LOCUS       sequence-309992-       16666 bp    DNA     circular SYN 15-APR-2025
DEFINITION  OsBiP1 promoter driven Cas9-tRNA system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 16666)
  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 16666)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: pGEL052
FEATURES             Location/Qualifiers
     source          1..16666
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(53..760)
                     /label=OCS terminator
                     /note="octopine synthase terminator"
     misc_RNA        complement(844..919)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             1246..1551
                     /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     1430..1449
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             3903..8006
                     /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             3903..8006
                     /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             8019..8039
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      8050..8298
                     /label=HSP terminator
                     /note="efficient transcription terminator from the 
                     Arabidopsis thaliana heat shock protein 18.2 gene (Nagaya 
                     et al., 2010)"
     misc_feature    8319..8343
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             9644..10273
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             10702..11775
                     /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      11841..12035
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     primer_bind     complement(12271..12293)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    12369..12509
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     12424..12443
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(12524..12541)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(12695..13283)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(12775..12794)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(13370..14164)
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     misc_feature    14589..14613
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     polyA_signal    14691..14865
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     promoter        complement(15955..16631)
                     /label=CaMV 35S promoter (enhanced)
                     /note="cauliflower mosaic virus 35S promoter with a 
                     duplicated enhancer region"
     primer_bind     complement(15998..16018)
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
ORIGIN
        1 attcttggag tagacgagag tgtcgtgctc caccatgggc gcgccgccgg tcctgctgag
       61 cctcgacatg ttgtcgcaaa attcgccctg gacccgccca acgatttgtc gtcactgtca
      121 aggtttgacc tgcacttcat ttggggccca catacaccaa aaaaatgctg cataattctc
      181 ggggcagcaa gtcggttacc cggccgccgt gctggaccgg gttgaatggt gcccgtaact
