LOCUS       sequence-330356-       11755 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for constituive spCas9 expression and tet-inducible 
            expression of sgRNA binding to the promoter of RRT8 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_RRT8
FEATURES             Location/Qualifiers
     source          1..11755
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    369..387
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        473..548
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      561..623
                     /label=RPR1 terminator
                     /note="transcription terminator for the S. cerevisiae RNase
                     P RNA gene"
     CDS             1758..2381
                     /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(1831..1850)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     2878..2895
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3012..3033
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3048..3053,3054..3071,3072..3078)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3086..3102
                     /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     3091..3113
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3110..3126
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3110..3126
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     3145..3165
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        3147..3165
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        3183..3581
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     primer_bind     3585..3601
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3585..3601
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             3613..7716
                     /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             3613..7716
                     /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             3613..7716
                     /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             7729..7749
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      7765..8012
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(7781..7799)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(8030..8049)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8031..8049)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8059..8076)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8059..8075)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8068..8090)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      8216..8671
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8303..8322)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8513..8534
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(8774..9448)
                     /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(9449..9730)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(9751..9770)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     9870..9892
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(9930..9948)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        10016..10120
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(10121..10189,10190..10981)
                     /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(10339..10358)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      11152..11740
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11641..11660
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gcctttttac ggttcctggc cttttgctgg ccttttgctc gggggatctg ccaattgaac
       61 ataacatggt agttacatat actagtaata tggttcggca cacattaaaa gtataaaaac
      121 tatctgaatt acgaattaca tatattggtc ataaaaatca atcaatcatc gtgtgtttta
      181 tatgtctctt atctaagtat aagaatatcc atagttaata ttcacttacg ctacctttta
      241 acctgtaatc attgtcaaca ggatatgtta acgacccaca ttgataaacg ctagtatttc
      301 tttttcctct tcttattggc cggctgtctc tatactcccc tatagtctgt ttcttttcgt
      361 ttcgattgtc cctatcagtg atagagatgg cgcacatggt acgctgtggt gctcgcggct
      421 gggaacgaaa ctctgggagc tgcgattggc aggggactta tgaaaatcag gggttttaga
      481 gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga
      541 gtcggtgctt ttttctcgag ccatatccaa cttccaattt aatctttctt ttttaatttt
      601 cacttatttg cgatacagaa agaaaaaagc gatagtaact attgaatttt gtttggattt
      661 ggttagatta gatatggttt ctctttatat ttacatgcta aaaatgggct acaccagaga
      721 tacataatta gatatatata cgccagtaca ccttatcggc ccaagccttg tcccaaggca
