LOCUS       sequence-193247-       11015 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cas9 driven by PGK1 promoter. gRNA can be cloned into the BplI 
            sites. HIS3MX marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11015)
  AUTHORS   
  TITLE     Cas9-mediated gene editing in Saccharomyces cerevisiae
  JOURNAL   Protocol Exchange (2017)
REFERENCE   2  (bases 1 to 11015)
  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: "Protocol 
            Exchange (2017)"
COMMENT     Sequence Label: pJH2970
FEATURES             Location/Qualifiers
     source          1..11015
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     terminator      complement(284..481)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(487..1149)
                     /codon_start=1
                     /gene="S. cerevisiae HIS3"
                     /product="imidazoleglycerol-phosphate dehydratase, required
                     for histidine biosynthesis"
                     /label=HIS3
                     /note="yeast auxotrophic marker"
                     /translation="MTEQKALVKRITNETKIQIAISLKGGPLAIEHSIFPEKEAEAVAE
                     QATQSQVINVHTGIGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHTTEDCGIALGQAF
                     KEALGAVRGVKRFGSGFAPLDEALSRAVVDLSNRPYAVVELGLQREKVGDLSCEMIPHF
                     LESFAEASRITLHVDCLRGKNDHHRSESAFKALAVAIREATSPNGTNDVPSTKGVLM"
     promoter        complement(1150..1493)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             2511..6614
                     /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             2511..6614
                     /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             6627..6647
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6651..6671
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6675..6695
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6696..6761
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     terminator      6780..7027
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(6796..6814)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(7087..7542)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7224..7245)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7436..7455
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7668..7690
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7682..7699
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7683..7699
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7706..7725
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7706..7724
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     7757..7773
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7757..7773
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     promoter        7805..8073
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        8101..8175
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      8180..8199
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     promoter        complement(8242..8260)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(8281..8297)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8281..8297)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8294..8316)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8305..8321
                     /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)."
     promoter        complement(join(8329..8335,8336..8353,8354..8359))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8374..8395
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(8512..8529)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8683..9271)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8763..8782)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9442..10233,10234..10302))
                     /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     10065..10084
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10303..10407)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    10444..10947
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     10988..11006
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accataccac cttttcaatt catcattttt tttttattct tttttttgat ttcgaattcg
      241 agctcgttta aactggatgg cggcgttagt atcgaatcga cagcagtata gcgaccagca
      301 ttcacatacg attgacgcat gatattactt tctgcgcact taacttcgca tctgggcaga