      241 ttcggtagag cggacggcca atactcaact tcaaggaatc tcacccatgc gcgccggcgg
      301 ggaaccggag ttcccttcag tgagcgttat tagttcgccg ctcggtgtgt cgtagatact
      361 agcccctggg gcacttttga aatttgaata agatttatgt aatcagtctt ttaggtttga
      421 ccggttctgc cgcttttttt aaaattggat ttgtaataat aaaacgcaat tgtttgttat
      481 tgtggcgctc tatcatagat gtcgctataa acctattcag cacaatatat tgttttcatt
      541 ttaatattgt acatataagt agtagggtac aatcagtaaa ttgaacggag aatattattc
      601 ataaaaatac gatagtaacg ggtgatatat tcattagaat gaaccgaaac cggcggtaag
      661 gatctgagct acacatgctc aggtttttta caacgtgcac aacagaattg aaagcaaata
      721 tcatgcgatc ataggcgtct cgcatatctc attaaagcag ggtacctgca ccagccggga
      781 atcgaacccg ggtctgtacc gtggcagggt actattctac cactagacca ctggtgcttt
      841 gttgcaccga ctcggtgcca ctttttcaag ttgataacgg actagcctta ttttaacttg
      901 ctatttctag ctctaaaact gagaccaagc cagataacag tatgcgtatt tgcgcgctga
      961 tttttgcggt ataagaatat atactgatat gtatacccga agtatgtcaa aaagaggtat
     1021 gctatgaagc agcgtattac agtgacagtt gacagcgaca gctatcagtt gctcaaggca
     1081 tatatgatgt caatatctcc ggtctggtaa gcacaaccat gcagaatgaa gcccgtcgtc
     1141 tgcgtgccga acgctggaaa gcggaaaatc aggaagggat ggctgaggtc gcccggttta
     1201 ttgaaatgaa cggctctttt gctgacgaga acaggggctg gtgaaatgca gtttaaggtt
     1261 tacacctata aaagagagag ccgttatcgt ctgtttgtgg atgtacagag tgatattatt
     1321 gacacgcccg ggcgacggat ggtgatcccc ctggccagtg cacgtctgct gtcagataaa
     1381 gtctcccgtg aactttaccc ggtggtgcat atcggggatg aaagctggcg catgatgacc
     1441 accgatatgg ccagtgtgcc ggtttccgtt atcggggaag aagtggctga tctcagccac
     1501 cgcgaaaatg acatcaaaaa cgccattaac ctgatgttct ggggaatata aggtctcctg
     1561 caccagccgg gaatcgaacc cgggtctgta ccgtggcagg gtactattct accactagac
     1621 cactggtgct ttgttactag tagctgcaaa cataacaaat atacctcaga atcaataaac
     1681 taggctaaaa gggaactgaa gagaatcaac agactgtagt ctctgagcag taactacaca
     1741 aaaacatatg atagaacaat ctggtaaaaa catatgcagt atgttctaca ccacaaattt
     1801 attacataca acaattacat ctatcagcgt gaagaacgca tgtcaaatag tttctttcaa
     1861 tttttcatac accaaaacaa aattcatggg ctaaaacccc aagactttaa attgcataaa
     1921 tactgacaag tggcaacaaa agctgatttt aagggtatgt aaacctacca gtaaggtgga
     1981 cgttgacagg gcaaacaaat tagaattttg cactgatact aaaatgggct tccaaaccct
     2041 aataggcact agatcagact gcacacacat acaactacta atgggaaaac taggtcagca
     2101 tatacattca acgccgtaaa ttggcataga aaacttcatc atcacagatg aaaccaagca
     2161 gcttgtattt cttatgtggt tataaagggg agcacttgcc attgctgtta caagacatac
     2221 atttatgtag attaataatg attttccatg gaatccacaa gcatttgtaa gcgaaaacga
     2281 agcaaacatt acaaaattcc caagtatcac taaagcagaa tagcagatgc aagtaacatg
     2341 gcacctatta gtaacctaag ccgtagcaaa cttcacactc cacgtctcca gaacaaataa
     2401 aagctctgca acgacctaaa gttatgcgaa aagtgcaaaa tccccaacaa gcgccacttc
     2461 gtcatgtgaa ccgaaccagc caaaatctca aacctcccct ccccttctcg attctcataa
