LOCUS       sequence-329611-       11755 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for constituive spCas9 and tet-inducible LHS1 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_LHS1
FEATURES             Location/Qualifiers
     source          1..11755
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(266..285)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     385..407
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(445..463)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        531..635
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(636..704,705..1496)
                     /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(854..873)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1667..2255
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2156..2175
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    2639..2657
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        2743..2818
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      2831..2893
                     /label=RPR1 terminator
                     /note="transcription terminator for the S. cerevisiae RNase
                     P RNA gene"
     CDS             4028..4651
                     /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(4101..4120)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     5148..5165
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5282..5303
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5318..5323,5324..5341,5342..5348)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5356..5372
                     /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     5361..5383
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5380..5396
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5380..5396
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5415..5435
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5417..5435
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5453..5851
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     primer_bind     5855..5871
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5855..5871
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             5883..9986
                     /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             5883..9986
                     /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             5883..9986
                     /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             9999..10019
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      10035..10282
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(10051..10069)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(10300..10319)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(10301..10319)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(10329..10346)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(10329..10345)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10338..10360)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      10486..10941
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10573..10592)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     10783..10804
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(11044..11718)
                     /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(join(11719..11755,1..245))
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
ORIGIN
        1 cacgtgctca atagtcacca atgccctccc tcttggccct ctccttttct tttttcgacc
       61 gaattaattc ttaatcggca aaaaaagaaa agctccggat caagattgta cgtaaggtga
      121 caagctattt ttcaataaag aatatcttcc actactgcca tctggcgtca taactgcaaa
      181 gtacacatat attacgatgc tgttctatta aatgcttcct atattatata tatagtaatg
      241 tcgttgatct atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc
      301 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
      361 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
      421 caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca
      481 tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
      541 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
      601 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
      661 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
      721 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
      781 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
      841 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
