LOCUS       sequence-170411-       11117 bp    DNA     circular SYN 14-APR-2025
DEFINITION  All-in-one CRISPR plasmid for integrating, deleting, or replacing a 
            gene in S. cerevisiae. High-copy, URA3 plasmid contains human 
            codon-optimized Cas9 and a guide RNA targeting ARS416d sequence 
            TAGTGCACTTACCCCACGTT in yeast chromosome 4..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11117)
  AUTHORS   Reider Apel A, d'Espaux L, Wehrs M, Sachs D, Li RA, Tong GJ, Garber 
            M, Nnadi O, Zhuang W, Hillson NJ, Keasling JD, Mukhopadhyay A
  TITLE     A Cas9-based toolkit to program gene expression in Saccharomyces 
            cerevisiae.
  JOURNAL   Nucleic Acids Res. 2017 Jan 9;45(1):496-508. doi: 
            10.1093/nar/gkw1023. Epub 2016 Nov 28.
  PUBMED    27899650
REFERENCE   2  (bases 1 to 11117)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1093/nar/gkw1023"; journalName: "Nucleic Acids Res"; date: 
            "2017-01-9- 9"; volume: "45"; issue: "1"; pages: "496-508"
COMMENT     Sequence Label: p426_Cas9_gRNA-ARS416d
FEATURES             Location/Qualifiers
     source          1..11117
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(10..28)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     rep_origin      69..1411
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    401..448
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     promoter        1438..1542
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1543..1611,1612..2403)
                     /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(1761..1780)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2574..3162
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3063..3082
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3316..3333
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        join(3486..3491,3492..3509,3510..3516)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3524..3540
                     /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     3529..3551
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3548..3564
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3548..3564
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     3583..3603
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        3585..3603
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     misc_RNA        3830..3905
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3991..4695
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 promoter
                     /note="promoter for alcohol dehydrogenase 1"
     CDS             4705..8808
                     /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             4705..8808
                     /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             4705..8808
                     /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             8821..8841
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      8857..9104
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(9122..9141)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(9123..9141)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(9151..9168)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(9151..9167)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9160..9182)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9312..9767
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9399..9418)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9609..9630
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(9898..10701)
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(10702..10922)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(10948..10967)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     11067..11089
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttagtatga tccaatatca aaggaaatga tagcattgaa ggatgagact aatccaattg
      121 aggagtggca gcatatagaa cagctaaagg gtagtgctga aggaagcata cgataccccg
      181 catggaatgg gataatatca caggaggtac tagactacct ttcatcctac ataaatagac
      241 gcatataagt acgcatttaa gcataaacac gcactatgcc gttcttctca tgtatatata
      301 tatacaggca acacgcagat ataggtgcga cgtgaacagt gagctgtatg tgcgcagctc
      361 gcgttgcatt ttcggaagcg ctcgttttcg gaaacgcttt gaagttccta ttccgaagtt
