LOCUS       sequence-329132-       11755 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for constituive spCas9 and tet-inducible YBL055C targeting 
            sgRNA expression for double stranded break formation in yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11755)
  TITLE     Burgess Lab Plasmids
REFERENCE   2  (bases 1 to 11755)
  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"
COMMENT     Sequence Label: pNSU299_YBL055C
FEATURES             Location/Qualifiers
     source          1..11755
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        1..76
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      89..151
                     /label=RPR1 terminator
                     /note="transcription terminator for the S. cerevisiae RNase
                     P RNA gene"
     CDS             1286..1909
                     /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(1359..1378)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     2406..2423
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2540..2561
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2576..2581,2582..2599,2600..2606)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2614..2630
                     /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     2619..2641
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2638..2654
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2638..2654
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2673..2693
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2675..2693
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        2711..3109
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     primer_bind     3113..3129
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3113..3129
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             3141..7244
                     /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             3141..7244
                     /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             3141..7244
                     /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             7257..7277
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      7293..7540
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(7309..7327)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(7558..7577)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7559..7577)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(7587..7604)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7587..7603)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7596..7618)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      7744..8199
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7831..7850)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8041..8062
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(8302..8976)
                     /codon_start=1
                     /gene="S. cerevisiae TRP1"
                     /product="phosphoribosylanthranilate isomerase, required 
                     for tryptophan biosynthesis"
                     /label=TRP1
                     /note="yeast auxotrophic marker"
                     /translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
                     RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
                     WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
                     VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
                     KK"
     promoter        complement(8977..9258)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(9279..9298)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     9398..9420
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(9458..9476)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     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"
     protein_bind    11652..11670
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
ORIGIN
        1 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt
       61 ggcaccgagt cggtgctttt ttctcgagcc atatccaact tccaatttaa tctttctttt
