LOCUS       sequence-330355-       11755 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for constituive spCas9 expression and tet-inducible 
            expression of an sgRNA targeting an intergenic site near CPR1 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_CPR1_2
FEATURES             Location/Qualifiers
     source          1..11755
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(71..352)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(373..392)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     492..514
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(552..570)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        638..742
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(743..811,812..1603)
                     /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(961..980)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1774..2362
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2263..2282
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    2746..2764
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        2850..2925
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      2938..3000
                     /label=RPR1 terminator
                     /note="transcription terminator for the S. cerevisiae RNase
                     P RNA gene"
     CDS             4135..4758
                     /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(4208..4227)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     5255..5272
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5389..5410
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5425..5430,5431..5448,5449..5455)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5463..5479
                     /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     5468..5490
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5487..5503
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5487..5503
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5522..5542
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5524..5542
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5560..5958
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     primer_bind     5962..5978
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5962..5978
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             5990..10093
                     /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             5990..10093
                     /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             5990..10093
                     /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             10106..10126
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      10142..10389
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(10158..10176)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(10407..10426)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(10408..10426)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(10436..10453)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(10436..10452)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10445..10467)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      10593..11048
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10680..10699)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     10890..10911
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(11151..11755,1..70))
                     /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"
ORIGIN
        1 cctctgtgct ctgcaagccg caaactttca ccaatggacc agaactacct gtgaaattaa
       61 taacagacat actccaagct gcctttgtgt gcttaatcac gtatactcac gtgctcaata
      121 gtcaccaatg ccctccctct tggccctctc cttttctttt ttcgaccgaa ttaattctta
      181 atcggcaaaa aaagaaaagc tccggatcaa gattgtacgt aaggtgacaa gctatttttc
      241 aataaagaat atcttccact actgccatct ggcgtcataa ctgcaaagta cacatatatt
      301 acgatgctgt tctattaaat gcttcctata ttatatatat agtaatgtcg ttgatctatg
      361 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc
      421 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
      481 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
      541 gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
      601 ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
      661 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
      721 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