      421 cctattctct agaaagtata ggaacttcag agcgcttttg aaaaccaaaa gcgctctgaa
      481 gacgcacttt caaaaaacca aaaacgcacc ggactgtaac gagctactaa aatattgcga
      541 ataccgcttc cacaaacatt gctcaaaagt atctctttgc tatatatctc tgtgctatat
      601 ccctatataa cctacccatc cacctttcgc tccttgaact tgcatctaaa ctcgacctct
      661 acatttttta tgtttatctc tagtattact ctttagacaa aaaaattgta gtaagaacta
      721 ttcatagagt gaatcgaaaa caatacgaaa atgtaaacat ttcctatacg tagtatatag
      781 agacaaaata gaagaaaccg ttcataattt tctgaccaat gaagaatcat caacgctatc
      841 actttctgtt cacaaagtat gcgcaatcca catcggtata gaatataatc ggggatgcct
      901 ttatcttgaa aaaatgcacc cgcagcttcg ctagtaatca gtaaacgcgg gaagtggagt
      961 caggcttttt ttatggaaga gaaaatagac accaaagtag ccttcttcta accttaacgg
     1021 acctacagtg caaaaagtta tcaagagact gcattataga gcgcacaaag gagaaaaaaa
     1081 gtaatctaag atgctttgtt agaaaaatag cgctctcggg atgcattttt gtagaacaaa
     1141 aaagaagtat agattctttg ttggtaaaat agcgctctcg cgttgcattt ctgttctgta
     1201 aaaatgcagc tcagattctt tgtttgaaaa attagcgctc tcgcgttgca tttttgtttt
     1261 acaaaaatga agcacagatt cttcgttggt aaaatagcgc tttcgcgttg catttctgtt
     1321 ctgtaaaaat gcagctcaga ttctttgttt gaaaaattag cgctctcgcg ttgcattttt
     1381 gttctacaaa atgaagcaca gatgcttcgt tcaggtggca cttttcgggg aaatgtgcgc
     1441 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     1501 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     1561 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     1621 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     1681 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     1741 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     1801 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     1861 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     1921 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     1981 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     2041 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     2101 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     2161 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     2221 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     2281 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     2341 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     2401 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     2461 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     2521 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     2581 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     2641 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     2701 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     2761 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     2821 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     2881 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     2941 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     3001 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     3061 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     3121 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     3181 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     3241 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     3301 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa
     3361 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
     3421 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt acctcactca ttaggcaccc
     3481 caggctttac actttatgct tccggctcct atgttgtgtg gaattgtgag cggataacaa
     3541 tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa ccctcactaa
     3601 agggaacaaa agctggagct agtatactct ttcttcaaca attaaatact ctcggtagcc
     3661 aagttggttt aaggcgcaag actgtaattt atcactacga aatcttgaga tcgggcgttc
     3721 gactcgcccc cgggagagat ggccggcatg gtcccagcct cctcgctggc gccggctggg
     3781 caacaccttc gggtggcgaa tgggactttt agtgcactta ccccacgttg ttttagagct
     3841 agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc
     3901 ggtgcttttt ttattttttg tcactattgt tatgtaaaat gccacctctg acagtatgga
     3961 acgcaaactt ctgtctagtg gatagtcgac atccttttgt tgtttccggg tgtacaatat
     4021 ggacttcctc ttttctggca accaaaccca tacatcggga ttcctataat accttcgttg
     4081 gtctccctaa catgtaggtg gcggagggga gatatacaat agaacagata ccagacaaga
     4141 cataatgggc taaacaagac tacaccaatt acactgcctc attgatggtg gtacataacg
