LOCUS       sequence-263114-       11274 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pAEF5 plasmid with two gRNA inserted.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11274)
  AUTHORS   Agier N, Fleiss A, Delmas S, Fischer G
  TITLE     A Versatile Protocol to Generate Translocations in Yeast Genomes 
            Using CRISPR/Cas9.
  JOURNAL   Methods Mol Biol. 2021;2196:181-198. doi: 
            10.1007/978-1-0716-0868-5_14.
  PUBMED    32889721
REFERENCE   2  (bases 1 to 11274)
  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"; journalName: "Methods Mol
            Biol."; date: "2021"; volume: "2196:181-198. doi"; pages: " 
            10.1007/978-1-0716-0868-5_14"
COMMENT     Sequence Label: pAEF6
FEATURES             Location/Qualifiers
     source          1..11274
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     260..282
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(320..338)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     rep_origin      379..1721
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    711..758
                     /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        1748..1852
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1853..1921,1922..2713)
                     /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(2071..2090)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2884..3472
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3373..3392
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3626..3643
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3760..3781
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3796..3801,3802..3819,3820..3826)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3834..3850
                     /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     3839..3861
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3858..3874
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3858..3874
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     3893..3913
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        3895..3913
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        3931..4329
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     primer_bind     4333..4349
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4333..4349
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             4361..8464
                     /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             4361..8464
                     /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             4361..8464
                     /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             8477..8497
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      8513..8760
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(8529..8547)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     promoter        8782..9050
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        9071..9145
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      9150..9169
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     promoter        9192..9460
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        9481..9555
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      9560..9579
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     primer_bind     complement(9601..9620)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(9602..9620)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(9630..9647)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(9630..9646)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9639..9661)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     gene            join(9751..11274,1..52)
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     promoter        9751..10094
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             10095..11123
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             10095..11123
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     terminator      join(11129..11274,1..52)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
ORIGIN
        1 gcagaaagta atatcatgcg tcaatcgtat gtgaatgctg gtcgctatac tgctgtcgat
       61 tcgatactaa cgccgccatc cagtgtcgaa aacgagctcg aattcatcga tgatatcaga
      121 tccactagtg gcctatgcgg cccaatctgc tctgatgccg catagttaag ccagccccga
      181 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
      241 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