      361 tgatgtcgag gcgaaaaaaa atataaatca cgctaacatt tgattaaaat agaacaacta
      421 caatataaaa aaactataca aatgacaagt tcttgaaaac aagaatcttt ttattgtcag
      481 tactgactac ataagaacac ctttggtgga gggaacatcg ttggtaccat tgggcgaggt
      541 ggcttctctt atggcaaccg caagagcctt gaacgcactc tcactacggt gatgatcatt
      601 cttgcctcgc agacaatcaa cgtggagggt aattctgcta gcctctgcaa agctttcaag
      661 aaaatgcggg atcatctcgc aagagagatc tcctactttc tccctttgca aaccaagttc
      721 gacaactgcg tacggcctgt tcgaaagatc taccaccgct ctggaaagtg cctcatccaa
      781 aggcgcaaat cctgatccaa acctttttac tccacgcacg gcccctaggg cctctttaaa
      841 agcttgaccg agagcaatcc cgcagtcttc agtggtgtga tggtcgtcta tgtgtaagtc
      901 accaatgcac tcaacgatta gcgaccagcc ggaatgcttg gccagagcat gtatcatatg
      961 gtccagaaac cctatacctg tgtggacgtt aatcacttgc gattgtgtgg cctgttctgc
     1021 tactgcttct gcctcttttt ctgggaagat cgagtgctct atcgctaggg gaccaccctt
     1081 taaagagatc gcaatctgaa tcttggtttc atttgtaata cgctttacta gggctttctg
     1141 ctctgtcatg gttgtttatg ttcggatgtg atgtgagaac tgtatcctag caagatttta
     1201 aaaggaagta tatgaaagaa gaacctcagt ggcaaatcct aaccttttat atttctctac
     1261 aggggcgcgg cgtggggaca attcaacgcg tctgtgaggg gagcgtttcc ctgctcgcag
     1321 gtccgcagcg aggagccgta atttttgctt cgcgccgtgc ggccatcaaa atgtatggat
     1381 gcaaatgatt atacatgggg atgtatgggc taaatgtacg ggcgacagtc acatcatgcc
     1441 cctgagctgc gcacgtcaag actgtcaagg agggtattct gggcctccat gtcgctggcc
     1501 gggtgacccg gcggggaaag taccttcaaa gaatggggtc ttatcttgtt ttgcaagtac
     1561 cactgagcag gataataata gaaatgataa tatactatag tagagataac gtcgatgact
     1621 tcccatactg taattgcttt tagttgtgta tttttagtgt gcaagtttct gtaaatcgat
     1681 taattttttt ttctttcctc tttttattaa ccttaatttt tattttagat tcctgacttc
     1741 aactcaagac gcacagatat tataacatct gcataatagg catttgcaag aattactcgt
     1801 gagtaaggaa agagtgagga actatcgcat acctgcattt aaagatgccg atttgggcgc
     1861 gaatccttta ttttggcttc accctcatac tattatcagg gccagaaaaa ggaagtgttt
     1921 ccctccttct tgaattgatg ttaccctcat aaagcacgtg gcctcttatc gagaaagaaa
     1981 ttaccgtcgc tcgtgatttg tttgcaaaaa gaacaaaact gaaaaaaccc agacacgctc
     2041 gacttcctgt cttcctattg attgcagctt ccaatttcgt cacacaacaa ggtcctagcg
     2101 acggctcaca ggttttgtaa caagcaatcg aaggttctgg aatggcggga aagggtttag
     2161 taccacatgc tatgatgccc actgtgatct ccagagcaaa gttcgttcga tcgtactgtt
     2221 actctctctc tttcaaacag aattgtccga atcgtgtgac aacaacagcc tgttctcaca
     2281 cactcttttc ttctaaccaa gggggtggtt tagtttagta gaacctcgtg aaacttacat
     2341 ttacatatat ataaacttgc ataaattggt caatgcaaga aatacatatt tggtcttttc
     2401 taattcgtag tttttcaagt tcttagatgc tttctttttc tcttttttac agatcatcaa
     2461 ggaagtaatt atctactttt tacaacaaat ataaaacacc ggatctcaaa atggataaaa
     2521 agtatagtat tggtttagat attggtacta actctgtggg ttgggcagtt atcactgacg
     2581 aatataaagt tccatcaaag aaatttaagg tgttaggtaa cactgacaga cactcaataa
     2641 aaaagaatct tatcggtgct cttttgttcg actccggtga aactgccgag gctacacgtt
     2701 taaaaagaac agcaagaaga agatataccc gtagaaaaaa tagaatatgt tatttacaag
     2761 aaatcttttc taatgaaatg gctaaagttg atgattcctt tttccataga ttggaagagt
     2821 catttttggt tgaagaagac aaaaagcatg agagacatcc aatctttggg aatatagttg
     2881 atgaagtggc ttaccatgaa aaatatccta ccatttatca tttaagaaag aaattggtag
     2941 attcaactga taaagctgac cttagattaa tctatttagc acttgcccat atgattaaat
     3001 ttagaggtca ttttttgatt gaaggtgatt tgaacccaga taattctgac gtggataaat
     3061 tatttattca attagtccaa acctacaacc aattatttga ggaaaatcca attaatgcta
     3121 gtggtgtcga tgccaaagct atattatcag ccagattatc aaaatctaga cgtttggaaa
     3181 atttgattgc ccaattgcca ggagaaaaaa agaatggatt atttggaaac ttgatcgcat
     3241 tatcattggg tttgacacca aattttaaat ctaattttga tttagctgaa gatgctaaat
     3301 tacaattatc aaaagacacc tatgacgacg atttggacaa tttacttgct caaattggtg
     3361 atcaatatgc agatttgttc ttagctgcta aaaacttatc tgatgctatt ttgttgtctg
     3421 atattttgag agtgaacaca gaaataacca aagctccatt atcagcatct atgatcaaac
     3481 gttatgatga acaccatcag gatttgactt tattgaaagc tttggtgaga caacaattgc
     3541 cagagaagta taaagaaatc tttttcgatc aatctaaaaa cgggtatgca ggttatattg
     3601 atgggggtgc ctcccaagag gaattttaca aatttataaa acctatttta gaaaagatgg
     3661 atgggactga ggaacttttg gtcaaattga acagagaaga tttgttacgt aaacagagaa
     3721 cttttgataa tggtagtata cctcaccaaa ttcatttggg tgagttgcat gcaattttaa