     4201 aactaatact gtagccctag acttgatagc catcatcata tcgaagtttc actacccttt
     4261 ttccatttgc catctattga agtaataata ggcgcatgca acttcttttc tttttttttc
     4321 ttttctctct cccccgttgt tgtctcacca tatccgcaat gacaaaaaaa tgatggaaga
     4381 cactaaagga aaaaattaac gacaaagaca gcaccaacag atgtcgttgt tccagagctg
     4441 atgaggggta tctcgaagca cacgaaactt tttccttcct tcattcacgc acactactct
     4501 ctaatgagca acggtatacg gccttccttc cagttacttg aatttgaaat aaaaaaaagt
     4561 ttgctgtctt gctatcaagt ataaatagac ctgcaattat taatcttttg tttcctcgtc
     4621 attgttctcg ttccctttct tccttgtttc tttttctgca caatatttca agctatacca
     4681 agcatacaat caacttggaa aaaaatggac aagaagtact ccattgggct cgatatcggc
     4741 acaaacagcg tcggttgggc cgtcattacg gacgagtaca aggtgccgag caaaaaattc
     4801 aaagttctgg gcaataccga tcgccacagc ataaagaaga acctcattgg cgccctcctg
     4861 ttcgactccg gggagacggc cgaagccacg cggctcaaaa gaacagcacg gcgcagatat
     4921 acccgcagaa agaatcggat ctgctacctg caggagatct ttagtaatga gatggctaag
     4981 gtggatgact ctttcttcca taggctggag gagtcctttt tggtggagga ggataaaaag
     5041 cacgagcgcc acccaatctt tggcaatatc gtggacgagg tggcgtacca tgaaaagtac
     5101 ccaaccatat atcatctgag gaagaagctt gtagacagta ctgataaggc tgacttgcgg
     5161 ttgatctatc tcgcgctggc gcatatgatc aaatttcggg gacacttcct catcgagggg
     5221 gacctgaacc cagacaacag cgatgtcgac aaactcttta tccaactggt tcagacttac
     5281 aatcagcttt tcgaagagaa cccgatcaac gcatccggag ttgacgccaa agcaatcctg
     5341 agcgctaggc tgtccaaatc ccggcggctc gaaaacctca tcgcacagct ccctggggag
     5401 aagaagaacg gcctgtttgg taatcttatc gccctgtcac tcgggctgac ccccaacttt
     5461 aaatctaact tcgacctggc cgaagatgcc aagcttcaac tgagcaaaga cacctacgat
     5521 gatgatctcg acaatctgct ggcccagatc ggcgaccagt acgcagacct ttttttggcg
     5581 gcaaagaacc tgtcagacgc cattctgctg agtgatattc tgcgagtgaa cacggagatc
     5641 accaaagctc cgctgagcgc tagtatgatc aagcgctatg atgagcacca ccaagacttg
     5701 actttgctga aggcccttgt cagacagcaa ctgcctgaga agtacaagga aattttcttc
     5761 gatcagtcta aaaatggcta cgccggatac attgacggcg gagcaagcca ggaggaattt
     5821 tacaaattta ttaagcccat cttggaaaaa atggacggca ccgaggagct gctggtaaag
     5881 cttaacagag aagatctgtt gcgcaaacag cgcactttcg acaatggaag catcccccac
     5941 cagattcacc tgggcgaact gcacgctatc ctcaggcggc aagaggattt ctaccccttt
     6001 ttgaaagata acagggaaaa gattgagaaa atcctcacat ttcggatacc ctactatgta
     6061 ggccccctcg cccggggaaa ttccagattc gcgtggatga ctcgcaaatc agaagagacc
     6121 atcactccct ggaacttcga ggaagtcgtg gataaggggg cctctgccca gtccttcatc
     6181 gaaaggatga ctaactttga taaaaatctg cctaacgaaa aggtgcttcc taaacactct
     6241 ctgctgtacg agtacttcac agtttataac gagctcacca aggtcaaata cgtcacagaa
     6301 gggatgagaa agccagcatt cctgtctgga gagcagaaga aagctatcgt ggacctcctc
     6361 ttcaagacga accggaaagt taccgtgaaa cagctcaaag aagactattt caaaaagatt
     6421 gaatgtttcg actctgttga aatcagcgga gtggaggatc gcttcaacgc atccctggga
     6481 acgtatcacg atctcctgaa aatcattaaa gacaaggact tcctggacaa tgaggagaac
     6541 gaggacattc ttgaggacat tgtcctcacc cttacgttgt ttgaagatag ggagatgatt
     6601 gaagaacgct tgaaaactta cgctcatctc ttcgacgaca aagtcatgaa acagctcaag
     6661 aggcgccgat atacaggatg ggggcggctg tcaagaaaac tgatcaatgg gatccgagac
     6721 aagcagagtg gaaagacaat cctggatttt cttaagtccg atggatttgc caaccggaac
     6781 ttcatgcagt tgatccatga tgactctctc acctttaagg aggacatcca gaaagcacaa
     6841 gtttctggcc agggggacag tcttcacgag cacatcgcta atcttgcagg tagcccagct
     6901 atcaaaaagg gaatactgca gaccgttaag gtcgtggatg aactcgtcaa agtaatggga
     6961 aggcataagc ccgagaatat cgttatcgag atggcccgag agaaccaaac tacccagaag
     7021 ggacagaaga acagtaggga aaggatgaag aggattgaag agggtataaa agaactgggg
     7081 tcccaaatcc ttaaggaaca cccagttgaa aacacccagc ttcagaatga gaagctctac
     7141 ctgtactacc tgcagaacgg cagggacatg tacgtggatc aggaactgga catcaatcgg
     7201 ctctccgact acgacgtgga tcatatcgtg ccccagtctt ttctcaaaga tgattctatt
     7261 gataataaag tgttgacaag atccgataaa aatagaggga agagtgataa cgtcccctca
     7321 gaagaagttg tcaagaaaat gaaaaattat tggcggcagc tgctgaacgc caaactgatc
     7381 acacaacgga agttcgataa tctgactaag gctgaacgag gtggcctgtc tgagttggat
     7441 aaagccggct tcatcaaaag gcagcttgtt gagacacgcc agatcaccaa gcacgtggcc
     7501 caaattctcg attcacgcat gaacaccaag tacgatgaaa atgacaaact gattcgagag
     7561 gtgaaagtta ttactctgaa gtctaagctg gtctcagatt tcagaaagga ctttcagttt
