LOCUS       sequence-403767-       11591 bp    DNA     circular SYN 14-APR-2025
DEFINITION  TEF1p-Cas9(noBsmBI)-CYC1t and tRNA-GFP-SUP4t with CEN-URA3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11591)
  AUTHORS   Zhao Y, Coelho C, Lauer S, Majewski M, Laurent JM, Brosh R, Boeke JD
  TITLE     CREEPY: CRISPR-mediated editing of synthetic episomes in yeast.
  JOURNAL   Nucleic Acids Res. 2023 Jun 16:gkad491. doi: 10.1093/nar/gkad491.
  PUBMED    37326023
REFERENCE   2  (bases 1 to 11591)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nucleic 
            Acids Res. 2023 Jun 16:gkad491. doi: 10.1093/nar/gkad491."
COMMENT     Sequence Label: pYZ959
FEATURES             Location/Qualifiers
     source          1..11591
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..24,25..31)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    39..55
                     /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     44..66
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     63..79
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     63..79
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     98..118
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        100..118
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        136..534
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     primer_bind     538..554
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     538..554
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             566..4669
                     /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             566..4669
                     /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             566..4669
                     /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             4682..4702
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      4718..4965
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(4734..4752)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     RBS             5627..5638
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             5645..6358
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      6370..6441
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      6457..6484
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_RNA        6498..6573
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(7011..7030)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7012..7030)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(7040..7057)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7040..7056)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7049..7071)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      7197..7652
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7284..7303)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7494..7515
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(7783..8586)
                     /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(8587..8802)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(8826..8845)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(8945..8963)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    9004..9507
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        9544..9648
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9649..9717,9718..10509)
                     /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(9867..9886)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10680..11268
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11169..11188
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11422..11439
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11556..11577
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca
       61 cacaggaaac agctatgacc atgattacgc caagcgcgca attaaccctc actaaaggga
      121 acaaaagctg gagctcatag cttcaaaatg tttctactcc ttttttactc ttccagattt
      181 tctcggactc cgcgcatcgc cgtaccactt caaaacaccc aagcacagca tactaaattt
      241 cccctctttc ttcctctagg gtgtcgttaa ttacccgtac taaaggtttg gaaaagaaaa
      301 aagagaccgc ctcgtttctt tttcttcgtc gaaaaaggca ataaaaattt ttatcacgtt
      361 tctttttctt gaaaattttt tttttgattt ttttctcttt cgatgacctc ccattgatat
      421 ttaagttaat aaacggtctt caatttctca agtttcagtt tcatttttct tgttctatta
      481 caactttttt tacttcttgc tcattagaaa gaaagcatag caatctaatc taagttttct
      541 agaactagtg gatcccccgg gaaaaatgga caagaagtac tccattgggc tcgatatcgg