      901 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
      961 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     1021 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     1081 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     1141 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     1201 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     1261 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     1321 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     1381 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     1441 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     1501 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     1561 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     1621 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     1681 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     1741 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     1801 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     1861 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     1921 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     1981 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     2041 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     2101 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     2161 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     2221 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     2281 ggttcctggc cttttgctgg ccttttgctc gggggatctg ccaattgaac ataacatggt
     2341 agttacatat actagtaata tggttcggca cacattaaaa gtataaaaac tatctgaatt
     2401 acgaattaca tatattggtc ataaaaatca atcaatcatc gtgtgtttta tatgtctctt
     2461 atctaagtat aagaatatcc atagttaata ttcacttacg ctacctttta acctgtaatc
     2521 attgtcaaca ggatatgtta acgacccaca ttgataaacg ctagtatttc tttttcctct
     2581 tcttattggc cggctgtctc tatactcccc tatagtctgt ttcttttcgt ttcgattgtc
     2641 cctatcagtg atagagatgg cgcacatggt acgctgtggt gctcgcggct gggaacgaaa
     2701 ctctgggagc tgcgattggc agggcttgcg gagaaaccac aagttttaga gctagaaata
     2761 gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt
     2821 ttttctcgag ccatatccaa cttccaattt aatctttctt ttttaatttt cacttatttg
     2881 cgatacagaa agaaaaaagc gatagtaact attgaatttt gtttggattt ggttagatta
     2941 gatatggttt ctctttatat ttacatgcta aaaatgggct acaccagaga tacataatta
     3001 gatatatata cgccagtaca ccttatcggc ccaagccttg tcccaaggca gcgttttgtt
     3061 cttggaaacg ctgccctaca cgttcgctat gcttcaagaa cttttctgag cacttcatga
     3121 tgcatgtttg ttccttattg gttagctttg atgttgtgaa gtcattgaca cagtctgtga
     3181 aacatctttc taccagatta gagtacaaac gcatgaaatc cttcatttgc ttttgttcca
     3241 ctactttttg gaactcttgt tgttctttcc tgcaggtagt cgtgcaatgt atgactttaa
     3301 gatttgtgag caggaagaaa agggagaatc ttctaacgat aaacccttga aaaactgggt
     3361 agactacgct atgttgagtt gctacgcagg ctgcacaatt acacgagaat gctcccgcct
     3421 aggatttaag gctaagggac gtgcaatgca gacgacagat ctaaatgacc gtgtcggtga
     3481 agtgttcgcc aaacttttcg gttaacacat gcagtgatgc acgcgcgatg gtgctaagtt
     3541 acatatatat atatatgtat agccatagtg atgtctaagt aacctttatg gtatatttct
     3601 taatgtggaa agatactagc gcgcgcaccc acacacaagc ttcgtctttt cttgaagaaa
     3661 agaggaagct cgctaaatgg gattccactt tccgttccct gccagctgat ggaaaaaggt
     3721 tagtggaacg atgaagaata aaaagagaga tccactgagg tgaaatttca gctgacagcg
     3781 agtttcatga tcgtgatgaa caatggtaac gagttgtggc tgttgccagg gagggtggtt
     3841 ctcaactttt aatgtatggc caaatcgcta cttgggtttg ttatataaca aagaagaaat
     3901 aatgaactga ttctcttcct ccttcttgtc ctttcttaat tctgttgtaa ttaccttcct
     3961 ttgtaatttt ttttgtaatt attcttctta ataatccaaa caaacacaca tattacaata
     4021 tattaaaatg tctagattag ataaaagtaa agtgattaac agcgcattag agctgcttaa
     4081 tgaggtcgga atcgaaggtt taacaacccg taaactcgcc cagaagctag gtgtagagca
     4141 gcctacattg tattggcatg taaaaaataa gcgggctttg ctcgacgcct tagccattga
     4201 gatgttagat aggcaccata ctcacttttg ccctttagaa ggggaaagct ggcaagattt
     4261 tttacgtaat aacgctaaaa gttttagatg tgctttacta agtcatcgcg atggagcaaa
     4321 agtacattta ggtacacggc ctacagaaaa acagtatgaa actctcgaaa atcaattagc
     4381 ctttttatgc caacaaggtt tttcactaga gaatgcatta tatgcactca gcgctgtggg
     4441 gcattttact ttaggttgcg tattggaaga tcaagagcat caagtcgcta aagaagaaag
     4501 ggaaacacct actactgata gtatgccgcc attattacga caagctatcg aattatttga
     4561 tcaccaaggt gcagagccag ccttcttatt cggccttgaa ttgatcatat gcggattaga