     7621 tataaggtga gagagatcaa caattaccac catgcgcatg atgcctacct gaatgcagtg
     7681 gtaggcactg cacttatcaa aaaatatccc aagcttgaat ctgaatttgt ttacggagac
     7741 tataaagtgt acgatgttag gaaaatgatc gcaaagtctg agcaggaaat aggcaaggcc
     7801 accgctaagt acttctttta cagcaatatt atgaattttt tcaagaccga gattacactg
     7861 gccaatggag agattcggaa gcgaccactt atcgaaacaa acggagaaac aggagaaatc
     7921 gtgtgggaca agggtaggga tttcgcgaca gtccggaagg tcctgtccat gccgcaggtg
     7981 aacatcgtta aaaagaccga agtacagacc ggaggcttct ccaaggaaag tatcctcccg
     8041 aaaaggaaca gcgacaagct gatcgcacgc aaaaaagatt gggaccccaa gaaatacggc
     8101 ggattcgatt ctcctacagt cgcttacagt gtactggttg tggccaaagt ggagaaaggg
     8161 aagtctaaaa aactcaaaag cgtcaaggaa ctgctgggca tcacaatcat ggagcgatca
     8221 agcttcgaaa aaaaccccat cgactttctc gaggcgaaag gatataaaga ggtcaaaaaa
     8281 gacctcatca ttaagcttcc caagtactct ctctttgagc ttgaaaacgg ccggaaacga
     8341 atgctcgcta gtgcgggcga gctgcagaaa ggtaacgagc tggcactgcc ctctaaatac
     8401 gttaatttct tgtatctggc cagccactat gaaaagctca aagggtctcc cgaagataat
     8461 gagcagaagc agctgttcgt ggaacaacac aaacactacc ttgatgagat catcgagcaa
     8521 ataagcgaat tctccaaaag agtgatcctc gccgacgcta acctcgataa ggtgctttct
     8581 gcttacaata agcacaggga taagcccatc agggagcagg cagaaaacat tatccacttg
     8641 tttactctga ccaacttggg cgcgcctgca gccttcaagt acttcgacac caccatagac
     8701 agaaagcggt acacctctac aaaggaggtc ctggacgcca cactgattca tcagtcaatt
     8761 acggggctct atgaaacaag aatcgacctc tctcagctcg gtggagacag cagggctgac
     8821 cccaagaaga agaggaaggt gtgatctctt ctcgagtcat gtaattagtt atgtcacgct
     8881 tacgttcacg ccctcccccc acatccgctc taaccgaaaa ggaaggagtt agacaacctg
     8941 aagtctaggt ccctatttat ttttttatag ttatgttagt attaagaacg ttatttatat
     9001 ttcaaatttt tctttttttt ctgtacagac gcgtgtacgc atgtaacatt atactgaaaa
     9061 ccttgcttga gaaggttttg ggacgctcga aggctttaat ttgcggccgg tacccaattc
     9121 gccctatagt gagtcgtatt acgcgcgctc actggccgtc gttttacaac gtcgtgactg
     9181 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg
     9241 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg
     9301 cgaatggcgc gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     9361 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     9421 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     9481 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     9541 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     9601 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     9661 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     9721 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcct gatgcggtat
     9781 tttctcctta cgcatctgtg cggtatttca caccgcatag ggtaataact gatataatta
     9841 aattgaagct ctaatttgtg agtttagtat acatgcattt acttataata cagtttttta
     9901 gttttgctgg ccgcatcttc tcaaatatgc ttcccagcct gcttttctgt aacgttcacc
     9961 ctctacctta gcatcccttc cctttgcaaa tagtcctctt ccaacaataa taatgtcaga
    10021 tcctgtagag accacatcat ccacggttct atactgttga cccaatgcgt ctcccttgtc
    10081 atctaaaccc acaccgggtg tcataatcaa ccaatcgtaa ccttcatctc ttccacccat
    10141 gtctctttga gcaataaagc cgataacaaa atctttgtcg ctcttcgcaa tgtcaacagt
    10201 acccttagta tattctccag tagataggga gcccttgcat gacaattctg ctaacatcaa
    10261 aaggcctcta ggttcctttg ttacttcttc tgccgcctgc ttcaaaccgc taacaatacc
    10321 tgggcccacc acaccgtgtg cattcgtaat gtctgcccat tctgctattc tgtatacacc
    10381 cgcagagtac tgcaatttga ctgtattacc aatgtcagca aattttctgt cttcgaagag
    10441 taaaaaattg tacttggcgg ataatgcctt tagcggctta actgtgccct ccatggaaaa
    10501 atcagtcaag atatccacat gtgtttttag taaacaaatt ttgggaccta atgcttcaac
    10561 taactccagt aattccttgg tggtacgaac atccaatgaa gcacacaagt ttgtttgctt
    10621 ttcgtgcatg atattaaata gcttggcagc aacaggacta ggatgagtag cagcacgttc
    10681 cttatatgta gctttcgaca tgatttatct tcgtttcctg caggtttttg ttctgtgcag
    10741 ttgggttaag aatactgggc aatttcatgt ttcttcaaca ctacatatgc gtatatatac
    10801 caatctaagt ctgtgctcct tccttcgttc ttccttctgt tcggagatta ccgaatcaaa
    10861 aaaatttcaa ggaaaccgaa atcaaaaaaa agaataaaaa aaaaatgatg aattgaattg
    10921 aaaagctgtg gtatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
    10981 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
    11041 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
    11101 atcaccgaaa cgcgcga
//