     2521 cgcaatagaa gtatagagaa gggattgctt cacgggacgc acctttgatc ccgagggacg
     2581 tctggccgga tccgaccgcg gtgagcgcgt gctcgatctg caccagcttc cccgacgggc
     2641 tgcaaacgaa atcgccacaa aaaccacaga tgacgaaacc gaaaccgcac cagatcacgc
     2701 ccgatctccg ggcgagggag gcgcaaggca gcacatacct gaaggtggtg agggagaagg
     2761 agtactggct gtctcccatg gccgccgacg cgcaggccta cctgtgagct gctgctcttc
     2821 gtgtctcgcg gcggaagagg aggcgaccga gcgagcgatt gcggcgagac ggcgtgggaa
     2881 gggagaggga ggcagagcac aactcgccaa ggaagaagag gagagagaga aagagactgg
     2941 atggatggat tgctccgcaa gaaggtggtg ggtcttgggc cggaaatctg gcccatctgt
     3001 tattgggtca gcccatgact cacccacctc cccgtcacgt cgtcttcctc ctcataactt
     3061 cttccccacg ggccaacggc tgcgcgtctt cccgttcgtc ctcctgtccg gtccgagtcc
     3121 ctctgacgac gcgactttcc ccacgccggg atcgagctcc ggggagggga gctgcgagct
     3181 ggtacgtacg cttgatttct tcccttgcca ccgcgatatt ccgatgtatc cgtgatgtgt
     3241 cgttcatggg tagatcggct agggatttgg cggcgttgag gaaagcgatg gctgccgggt
     3301 ggggggaatt gttgtattag actctcgaag ctgatatctg gtgtggattt gcggtggaag
     3361 ccatggtcgg aggcccagtg ccttccggga gttgttgggt tatcgggagc ccggagggca
     3421 gtgcggctcg ccgactgctg ccatgatccc cgtcggcaaa atctccagat tgggggagat
     3481 ggggggtagg aaccatttgg attagtagtg tatacacggg gtttcgttta ttatatttag
     3541 agcgctctac atgtagacag cacacagcag ctttaacgta tggatggtgt atcgcaggga
     3601 atgtatctcg aagacattgg attaggctat tattacattt agagaaccag attgggtttg
     3661 cgataactag aaattgagtc tgttatgaat ttattgatgg agcggacagg gaattacgat
     3721 tactatctgt ttagcttttc agttgtgaat gatgattatt tcaatttagc taatcaaggt
     3781 tcctcaatca aaccgttcta actttttcta tttatgctct tggttacatt ttgacagaaa
     3841 cactttgtca tatcagtcaa cagtagagag gagacgagcc tgcaggtaga tcgctcgtcg
     3901 acatggataa gaagtactct atcggactcg atatcggaac taactctgtg ggatgggctg
     3961 tgatcaccga tgagtacaag gtgccatcta agaagttcaa ggttctcgga aacaccgata
     4021 ggcactctat caagaaaaac cttatcggtg ctctcctctt cgattctggt gaaactgctg
     4081 aggctaccag actcaagaga accgctagaa gaaggtacac cagaagaaag aacaggatct
     4141 gctacctcca agagatcttc tctaacgaga tggctaaagt ggatgattca ttcttccaca
     4201 ggctcgaaga gtcattcctc gtggaagaag ataagaagca cgagaggcac cctatcttcg
     4261 gaaacatcgt tgatgaggtg gcataccacg agaagtaccc tactatctac cacctcagaa
     4321 agaagctcgt tgattctact gataaggctg atctcaggct catctacctc gctctcgctc
     4381 acatgatcaa gttcagagga cacttcctca tcgagggtga tctcaaccct gataactctg
     4441 atgtggataa gttgttcatc cagctcgtgc agacctacaa ccagcttttc gaagagaacc
     4501 ctatcaacgc ttcaggtgtg gatgctaagg ctatcctctc tgctaggctc tctaagtcaa
     4561 gaaggcttga gaacctcatt gctcagctcc ctggtgagaa gaagaacgga cttttcggaa
     4621 acttgatcgc tctctctctc ggactcaccc ctaacttcaa gtctaacttc gatctcgctg
     4681 aggatgcaaa gctccagctc tcaaaggata cctacgatga tgatctcgat aacctcctcg
     4741 ctcagatcgg agatcagtac gctgatttgt tcctcgctgc taagaacctc tctgatgcta
     4801 tcctcctcag tgatatcctc agagtgaaca ccgagatcac caaggctcca ctctcagctt