      121 ttaattttca cttatttgcg atacagaaag aaaaaagcga tagtaactat tgaattttgt
      181 ttggatttgg ttagattaga tatggtttct ctttatattt acatgctaaa aatgggctac
      241 accagagata cataattaga tatatatacg ccagtacacc ttatcggccc aagccttgtc
      301 ccaaggcagc gttttgttct tggaaacgct gccctacacg ttcgctatgc ttcaagaact
      361 tttctgagca cttcatgatg catgtttgtt ccttattggt tagctttgat gttgtgaagt
      421 cattgacaca gtctgtgaaa catctttcta ccagattaga gtacaaacgc atgaaatcct
      481 tcatttgctt ttgttccact actttttgga actcttgttg ttctttcctg caggtagtcg
      541 tgcaatgtat gactttaaga tttgtgagca ggaagaaaag ggagaatctt ctaacgataa
      601 acccttgaaa aactgggtag actacgctat gttgagttgc tacgcaggct gcacaattac
      661 acgagaatgc tcccgcctag gatttaaggc taagggacgt gcaatgcaga cgacagatct
      721 aaatgaccgt gtcggtgaag tgttcgccaa acttttcggt taacacatgc agtgatgcac
      781 gcgcgatggt gctaagttac atatatatat atatgtatag ccatagtgat gtctaagtaa
      841 cctttatggt atatttctta atgtggaaag atactagcgc gcgcacccac acacaagctt
      901 cgtcttttct tgaagaaaag aggaagctcg ctaaatggga ttccactttc cgttccctgc
      961 cagctgatgg aaaaaggtta gtggaacgat gaagaataaa aagagagatc cactgaggtg
     1021 aaatttcagc tgacagcgag tttcatgatc gtgatgaaca atggtaacga gttgtggctg
     1081 ttgccaggga gggtggttct caacttttaa tgtatggcca aatcgctact tgggtttgtt
     1141 atataacaaa gaagaaataa tgaactgatt ctcttcctcc ttcttgtcct ttcttaattc
     1201 tgttgtaatt accttccttt gtaatttttt ttgtaattat tcttcttaat aatccaaaca
     1261 aacacacata ttacaatata ttaaaatgtc tagattagat aaaagtaaag tgattaacag
     1321 cgcattagag ctgcttaatg aggtcggaat cgaaggttta acaacccgta aactcgccca
     1381 gaagctaggt gtagagcagc ctacattgta ttggcatgta aaaaataagc gggctttgct
     1441 cgacgcctta gccattgaga tgttagatag gcaccatact cacttttgcc ctttagaagg
     1501 ggaaagctgg caagattttt tacgtaataa cgctaaaagt tttagatgtg ctttactaag
     1561 tcatcgcgat ggagcaaaag tacatttagg tacacggcct acagaaaaac agtatgaaac
     1621 tctcgaaaat caattagcct ttttatgcca acaaggtttt tcactagaga atgcattata
     1681 tgcactcagc gctgtggggc attttacttt aggttgcgta ttggaagatc aagagcatca
     1741 agtcgctaaa gaagaaaggg aaacacctac tactgatagt atgccgccat tattacgaca
     1801 agctatcgaa ttatttgatc accaaggtgc agagccagcc ttcttattcg gccttgaatt
     1861 gatcatatgc ggattagaaa aacaacttaa atgtgaaagt gggtcttaag tctgaagaat
     1921 gaatgatttg atgatttctt tttccctcca tttttcttac tgaatatatc aatgatatag
     1981 acttgtatag tttattattt caaattaagt agctatatat agtcaagata acgtttgttt
     2041 gacacgatta cattattcgt cgacatcttt tttcagcctg tcgtggtagc aatttgagga
     2101 gtattattaa ttgaataggt tcattttgcg ctcgcataaa cagttttcgt cagggacagt
     2161 atgttggaat gagtggtaat taatggtgac atgacatgtt atagcaataa ccttgatgtt
     2221 tacatcgtag tttaatgtac accccgcgaa ttcgttcaag taggagtgca ccaattgcaa
     2281 agggaaaagc tgaatgggca gttcgaaccg cggtctttcc tgcgttatcc cctgattctg
     2341 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     2401 agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc
     2461 ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg
     2521 gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac
     2581 actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag
     2641 gaaacagcta tgaccatgat tacgccaagc gcgcaattaa ccctcactaa agggaacaaa
     2701 agctggagct catagcttca aaatgtttct actccttttt tactcttcca gattttctcg
     2761 gactccgcgc atcgccgtac cacttcaaaa cacccaagca cagcatacta aatttcccct
     2821 ctttcttcct ctagggtgtc gttaattacc cgtactaaag gtttggaaaa gaaaaaagag
     2881 accgcctcgt ttctttttct tcgtcgaaaa aggcaataaa aatttttatc acgtttcttt
     2941 ttcttgaaaa tttttttttt gatttttttc tctttcgatg acctcccatt gatatttaag
     3001 ttaataaacg gtcttcaatt tctcaagttt cagtttcatt tttcttgttc tattacaact
     3061 ttttttactt cttgctcatt agaaagaaag catagcaatc taatctaagt tttctagaac
     3121 tagtggatcc cccgggaaaa atggacaaga agtactccat tgggctcgat atcggcacaa
     3181 acagcgtcgg ttgggccgtc attacggacg agtacaaggt gccgagcaaa aaattcaaag
     3241 ttctgggcaa taccgatcgc cacagcataa agaagaacct cattggcgcc ctcctgttcg
     3301 actccgggga gacggccgaa gccacgcggc tcaaaagaac agcacggcgc agatataccc
     3361 gcagaaagaa tcggatctgc tacctgcagg agatctttag taatgagatg gctaaggtgg