      781 gcgttttgtt cttggaaacg ctgccctaca cgttcgctat gcttcaagaa cttttctgag
      841 cacttcatga tgcatgtttg ttccttattg gttagctttg atgttgtgaa gtcattgaca
      901 cagtctgtga aacatctttc taccagatta gagtacaaac gcatgaaatc cttcatttgc
      961 ttttgttcca ctactttttg gaactcttgt tgttctttcc tgcaggtagt cgtgcaatgt
     1021 atgactttaa gatttgtgag caggaagaaa agggagaatc ttctaacgat aaacccttga
     1081 aaaactgggt agactacgct atgttgagtt gctacgcagg ctgcacaatt acacgagaat
     1141 gctcccgcct aggatttaag gctaagggac gtgcaatgca gacgacagat ctaaatgacc
     1201 gtgtcggtga agtgttcgcc aaacttttcg gttaacacat gcagtgatgc acgcgcgatg
     1261 gtgctaagtt acatatatat atatatgtat agccatagtg atgtctaagt aacctttatg
     1321 gtatatttct taatgtggaa agatactagc gcgcgcaccc acacacaagc ttcgtctttt
     1381 cttgaagaaa agaggaagct cgctaaatgg gattccactt tccgttccct gccagctgat
     1441 ggaaaaaggt tagtggaacg atgaagaata aaaagagaga tccactgagg tgaaatttca
     1501 gctgacagcg agtttcatga tcgtgatgaa caatggtaac gagttgtggc tgttgccagg
     1561 gagggtggtt ctcaactttt aatgtatggc caaatcgcta cttgggtttg ttatataaca
     1621 aagaagaaat aatgaactga ttctcttcct ccttcttgtc ctttcttaat tctgttgtaa
     1681 ttaccttcct ttgtaatttt ttttgtaatt attcttctta ataatccaaa caaacacaca
     1741 tattacaata tattaaaatg tctagattag ataaaagtaa agtgattaac agcgcattag
     1801 agctgcttaa tgaggtcgga atcgaaggtt taacaacccg taaactcgcc cagaagctag
     1861 gtgtagagca gcctacattg tattggcatg taaaaaataa gcgggctttg ctcgacgcct
     1921 tagccattga gatgttagat aggcaccata ctcacttttg ccctttagaa ggggaaagct
     1981 ggcaagattt tttacgtaat aacgctaaaa gttttagatg tgctttacta agtcatcgcg
     2041 atggagcaaa agtacattta ggtacacggc ctacagaaaa acagtatgaa actctcgaaa
     2101 atcaattagc ctttttatgc caacaaggtt tttcactaga gaatgcatta tatgcactca
     2161 gcgctgtggg gcattttact ttaggttgcg tattggaaga tcaagagcat caagtcgcta
     2221 aagaagaaag ggaaacacct actactgata gtatgccgcc attattacga caagctatcg
     2281 aattatttga tcaccaaggt gcagagccag ccttcttatt cggccttgaa ttgatcatat
     2341 gcggattaga aaaacaactt aaatgtgaaa gtgggtctta agtctgaaga atgaatgatt
     2401 tgatgatttc tttttccctc catttttctt actgaatata tcaatgatat agacttgtat
     2461 agtttattat ttcaaattaa gtagctatat atagtcaaga taacgtttgt ttgacacgat
     2521 tacattattc gtcgacatct tttttcagcc tgtcgtggta gcaatttgag gagtattatt
     2581 aattgaatag gttcattttg cgctcgcata aacagttttc gtcagggaca gtatgttgga
     2641 atgagtggta attaatggtg acatgacatg ttatagcaat aaccttgatg tttacatcgt
     2701 agtttaatgt acaccccgcg aattcgttca agtaggagtg caccaattgc aaagggaaaa
     2761 gctgaatggg cagttcgaac cgcggtcttt cctgcgttat cccctgattc tgtggataac
     2821 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     2881 gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt
     2941 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
     3001 cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg
     3061 cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc
     3121 tatgaccatg attacgccaa gcgcgcaatt aaccctcact aaagggaaca aaagctggag
     3181 ctcatagctt caaaatgttt ctactccttt tttactcttc cagattttct cggactccgc
     3241 gcatcgccgt accacttcaa aacacccaag cacagcatac taaatttccc ctctttcttc
     3301 ctctagggtg tcgttaatta cccgtactaa aggtttggaa aagaaaaaag agaccgcctc
     3361 gtttcttttt cttcgtcgaa aaaggcaata aaaattttta tcacgtttct ttttcttgaa
     3421 aatttttttt ttgatttttt tctctttcga tgacctccca ttgatattta agttaataaa
     3481 cggtcttcaa tttctcaagt ttcagtttca tttttcttgt tctattacaa ctttttttac
     3541 ttcttgctca ttagaaagaa agcatagcaa tctaatctaa gttttctaga actagtggat
     3601 cccccgggaa aaatggacaa gaagtactcc attgggctcg atatcggcac aaacagcgtc
     3661 ggttgggccg tcattacgga cgagtacaag gtgccgagca aaaaattcaa agttctgggc
     3721 aataccgatc gccacagcat aaagaagaac ctcattggcg ccctcctgtt cgactccggg
     3781 gagacggccg aagccacgcg gctcaaaaga acagcacggc gcagatatac ccgcagaaag
     3841 aatcggatct gctacctgca ggagatcttt agtaatgaga tggctaaggt ggatgactct
     3901 ttcttccata ggctggagga gtcctttttg gtggaggagg ataaaaagca cgagcgccac
     3961 ccaatctttg gcaatatcgt ggacgaggtg gcgtaccatg aaaagtaccc aaccatatat
     4021 catctgagga agaagcttgt agacagtact gataaggctg acttgcggtt gatctatctc
     4081 gcgctggcgc atatgatcaa atttcgggga cacttcctca tcgaggggga cctgaaccca
     4141 gacaacagcg atgtcgacaa actctttatc caactggttc agacttacaa tcagcttttc
     4201 gaagagaacc cgatcaacgc atccggagtt gacgccaaag caatcctgag cgctaggctg
     4261 tccaaatccc ggcggctcga aaacctcatc gcacagctcc ctggggagaa gaagaacggc
