LOCUS       sequence-201371-       13951 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TetR inducible dCas9-VPR plasmid with pRPR-NotI-tRPR1 Csy4-ready 
            site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13951)
  AUTHORS   Ferreira R, Skrekas C, Nielsen J, David F
  TITLE     Multiplexed CRISPR/Cas9 Genome Editing and Gene Regulation using 
            Csy4 in Saccharomyces cerevisiae.
  JOURNAL   ACS Synth Biol. 2017 Nov 21. doi: 10.1021/acssynbio.7b00259.
  PUBMED    29161506
REFERENCE   2  (bases 1 to 13951)
  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: "ACS Synth 
            Biol. 2017 Nov 21. doi: 10.1021/acssynbio.7b00259."
COMMENT     Sequence Label: dCas9v2
FEATURES             Location/Qualifiers
     source          1..13951
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        419..523
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(524..592,593..1384)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(742..761)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1555..2143
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2044..2063
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    2538..2556
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     terminator      2629..2691
                     /label=RPR1 terminator
                     /note="transcription terminator for the S. cerevisiae RNase
                     P RNA gene"
     CDS             3826..4449
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(3899..3918)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     4946..4963
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5080..5101
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5116..5121,5122..5139,5140..5146)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5154..5170
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     5159..5181
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5178..5194
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5178..5194
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5213..5233
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5215..5233
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5251..5649
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     CDS             5672..5692
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(5702..5728,5729..5731,5732..8215,8216..8221,8222..8287,
                     8288..8290,8291..9805)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=Cas9m4
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the mutations D10A, D839A, 
                     H840A, and N863A (Mali et al., 2013)"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             join(5702..5728,5729..5731,5732..8218,8219..8221,
                     8222..9805)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             5702..9805
                     /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="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             9818..9838
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             9863..10012
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     CDS             10037..10057
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             10487..10843
                     /codon_start=1
                     /product="transcriptional activation domain of human RelA, 
                     also known as p65 (O'Shea and Perkins, 2008)"
                     /label=RelA (p65) AD
                     /translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS
                     EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD
                     EDFSSIADMDFSALL"
     CDS             10862..11431
                     /codon_start=1
                     /product="transcriptional activation domain from the human 