      601 cacaaacagc gtcggttggg ccgtcattac ggacgagtac aaggtgccga gcaaaaaatt
      661 caaagttctg ggcaataccg atcgccacag cataaagaag aacctcattg gcgccctcct
      721 gttcgactcc ggggaaacgg ccgaagccac gcggctcaaa agaacagcac ggcgcagata
      781 tacccgcaga aagaatcgga tctgctacct gcaggagatc tttagtaatg agatggctaa
      841 ggtggatgac tctttcttcc ataggctgga ggagtccttt ttggtggagg aggataaaaa
      901 gcacgagcgc cacccaatct ttggcaatat cgtggacgag gtggcgtacc atgaaaagta
      961 cccaaccata tatcatctga ggaagaagct tgtagacagt actgataagg ctgacttgcg
     1021 gttgatctat ctcgcgctgg cgcatatgat caaatttcgg ggacacttcc tcatcgaggg
     1081 ggacctgaac ccagacaaca gcgatgtcga caaactcttt atccaactgg ttcagactta
     1141 caatcagctt ttcgaagaga acccgatcaa cgcatccgga gttgacgcca aagcaatcct
     1201 gagcgctagg ctgtccaaat cccggcggct cgaaaacctc atcgcacagc tccctgggga
     1261 gaagaagaac ggcctgtttg gtaatcttat cgccctgtca ctcgggctga cccccaactt
     1321 taaatctaac ttcgacctgg ccgaagatgc caagcttcaa ctgagcaaag acacctacga
     1381 tgatgatctc gacaatctgc tggcccagat cggcgaccag tacgcagacc tttttttggc
     1441 ggcaaagaac ctgtcagacg ccattctgct gagtgatatt ctgcgagtga acacggagat
     1501 caccaaagct ccgctgagcg ctagtatgat caagcgctat gatgagcacc accaagactt
     1561 gactttgctg aaggcccttg tcagacagca actgcctgag aagtacaagg aaattttctt
     1621 cgatcagtct aaaaatggct acgccggata cattgacggc ggagcaagcc aggaggaatt
     1681 ttacaaattt attaagccca tcttggaaaa aatggacggc accgaggagc tgctggtaaa
     1741 gcttaacaga gaagatctgt tgcgcaaaca gcgcactttc gacaatggaa gcatccccca
     1801 ccagattcac ctgggcgaac tgcacgctat cctcaggcgg caagaggatt tctacccctt
     1861 tttgaaagat aacagggaaa agattgagaa aatcctcaca tttcggatac cctactatgt
     1921 aggccccctc gcccggggaa attccagatt cgcgtggatg actcgcaaat cagaagagac
     1981 catcactccc tggaacttcg aggaagtcgt ggataagggg gcctctgccc agtccttcat
     2041 cgaaaggatg actaactttg ataaaaatct gcctaacgaa aaggtgcttc ctaaacactc
     2101 tctgctgtac gagtacttca cagtttataa cgagctcacc aaggtcaaat acgtcacaga
     2161 agggatgaga aagccagcat tcctgtctgg agagcagaag aaagctatcg tggacctcct
     2221 cttcaagacg aaccggaaag ttaccgtgaa acagctcaaa gaagactatt tcaaaaagat
     2281 tgaatgtttc gactctgttg aaatcagcgg agtggaggat cgcttcaacg catccctggg
     2341 aacgtatcac gatctcctga aaatcattaa agacaaggac ttcctggaca atgaggagaa
     2401 cgaggacatt cttgaggaca ttgtcctcac ccttacgttg tttgaagata gggagatgat
     2461 tgaagaacgc ttgaaaactt acgctcatct cttcgacgac aaagtcatga aacagctcaa
     2521 gaggcgccga tatacaggat gggggcggct gtcaagaaaa ctgatcaatg ggatccgaga
     2581 caagcagagt ggaaagacaa tcctggattt tcttaagtcc gatggatttg ccaaccggaa
     2641 cttcatgcag ttgatccatg atgactctct cacctttaag gaggacatcc agaaagcaca
     2701 agtttctggc cagggggaca gtcttcacga gcacatcgct aatcttgcag gtagcccagc
     2761 tatcaaaaag ggaatactgc agaccgttaa ggtcgtggat gaactcgtca aagtaatggg
     2821 aaggcataag cccgagaata tcgttatcga gatggcccga gagaaccaaa ctacccagaa
     2881 gggacagaag aacagtaggg aaaggatgaa gaggattgaa gagggtataa aagaactggg
     2941 gtcccaaatc cttaaggaac acccagttga aaacacccag cttcagaatg agaagctcta
     3001 cctgtactac ctgcagaacg gcagggacat gtacgtggat caggaactgg acatcaatcg
     3061 gctctccgac tacgacgtgg atcatatcgt gccccagtct tttctcaaag atgattctat
     3121 tgataataaa gtgttgacaa gatccgataa aaatagaggg aagagtgata acgtcccctc
     3181 agaagaagtt gtcaagaaaa tgaaaaatta ttggcggcag ctgctgaacg ccaaactgat
     3241 cacacaacgg aagttcgata atctgactaa ggctgaacga ggtggcctgt ctgagttgga
     3301 taaagccggc ttcatcaaaa ggcagcttgt tgagacacgc cagatcacca agcacgtggc
     3361 ccaaattctc gattcacgca tgaacaccaa gtacgatgaa aatgacaaac tgattcgaga
     3421 ggtgaaagtt attactctga agtctaagct ggtctcagat ttcagaaagg actttcagtt
     3481 ttataaggtg agagagatca acaattacca ccatgcgcat gatgcctacc tgaatgcagt
     3541 ggtaggcact gcacttatca aaaaatatcc caagcttgaa tctgaatttg tttacggaga
     3601 ctataaagtg tacgatgtta ggaaaatgat cgcaaagtct gagcaggaaa taggcaaggc
     3661 caccgctaag tacttctttt acagcaatat tatgaatttt ttcaagaccg agattacact
     3721 ggccaatgga gagattcgga agcgaccact tatcgaaaca aacggagaaa caggagaaat
     3781 cgtgtgggac aagggtaggg atttcgcgac agtccggaag gtcctgtcca tgccgcaggt
     3841 gaacatcgtt aaaaagaccg aagtacagac cggaggcttc tccaaggaaa gtatcctccc
     3901 gaaaaggaac agcgacaagc tgatcgcacg caaaaaagat tgggacccca agaaatacgg
     3961 cggattcgat tctcctacag tcgcttacag tgtactggtt gtggccaaag tggagaaagg
     4021 gaagtctaaa aaactcaaaa gcgtcaagga actgctgggc atcacaatca tggagcgatc
     4081 aagcttcgaa aaaaacccca tcgactttct cgaggcgaaa ggatataaag aggtcaaaaa