     4861 ctatgatcaa gagatacgat gagcaccacc aggatctcac acttctcaag gctcttgtta
     4921 gacagcagct cccagagaag tacaaagaga ttttcttcga tcagtctaag aacggatacg
     4981 ctggttacat cgatggtggt gcatctcaag aagagttcta caagttcatc aagcctatcc
     5041 tcgagaagat ggatggaacc gaggaactcc tcgtgaagct caatagagag gatcttctca
     5101 gaaagcagag gaccttcgat aacggatcta tccctcatca gatccacctc ggagagttgc
     5161 acgctatcct tagaaggcaa gaggatttct acccattcct caaggataac agggaaaaga
     5221 ttgagaagat tctcaccttc agaatccctt actacgtggg acctctcgct agaggaaact
     5281 caagattcgc ttggatgacc agaaagtctg aggaaaccat caccccttgg aacttcgaag
     5341 aggtggtgga taagggtgct agtgctcagt ctttcatcga gaggatgacc aacttcgata
     5401 agaaccttcc aaacgagaag gtgctcccta agcactcttt gctctacgag tacttcaccg
     5461 tgtacaacga gttgaccaag gttaagtacg tgaccgaggg aatgaggaag cctgcttttt
     5521 tgtcaggtga gcaaaagaag gctatcgttg atctcttgtt caagaccaac agaaaggtga
     5581 ccgtgaagca gctcaaagag gattacttca agaaaatcga gtgcttcgat tcagttgaga
     5641 tttctggtgt tgaggatagg ttcaacgcat ctctcggaac ctaccacgat ctcctcaaga
     5701 tcattaagga taaggatttc ttggataacg aggaaaacga ggatatcttg gaggatatcg
     5761 ttcttaccct caccctcttt gaagatagag agatgattga agaaaggctc aagacctacg
     5821 ctcatctctt cgatgataag gtgatgaagc agttgaagag aagaagatac actggttggg
     5881 gaaggctctc aagaaagctc attaacggaa tcagggataa gcagtctgga aagacaatcc
     5941 ttgatttcct caagtctgat ggattcgcta acagaaactt catgcagctc atccacgatg
     6001 attctctcac ctttaaagag gatatccaga aggctcaggt ttcaggacag ggtgatagtc
     6061 tccatgagca tatcgctaac ctcgctggat ctcctgcaat caagaaggga atcctccaga
     6121 ctgtgaaggt tgtggatgag ttggtgaagg tgatgggaag gcataagcct gagaacatcg
     6181 tgatcgaaat ggctagagag aaccagacca ctcagaaggg acagaagaac tctagggaaa
     6241 ggatgaagag gatcgaggaa ggtatcaaag agcttggatc tcagatcctc aaagagcacc
     6301 ctgttgagaa cactcagctc cagaatgaga agctctacct ctactacctc cagaacggaa
     6361 gggatatgta tgtggatcaa gagttggata tcaacaggct ctctgattac gatgttgatc
     6421 atatcgtgcc acagtcattc ttgaaggatg attctatcga taacaaggtg ctcaccaggt
     6481 ctgataagaa caggggtaag agtgataacg tgccaagtga agaggttgtg aagaaaatga
     6541 agaactattg gaggcagctc ctcaacgcta agctcatcac tcagagaaag ttcgataact
     6601 tgactaaggc tgagagggga ggactctctg aattggataa ggcaggattc atcaagaggc
     6661 agcttgtgga aaccaggcag atcactaagc acgttgcaca gatcctcgat tctaggatga
     6721 acaccaagta cgatgagaac gataagttga tcagggaagt gaaggttatc accctcaagt
     6781 caaagctcgt gtctgatttc agaaaggatt tccaattcta caaggtgagg gaaatcaaca
     6841 actaccacca cgctcacgat gcttacctta acgctgttgt tggaaccgct ctcatcaaga
     6901 agtatcctaa gctcgagtca gagttcgtgt acggtgatta caaggtgtac gatgtgagga
     6961 agatgatcgc taagtctgag caagagatcg gaaaggctac cgctaagtat ttcttctact
     7021 ctaacatcat gaatttcttc aagaccgaga ttaccctcgc taacggtgag atcagaaaga
     7081 ggccactcat cgagacaaac ggtgaaacag gtgagatcgt gtgggataag ggaagggatt
     7141 tcgctaccgt tagaaaggtg ctctctatgc cacaggtgaa catcgttaag aaaaccgagg