                     herpesvirus 4 (Epstein-Barr virus) replication and 
                     transcription activator Rta/BRLF1 (Hardwick et al., 1992; 
                     Chavez et al., 2015)"
                     /label=Rta AD
                     /translation="RDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWAN
                     RPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKAL
                     REMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEIL
                     DTFLNDECLLHAMHISTGLSIFDTSLF"
     protein_bind    complement(11436..11460)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(11493..11509)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11494..11510)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      11512..11759
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(11528..11546)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(11777..11796)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(11778..11796)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(11806..11823)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(11806..11822)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11815..11837)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      11963..12418
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(12050..12069)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     12260..12281
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(12549..13352)
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(13353..13568)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(13592..13611)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     13711..13733
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(13771..13789)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    join(13830..13951,1..382)
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
ORIGIN
        1 gtagaagagt tacggaatga agaaaaaaaa ataaacaaag gtttaaaaaa tttcaacaaa
       61 aagcgtactt tacatatata tttattagac aagaaaagca gattaaatag atatacattc
      121 gattaacgat aagtaaaatg taaaatcaca ggattttcgt gtgtggtctt ctacacagac
      181 aagatgaaac aattcggcat taatacctga gagcaggaag agcaagataa aaggtagtat
      241 ttgttggcga tccccctaga gtcttttaca tcttcggaaa acaaaaacta ttttttcttt
      301 aatttctttt tttactttct atttttaatt tatatattta tattaaaaaa tttaaattat
      361 aattattttt atagcacgtg atgaaaagga cccaggtggc acttttcggg gaaatgtgcg
      421 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
      481 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
      541 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
      601 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
      661 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
      721 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
      781 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
      841 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
      901 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
      961 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     1021 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     1081 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     1141 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     1201 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     1261 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     1321 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     1381 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     1441 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     1501 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     1561 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     1621 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     1681 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     1741 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     1801 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     1861 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     1921 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     1981 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     2041 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     2101 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     2161 ctttttacgg ttcctggcct tttgctggcc ttttgctggc cttttgctcg ggggatctgc