     4141 agacctcatc attaagcttc ccaagtactc tctctttgag cttgaaaacg gccggaaacg
     4201 aatgctcgct agtgcgggcg agctgcagaa aggtaacgag ctggcactgc cctctaaata
     4261 cgttaatttc ttgtatctgg ccagccacta tgaaaagctc aaagggtctc ccgaagataa
     4321 tgagcagaag cagctgttcg tggaacaaca caaacactac cttgatgaga tcatcgagca
     4381 aataagcgaa ttctccaaaa gagtgatcct cgccgacgct aacctcgata aggtgctttc
     4441 tgcttacaat aagcacaggg ataagcccat cagggagcag gcagaaaaca ttatccactt
     4501 gtttactctg accaacttgg gcgcgcctgc agccttcaag tacttcgaca ccaccataga
     4561 cagaaagcgg tacacctcta caaaggaggt cctggacgcc acactgattc atcagtcaat
     4621 tacggggctc tatgaaacaa gaatcgacct ctctcagctc ggtggagaca gcagggctga
     4681 ccccaagaag aagaggaagg tgtgatctct tctcgagtca tgtaattagt tatgtcacgc
     4741 ttacattcac gccctccccc cacatccgct ctaaccgaaa aggaaggagt tagacaacct
     4801 gaagtctagg tccctattta tttttttata gttatgttag tattaagaac gttatttata
     4861 tttcaaattt ttcttttttt tctgtacaga cgcgtgtacg catgtaacat tatactgaaa
     4921 accttgcttg agaaggtttt gggacgctcg aaggctttaa tttgcgcatg catcaacaac
     4981 agaggacata tgccctacct ccatgatcta ctgacgtccc tgaggctgca attcatgtaa
     5041 tggggcagta tccgcggcaa gtcctagtgc aatggcggtt ttttaccctc gttctgaaga
     5101 agaggcgacg cgggtgcggt catcactaat gtggaaattg gtaagactct cgggcctccg
     5161 cctttaggcg gtgcttactc tttcataaag gggctgttag ttatgcaacg atgtgcttca
     5221 gtattacatt ttttgccttc aacgccttga ttgttctatt tttgctaata ataaatctat
     5281 ttcatcggac taaaagtcca ttagttgtaa gcggatttag ctcagttggg agagcgccag
     5341 actgaagaaa aacttcggtc aagtcatctg gaggtcctgt gttcgatcca cagaattcgc
     5401 agatggccgg catggtccca gcctcctcgc tggcgccggc tgggcaacac cttcgggtgg
     5461 cgaatgggac ttgagacgga aagtgaaacg tgatttcatg cgtcattttg aacattttgt
     5521 aaatcttatt taataatgtg tgcggcaatt cacatttaat ttatgaatgt tttcttaaca
     5581 tcgcggcaac tcaagaaacg gcaggttcgg atcttagcta ctagagaaag aggagaaata
     5641 ctagatgcgt aaaggcgaag agctgttcac tggtgtcgtc cctattctgg tggaactgga
     5701 tggtgatgtc aacggtcata agttttccgt gcgtggcgag ggtgaaggtg acgcaactaa
     5761 tggtaaactg acgctgaagt tcatctgtac tactggtaaa ctgccggttc cttggccgac
     5821 tctggtaacg acgctgactt atggtgttca gtgctttgct cgttatccgg accatatgaa
     5881 gcagcatgac ttcttcaagt ccgccatgcc ggaaggctat gtgcaggaac gcacgatttc
     5941 ctttaaggat gacggcacgt acaaaacgcg tgcggaagtg aaatttgaag gcgataccct
     6001 ggtaaaccgc attgagctga aaggcattga ctttaaagag gacggcaata tcctgggcca
     6061 taagctggaa tacaatttta acagccacaa tgtttacatc accgccgata aacaaaaaaa
     6121 tggcattaaa gcgaatttta aaattcgcca caacgtggag gatggcagcg tgcagctggc
     6181 tgatcactac cagcaaaaca ctccaatcgg tgatggtcct gttctgctgc cagacaatca
     6241 ctatctgagc acgcaaagcg ttctgtctaa agatccgaac gagaaacgcg atcatatggt
     6301 tctgctggag ttcgtaaccg cagcgggcat cacgcatggt atggatgaac tgtacaaatg
     6361 accaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt
     6421 tgtttgtcgg tgaacgctct ctactagagt cacactggct caccttcggg tgggcctttc
     6481 tgcgtttata cgtctcagtt ttagagctag aaatagcaag ttaaaataag gctagtccgt
     6541 tatcaacttg aaaaagtggc accgagtcgg tgcttttttt attttttgtc actattgtta
     6601 tgtaaaatgc cacctctgac agtatggaac gcaaacttct gtctagtgga tagctggaca
     6661 ttccaggcag tgcctctgcc gccggacccc tctcgtgatt gggtagctgg acatgccctt
     6721 gtaagatata acaagagcct gcctgtctaa tgatctcacg gcgaaagtcg gggagacagc
     6781 agcggctgca gacattatac cgcaacaaca ctaaggtgag ataactccgt acattgacta
     6841 cgcgttcctc tagaccttac ttgaccggat acagtgtctt tgacacgttt atgggttaca
     6901 gcaatcacat ccaagactgg ctatgcacga agcaactctt gagtgttaaa atgttgaccc
     6961 ctgtatttgg gatgcgggta gtagatgagt gcaggatggt acccaattcg ccctatagtg
     7021 agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg
     7081 gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg
     7141 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggacgc
     7201 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     7261 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     7321 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     7381 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     7441 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     7501 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     7561 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     7621 gaattttaac aaaatattaa cgcttacaat ttcctgatgc ggtattttct ccttacgcat
     7681 ctgtgcggta tttcacaccg catagggtaa taactgatat aattaaattg aagctctaat