     4321 ctgtttggta atcttatcgc cctgtcactc gggctgaccc ccaactttaa atctaacttc
     4381 gacctggccg aagatgccaa gcttcaactg agcaaagaca cctacgatga tgatctcgac
     4441 aatctgctgg cccagatcgg cgaccagtac gcagaccttt ttttggcggc aaagaacctg
     4501 tcagacgcca ttctgctgag tgatattctg cgagtgaaca cggagatcac caaagctccg
     4561 ctgagcgcta gtatgatcaa gcgctatgat gagcaccacc aagacttgac tttgctgaag
     4621 gcccttgtca gacagcaact gcctgagaag tacaaggaaa ttttcttcga tcagtctaaa
     4681 aatggctacg ccggatacat tgacggcgga gcaagccagg aggaatttta caaatttatt
     4741 aagcccatct tggaaaaaat ggacggcacc gaggagctgc tggtaaagct taacagagaa
     4801 gatctgttgc gcaaacagcg cactttcgac aatggaagca tcccccacca gattcacctg
     4861 ggcgaactgc acgctatcct caggcggcaa gaggatttct accccttttt gaaagataac
     4921 agggaaaaga ttgagaaaat cctcacattt cggataccct actatgtagg ccccctcgcc
     4981 cggggaaatt ccagattcgc gtggatgact cgcaaatcag aagagaccat cactccctgg
     5041 aacttcgagg aagtcgtgga taagggggcc tctgcccagt ccttcatcga aaggatgact
     5101 aactttgata aaaatctgcc taacgaaaag gtgcttccta aacactctct gctgtacgag
     5161 tacttcacag tttataacga gctcaccaag gtcaaatacg tcacagaagg gatgagaaag
     5221 ccagcattcc tgtctggaga gcagaagaaa gctatcgtgg acctcctctt caagacgaac
     5281 cggaaagtta ccgtgaaaca gctcaaagaa gactatttca aaaagattga atgtttcgac
     5341 tctgttgaaa tcagcggagt ggaggatcgc ttcaacgcat ccctgggaac gtatcacgat
     5401 ctcctgaaaa tcattaaaga caaggacttc ctggacaatg aggagaacga ggacattctt
     5461 gaggacattg tcctcaccct tacgttgttt gaagataggg agatgattga agaacgcttg
     5521 aaaacttacg ctcatctctt cgacgacaaa gtcatgaaac agctcaagag gcgccgatat
     5581 acaggatggg ggcggctgtc aagaaaactg atcaatggga tccgagacaa gcagagtgga
     5641 aagacaatcc tggattttct taagtccgat ggatttgcca accggaactt catgcagttg
     5701 atccatgatg actctctcac ctttaaggag gacatccaga aagcacaagt ttctggccag
     5761 ggggacagtc ttcacgagca catcgctaat cttgcaggta gcccagctat caaaaaggga
     5821 atactgcaga ccgttaaggt cgtggatgaa ctcgtcaaag taatgggaag gcataagccc
     5881 gagaatatcg ttatcgagat ggcccgagag aaccaaacta cccagaaggg acagaagaac
     5941 agtagggaaa ggatgaagag gattgaagag ggtataaaag aactggggtc ccaaatcctt
     6001 aaggaacacc cagttgaaaa cacccagctt cagaatgaga agctctacct gtactacctg
     6061 cagaacggca gggacatgta cgtggatcag gaactggaca tcaatcggct ctccgactac
     6121 gacgtggatc atatcgtgcc ccagtctttt ctcaaagatg attctattga taataaagtg
     6181 ttgacaagat ccgataaaaa tagagggaag agtgataacg tcccctcaga agaagttgtc
     6241 aagaaaatga aaaattattg gcggcagctg ctgaacgcca aactgatcac acaacggaag
     6301 ttcgataatc tgactaaggc tgaacgaggt ggcctgtctg agttggataa agccggcttc
     6361 atcaaaaggc agcttgttga gacacgccag atcaccaagc acgtggccca aattctcgat
     6421 tcacgcatga acaccaagta cgatgaaaat gacaaactga ttcgagaggt gaaagttatt
     6481 actctgaagt ctaagctggt ctcagatttc agaaaggact ttcagtttta taaggtgaga
     6541 gagatcaaca attaccacca tgcgcatgat gcctacctga atgcagtggt aggcactgca
     6601 cttatcaaaa aatatcccaa gcttgaatct gaatttgttt acggagacta taaagtgtac
     6661 gatgttagga aaatgatcgc aaagtctgag caggaaatag gcaaggccac cgctaagtac
     6721 ttcttttaca gcaatattat gaattttttc aagaccgaga ttacactggc caatggagag
     6781 attcggaagc gaccacttat cgaaacaaac ggagaaacag gagaaatcgt gtgggacaag
     6841 ggtagggatt tcgcgacagt ccggaaggtc ctgtccatgc cgcaggtgaa catcgttaaa
     6901 aagaccgaag tacagaccgg aggcttctcc aaggaaagta tcctcccgaa aaggaacagc
     6961 gacaagctga tcgcacgcaa aaaagattgg gaccccaaga aatacggcgg attcgattct
     7021 cctacagtcg cttacagtgt actggttgtg gccaaagtgg agaaagggaa gtctaaaaaa
     7081 ctcaaaagcg tcaaggaact gctgggcatc acaatcatgg agcgatcaag cttcgaaaaa
     7141 aaccccatcg actttctcga ggcgaaagga tataaagagg tcaaaaaaga cctcatcatt
     7201 aagcttccca agtactctct ctttgagctt gaaaacggcc ggaaacgaat gctcgctagt
     7261 gcgggcgagc tgcagaaagg taacgagctg gcactgccct ctaaatacgt taatttcttg
     7321 tatctggcca gccactatga aaagctcaaa gggtctcccg aagataatga gcagaagcag
     7381 ctgttcgtgg aacaacacaa acactacctt gatgagatca tcgagcaaat aagcgaattc
     7441 tccaaaagag tgatcctcgc cgacgctaac ctcgataagg tgctttctgc ttacaataag
     7501 cacagggata agcccatcag ggagcaggca gaaaacatta tccacttgtt tactctgacc
     7561 aacttgggcg cgcctgcagc cttcaagtac ttcgacacca ccatagacag aaagcggtac
     7621 acctctacaa aggaggtcct ggacgccaca ctgattcatc agtcaattac ggggctctat
     7681 gaaacaagaa tcgacctctc tcagctcggt ggagacagca gggctgaccc caagaagaag
     7741 aggaaggtgt gatctcttct cgagtcatgt aattagttat gtcacgctta cattcacgcc
     7801 ctccccccac atccgctcta accgaaaagg aaggagttag acaacctgaa gtctaggtcc