     3421 atgactcttt cttccatagg ctggaggagt cctttttggt ggaggaggat aaaaagcacg
     3481 agcgccaccc aatctttggc aatatcgtgg acgaggtggc gtaccatgaa aagtacccaa
     3541 ccatatatca tctgaggaag aagcttgtag acagtactga taaggctgac ttgcggttga
     3601 tctatctcgc gctggcgcat atgatcaaat ttcggggaca cttcctcatc gagggggacc
     3661 tgaacccaga caacagcgat gtcgacaaac tctttatcca actggttcag acttacaatc
     3721 agcttttcga agagaacccg atcaacgcat ccggagttga cgccaaagca atcctgagcg
     3781 ctaggctgtc caaatcccgg cggctcgaaa acctcatcgc acagctccct ggggagaaga
     3841 agaacggcct gtttggtaat cttatcgccc tgtcactcgg gctgaccccc aactttaaat
     3901 ctaacttcga cctggccgaa gatgccaagc ttcaactgag caaagacacc tacgatgatg
     3961 atctcgacaa tctgctggcc cagatcggcg accagtacgc agaccttttt ttggcggcaa
     4021 agaacctgtc agacgccatt ctgctgagtg atattctgcg agtgaacacg gagatcacca
     4081 aagctccgct gagcgctagt atgatcaagc gctatgatga gcaccaccaa gacttgactt
     4141 tgctgaaggc ccttgtcaga cagcaactgc ctgagaagta caaggaaatt ttcttcgatc
     4201 agtctaaaaa tggctacgcc ggatacattg acggcggagc aagccaggag gaattttaca
     4261 aatttattaa gcccatcttg gaaaaaatgg acggcaccga ggagctgctg gtaaagctta
     4321 acagagaaga tctgttgcgc aaacagcgca ctttcgacaa tggaagcatc ccccaccaga
     4381 ttcacctggg cgaactgcac gctatcctca ggcggcaaga ggatttctac ccctttttga
     4441 aagataacag ggaaaagatt gagaaaatcc tcacatttcg gataccctac tatgtaggcc
     4501 ccctcgcccg gggaaattcc agattcgcgt ggatgactcg caaatcagaa gagaccatca
     4561 ctccctggaa cttcgaggaa gtcgtggata agggggcctc tgcccagtcc ttcatcgaaa
     4621 ggatgactaa ctttgataaa aatctgccta acgaaaaggt gcttcctaaa cactctctgc
     4681 tgtacgagta cttcacagtt tataacgagc tcaccaaggt caaatacgtc acagaaggga
     4741 tgagaaagcc agcattcctg tctggagagc agaagaaagc tatcgtggac ctcctcttca
     4801 agacgaaccg gaaagttacc gtgaaacagc tcaaagaaga ctatttcaaa aagattgaat
     4861 gtttcgactc tgttgaaatc agcggagtgg aggatcgctt caacgcatcc ctgggaacgt
     4921 atcacgatct cctgaaaatc attaaagaca aggacttcct ggacaatgag gagaacgagg
     4981 acattcttga ggacattgtc ctcaccctta cgttgtttga agatagggag atgattgaag
     5041 aacgcttgaa aacttacgct catctcttcg acgacaaagt catgaaacag ctcaagaggc
     5101 gccgatatac aggatggggg cggctgtcaa gaaaactgat caatgggatc cgagacaagc
     5161 agagtggaaa gacaatcctg gattttctta agtccgatgg atttgccaac cggaacttca
     5221 tgcagttgat ccatgatgac tctctcacct ttaaggagga catccagaaa gcacaagttt
     5281 ctggccaggg ggacagtctt cacgagcaca tcgctaatct tgcaggtagc ccagctatca
     5341 aaaagggaat actgcagacc gttaaggtcg tggatgaact cgtcaaagta atgggaaggc
     5401 ataagcccga gaatatcgtt atcgagatgg cccgagagaa ccaaactacc cagaagggac
     5461 agaagaacag tagggaaagg atgaagagga ttgaagaggg tataaaagaa ctggggtccc
     5521 aaatccttaa ggaacaccca gttgaaaaca cccagcttca gaatgagaag ctctacctgt
     5581 actacctgca gaacggcagg gacatgtacg tggatcagga actggacatc aatcggctct
     5641 ccgactacga cgtggatcat atcgtgcccc agtcttttct caaagatgat tctattgata
     5701 ataaagtgtt gacaagatcc gataaaaata gagggaagag tgataacgtc ccctcagaag
     5761 aagttgtcaa gaaaatgaaa aattattggc ggcagctgct gaacgccaaa ctgatcacac
     5821 aacggaagtt cgataatctg actaaggctg aacgaggtgg cctgtctgag ttggataaag
     5881 ccggcttcat caaaaggcag cttgttgaga cacgccagat caccaagcac gtggcccaaa
     5941 ttctcgattc acgcatgaac accaagtacg atgaaaatga caaactgatt cgagaggtga
     6001 aagttattac tctgaagtct aagctggtct cagatttcag aaaggacttt cagttttata
     6061 aggtgagaga gatcaacaat taccaccatg cgcatgatgc ctacctgaat gcagtggtag
     6121 gcactgcact tatcaaaaaa tatcccaagc ttgaatctga atttgtttac ggagactata
     6181 aagtgtacga tgttaggaaa atgatcgcaa agtctgagca ggaaataggc aaggccaccg
     6241 ctaagtactt cttttacagc aatattatga attttttcaa gaccgagatt acactggcca
     6301 atggagagat tcggaagcga ccacttatcg aaacaaacgg agaaacagga gaaatcgtgt
     6361 gggacaaggg tagggatttc gcgacagtcc ggaaggtcct gtccatgccg caggtgaaca
     6421 tcgttaaaaa gaccgaagta cagaccggag gcttctccaa ggaaagtatc ctcccgaaaa
     6481 ggaacagcga caagctgatc gcacgcaaaa aagattggga ccccaagaaa tacggcggat
     6541 tcgattctcc tacagtcgct tacagtgtac tggttgtggc caaagtggag aaagggaagt
     6601 ctaaaaaact caaaagcgtc aaggaactgc tgggcatcac aatcatggag cgatcaagct
     6661 tcgaaaaaaa ccccatcgac tttctcgagg cgaaaggata taaagaggtc aaaaaagacc
