga
    14881 agctaattat aacaagacga actccaattc actgttcctt gcattctaaa accttaaata
    14941 ccagaaaaca gctttttcaa agttgttttc aaagttggcg tataacatag tatcgacgga
    15001 gccgattttg aaaccgcggt gatcacaggc agcaacgctc tgtcatcgtt acaatcaaca
    15061 tgctaccctc cgcgagatca tccgtgtttc aaacccggca gcttagttgc cgttcttccg
    15121 aatagcatcg gtaacatgag caaagtctgc cgccttacaa cggctctccc gctgacgccg
    15181 tcccggactg atgggctgcc tgtatcgagt ggtgattttg tgccgagctg ccggtcgggg
    15241 agctgttggc tggctggtgg caggatatat tgtggtgtaa acaaattgac gcttagacaa
    15301 cttaataaca cattgcggac gtttttaatg tactgaatta acgccgaatt aattcggggg
    15361 atctggattt tagtactgga ttttggtttt aggaattaga aattttattg atagaagtat
    15421 tttacaaata caaatacata ctaagggttt cttatatgct caacacatga gcgaaaccct
    15481 ataggaaccc taattccctt atctgggaac tactcacaca ttattatgga gaaaatagag
    15541 cttgtcgatc gttaattaat cacctccaag atcccggacg agtgctgggg cgtcggtttc
    15601 cactatcggc gagtacttct acacagccat cggtccagac ggccgcgctt ctgcgggcga
    15661 tttgtgtacg cccgacagtc ccggctccgg atcggacgat tgcgtcgcat cgaccctgcg
    15721 cccaagctgc atcatcgaaa ttgccgtcaa ccaagctctg atagagttgg tcaagaccaa
    15781 tgcggagcat atacgcccgg agccgcggcg atcctgcaag ctccggatgc ctccgctcga
    15841 agtagcgcgt ctgctgctcc atacaagcca accacggcct ccagaagaag atgttggcga
    15901 cctcgtattg ggaatccccg aacatcgcct cgctccagtc aatgaccgct gttatgcggc
    15961 cattgtccgt caggacattg ttggagccga aatccgcgtg cacgaggtgc cggacttcgg
    16021 ggcagtcctc ggcccaaagc atcagctcat cgagagcctg cgcgacggac gcactgacgg
    16081 tgtcgtccat cacagtttgc cagtgataca catggggatc agcaatcgcg catatgaaat
    16141 cacgccatgt agtgtattga ccgattcctt gcggtccgaa tgggccgaac ccgctcgtct
    16201 ggctaagatc ggccgcagcg atcgcatcca tggcctccgc gaccggctgc agaacagcgg
    16261 gcagttcggt ttcaggcagg tcttgcaacg tgacaccctg tgcacggcgg gagatgcaat
    16321 aggtcaggct ctcgctgaat tccccaatgt caagcacttc cggaatcggg agcgcggccg
    16381 atgcaaagtg ccgataaaca taacgatctt tgtagaaacc atcggcgcag ctatttaccc
    16441 gcaggacata tccacgccct cctacatcga agctgaaagc acgagattct tcgccctccg
    16501 agagctgcat caggtcggac acgctgtcga acttttcgat cagaaacttc tcgacagacg
    16561 tcgcggtgag ttcaggcttt ttcatcctag gagagataga tttgtagaga gagactggtg
    16621 atttcagcgt gtcctctcca aatgaaatga acttccttat atagaggaag ggtcttgcga
    16681 aggatagtgg gattgtgcgt catcccttac gtcagtggag atatcacatc aatccacttg
    16741 ctttgaagac gtggttggaa cgtcttcttt ttccacgatg ctcctcgtgg gtgggggtcc
    16801 atctttggga ccactgtcgg cagaggcatc ttgaacgata gcctttcctt tatcgcaatg
    16861 atggcatttg taggtgccac cttccttttc tactgtcctt ttgatgaagt gacagatagc
    16921 tgggcaatgg aatccgagga ggtttcccga tattaccctt tgttgaaaag tctcaatagc
    16981 cctttggtct tctgagactg tatctttgat attcttggag tagacgagag tgtcgtgctc
    17041 caccatgttc acatcaatcc acttgctttg aagacgtggt tggaacgtct tctttttcca
    17101 cgatgctcct cgtgggtggg ggtccatctt tgggaccact gtcggcagag gcatcttgaa
    17161 cgatagcctt tcctttatcg caatgatggc atttgtaggt gccaccttcc ttttctactg
    17221 tccttttgat
//