     3781 gaagacaaga agatttttat ccatttttaa aagataatag agaaaaaatc gagaaaattt
     3841 taacctttag aattccatac tatgttgggc ctttggctag aggtaattca agatttgcct
     3901 ggatgacacg taaatcagaa gaaactatta ccccttggaa ttttgaagag gttgttgata
     3961 aaggagcatc agcacagagt tttattgaaa gaatgaccaa tttcgataaa aacttaccaa
     4021 atgaaaaagt tttaccaaaa cattccttgt tatacgaata ttttactgtt tacaatgaac
     4081 ttacaaaggt taaatatgtt actgaaggta tgcgtaagcc agccttttta tctggagaac
     4141 agaaaaaggc aatagttgat ttattgttta aaacaaatag aaaagttact gttaaacaat
     4201 taaaagaaga ttactttaag aaaattgaat gttttgattc agttgaaatc agtggtgttg
     4261 aagacagatt taatgctagt ttaggaactt accatgattt acttaaaatt atcaaagata
     4321 aagatttctt ggataacgaa gaaaatgaag acattttaga agacattgtt ttaaccttaa
     4381 ctttattcga agatagagag atgattgaag aacgtttgaa gacttatgca catttgtttg
     4441 acgataaagt gatgaaacag ttgaaaagaa gacgttatac tggatggggt agattgtctc
     4501 gtaaattgat caatggaatt agagataaac aaagtggtaa aactatcttg gactttttga
     4561 aatctgacgg atttgctaat agaaatttca tgcaattgat ccacgacgat agtttgacat
     4621 ttaaagaaga catccaaaag gcccaagtga gtgggcaagg tgattcatta catgaacata
     4681 ttgcaaattt agccggatct cctgctatta agaaagggat attacaaact gttaaagttg
     4741 tggatgaatt agtgaaagta atgggaagac ataaacctga aaacattgtc attgagatgg
     4801 caagagaaaa tcaaactaca caaaaaggac agaaaaatag tagagaacgt atgaaaagaa
     4861 tagaagaggg tattaaagaa ttgggtagtc aaatattgaa agaacaccca gtggaaaata
     4921 cccagttgca aaatgaaaaa ttatatcttt actaccttca aaatggacgt gatatgtatg
     4981 ttgatcagga attagatata aatagacttt cagattatga tgtagatcat atagttccac
     5041 aatctttctt gaaagatgat tccatagaca ataaagtatt aactagaagt gataaaaata
     5101 gaggtaaaag tgataatgtc ccaagtgagg aagtcgtcaa aaagatgaaa aattactggc
     5161 gtcaactttt gaatgctaaa ttaattactc aaagaaaatt tgataatttg actaaagcag
     5221 aaagaggtgg gctttctgaa ttagataaag ccgggttcat taaaagacaa ttggtcgaaa
     5281 ctagacaaat tactaaacat gttgcccaaa ttttagattc ccgtatgaac actaagtatg
     5341 acgaaaatga taagttaata cgtgaggtta aagtcattac tttaaaatca aaacttgtct
     5401 ctgatttcag aaaggatttc caattctata aagttagaga aattaataat tatcatcatg
     5461 ctcatgatgc atatttgaat gctgtagttg gaactgcttt aatcaagaaa taccctaaat
     5521 tagaatctga atttgtatat ggtgattaca aagtctatga tgttagaaag atgattgcta
     5581 aatcagaaca agaaattggt aaagctacag ctaaatactt cttttactct aacattatga
     5641 atttctttaa aacagaaatt actttggcaa acggtgaaat tagaaaaaga cctcttattg
     5701 aaacaaatgg tgagactgga gagatagttt gggacaaagg gcgtgatttc gctactgtta
     5761 gaaaagtttt atcaatgcca caagttaaca ttgtaaagaa aacagaggtt caaactggtg
     5821 gtttctcaaa agaaagtatt ttgcctaaaa gaaatagtga taaattgatt gccagaaaaa
     5881 aggattggga tccaaagaaa tatggtggtt tcgactcacc aaccgtagcc tattctgttt
     5941 tggttgtggc aaaggttgaa aagggtaaaa gtaaaaagct taaatcagta aaagaacttt
     6001 tgggtattac aataatggaa agaagttcct ttgaaaagaa ccctattgat tttttggaag
     6061 ctaaaggtta taaggaagta aagaaggact taataatcaa attgcctaaa tattctttat
     6121 ttgaattaga aaatgggaga aaaagaatgt tggcttctgc tggagaattg caaaagggta
     6181 atgaattagc attgccttcc aaatatgtta acttcttgta tttagcttca cactatgaaa
     6241 agttgaaagg gtcaccagaa gataacgagc aaaaacaatt atttgttgaa caacacaaac
     6301 actacttaga tgagattata gaacaaatta gtgaattcag taaaagagtg atattagctg
     6361 atgcaaattt agataaagtt ttgtcagcct ataacaaaca tagagataag ccaattagag
     6421 aacaagcaga aaacattatt cacttattta cccttaccaa tttaggagca cctgctgctt
     6481 tcaagtattt tgatacaaca attgatcgta aaagatatac ctcaacaaaa gaagtcttag
     6541 acgccacctt aattcatcaa tcaatcactg gattgtatga gacaagaatt gatttgtctc
     6601 aattgggtgg tgatgaaggg gctgatccta agaagaaaag aaaagttgat ccaaagaaaa
     6661 agcgtaaggt ggatcctaag aaaaagagaa aggttgacta caaagaccat gacggtgatt
     6721 ataaagatca tgacatcgac tacaaggatg acgatgacaa gtgataatct cttctcgagt
     6781 catgtaatta gttatgtcac gcttacattc acgccctccc cccacatccg ctctaaccga
     6841 aaaggaagga gttagacaac ctgaagtcta ggtccctatt tattttttta tagttatgtt
     6901 agtattaaga acgttattta tatttcaaat ttttcttttt tttctgtaca gacgcgtgta
     6961 cgcatgtaac attatactga aaaccttgct tgagaaggtt ttgggacgct cgaaggcttt
     7021 aatttgcggc cggtactgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc
     7081 atcaggaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     7141 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