     7201 tgcagaccgg tggattctct aaagagtcta tcctccctaa gaggaactct gataagctca
     7261 ttgctaggaa gaaggattgg gaccctaaga aatacggtgg tttcgattct cctaccgtgg
     7321 cttactctgt tctcgttgtg gctaaggttg agaagggaaa gagtaagaag ctcaagtctg
     7381 ttaaggaact tctcggaatc actatcatgg aaaggtcatc tttcgagaag aacccaatcg
     7441 atttcctcga ggctaaggga tacaaagagg ttaagaagga tctcatcatc aagctcccaa
     7501 agtactcact cttcgaactc gagaacggta gaaagaggat gctcgcttct gctggtgagc
     7561 ttcaaaaggg aaacgagctt gctctcccat ctaagtacgt taactttctt tacctcgctt
     7621 ctcactacga gaagttgaag ggatctccag aagataacga gcagaagcaa cttttcgttg
     7681 agcagcacaa gcactacttg gatgagatca tcgagcagat ctctgagttc tctaaaaggg
     7741 tgatcctcgc tgatgcaaac ctcgataagg tgttgtctgc ttacaacaag cacagagata
     7801 agcctatcag ggaacaggca gagaacatca tccatctctt cacccttacc aacctcggtg
     7861 ctcctgctgc tttcaagtac ttcgatacaa ccatcgatag gaagagatac acctctacca
     7921 aagaagtgct cgatgctacc ctcatccatc agtctatcac tggactctac gagactagga
     7981 tcgatctctc acagctcggt ggtgattcaa gggctgatcc taagaagaag aggaaggttt
     8041 gaggatccat atgaagatga agatgaaata tttggtgtgt caaataaaaa gcttgtgtgc
     8101 ttaagtttgt gtttttttct tggcttgttg tgttatgaat ttgtggcttt ttctaatatt
     8161 aaatgaatgt aagatcacat tataatgaat aaacaaatgt ttctataatc cattgtgaat
     8221 gttttgttgg atctcttctg cagcatataa ctactgtatg tgctatggta tggactatgg
     8281 aatatgatta aagataagga gctcaaacta tcagtgtttg acaggatata ttggcgggta
     8341 aacctaagag aaaagagcgt ttattagaat aatcggatat ttaaaagggc gtgaaaaggt
     8401 ttatccgttc gtccatttgt atgtgcatgc caaccacagg gttcccctcg ggatcaaagt
     8461 actttgatcc aacccctccg ctgctatagt gcagtcggct tctgacgttc agtgcagccg
     8521 tcttctgaaa acgacatgtc gcacaagtcc taagttacgc gacaggctgc cgccctgccc
     8581 ttttcctggc gttttcttgt cgcgtgtttt agtcgcataa agtagaatac ttgcgactag
     8641 aaccggagac attacgccat gaacaagagc gccgccgctg gcctgctggg ctatgcccgc
     8701 gtcagcaccg acgaccagga cttgaccaac caacgggccg aactgcacgc ggccggctgc
     8761 accaagctgt tttccgagaa gatcaccggc accaggcgcg accgcccgga gctggccagg
     8821 atgcttgacc acctacgccc tggcgacgtt gtgacagtga ccaggctaga ccgcctggcc
     8881 cgcagcaccc gcgacctact ggacattgcc gagcgcatcc aggaggccgg cgcgggcctg
     8941 cgtagcctgg cagagccgtg ggccgacacc accacgccgg ccggccgcat ggtgttgacc
     9001 gtgttcgccg gcattgccga gttcgagcgt tccctaatca tcgaccgcac ccggagcggg
     9061 cgcgaggccg ccaaggcccg aggcgtgaag tttggccccc gccctaccct caccccggca
     9121 cagatcgcgc acgcccgcga gctgatcgac caggaaggcc gcaccgtgaa agaggcggct
     9181 gcactgcttg gcgtgcatcg ctcgaccctg taccgcgcac ttgagcgcag cgaggaagtg
     9241 acgcccaccg aggccaggcg gcgcggtgcc ttccgtgagg acgcattgac cgaggccgac
     9301 gccctggcgg ccgccgagaa tgaacgccaa gaggaacaag catgaaaccg caccaggacg
     9361 gccaggacga accgtttttc attaccgaag agatcgaggc ggagatgatc gcggccgggt
     9421 acgtgttcga gccgcccgcg cacggctcaa ccgtgcggct gcatgaaatc ctggccggtt
     9481 tgtctgatgc caagctggcg gcctggccgg ccagcttggc cgctgaagaa accgagcgcc