     2221 caattgaaca taacatggta gttacatata ctagtaatat ggttcggcac acattaaaag
     2281 tataaaaact atctgaatta cgaattacat atattggtca taaaaatcaa tcaatcatcg
     2341 tgtgttttat atgtctctta tctaagtata agaatatcca tagttaatat tcacttacgc
     2401 taccttttaa cctgtaatca ttgtcaacag gatatgttaa cgacccacat tgataaacgc
     2461 tagtatttct ttttcctctt cttattggcc ggctgtctct atactcccct atagtctgtt
     2521 tcttttcgtt tcgattgtcc ctatcagtga tagagatggc gcacatggta cgctgtggtg
     2581 ctcgcggctg ggaacgaaac tctgggagct gcgattggca gcggccgccc atatccaact
     2641 tccaatttaa tctttctttt ttaattttca cttatttgcg atacagaaag aaaaaagcga
     2701 tagtaactat tgaattttgt ttggatttgg ttagattaga tatggtttct ctttatattt
     2761 acatgctaaa aatgggctac accagagata cataattaga tatatatacg ccagtacacc
     2821 ttatcggccc aagccttgtc ccaaggcagc gttttgttct tggaaacgct gccctacacg
     2881 ttcgctatgc ttcaagaact tttctgagca cttcatgatg catgtttgtt ccttattggt
     2941 tagctttgat gttgtgaagt cattgacaca gtctgtgaaa catctttcta ccagattaga
     3001 gtacaaacgc atgaaatcct tcatttgctt ttgttccact actttttgga actcttgttg
     3061 ttctttcctg caggtagtcg tgcaatgtat gactttaaga tttgtgagca ggaagaaaag
     3121 ggagaatctt ctaacgataa acccttgaaa aactgggtag actacgctat gttgagttgc
     3181 tacgcaggct gcacaattac acgagaatgc tcccgcctag gatttaaggc taagggacgt
     3241 gcaatgcaga cgacagatct aaatgaccgt gtcggtgaag tgttcgccaa acttttcggt
     3301 taacacatgc agtgatgcac gcgcgatggt gctaagttac atatatatat atatgtatag
     3361 ccatagtgat gtctaagtaa cctttatggt atatttctta atgtggaaag atactagcgc
     3421 gcgcacccac acacaagctt cgtcttttct tgaagaaaag aggaagctcg ctaaatggga
     3481 ttccactttc cgttccctgc cagctgatgg aaaaaggtta gtggaacgat gaagaataaa
     3541 aagagagatc cactgaggtg aaatttcagc tgacagcgag tttcatgatc gtgatgaaca
     3601 atggtaacga gttgtggctg ttgccaggga gggtggttct caacttttaa tgtatggcca
     3661 aatcgctact tgggtttgtt atataacaaa gaagaaataa tgaactgatt ctcttcctcc
     3721 ttcttgtcct ttcttaattc tgttgtaatt accttccttt gtaatttttt ttgtaattat
     3781 tcttcttaat aatccaaaca aacacacata ttacaatata ttaaaatgtc tagattagat
     3841 aaaagtaaag tgattaacag cgcattagag ctgcttaatg aggtcggaat cgaaggttta
     3901 acaacccgta aactcgccca gaagctaggt gtagagcagc ctacattgta ttggcatgta
     3961 aaaaataagc gggctttgct cgacgcctta gccattgaga tgttagatag gcaccatact
     4021 cacttttgcc ctttagaagg ggaaagctgg caagattttt tacgtaataa cgctaaaagt
     4081 tttagatgtg ctttactaag tcatcgcgat ggagcaaaag tacatttagg tacacggcct
     4141 acagaaaaac agtatgaaac tctcgaaaat caattagcct ttttatgcca acaaggtttt
     4201 tcactagaga atgcattata tgcactcagc gctgtggggc attttacttt aggttgcgta
     4261 ttggaagatc aagagcatca agtcgctaaa gaagaaaggg aaacacctac tactgatagt
     4321 atgccgccat tattacgaca agctatcgaa ttatttgatc accaaggtgc agagccagcc
     4381 ttcttattcg gccttgaatt gatcatatgc ggattagaaa aacaacttaa atgtgaaagt
     4441 gggtcttaag tctgaagaat gaatgatttg atgatttctt tttccctcca tttttcttac
     4501 tgaatatatc aatgatatag acttgtatag tttattattt caaattaagt agctatatat
     4561 agtcaagata acgtttgttt gacacgatta cattattcgt cgacatcttt tttcagcctg
     4621 tcgtggtagc aatttgagga gtattattaa ttgaataggt tcattttgcg ctcgcataaa
     4681 cagttttcgt cagggacagt atgttggaat gagtggtaat taatggtgac atgacatgtt
     4741 atagcaataa ccttgatgtt tacatcgtag tttaatgtac accccgcgaa ttcgttcaag