      781 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
      841 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
      901 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
      961 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     1021 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     1081 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     1141 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     1201 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     1261 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     1321 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     1381 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     1441 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     1501 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     1561 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     1621 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     1681 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     1741 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     1801 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     1861 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     1921 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     1981 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     2041 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     2101 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     2161 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     2221 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     2281 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     2341 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     2401 ttgctggcct tttgctcggg ggatctgcca attgaacata acatggtagt tacatatact
     2461 agtaatatgg ttcggcacac attaaaagta taaaaactat ctgaattacg aattacatat
     2521 attggtcata aaaatcaatc aatcatcgtg tgttttatat gtctcttatc taagtataag
     2581 aatatccata gttaatattc acttacgcta ccttttaacc tgtaatcatt gtcaacagga
     2641 tatgttaacg acccacattg ataaacgcta gtatttcttt ttcctcttct tattggccgg
     2701 ctgtctctat actcccctat agtctgtttc ttttcgtttc gattgtccct atcagtgata
     2761 gagatggcgc acatggtacg ctgtggtgct cgcggctggg aacgaaactc tgggagctgc
     2821 gattggcagc ttaacaggca taacctagcg ttttagagct agaaatagca agttaaaata
     2881 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tctcgagcca
     2941 tatccaactt ccaatttaat ctttcttttt taattttcac ttatttgcga tacagaaaga
     3001 aaaaagcgat agtaactatt gaattttgtt tggatttggt tagattagat atggtttctc
     3061 tttatattta catgctaaaa atgggctaca ccagagatac ataattagat atatatacgc
     3121 cagtacacct tatcggccca agccttgtcc caaggcagcg ttttgttctt ggaaacgctg
     3181 ccctacacgt tcgctatgct tcaagaactt ttctgagcac ttcatgatgc atgtttgttc
     3241 cttattggtt agctttgatg ttgtgaagtc attgacacag tctgtgaaac atctttctac
     3301 cagattagag tacaaacgca tgaaatcctt catttgcttt tgttccacta ctttttggaa
     3361 ctcttgttgt tctttcctgc aggtagtcgt gcaatgtatg actttaagat ttgtgagcag
     3421 gaagaaaagg gagaatcttc taacgataaa cccttgaaaa actgggtaga ctacgctatg
     3481 ttgagttgct acgcaggctg cacaattaca cgagaatgct cccgcctagg atttaaggct
     3541 aagggacgtg caatgcagac gacagatcta aatgaccgtg tcggtgaagt gttcgccaaa
     3601 cttttcggtt aacacatgca gtgatgcacg cgcgatggtg ctaagttaca tatatatata
     3661 tatgtatagc catagtgatg tctaagtaac ctttatggta tatttcttaa tgtggaaaga
     3721 tactagcgcg cgcacccaca cacaagcttc gtcttttctt gaagaaaaga ggaagctcgc
     3781 taaatgggat tccactttcc gttccctgcc agctgatgga aaaaggttag tggaacgatg
     3841 aagaataaaa agagagatcc actgaggtga aatttcagct gacagcgagt ttcatgatcg
     3901 tgatgaacaa tggtaacgag ttgtggctgt tgccagggag ggtggttctc aacttttaat
     3961 gtatggccaa atcgctactt gggtttgtta tataacaaag aagaaataat gaactgattc
     4021 tcttcctcct tcttgtcctt tcttaattct gttgtaatta ccttcctttg taattttttt
     4081 tgtaattatt cttcttaata atccaaacaa acacacatat tacaatatat taaaatgtct
     4141 agattagata aaagtaaagt gattaacagc gcattagagc tgcttaatga ggtcggaatc
     4201 gaaggtttaa caacccgtaa actcgcccag aagctaggtg tagagcagcc tacattgtat
     4261 tggcatgtaa aaaataagcg ggctttgctc gacgccttag ccattgagat gttagatagg
     4321 caccatactc acttttgccc tttagaaggg gaaagctggc aagatttttt acgtaataac
     4381 gctaaaagtt ttagatgtgc tttactaagt catcgcgatg gagcaaaagt acatttaggt
     4441 acacggccta cagaaaaaca gtatgaaact ctcgaaaatc aattagcctt tttatgccaa
     4501 caaggttttt cactagagaa tgcattatat gcactcagcg ctgtggggca ttttacttta
     4561 ggttgcgtat tggaagatca agagcatcaa gtcgctaaag aagaaaggga aacacctact