     9541 gccgtctaaa aaggtgatgt gtatttgagt aaaacagctt gcgtcatgcg gtcgctgcgt
     9601 atatgatgcg atgagtaaat aaacaaatac gcaaggggaa cgcatgaagg ttatcgctgt
     9661 acttaaccag aaaggcgggt caggcaagac gaccatcgca acccatctag cccgcgccct
     9721 gcaactcgcc ggggccgatg ttctgttagt cgattccgat ccccagggca gtgcccgcga
     9781 ttgggcggcc gtgcgggaag atcaaccgct aaccgttgtc ggcatcgacc gcccgacgat
     9841 tgaccgcgac gtgaaggcca tcggccggcg cgacttcgta gtgatcgacg gagcgcccca
     9901 ggcggcggac ttggctgtgt ccgcgatcaa ggcagccgac ttcgtgctga ttccggtgca
     9961 gccaagccct tacgacatat gggccaccgc cgacctggtg gagctggtta agcagcgcat
    10021 tgaggtcacg gatggaaggc tacaagcggc ctttgtcgtg tcgcgggcga tcaaaggcac
    10081 gcgcatcggc ggtgaggttg ccgaggcgct ggccgggtac gagctgccca ttcttgagtc
    10141 ccgtatcacg cagcgcgtga gctacccagg cactgccgcc gccggcacaa ccgttcttga
    10201 atcagaaccc gagggcgacg ctgcccgcga ggtccaggcg ctggccgctg aaattaaatc
    10261 aaaactcatt tgagttaatg aggtaaagag aaaatgagca aaagcacaaa cacgctaagt
    10321 gccggccgtc cgagcgcacg cagcagcaag gctgcaacgt tggccagcct ggcagacacg
    10381 ccagccatga agcgggtcaa ctttcagttg ccggcggagg atcacaccaa gctgaagatg
    10441 tacgcggtac gccaaggcaa gaccattacc gagctgctat ctgaatacat cgcgcagcta
    10501 ccagagtaaa tgagcaaatg aataaatgag tagatgaatt ttagcggcta aaggaggcgg
    10561 catggaaaat caagaacaac caggcaccga cgccgtggaa tgccccatgt gtggaggaac
    10621 gggcggttgg ccaggcgtaa gcggctgggt tgcctgccgg ccctgcaatg gcactggaac
    10681 ccccaagccc gaggaatcgg cgtgagcggt cgcaaaccat ccggcccggt acaaatcggc
    10741 gcggcgctgg gtgatgacct ggtggagaag ttgaaggccg cgcaggccgc ccagcggcaa
    10801 cgcatcgagg cagaagcacg ccccggtgaa tcgtggcaag cggccgctga tcgaatccgc
    10861 aaagaatccc ggcaaccgcc ggcagccggt gcgccgtcga ttaggaagcc gcccaagggc
    10921 gacgagcaac cagatttttt cgttccgatg ctctatgacg tgggcacccg cgatagtcgc
    10981 agcatcatgg acgtggccgt tttccgtctg tcgaagcgtg accgacgagc tggcgaggtg
    11041 atccgctacg agcttccaga cgggcacgta gaggtttccg cagggccggc cggcatggcc
    11101 agtgtgtggg attacgacct ggtactgatg gcggtttccc atctaaccga atccatgaac
    11161 cgataccggg aagggaaggg agacaagccc ggccgcgtgt tccgtccaca cgttgcggac
    11221 gtactcaagt tctgccggcg agccgatggc ggaaagcaga aagacgacct ggtagaaacc
    11281 tgcattcggt taaacaccac gcacgttgcc atgcagcgta cgaagaaggc caagaacggc
    11341 cgcctggtga cggtatccga gggtgaagcc ttgattagcc gctacaagat cgtaaagagc
    11401 gaaaccgggc ggccggagta catcgagatc gagctagctg attggatgta ccgcgagatc
    11461 acagaaggca agaacccgga cgtgctgacg gttcaccccg attacttttt gatcgatccc
    11521 ggcatcggcc gttttctcta ccgcctggca cgccgcgccg caggcaaggc agaagccaga
    11581 tggttgttca agacgatcta cgaacgcagt ggcagcgccg gagagttcaa gaagttctgt
    11641 ttcaccgtgc gcaagctgat cgggtcaaat gacctgccgg agtacgattt gaaggaggag
    11701 gcggggcagg ctggcccgat cctagtcatg cgctaccgca acctgatcga gggcgaagca
    11761 tccgccggtt cctaatgtac ggagcagatg ctagggcaaa ttgccctagc aggggaaaaa
    11821 ggtcgaaaag gcctctttcc tgtggatagc acgtacattg ggaacccaaa gccgtacatt