     4801 taggagtgca ccaattgcaa agggaaaagc tgaatgggca gttcgaaccg cggtctttcc
     4861 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     4921 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc
     4981 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
     5041 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca
     5101 ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag
     5161 cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa
     5221 ccctcactaa agggaacaaa agctggagct catagcttca aaatgtttct actccttttt
     5281 tactcttcca gattttctcg gactccgcgc atcgccgtac cacttcaaaa cacccaagca
     5341 cagcatacta aatttcccct ctttcttcct ctagggtgtc gttaattacc cgtactaaag
     5401 gtttggaaaa gaaaaaagag accgcctcgt ttctttttct tcgtcgaaaa aggcaataaa
     5461 aatttttatc acgtttcttt ttcttgaaaa tttttttttt gatttttttc tctttcgatg
     5521 acctcccatt gatatttaag ttaataaacg gtcttcaatt tctcaagttt cagtttcatt
     5581 tttcttgttc tattacaact ttttttactt cttgctcatt agaaagaaag catagcaatc
     5641 taatctaagt tttctagaga tgtctagagc cccaaagaag aagagaaaag ttagacccgg
     5701 gatggacaag aagtactcca ttgggctcgc tatcggcaca aacagcgtcg gctgggccgt
     5761 cattacggac gagtacaagg tgccgagcaa aaaattcaaa gttctgggca ataccgatcg
     5821 ccacagcata aagaagaacc tcattggcgc cctcctgttc gactccgggg agacggccga
     5881 agccacgcgg ctcaaaagaa cagcacggcg cagatatacc cgcagaaaga atcggatctg
     5941 ctacctgcag gagatcttta gtaatgagat ggctaaggtg gatgactctt tcttccatag
     6001 gctggaggag tcctttttgg tggaggagga taaaaagcac gagcgccacc caatctttgg
     6061 caatatcgtg gacgaggtgg cgtaccatga aaagtaccca accatatatc atctgaggaa
     6121 gaagcttgta gacagtactg ataaggctga cttgcggttg atctatctcg cgctggcgca
     6181 tatgatcaaa tttcggggac acttcctcat cgagggggac ctgaacccag acaacagcga
     6241 tgtcgacaaa ctctttatcc aactggttca gacttacaat cagcttttcg aagagaaccc
     6301 gatcaacgca tccggagttg acgccaaagc aatcctgagc gctaggctgt ccaaatcccg
     6361 gcggctcgaa aacctcatcg cacagctccc tggggagaag aagaacggcc tgtttggtaa
     6421 tcttatcgcc ctgtcactcg ggctgacccc caactttaaa tctaacttcg acctggccga
     6481 agatgccaag cttcaactga gcaaagacac ctacgatgat gatctcgaca atctgctggc
     6541 ccagatcggc gaccagtacg cagacctttt tttggcggca aagaacctgt cagacgccat
     6601 tctgctgagt gatattctgc gagtgaacac ggagatcacc aaagctccgc tgagcgctag
     6661 tatgatcaag cgctatgatg agcaccacca agacttgact ttgctgaagg cccttgtcag
     6721 acagcaactg cctgagaagt acaaggaaat tttcttcgat cagtctaaaa atggctacgc
     6781 cggatacatt gacggcggag caagccagga ggaattttac aaatttatta agcccatctt
     6841 ggaaaaaatg gacggcaccg aggagctgct ggtaaagctt aacagagaag atctgttgcg
     6901 caaacagcgc actttcgaca atggaagcat cccccaccag attcacctgg gcgaactgca
     6961 cgctatcctc aggcggcaag aggatttcta cccctttttg aaagataaca gggaaaagat
     7021 tgagaaaatc ctcacatttc ggatacccta ctatgtaggc cccctcgccc ggggaaattc
     7081 cagattcgcg tggatgactc gcaaatcaga agagaccatc actccctgga acttcgagga
     7141 agtcgtggat aagggggcct ctgcccagtc cttcatcgaa aggatgacta actttgataa
     7201 aaatctgcct aacgaaaagg tgcttcctaa acactctctg ctgtacgagt acttcacagt
     7261 ttataacgag ctcaccaagg tcaaatacgt cacagaaggg atgagaaagc cagcattcct
     7321 gtctggagag cagaagaaag ctatcgtgga cctcctcttc aagacgaacc ggaaagttac
     7381 cgtgaaacag ctcaaagaag actatttcaa aaagattgaa tgtttcgact ctgttgaaat
     7441 cagcggagtg gaggatcgct tcaacgcatc cctgggaacg tatcacgatc tcctgaaaat
     7501 cattaaagac aaggacttcc tggacaatga ggagaacgag gacattcttg aggacattgt
     7561 cctcaccctt acgttgtttg aagataggga gatgattgaa gaacgcttga aaacttacgc
     7621 tcatctcttc gacgacaaag tcatgaaaca gctcaagagg cgccgatata caggatgggg
     7681 gcggctgtca agaaaactga tcaatgggat ccgagacaag cagagtggaa agacaatcct
     7741 ggattttctt aagtccgatg gatttgccaa ccggaacttc atgcagttga tccatgatga
     7801 ctctctcacc tttaaggagg acatccagaa agcacaagtt tctggccagg gggacagtct