     6721 tcatcattaa gcttcccaag tactctctct ttgagcttga aaacggccgg aaacgaatgc
     6781 tcgctagtgc gggcgagctg cagaaaggta acgagctggc actgccctct aaatacgtta
     6841 atttcttgta tctggccagc cactatgaaa agctcaaagg gtctcccgaa gataatgagc
     6901 agaagcagct gttcgtggaa caacacaaac actaccttga tgagatcatc gagcaaataa
     6961 gcgaattctc caaaagagtg atcctcgccg acgctaacct cgataaggtg ctttctgctt
     7021 acaataagca cagggataag cccatcaggg agcaggcaga aaacattatc cacttgttta
     7081 ctctgaccaa cttgggcgcg cctgcagcct tcaagtactt cgacaccacc atagacagaa
     7141 agcggtacac ctctacaaag gaggtcctgg acgccacact gattcatcag tcaattacgg
     7201 ggctctatga aacaagaatc gacctctctc agctcggtgg agacagcagg gctgacccca
     7261 agaagaagag gaaggtgtga tctcttctcg agtcatgtaa ttagttatgt cacgcttaca
     7321 ttcacgccct ccccccacat ccgctctaac cgaaaaggaa ggagttagac aacctgaagt
     7381 ctaggtccct atttattttt ttatagttat gttagtatta agaacgttat ttatatttca
     7441 aatttttctt ttttttctgt acagacgcgt gtacgcatgt aacattatac tgaaaacctt
     7501 gcttgagaag gttttgggac gctcgaaggc tttaatttgc ggccggtacc caattcgccc
     7561 tatagtgagt cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa
     7621 aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt
     7681 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa
     7741 tggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     7801 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     7861 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     7921 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     7981 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     8041 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     8101 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     8161 ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc ctgatgcggt attttctcct
     8221 tacgcatctg tgcggtattt cacaccgcat agatcggcaa gtgcacaaac aatacttaaa
     8281 taaatactac tcagtaataa cctatttctt agcatttttg acgaaatttg ctattttgtt
     8341 agagtctttt acaccatttg tctccacacc tccgcttaca tcaacaccaa taacgccatt
     8401 taatctaagc gcatcaccaa cattttctgg cgtcagtcca ccagctaaca taaaatgtaa
     8461 gctttcgggg ctctcttgcc ttccaaccca gtcagaaatc gagttccaat ccaaaagttc
     8521 acctgtccca cctgcttctg aatcaaacaa gggaataaac gaatgaggtt tctgtgaagc
     8581 tgcactgagt agtatgttgc agtcttttgg aaatacgagt cttttaataa ctggcaaacc
     8641 gaggaactct tggtattctt gccacgactc atctccatgc agttggacga tatcaatgcc
     8701 gtaatcattg accagagcca aaacatcctc cttaggttga ttacgaaaca cgccaaccaa
     8761 gtatttcgga gtgcctgaac tatttttata tgcttttaca agacttgaaa ttttccttgc
     8821 aataaccggg tcaattgttc tctttctatt gggcacacat ataataccca gcaagtcagc
     8881 atcggaatct agagcacatt ctgcggcctc tgtgctctgc aagccgcaaa ctttcaccaa
     8941 tggaccagaa ctacctgtga aattaataac agacatactc caagctgcct ttgtgtgctt
     9001 aatcacgtat actcacgtgc tcaatagtca ccaatgccct ccctcttggc cctctccttt
     9061 tcttttttcg accgaattaa ttcttaatcg gcaaaaaaag aaaagctccg gatcaagatt
     9121 gtacgtaagg tgacaagcta tttttcaata aagaatatct tccactactg ccatctggcg
     9181 tcataactgc aaagtacaca tatattacga tgctgttcta ttaaatgctt cctatattat
     9241 atatatagta atgtcgttga tctatggtgc actctcagta caatctgctc tgatgccgca
     9301 tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     9361 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     9421 ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta
     9481 taggttaatg tcatgataat aatggtttct tagacgtcag 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 gtgggtctcg 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 ctcgggggat ctgccaattg
    11341 aacataacat ggtagttaca tatactagta atatggttcg gcacacatta aaagtataaa
    11401 aactatctga attacgaatt acatatattg gtcataaaaa tcaatcaatc atcgtgtgtt
    11461 ttatatgtct cttatctaag tataagaata tccatagtta atattcactt acgctacctt
    11521 ttaacctgta atcattgtca acaggatatg ttaacgaccc acattgataa acgctagtat
    11581 ttctttttcc tcttcttatt ggccggctgt ctctatactc ccctatagtc tgtttctttt
    11641 cgtttcgatt gtccctatca gtgatagaga tggcgcacat ggtacgctgt ggtgctcgcg
    11701 gctgggaacg aaactctggg agctgcgatt ggcagctact ttgatagata ccacg
//