    11881 gggaaccgga acccgtacat tgggaaccca aagccgtaca ttgggaaccg gtcacacatg
    11941 taagtgactg atataaaaga gaaaaaaggc gatttttccg cctaaaactc tttaaaactt
    12001 attaaaactc ttaaaacccg cctggcctgt gcataactgt ctggccagcg cacagccgaa
    12061 gagctgcaaa aagcgcctac ccttcggtcg ctgcgctccc tacgccccgc cgcttcgcgt
    12121 cggcctatcg cggccgctgg ccgctcaaaa atggctggcc tacggccagg caatctacca
    12181 gggcgcggac aagccgcgcc gtcgccactc gaccgccggc gcccacatca aggcaccctg
    12241 cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggaaacggt
    12301 cacagcttgt ctgtaagcgg atgccgggag cagacaagcg cgtcagcggg tgttggcggg
    12361 tgtcggggcg cagccatgac ccagtcacgt agcgatagcg gagtgtatac tggcttaact
    12421 atgcggcatc agagcagatt gtactgagag tgcaccatat gcggtgtgaa ataccgcaca
    12481 gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc actgactcgc
    12541 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
    12601 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
    12661 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
    12721 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
    12781 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
    12841 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
    12901 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
    12961 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
    13021 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
    13081 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag
    13141 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
    13201 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
    13261 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
    13321 agtggaacga aaactcacgt taagggattt tggtcatgca ttctaggtac taaaacaatt
    13381 catccagtaa aatataatat tttattttct cccaatcagg cttgatcccc agtaagtcaa
    13441 aaaatagctc gacatactgt tcttccccga tatcctccct gatcgaccgg acgcagaagg
    13501 caatgtcata ccacttgtcc gccctgccgc ttctcccaag atcaataaag ccacttactt
    13561 tgccatcttt cacaaagatg ttgctgtctc ccaggtcgcc gtgggaaaag acaagttcct
    13621 cttcgggctt ttccgtcttt aaaaaatcat acagctcgcg cggatcttta aatggagtgt
    13681 cttcttccca gttttcgcaa tccacatcgg ccagatcgtt attcagtaag taatccaatt
    13741 cggctaagcg gctgtctaag ctattcgtat agggacaatc cgatatgtcg atggagtgaa
    13801 agagcctgat gcactccgca tacagctcga taatcttttc agggctttgt tcatcttcat
    13861 actcttccga gcaaaggacg ccatcggcct cactcatgag cagattgctc cagccatcat
    13921 gccgttcaaa gtgcaggacc tttggaacag gcagctttcc ttccagccat agcatcatgt
    13981 ccttttcccg ttccacatca taggtggtcc ctttataccg gctgtccgtc atttttaaat
    14041 ataggttttc attttctccc accagcttat ataccttagc aggagacatt ccttccgtat
    14101 cttttacgca gcggtatttt tcgatcagtt ttttcaattc cggtgatatt ctcattttag
    14161 ccatttatta tttccttcct cttttctaca gtatttaaag ataccccaag aagctaatta
    14221 taacaagacg aactccaatt cactgttcct tgcattctaa aaccttaaat accagaaaac
    14281 agctttttca aagttgtttt caaagttggc gtataacata gtatcgacgg agccgatttt