     4621 actgatagta tgccgccatt attacgacaa gctatcgaat tatttgatca ccaaggtgca
     4681 gagccagcct tcttattcgg ccttgaattg atcatatgcg gattagaaaa acaacttaaa
     4741 tgtgaaagtg ggtcttaagt ctgaagaatg aatgatttga tgatttcttt ttccctccat
     4801 ttttcttact gaatatatca atgatataga cttgtatagt ttattatttc aaattaagta
     4861 gctatatata gtcaagataa cgtttgtttg acacgattac attattcgtc gacatctttt
     4921 ttcagcctgt cgtggtagca atttgaggag tattattaat tgaataggtt cattttgcgc
     4981 tcgcataaac agttttcgtc agggacagta tgttggaatg agtggtaatt aatggtgaca
     5041 tgacatgtta tagcaataac cttgatgttt acatcgtagt ttaatgtaca ccccgcgaat
     5101 tcgttcaagt aggagtgcac caattgcaaa gggaaaagct gaatgggcag ttcgaaccgc
     5161 ggtctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     5221 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     5281 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     5341 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
     5401 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
     5461 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg
     5521 cgcaattaac cctcactaaa gggaacaaaa gctggagctc atagcttcaa aatgtttcta
     5581 ctcctttttt actcttccag attttctcgg actccgcgca tcgccgtacc acttcaaaac
     5641 acccaagcac agcatactaa atttcccctc tttcttcctc tagggtgtcg ttaattaccc
     5701 gtactaaagg tttggaaaag aaaaaagaga ccgcctcgtt tctttttctt cgtcgaaaaa
     5761 ggcaataaaa atttttatca cgtttctttt tcttgaaaat tttttttttg atttttttct
     5821 ctttcgatga cctcccattg atatttaagt taataaacgg tcttcaattt ctcaagtttc
     5881 agtttcattt ttcttgttct attacaactt tttttacttc ttgctcatta gaaagaaagc
     5941 atagcaatct aatctaagtt ttctagaact agtggatccc ccgggaaaaa tggacaagaa
     6001 gtactccatt gggctcgata tcggcacaaa cagcgtcggt tgggccgtca ttacggacga
     6061 gtacaaggtg ccgagcaaaa aattcaaagt tctgggcaat accgatcgcc acagcataaa
     6121 gaagaacctc attggcgccc tcctgttcga ctccggggag acggccgaag ccacgcggct
     6181 caaaagaaca gcacggcgca gatatacccg cagaaagaat cggatctgct acctgcagga
     6241 gatctttagt aatgagatgg ctaaggtgga tgactctttc ttccataggc tggaggagtc
     6301 ctttttggtg gaggaggata aaaagcacga gcgccaccca atctttggca atatcgtgga
     6361 cgaggtggcg taccatgaaa agtacccaac catatatcat ctgaggaaga agcttgtaga
     6421 cagtactgat aaggctgact tgcggttgat ctatctcgcg ctggcgcata tgatcaaatt
     6481 tcggggacac ttcctcatcg agggggacct gaacccagac aacagcgatg tcgacaaact
     6541 ctttatccaa ctggttcaga cttacaatca gcttttcgaa gagaacccga tcaacgcatc
     6601 cggagttgac gccaaagcaa tcctgagcgc taggctgtcc aaatcccggc ggctcgaaaa
     6661 cctcatcgca cagctccctg gggagaagaa gaacggcctg tttggtaatc ttatcgccct
     6721 gtcactcggg ctgaccccca actttaaatc taacttcgac ctggccgaag atgccaagct
     6781 tcaactgagc aaagacacct acgatgatga tctcgacaat ctgctggccc agatcggcga
     6841 ccagtacgca gacctttttt tggcggcaaa gaacctgtca gacgccattc tgctgagtga
     6901 tattctgcga gtgaacacgg agatcaccaa agctccgctg agcgctagta tgatcaagcg
     6961 ctatgatgag caccaccaag acttgacttt gctgaaggcc cttgtcagac agcaactgcc
     7021 tgagaagtac aaggaaattt tcttcgatca gtctaaaaat ggctacgccg gatacattga
     7081 cggcggagca agccaggagg aattttacaa atttattaag cccatcttgg aaaaaatgga
     7141 cggcaccgag gagctgctgg taaagcttaa cagagaagat ctgttgcgca aacagcgcac
     7201 tttcgacaat ggaagcatcc cccaccagat tcacctgggc gaactgcacg ctatcctcag
     7261 gcggcaagag gatttctacc cctttttgaa agataacagg gaaaagattg agaaaatcct
     7321 cacatttcgg ataccctact atgtaggccc cctcgcccgg ggaaattcca gattcgcgtg
     7381 gatgactcgc aaatcagaag agaccatcac tccctggaac ttcgaggaag tcgtggataa
     7441 gggggcctct gcccagtcct tcatcgaaag gatgactaac tttgataaaa atctgcctaa
     7501 cgaaaaggtg cttcctaaac actctctgct gtacgagtac ttcacagttt ataacgagct
     7561 caccaaggtc aaatacgtca cagaagggat gagaaagcca gcattcctgt ctggagagca
     7621 gaagaaagct atcgtggacc tcctcttcaa gacgaaccgg aaagttaccg tgaaacagct
     7681 caaagaagac tatttcaaaa agattgaatg tttcgactct gttgaaatca gcggagtgga
     7741 ggatcgcttc aacgcatccc tgggaacgta tcacgatctc ctgaaaatca ttaaagacaa
     7801 ggacttcctg gacaatgagg agaacgagga cattcttgag gacattgtcc tcacccttac
     7861 gttgtttgaa gatagggaga tgattgaaga acgcttgaaa acttacgctc atctcttcga
     7921 cgacaaagtc atgaaacagc tcaagaggcg ccgatataca ggatgggggc ggctgtcaag
     7981 aaaactgatc aatgggatcc gagacaagca gagtggaaag acaatcctgg attttcttaa
     8041 gtccgatgga tttgccaacc ggaacttcat gcagttgatc catgatgact ctctcacctt
     8101 taaggaggac atccagaaag cacaagtttc tggccagggg gacagtcttc acgagcacat