     7861 ctatttattt ttttatagtt atgttagtat taagaacgtt atttatattt caaatttttc
     7921 ttttttttct gtacagacgc gtgtacgcat gtaacattat actgaaaacc ttgcttgaga
     7981 aggttttggg acgctcgaag gctttaattt gcggccggta cccaattcgc cctatagtga
     8041 gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg
     8101 cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga
     8161 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggacgcg
     8221 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     8281 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     8341 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     8401 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     8461 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     8521 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     8581 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     8641 aattttaaca aaatattaac gcttacaatt tcctgatgcg gtattttctc cttacgcatc
     8701 tgtgcggtat ttcacaccgc atagatcggc aagtgcacaa acaatactta aataaatact
     8761 actcagtaat aacctatttc ttagcatttt tgacgaaatt tgctattttg ttagagtctt
     8821 ttacaccatt tgtctccaca cctccgctta catcaacacc aataacgcca tttaatctaa
     8881 gcgcatcacc aacattttct ggcgtcagtc caccagctaa cataaaatgt aagctttcgg
     8941 ggctctcttg ccttccaacc cagtcagaaa tcgagttcca atccaaaagt tcacctgtcc
     9001 cacctgcttc tgaatcaaac aagggaataa acgaatgagg tttctgtgaa gctgcactga
     9061 gtagtatgtt gcagtctttt ggaaatacga gtcttttaat aactggcaaa ccgaggaact
     9121 cttggtattc ttgccacgac tcatctccat gcagttggac gatatcaatg ccgtaatcat
     9181 tgaccagagc caaaacatcc tccttaggtt gattacgaaa cacgccaacc aagtatttcg
     9241 gagtgcctga actattttta tatgctttta caagacttga aattttcctt gcaataaccg
     9301 ggtcaattgt tctctttcta ttgggcacac atataatacc cagcaagtca gcatcggaat
     9361 ctagagcaca ttctgcggcc tctgtgctct gcaagccgca aactttcacc aatggaccag
     9421 aactacctgt gaaattaata acagacatac tccaagctgc ctttgtgtgc ttaatcacgt
     9481 atactcacgt gctcaatagt caccaatgcc ctccctcttg gccctctcct tttctttttt
     9541 cgaccgaatt aattcttaat cggcaaaaaa agaaaagctc cggatcaaga ttgtacgtaa
     9601 ggtgacaagc tatttttcaa taaagaatat cttccactac tgccatctgg cgtcataact
     9661 gcaaagtaca catatattac gatgctgttc tattaaatgc ttcctatatt atatatatag
     9721 taatgtcgtt gatctatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     9781 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     9841 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     9901 gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa
     9961 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
    10021 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
    10081 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
    10141 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
    10201 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
    10261 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
    10321 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
    10381 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
    10441 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
    10501 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
    10561 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
    10621 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
    10681 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
    10741 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
    10801 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
    10861 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
    10921 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
    10981 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
    11041 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
    11101 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
    11161 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
    11221 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
    11281 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
    11341 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
    11401 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
    11461 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
    11521 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
    11581 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
    11641 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
    11701 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcg
//