     4621 aaaacaactt aaatgtgaaa gtgggtctta agtctgaaga atgaatgatt tgatgatttc
     4681 tttttccctc catttttctt actgaatata tcaatgatat agacttgtat agtttattat
     4741 ttcaaattaa gtagctatat atagtcaaga taacgtttgt ttgacacgat tacattattc
     4801 gtcgacatct tttttcagcc tgtcgtggta gcaatttgag gagtattatt aattgaatag
     4861 gttcattttg cgctcgcata aacagttttc gtcagggaca gtatgttgga atgagtggta
     4921 attaatggtg acatgacatg ttatagcaat aaccttgatg tttacatcgt agtttaatgt
     4981 acaccccgcg aattcgttca agtaggagtg caccaattgc aaagggaaaa gctgaatggg
     5041 cagttcgaac cgcggtcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     5101 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     5161 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     5221 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
     5281 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc
     5341 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg
     5401 attacgccaa gcgcgcaatt aaccctcact aaagggaaca aaagctggag ctcatagctt
     5461 caaaatgttt ctactccttt tttactcttc cagattttct cggactccgc gcatcgccgt
     5521 accacttcaa aacacccaag cacagcatac taaatttccc ctctttcttc ctctagggtg
     5581 tcgttaatta cccgtactaa aggtttggaa aagaaaaaag agaccgcctc gtttcttttt
     5641 cttcgtcgaa aaaggcaata aaaattttta tcacgtttct ttttcttgaa aatttttttt
     5701 ttgatttttt tctctttcga tgacctccca ttgatattta agttaataaa cggtcttcaa
     5761 tttctcaagt ttcagtttca tttttcttgt tctattacaa ctttttttac ttcttgctca
     5821 ttagaaagaa agcatagcaa tctaatctaa gttttctaga actagtggat cccccgggaa
     5881 aaatggacaa gaagtactcc attgggctcg atatcggcac aaacagcgtc ggttgggccg
     5941 tcattacgga cgagtacaag gtgccgagca aaaaattcaa agttctgggc aataccgatc
     6001 gccacagcat aaagaagaac ctcattggcg ccctcctgtt cgactccggg gagacggccg
     6061 aagccacgcg gctcaaaaga acagcacggc gcagatatac ccgcagaaag aatcggatct
     6121 gctacctgca ggagatcttt agtaatgaga tggctaaggt ggatgactct ttcttccata
     6181 ggctggagga gtcctttttg gtggaggagg ataaaaagca cgagcgccac ccaatctttg
     6241 gcaatatcgt ggacgaggtg gcgtaccatg aaaagtaccc aaccatatat catctgagga
     6301 agaagcttgt agacagtact gataaggctg acttgcggtt gatctatctc gcgctggcgc
     6361 atatgatcaa atttcgggga cacttcctca tcgaggggga cctgaaccca gacaacagcg
     6421 atgtcgacaa actctttatc caactggttc agacttacaa tcagcttttc gaagagaacc
     6481 cgatcaacgc atccggagtt gacgccaaag caatcctgag cgctaggctg tccaaatccc
     6541 ggcggctcga aaacctcatc gcacagctcc ctggggagaa gaagaacggc ctgtttggta
     6601 atcttatcgc cctgtcactc gggctgaccc ccaactttaa atctaacttc gacctggccg
     6661 aagatgccaa gcttcaactg agcaaagaca cctacgatga tgatctcgac aatctgctgg
     6721 cccagatcgg cgaccagtac gcagaccttt ttttggcggc aaagaacctg tcagacgcca
     6781 ttctgctgag tgatattctg cgagtgaaca cggagatcac caaagctccg ctgagcgcta
     6841 gtatgatcaa gcgctatgat gagcaccacc aagacttgac tttgctgaag gcccttgtca
     6901 gacagcaact gcctgagaag tacaaggaaa ttttcttcga tcagtctaaa aatggctacg
     6961 ccggatacat tgacggcgga gcaagccagg aggaatttta caaatttatt aagcccatct
     7021 tggaaaaaat ggacggcacc gaggagctgc tggtaaagct taacagagaa gatctgttgc
     7081 gcaaacagcg cactttcgac aatggaagca tcccccacca gattcacctg ggcgaactgc
     7141 acgctatcct caggcggcaa gaggatttct accccttttt gaaagataac agggaaaaga
     7201 ttgagaaaat cctcacattt cggataccct actatgtagg ccccctcgcc cggggaaatt
     7261 ccagattcgc gtggatgact cgcaaatcag aagagaccat cactccctgg aacttcgagg
     7321 aagtcgtgga taagggggcc tctgcccagt ccttcatcga aaggatgact aactttgata
     7381 aaaatctgcc taacgaaaag gtgcttccta aacactctct gctgtacgag tacttcacag
     7441 tttataacga gctcaccaag gtcaaatacg tcacagaagg gatgagaaag ccagcattcc
     7501 tgtctggaga gcagaagaaa gctatcgtgg acctcctctt caagacgaac cggaaagtta
     7561 ccgtgaaaca gctcaaagaa gactatttca aaaagattga atgtttcgac tctgttgaaa
     7621 tcagcggagt ggaggatcgc ttcaacgcat ccctgggaac gtatcacgat ctcctgaaaa
     7681 tcattaaaga caaggacttc ctggacaatg aggagaacga ggacattctt gaggacattg
     7741 tcctcaccct tacgttgttt gaagataggg agatgattga agaacgcttg aaaacttacg
     7801 ctcatctctt cgacgacaaa gtcatgaaac agctcaagag gcgccgatat acaggatggg
     7861 ggcggctgtc aagaaaactg atcaatggga tccgagacaa gcagagtgga aagacaatcc
     7921 tggattttct taagtccgat ggatttgcca accggaactt catgcagttg atccatgatg
     7981 actctctcac ctttaaggag gacatccaga aagcacaagt ttctggccag ggggacagtc
     8041 ttcacgagca catcgctaat cttgcaggta gcccagctat caaaaaggga atactgcaga
     8101 ccgttaaggt cgtggatgaa ctcgtcaaag taatgggaag gcataagccc gagaatatcg