     7201 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
     7261 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
     7321 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     7381 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     7441 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     7501 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtccattcgc cattcaggct
     7561 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa
     7621 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
     7681 ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat tggagctcca
     7741 ccgcggtggc ggccgctcta gaactagtgg atcccccggg ctgcaggaat tcgatatcaa
     7801 gctttctttg aaaagataat gtatgattat gctttcactc atatttatac agaaacttga
     7861 tgttttcttt cgagtatata caaggtgatt acatgtacgt ttgaagtaca actctagatt
     7921 ttgtagtgcc ctcttgggct agcggtaaag gtgcgcattt tttcacaccc tacaatgttc
     7981 tgttcaaaag attttggtca aacgctgtag aagtgaaagt tggtgcgcat gtttcggcgt
     8041 tcgaaacttc tccgcagtga aagataaatg atcctagatg cgagatatcc tcatgcctag
     8101 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt
     8161 ggcaccgagt cggtggtgct ttttttgttt tttatgtcta ccgtcgacct cgaggggggg
     8221 cccggtaccc agcttttgtt ccctttagtg agggttaatt tcgagcttgg cgtaatcatg
     8281 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     8341 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     8401 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     8461 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     8521 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     8581 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     8641 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     8701 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     8761 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     8821 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     8881 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     8941 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     9001 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     9061 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     9121 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     9181 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     9241 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     9301 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     9361 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     9421 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     9481 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     9541 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     9601 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     9661 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     9721 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     9781 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     9841 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     9901 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     9961 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
    10021 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
    10081 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
    10141 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
    10201 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
    10261 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
    10321 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
    10381 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgggtcctt
    10441 ttcatcacgt gctataaaaa taattataat ttaaattttt taatataaat atataaatta
    10501 aaaatagaaa gtaaaaaaag aaattaaaga aaaaatagtt tttgttttcc gaagatgtaa
    10561 aagactctag ggggatcgcc aacaaatact accttttatc ttgctcttcc tgctctcagg
    10621 tattaatgcc gaattgtttc atcttgtctg tgtagaagac cacacacgaa aatcctgtga
    10681 ttttacattt tacttatcgt taatcgaatg tatatctatt taatctgctt ttcttgtcta
    10741 ataaatatat atgtaaagta cgctttttgt tgaaattttt taaacctttg tttatttttt
    10801 tttcttcatt ccgtaactct tctaccttct ttatttactt tctaaaatcc aaatacaaaa
    10861 cataaaaata aataaacaca gagtaaattc ccaaattatt ccatcattaa aagatacgag
    10921 gcgcgtgtaa gttacaggca agcgatccgt ctaagaaacc attattatca tgacattaac
    10981 ctataaaaat aggcgtatca cgaggccctt tcgtc
//