     7861 tcacgagcac atcgctaatc ttgcaggtag cccagctatc aaaaagggaa tactgcagac
     7921 cgttaaggtc gtggatgaac tcgtcaaagt aatgggaagg cataagcccg agaatatcgt
     7981 tatcgagatg gcccgagaga accaaactac ccagaaggga cagaagaaca gtagggaaag
     8041 gatgaagagg attgaagagg gtataaaaga actggggtcc caaatcctta aggaacaccc
     8101 agttgaaaac acccagcttc agaatgagaa gctctacctg tactacctgc agaacggcag
     8161 ggacatgtac gtggatcagg aactggacat caatcggctc tccgactacg acgtggatgc
     8221 tatcgtgccc cagtcttttc tcaaagatga ttctattgat aataaagtgt tgacaagatc
     8281 cgataaaaat agagggaaga gtgataacgt cccctcagaa gaagttgtca agaaaatgaa
     8341 aaattattgg cggcagctgc tgaacgccaa actgatcaca caacggaagt tcgataatct
     8401 gactaaggct gaacgaggtg gcctgtctga gttggataaa gccggcttca tcaaaaggca
     8461 gcttgttgag acacgccaga tcaccaagca cgtggcccaa attctcgatt cacgcatgaa
     8521 caccaagtac gatgaaaatg acaaactgat tcgagaggtg aaagttatta ctctgaagtc
     8581 taagctggtc tcagatttca gaaaggactt tcagttttat aaggtgagag agatcaacaa
     8641 ttaccaccat gcgcatgatg cctacctgaa tgcagtggta ggcactgcac ttatcaaaaa
     8701 atatcccaag cttgaatctg aatttgttta cggagactat aaagtgtacg atgttaggaa
     8761 aatgatcgca aagtctgagc aggaaatagg caaggccacc gctaagtact tcttttacag
     8821 caatattatg aattttttca agaccgagat tacactggcc aatggagaga ttcggaagcg
     8881 accacttatc gaaacaaacg gagaaacagg agaaatcgtg tgggacaagg gtagggattt
     8941 cgcgacagtc cggaaggtcc tgtccatgcc gcaggtgaac atcgttaaaa agaccgaagt
     9001 acagaccgga ggcttctcca aggaaagtat cctcccgaaa aggaacagcg acaagctgat
     9061 cgcacgcaaa aaagattggg accccaagaa atacggcgga ttcgattctc ctacagtcgc
     9121 ttacagtgta ctggttgtgg ccaaagtgga gaaagggaag tctaaaaaac tcaaaagcgt
     9181 caaggaactg ctgggcatca caatcatgga gcgatcaagc ttcgaaaaaa accccatcga
     9241 ctttctcgag gcgaaaggat ataaagaggt caaaaaagac ctcatcatta agcttcccaa
     9301 gtactctctc tttgagcttg aaaacggccg gaaacgaatg ctcgctagtg cgggcgagct
     9361 gcagaaaggt aacgagctgg cactgccctc taaatacgtt aatttcttgt atctggccag
     9421 ccactatgaa aagctcaaag ggtctcccga agataatgag cagaagcagc tgttcgtgga
     9481 acaacacaaa cactaccttg atgagatcat cgagcaaata agcgaattct ccaaaagagt
     9541 gatcctcgcc gacgctaacc tcgataaggt gctttctgct tacaataagc acagggataa
     9601 gcccatcagg gagcaggcag aaaacattat ccacttgttt actctgacca acttgggcgc
     9661 gcctgcagcc ttcaagtact tcgacaccac catagacaga aagcggtaca cctctacaaa
     9721 ggaggtcctg gacgccacac tgattcatca gtcaattacg gggctctatg aaacaagaat
     9781 cgacctctct cagctcggtg gagacagcag ggctgacccc aagaagaaga ggaaggtgga
     9841 ggccagcggt tccggacggg ctgacgcatt ggacgatttt gatctggata tgctgggaag
     9901 tgacgccctc gatgattttg accttgacat gcttggttcg gatgcccttg atgactttga
     9961 cctcgacatg ctcggcagtg acgcccttga tgatttcgac ctggacatgc tgattaactc
    10021 tagaagttcc ggatctccga aaaagaaacg caaagttggt agccagtacc tgcccgacac
    10081 cgacgaccgg caccggatcg aggaaaagcg gaagcggacc tacgagacat tcaagagcat
    10141 catgaagaag tcccccttca gcggccccac cgaccctaga cctccaccta gaagaatcgc
    10201 cgtgcccagc agatccagcg ccagcgtgcc aaaacctgcc ccccagcctt accccttcac
    10261 cagcagcctg agcaccatca actacgacga gttccctacc atggtgttcc ccagcggcca
    10321 gatctctcag gcctctgctc tggctccagc ccctcctcag gtgctgcctc aggctcctgc
    10381 tcctgcacca gctccagcca tggtgtctgc actggctcag gcaccagcac ccgtgcctgt
    10441 gctggctcct ggacctccac aggctgtggc tccaccagcc cctaaaccta cacaggccgg
    10501 cgagggcaca ctgtctgaag ctctgctgca gctgcagttc gacgacgagg atctgggagc
    10561 cctgctggga aacagcaccg atcctgccgt gttcaccgac ctggccagcg tggacaacag
    10621 cgagttccag cagctgctga accagggcat ccctgtggcc cctcacacca ccgagcccat
    10681 gctgatggaa taccccgagg ccatcacccg gctcgtgaca ggcgctcaga ggcctcctga
    10741 tccagctcct gcccctctgg gagcaccagg cctgcctaat ggactgctgt ctggcgacga
    10801 ggacttcagc tctatcgccg acatggactt ctccgcactg ctgggtagcg gatcgggatc