    14341 gaaaccgcgg tgatcacagg cagcaacgct ctgtcatcgt tacaatcaac atgctaccct
    14401 ccgcgagatc atccgtgttt caaacccggc agcttagttg ccgttcttcc gaatagcatc
    14461 ggtaacatga gcaaagtctg ccgccttaca acggctctcc cgctgacgcc gtcccggact
    14521 gatgggctgc ctgtatcgag tggtgatttt gtgccgagct gccggtcggg gagctgttgg
    14581 ctggctggtg gcaggatata ttgtggtgta aacaaattga cgcttagaca acttaataac
    14641 acattgcgga cgtttttaat gtactgaatt aacgccgaat taattcgggg gatctggatt
    14701 ttagtactgg attttggttt taggaattag aaattttatt gatagaagta ttttacaaat
    14761 acaaatacat actaagggtt tcttatatgc tcaacacatg agcgaaaccc tataggaacc
    14821 ctaattccct tatctgggaa ctactcacac attattatgg agaaaataga gcttgtcgat
    14881 cgttaattaa tcacctccaa gatcccggac gagtgctggg gcgtcggttt ccactatcgg
    14941 cgagtacttc tacacagcca tcggtccaga cggccgcgct tctgcgggcg atttgtgtac
    15001 gcccgacagt cccggctccg gatcggacga ttgcgtcgca tcgaccctgc gcccaagctg
    15061 catcatcgaa attgccgtca accaagctct gatagagttg gtcaagacca atgcggagca
    15121 tatacgcccg gagccgcggc gatcctgcaa gctccggatg cctccgctcg aagtagcgcg
    15181 tctgctgctc catacaagcc aaccacggcc tccagaagaa gatgttggcg acctcgtatt
    15241 gggaatcccc gaacatcgcc tcgctccagt caatgaccgc tgttatgcgg ccattgtccg
    15301 tcaggacatt gttggagccg aaatccgcgt gcacgaggtg ccggacttcg gggcagtcct
    15361 cggcccaaag catcagctca tcgagagcct gcgcgacgga cgcactgacg gtgtcgtcca
    15421 tcacagtttg ccagtgatac acatggggat cagcaatcgc gcatatgaaa tcacgccatg
    15481 tagtgtattg accgattcct tgcggtccga atgggccgaa cccgctcgtc tggctaagat
    15541 cggccgcagc gatcgcatcc atggcctccg cgaccggctg cagaacagcg ggcagttcgg
    15601 tttcaggcag gtcttgcaac gtgacaccct gtgcacggcg ggagatgcaa taggtcaggc
    15661 tctcgctgaa ttccccaatg tcaagcactt ccggaatcgg gagcgcggcc gatgcaaagt
    15721 gccgataaac ataacgatct ttgtagaaac catcggcgca gctatttacc cgcaggacat
    15781 atccacgccc tcctacatcg aagctgaaag cacgagattc ttcgccctcc gagagctgca
    15841 tcaggtcgga cacgctgtcg aacttttcga tcagaaactt ctcgacagac gtcgcggtga
    15901 gttcaggctt tttcatccta ggagagatag atttgtagag agagactggt gatttcagcg
    15961 tgtcctctcc aaatgaaatg aacttcctta tatagaggaa gggtcttgcg aaggatagtg
    16021 ggattgtgcg tcatccctta cgtcagtgga gatatcacat caatccactt gctttgaaga
    16081 cgtggttgga acgtcttctt tttccacgat gctcctcgtg ggtgggggtc catctttggg
    16141 accactgtcg gcagaggcat cttgaacgat agcctttcct ttatcgcaat gatggcattt
    16201 gtaggtgcca ccttcctttt ctactgtcct tttgatgaag tgacagatag ctgggcaatg
    16261 gaatccgagg aggtttcccg atattaccct ttgttgaaaa gtctcaatag ccctttggtc
    16321 ttctgagact gtatctttga tattcttgga gtagacgaga gtgtcgtgct ccaccatgtt
    16381 cacatcaatc cacttgcttt gaagacgtgg ttggaacgtc ttctttttcc acgatgctcc
    16441 tcgtgggtgg gggtccatct ttgggaccac tgtcggcaga ggcatcttga acgatagcct
    16501 ttcctttatc gcaatgatgg catttgtagg tgccaccttc cttttctact gtccttttga
    16561 tgaagtgaca gatagctggg caatggaatc cgaggaggtt tcccgatatt accctttgtt
    16621 gaaaagtctc aatagccctt tggtcttctg agactgtatc tttgat
//