     7741 ttgtgagttt agtatacatg catttactta taatacagtt ttttagtttt gctggccgca
     7801 tcttctcaaa tatgcttccc agcctgcttt tctgtaacgt tcaccctcta ccttagcatc
     7861 ccttcccttt gcaaatagtc ctcttccaac aataataatg tcagatcctg tagacaccac
     7921 atcatccacg gttctatact gttgacccaa tgcatctccc ttgtcatcta aacccacacc
     7981 gggtgtcata atcaaccaat cgtaaccttc atctcttcca cccatgtctc tttgagcaat
     8041 aaagccgata acaaaatctt tgtcgctctt cgcaatgtca acagtaccct tagtatattc
     8101 tccagtagat agggagccct tgcatgacaa ttctgctaac atcaaaaggc ctctaggttc
     8161 ctttgttact tcttctgccg cctgcttcaa accgctaaca atacctgggc ccaccacacc
     8221 gtgtgcattc gtaatgtctg cccattctgc tattctgtat acacccgcag agtactgcaa
     8281 tttgactgta ttaccaatgt cagcaaattt tctgtcttcg aagagtaaaa aattgtactt
     8341 ggcggataat gcctttagcg gcttaactgt gccctccatg gaaaaatcag tcaagatatc
     8401 cacatgtgtt tttagtaaac aaattttggg acctaatgct tcaactaact ccagtaattc
     8461 cttggtggta cgaacatcca atgaagcaca caagtttgtt tgcttttcgt gcatgatatt
     8521 aaatagcttg gcagcaacag gactaggatg agtagcagca cgttccttat atgtagcttt
     8581 cgacatgatt tatcttcgtt tcctgcaggt ttttgttctg tgcagttggg ttaagaatac
     8641 tgggcaattt catgtttctt caacactaca tatgcgtata tataccaatc taagtctgtg
     8701 ctccttcctt cgttcttcct tctgttcgga gattaccgaa tcaaaaaaat ttcaaggaaa
     8761 ccgaaatcaa aaaaaagaat aaaaaaaaaa tgatgaattg aaaaggtggt atggtgcact
     8821 ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc
     8881 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgaca
     8941 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     9001 acggatcgct tgcctgtaac ttacacgcgc ctcgtatctt ttaatgatgg aataatttgg
     9061 gaatttactc tgtgtttatt tatttttatg ttttgtattt ggattttaga aagtaaataa
     9121 agaaggtaga agagttacgg aatgaagaaa aaaaaataaa caaaggttta aaaaatttca
     9181 acaaaaagcg tactttacat atatatttat tagacaagaa aagcagatta aatagatata
     9241 cattcgatta acgataagta aaatgtaaaa tcacaggatt ttcgtgtgtg gtcttctaca
     9301 cagacaagat gaaacaattc ggcattaata cctgagagca ggaagagcaa gataaaaggt
     9361 agtatttgtt ggcgatcccc ctagagtctt ttacatcttc ggaaaacaaa aactattttt
     9421 tctttaattt ctttttttac tttctatttt taatttatat atttatatta aaaaatttaa
     9481 attataatta tttttatagc acgtgatgaa aaggacccag gtggcacttt tcggggaaat
     9541 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     9601 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     9661 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     9721 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     9781 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     9841 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     9901 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     9961 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
    10021 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
    10081 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
    10141 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
    10201 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
    10261 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
    10321 gctggctggt ttattgctga taaatctgga gccggtgagc gtggatctcg cggtatcatt
    10381 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
    10441 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
    10501 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
    10561 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
    10621 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
    10681 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
    10741 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
    10801 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
    10861 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
    10921 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
    10981 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
    11041 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
    11101 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
    11161 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
    11221 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
    11281 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
    11341 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
    11401 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata
    11461 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
    11521 cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag
    11581 gcaccccagg c
//