    10861 tcgggattcc agggaaggga tgtttttgcc gaagcctgag gccggctccg ctattagtga
    10921 cgtgtttgag ggccgcgagg tgtgccagcc aaaacgaatc cggccatttc atcctccagg
    10981 aagtccatgg gccaaccgcc cactccccgc cagcctcgca ccaacaccaa ccggtccagt
    11041 acatgagcca gtcgggtcac tgaccccggc accagtccct cagccactgg atccagcgcc
    11101 cgcagtgact cccgaggcca gtcacctgtt ggaggatccc gatgaagaga cgagccaggc
    11161 tgtcaaagcc cttcgggaga tggccgatac tgtgattccc cagaaggaag aggctgcaat
    11221 ctgtggccaa atggaccttt cccatccgcc cccaaggggc catctggatg agctgacaac
    11281 cacacttgag tccatgaccg aggatctgaa cctggactca cccctgaccc cggaattgaa
    11341 cgagattctg gataccttcc tgaacgacga gtgcctcttg catgccatgc atatcagcac
    11401 aggactgtcc atcttcgaca catctctgtt ttgaaaccca gctttcttgt acaaagtggt
    11461 gatgggctgc aggaattcga tatcaagctt atcgataccg tcgacctcga gtcatgtaat
    11521 tagttatgtc acgcttacat tcacgccctc cccccacatc cgctctaacc gaaaaggaag
    11581 gagttagaca acctgaagtc taggtcccta tttatttttt tatagttatg ttagtattaa
    11641 gaacgttatt tatatttcaa atttttcttt tttttctgta cagacgcgtg tacgcatgta
    11701 acattatact gaaaaccttg cttgagaagg ttttgggacg ctcgaaggct ttaatttgcg
    11761 gccggtaccc aattcgccct atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt
    11821 acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc
    11881 ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt
    11941 gcgcagcctg aatggcgaat ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
    12001 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
    12061 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
    12121 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
    12181 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
    12241 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
    12301 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
    12361 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttcc
    12421 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata gggtaataac
    12481 tgatataatt aaattgaagc tctaatttgt gagtttagta tacatgcatt tacttataat
    12541 acagtttttt agttttgctg gccgcatctt ctcaaatatg cttcccagcc tgcttttctg
    12601 taacgttcac cctctacctt agcatccctt ccctttgcaa atagtcctct tccaacaata
    12661 ataatgtcag atcctgtaga gaccacatca tccacggttc tatactgttg acccaatgcg
    12721 tctcccttgt catctaaacc cacaccgggt gtcataatca accaatcgta accttcatct
    12781 cttccaccca tgtctctttg agcaataaag ccgataacaa aatctttgtc gctcttcgca
    12841 atgtcaacag tacccttagt atattctcca gtagataggg agcccttgca tgacaattct
    12901 gctaacatca aaaggcctct aggttccttt gttacttctt ctgccgcctg cttcaaaccg
    12961 ctaacaatac ctgggcccac cacaccgtgt gcattcgtaa tgtctgccca ttctgctatt
    13021 ctgtatacac ccgcagagta ctgcaatttg actgtattac caatgtcagc aaattttctg
    13081 tcttcgaaga gtaaaaaatt gtacttggcg gataatgcct ttagcggctt aactgtgccc
    13141 tccatggaaa aatcagtcaa gatatccaca tgtgttttta gtaaacaaat tttgggacct
    13201 aatgcttcaa ctaactccag taattccttg gtggtacgaa catccaatga agcacacaag
    13261 tttgtttgct tttcgtgcat gatattaaat agcttggcag caacaggact aggatgagta
    13321 gcagcacgtt ccttatatgt agctttcgac atgatttatc ttcgtttcct gcaggttttt
    13381 gttctgtgca gttgggttaa gaatactggg caatttcatg tttcttcaac actacatatg
    13441 cgtatatata ccaatctaag tctgtgctcc ttccttcgtt cttccttctg ttcggagatt
    13501 accgaatcaa aaaaatttca aggaaaccga aatcaaaaaa aagaataaaa aaaaaatgat
    13561 gaattgaaaa ggtggtatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
    13621 gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
    13681 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
    13741 cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta
    13801 atgtcatgat aataatggtt tcttagacgg atcgcttgcc tgtaacttac acgcgcctcg
    13861 tatcttttaa tgatggaata atttgggaat ttactctgtg tttatttatt tttatgtttt
    13921 gtatttggat tttagaaagt aaataaagaa g
//