      301 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
      361 ataatggttt cttagtatga tccaatatca aaggaaatga tagcattgaa ggatgagact
      421 aatccaattg aggagtggca gcatatagaa cagctaaagg gtagtgctga aggaagcata
      481 cgataccccg catggaatgg gataatatca caggaggtac tagactacct ttcatcctac
      541 ataaatagac gcatataagt acgcatttaa gcataaacac gcactatgcc gttcttctca
      601 tgtatatata tatacaggca acacgcagat ataggtgcga cgtgaacagt gagctgtatg
      661 tgcgcagctc gcgttgcatt ttcggaagcg ctcgttttcg gaaacgcttt gaagttccta
      721 ttccgaagtt cctattctct agaaagtata ggaacttcag agcgcttttg aaaaccaaaa
      781 gcgctctgaa gacgcacttt caaaaaacca aaaacgcacc ggactgtaac gagctactaa
      841 aatattgcga ataccgcttc cacaaacatt gctcaaaagt atctctttgc tatatatctc
      901 tgtgctatat ccctatataa cctacccatc cacctttcgc tccttgaact tgcatctaaa
      961 ctcgacctct acatttttta tgtttatctc tagtattact ctttagacaa aaaaattgta
     1021 gtaagaacta ttcatagagt gaatcgaaaa caatacgaaa atgtaaacat ttcctatacg
     1081 tagtatatag agacaaaata gaagaaaccg ttcataattt tctgaccaat gaagaatcat
     1141 caacgctatc actttctgtt cacaaagtat gcgcaatcca catcggtata gaatataatc
     1201 ggggatgcct ttatcttgaa aaaatgcacc cgcagcttcg ctagtaatca gtaaacgcgg
     1261 gaagtggagt caggcttttt ttatggaaga gaaaatagac accaaagtag ccttcttcta
     1321 accttaacgg acctacagtg caaaaagtta tcaagagact gcattataga gcgcacaaag
     1381 gagaaaaaaa gtaatctaag atgctttgtt agaaaaatag cgctctcggg atgcattttt
     1441 gtagaacaaa aaagaagtat agattctttg ttggtaaaat agcgctctcg cgttgcattt
     1501 ctgttctgta aaaatgcagc tcagattctt tgtttgaaaa attagcgctc tcgcgttgca
     1561 tttttgtttt acaaaaatga agcacagatt cttcgttggt aaaatagcgc tttcgcgttg
     1621 catttctgtt ctgtaaaaat gcagctcaga ttctttgttt gaaaaattag cgctctcgcg
     1681 ttgcattttt gttctacaaa atgaagcaca gatgcttcgt tcaggtggca cttttcgggg
     1741 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     1801 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     1861 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     1921 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     1981 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     2041 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     2101 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     2161 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     2221 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     2281 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     2341 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     2401 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     2461 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     2521 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     2581 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     2641 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     2701 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     2761 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     2821 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     2881 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     2941 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     3001 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     3061 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     3121 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     3181 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     3241 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     3301 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     3361 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     3421 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     3481 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     3541 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     3601 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg agagacgccc
     3661 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
     3721 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca
     3781 ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag
     3841 cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa
     3901 ccctcactaa agggaacaaa agctggagct catagcttca aaatgtttct actccttttt
     3961 tactcttcca gattttctcg gactccgcgc atcgccgtac cacttcaaaa cacccaagca
     4021 cagcatacta aatttcccct ctttcttcct ctagggtgtc gttaattacc cgtactaaag
     4081 gtttggaaaa gaaaaaagag accgcctcgt ttctttttct tcgtcgaaaa aggcaataaa
     4141 aatttttatc acgtttcttt ttcttgaaaa tttttttttt gatttttttc tctttcgatg
     4201 acctcccatt gatatttaag ttaataaacg gtcttcaatt tctcaagttt cagtttcatt