     8161 ttatcgagat ggcccgagag aaccaaacta cccagaaggg acagaagaac agtagggaaa
     8221 ggatgaagag gattgaagag ggtataaaag aactggggtc ccaaatcctt aaggaacacc
     8281 cagttgaaaa cacccagctt cagaatgaga agctctacct gtactacctg cagaacggca
     8341 gggacatgta cgtggatcag gaactggaca tcaatcggct ctccgactac gacgtggatc
     8401 atatcgtgcc ccagtctttt ctcaaagatg attctattga taataaagtg ttgacaagat
     8461 ccgataaaaa tagagggaag agtgataacg tcccctcaga agaagttgtc aagaaaatga
     8521 aaaattattg gcggcagctg ctgaacgcca aactgatcac acaacggaag ttcgataatc
     8581 tgactaaggc tgaacgaggt ggcctgtctg agttggataa agccggcttc atcaaaaggc
     8641 agcttgttga gacacgccag atcaccaagc acgtggccca aattctcgat tcacgcatga
     8701 acaccaagta cgatgaaaat gacaaactga ttcgagaggt gaaagttatt actctgaagt
     8761 ctaagctggt ctcagatttc agaaaggact ttcagtttta taaggtgaga gagatcaaca
     8821 attaccacca tgcgcatgat gcctacctga atgcagtggt aggcactgca cttatcaaaa
     8881 aatatcccaa gcttgaatct gaatttgttt acggagacta taaagtgtac gatgttagga
     8941 aaatgatcgc aaagtctgag caggaaatag gcaaggccac cgctaagtac ttcttttaca
     9001 gcaatattat gaattttttc aagaccgaga ttacactggc caatggagag attcggaagc
     9061 gaccacttat cgaaacaaac ggagaaacag gagaaatcgt gtgggacaag ggtagggatt
     9121 tcgcgacagt ccggaaggtc ctgtccatgc cgcaggtgaa catcgttaaa aagaccgaag
     9181 tacagaccgg aggcttctcc aaggaaagta tcctcccgaa aaggaacagc gacaagctga
     9241 tcgcacgcaa aaaagattgg gaccccaaga aatacggcgg attcgattct cctacagtcg
     9301 cttacagtgt actggttgtg gccaaagtgg agaaagggaa gtctaaaaaa ctcaaaagcg
     9361 tcaaggaact gctgggcatc acaatcatgg agcgatcaag cttcgaaaaa aaccccatcg
     9421 actttctcga ggcgaaagga tataaagagg tcaaaaaaga cctcatcatt aagcttccca
     9481 agtactctct ctttgagctt gaaaacggcc ggaaacgaat gctcgctagt gcgggcgagc
     9541 tgcagaaagg taacgagctg gcactgccct ctaaatacgt taatttcttg tatctggcca
     9601 gccactatga aaagctcaaa gggtctcccg aagataatga gcagaagcag ctgttcgtgg
     9661 aacaacacaa acactacctt gatgagatca tcgagcaaat aagcgaattc tccaaaagag
     9721 tgatcctcgc cgacgctaac ctcgataagg tgctttctgc ttacaataag cacagggata
     9781 agcccatcag ggagcaggca gaaaacatta tccacttgtt tactctgacc aacttgggcg
     9841 cgcctgcagc cttcaagtac ttcgacacca ccatagacag aaagcggtac acctctacaa
     9901 aggaggtcct ggacgccaca ctgattcatc agtcaattac ggggctctat gaaacaagaa
     9961 tcgacctctc tcagctcggt ggagacagca gggctgaccc caagaagaag aggaaggtgt
    10021 gatctcttct cgagtcatgt aattagttat gtcacgctta cattcacgcc ctccccccac
    10081 atccgctcta accgaaaagg aaggagttag acaacctgaa gtctaggtcc ctatttattt
    10141 ttttatagtt atgttagtat taagaacgtt atttatattt caaatttttc ttttttttct
    10201 gtacagacgc gtgtacgcat gtaacattat actgaaaacc ttgcttgaga aggttttggg
    10261 acgctcgaag gctttaattt gcggccggta cccaattcgc cctatagtga gtcgtattac
    10321 gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa
    10381 cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc
    10441 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggacgcg ccctgtagcg
    10501 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
    10561 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
    10621 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
    10681 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
    10741 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
    10801 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
    10861 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
    10921 aaatattaac gcttacaatt tcctgatgcg gtattttctc cttacgcatc tgtgcggtat
    10981 ttcacaccgc atagatcggc aagtgcacaa acaatactta aataaatact actcagtaat
    11041 aacctatttc ttagcatttt tgacgaaatt tgctattttg ttagagtctt ttacaccatt
    11101 tgtctccaca cctccgctta catcaacacc aataacgcca tttaatctaa gcgcatcacc
    11161 aacattttct ggcgtcagtc caccagctaa cataaaatgt aagctttcgg ggctctcttg
    11221 ccttccaacc cagtcagaaa tcgagttcca atccaaaagt tcacctgtcc cacctgcttc
    11281 tgaatcaaac aagggaataa acgaatgagg tttctgtgaa gctgcactga gtagtatgtt
    11341 gcagtctttt ggaaatacga gtcttttaat aactggcaaa ccgaggaact cttggtattc
    11401 ttgccacgac tcatctccat gcagttggac gatatcaatg ccgtaatcat tgaccagagc
    11461 caaaacatcc tccttaggtt gattacgaaa cacgccaacc aagtatttcg gagtgcctga
    11521 actattttta tatgctttta caagacttga aattttcctt gcaataaccg ggtcaattgt
    11581 tctctttcta ttgggcacac atataatacc cagcaagtca gcatcggaat ctagagcaca
    11641 ttctgcggcc tctgtgctct gcaagccgca aactttcacc aatggaccag aactacctgt
    11701 gaaattaata acagacatac tccaagctgc ctttgtgtgc ttaatcacgt atact
//