     8161 cgctaatctt gcaggtagcc cagctatcaa aaagggaata ctgcagaccg ttaaggtcgt
     8221 ggatgaactc gtcaaagtaa tgggaaggca taagcccgag aatatcgtta tcgagatggc
     8281 ccgagagaac caaactaccc agaagggaca gaagaacagt agggaaagga tgaagaggat
     8341 tgaagagggt ataaaagaac tggggtccca aatccttaag gaacacccag ttgaaaacac
     8401 ccagcttcag aatgagaagc tctacctgta ctacctgcag aacggcaggg acatgtacgt
     8461 ggatcaggaa ctggacatca atcggctctc cgactacgac gtggatcata tcgtgcccca
     8521 gtcttttctc aaagatgatt ctattgataa taaagtgttg acaagatccg ataaaaatag
     8581 agggaagagt gataacgtcc cctcagaaga agttgtcaag aaaatgaaaa attattggcg
     8641 gcagctgctg aacgccaaac tgatcacaca acggaagttc gataatctga ctaaggctga
     8701 acgaggtggc ctgtctgagt tggataaagc cggcttcatc aaaaggcagc ttgttgagac
     8761 acgccagatc accaagcacg tggcccaaat tctcgattca cgcatgaaca ccaagtacga
     8821 tgaaaatgac aaactgattc gagaggtgaa agttattact ctgaagtcta agctggtctc
     8881 agatttcaga aaggactttc agttttataa ggtgagagag atcaacaatt accaccatgc
     8941 gcatgatgcc tacctgaatg cagtggtagg cactgcactt atcaaaaaat atcccaagct
     9001 tgaatctgaa tttgtttacg gagactataa agtgtacgat gttaggaaaa tgatcgcaaa
     9061 gtctgagcag gaaataggca aggccaccgc taagtacttc ttttacagca atattatgaa
     9121 ttttttcaag accgagatta cactggccaa tggagagatt cggaagcgac cacttatcga
     9181 aacaaacgga gaaacaggag aaatcgtgtg ggacaagggt agggatttcg cgacagtccg
     9241 gaaggtcctg tccatgccgc aggtgaacat cgttaaaaag accgaagtac agaccggagg
     9301 cttctccaag gaaagtatcc tcccgaaaag gaacagcgac aagctgatcg cacgcaaaaa
     9361 agattgggac cccaagaaat acggcggatt cgattctcct acagtcgctt acagtgtact
     9421 ggttgtggcc aaagtggaga aagggaagtc taaaaaactc aaaagcgtca aggaactgct
     9481 gggcatcaca atcatggagc gatcaagctt cgaaaaaaac cccatcgact ttctcgaggc
     9541 gaaaggatat aaagaggtca aaaaagacct catcattaag cttcccaagt actctctctt
     9601 tgagcttgaa aacggccgga aacgaatgct cgctagtgcg ggcgagctgc agaaaggtaa
     9661 cgagctggca ctgccctcta aatacgttaa tttcttgtat ctggccagcc actatgaaaa
     9721 gctcaaaggg tctcccgaag ataatgagca gaagcagctg ttcgtggaac aacacaaaca
     9781 ctaccttgat gagatcatcg agcaaataag cgaattctcc aaaagagtga tcctcgccga
     9841 cgctaacctc gataaggtgc tttctgctta caataagcac agggataagc ccatcaggga
     9901 gcaggcagaa aacattatcc acttgtttac tctgaccaac ttgggcgcgc ctgcagcctt
     9961 caagtacttc gacaccacca tagacagaaa gcggtacacc tctacaaagg aggtcctgga
    10021 cgccacactg attcatcagt caattacggg gctctatgaa acaagaatcg acctctctca
    10081 gctcggtgga gacagcaggg ctgaccccaa gaagaagagg aaggtgtgat ctcttctcga
    10141 gtcatgtaat tagttatgtc acgcttacat tcacgccctc cccccacatc cgctctaacc
    10201 gaaaaggaag gagttagaca acctgaagtc taggtcccta tttatttttt tatagttatg
    10261 ttagtattaa gaacgttatt tatatttcaa atttttcttt tttttctgta cagacgcgtg
    10321 tacgcatgta acattatact gaaaaccttg cttgagaagg ttttgggacg ctcgaaggct
    10381 ttaatttgcg gccggtaccc aattcgccct atagtgagtc gtattacgcg cgctcactgg
    10441 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg
    10501 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt
    10561 cccaacagtt gcgcagcctg aatggcgaat ggacgcgccc tgtagcggcg cattaagcgc
    10621 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
    10681 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
    10741 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
    10801 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
    10861 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
    10921 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
    10981 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
    11041 tacaatttcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
    11101 gatcggcaag tgcacaaaca atacttaaat aaatactact cagtaataac ctatttctta
    11161 gcatttttga cgaaatttgc tattttgtta gagtctttta caccatttgt ctccacacct
    11221 ccgcttacat caacaccaat aacgccattt aatctaagcg catcaccaac attttctggc
    11281 gtcagtccac cagctaacat aaaatgtaag ctttcggggc tctcttgcct tccaacccag
    11341 tcagaaatcg agttccaatc caaaagttca cctgtcccac ctgcttctga atcaaacaag
    11401 ggaataaacg aatgaggttt ctgtgaagct gcactgagta gtatgttgca gtcttttgga
    11461 aatacgagtc ttttaataac tggcaaaccg aggaactctt ggtattcttg ccacgactca
    11521 tctccatgca gttggacgat atcaatgccg taatcattga ccagagccaa aacatcctcc
    11581 ttaggttgat tacgaaacac gccaaccaag tatttcggag tgcctgaact atttttatat
    11641 gcttttacaa gacttgaaat tttccttgca ataaccgggt caattgttct ctttctattg
    11701 ggcacacata taatacccag caagtcagca tcggaatcta gagcacattc tgcgg
//