     4261 tttcttgttc tattacaact ttttttactt cttgctcatt agaaagaaag catagcaatc
     4321 taatctaagt tttctagaac tagtggatcc cccgggaaaa atggacaaga agtactccat
     4381 tgggctcgat atcggcacaa acagcgtcgg ttgggccgtc attacggacg agtacaaggt
     4441 gccgagcaaa aaattcaaag ttctgggcaa taccgatcgc cacagcataa agaagaacct
     4501 cattggcgcc ctcctgttcg actccgggga gacggccgaa gccacgcggc tcaaaagaac
     4561 agcacggcgc agatataccc gcagaaagaa tcggatctgc tacctgcagg agatctttag
     4621 taatgagatg gctaaggtgg atgactcttt cttccatagg ctggaggagt cctttttggt
     4681 ggaggaggat aaaaagcacg agcgccaccc aatctttggc aatatcgtgg acgaggtggc
     4741 gtaccatgaa aagtacccaa ccatatatca tctgaggaag aagcttgtag acagtactga
     4801 taaggctgac ttgcggttga tctatctcgc gctggcgcat atgatcaaat ttcggggaca
     4861 cttcctcatc gagggggacc tgaacccaga caacagcgat gtcgacaaac tctttatcca
     4921 actggttcag acttacaatc agcttttcga agagaacccg atcaacgcat ccggagttga
     4981 cgccaaagca atcctgagcg ctaggctgtc caaatcccgg cggctcgaaa acctcatcgc
     5041 acagctccct ggggagaaga agaacggcct gtttggtaat cttatcgccc tgtcactcgg
     5101 gctgaccccc aactttaaat ctaacttcga cctggccgaa gatgccaagc ttcaactgag
     5161 caaagacacc tacgatgatg atctcgacaa tctgctggcc cagatcggcg accagtacgc
     5221 agaccttttt ttggcggcaa agaacctgtc agacgccatt ctgctgagtg atattctgcg
     5281 agtgaacacg gagatcacca aagctccgct gagcgctagt atgatcaagc gctatgatga
     5341 gcaccaccaa gacttgactt tgctgaaggc ccttgtcaga cagcaactgc ctgagaagta
     5401 caaggaaatt ttcttcgatc agtctaaaaa tggctacgcc ggatacattg acggcggagc
     5461 aagccaggag gaattttaca aatttattaa gcccatcttg gaaaaaatgg acggcaccga
     5521 ggagctgctg gtaaagctta acagagaaga tctgttgcgc aaacagcgca ctttcgacaa
     5581 tggaagcatc ccccaccaga ttcacctggg cgaactgcac gctatcctca ggcggcaaga
     5641 ggatttctac ccctttttga aagataacag ggaaaagatt gagaaaatcc tcacatttcg
     5701 gataccctac tatgtaggcc ccctcgcccg gggaaattcc agattcgcgt ggatgactcg
     5761 caaatcagaa gagaccatca ctccctggaa cttcgaggaa gtcgtggata agggggcctc
     5821 tgcccagtcc ttcatcgaaa ggatgactaa ctttgataaa aatctgccta acgaaaaggt
     5881 gcttcctaaa cactctctgc tgtacgagta cttcacagtt tataacgagc tcaccaaggt
     5941 caaatacgtc acagaaggga tgagaaagcc agcattcctg tctggagagc agaagaaagc
     6001 tatcgtggac ctcctcttca agacgaaccg gaaagttacc gtgaaacagc tcaaagaaga
     6061 ctatttcaaa aagattgaat gtttcgactc tgttgaaatc agcggagtgg aggatcgctt
     6121 caacgcatcc ctgggaacgt atcacgatct cctgaaaatc attaaagaca aggacttcct
     6181 ggacaatgag gagaacgagg acattcttga ggacattgtc ctcaccctta cgttgtttga
     6241 agatagggag atgattgaag aacgcttgaa aacttacgct catctcttcg acgacaaagt
     6301 catgaaacag ctcaagaggc gccgatatac aggatggggg cggctgtcaa gaaaactgat
     6361 caatgggatc cgagacaagc agagtggaaa gacaatcctg gattttctta agtccgatgg
     6421 atttgccaac cggaacttca tgcagttgat ccatgatgac tctctcacct ttaaggagga
     6481 catccagaaa gcacaagttt ctggccaggg ggacagtctt cacgagcaca tcgctaatct
     6541 tgcaggtagc ccagctatca aaaagggaat actgcagacc gttaaggtcg tggatgaact
     6601 cgtcaaagta atgggaaggc ataagcccga gaatatcgtt atcgagatgg cccgagagaa
     6661 ccaaactacc cagaagggac agaagaacag tagggaaagg atgaagagga ttgaagaggg
     6721 tataaaagaa ctggggtccc aaatccttaa ggaacaccca gttgaaaaca cccagcttca
     6781 gaatgagaag ctctacctgt actacctgca gaacggcagg gacatgtacg tggatcagga
     6841 actggacatc aatcggctct ccgactacga cgtggatcat atcgtgcccc agtcttttct
     6901 caaagatgat tctattgata ataaagtgtt gacaagatcc gataaaaata gagggaagag
     6961 tgataacgtc ccctcagaag aagttgtcaa gaaaatgaaa aattattggc ggcagctgct
     7021 gaacgccaaa ctgatcacac aacggaagtt cgataatctg actaaggctg aacgaggtgg
     7081 cctgtctgag ttggataaag ccggcttcat caaaaggcag cttgttgaga cacgccagat
     7141 caccaagcac gtggcccaaa ttctcgattc acgcatgaac accaagtacg atgaaaatga
     7201 caaactgatt cgagaggtga aagttattac tctgaagtct aagctggtct cagatttcag
     7261 aaaggacttt cagttttata aggtgagaga gatcaacaat taccaccatg cgcatgatgc
     7321 ctacctgaat gcagtggtag gcactgcact tatcaaaaaa tatcccaagc ttgaatctga
     7381 atttgtttac ggagactata aagtgtacga tgttaggaaa atgatcgcaa agtctgagca
     7441 ggaaataggc aaggccaccg ctaagtactt cttttacagc aatattatga attttttcaa
     7501 gaccgagatt acactggcca atggagagat tcggaagcga ccacttatcg aaacaaacgg
     7561 agaaacagga gaaatcgtgt gggacaaggg tagggatttc gcgacagtcc ggaaggtcct
     7621 gtccatgccg caggtgaaca tcgttaaaaa gaccgaagta cagaccggag gcttctccaa
     7681 ggaaagtatc ctcccgaaaa ggaacagcga caagctgatc gcacgcaaaa aagattggga
     7741 ccccaagaaa tacggcggat tcgattctcc tacagtcgct tacagtgtac tggttgtggc
     7801 caaagtggag aaagggaagt ctaaaaaact caaaagcgtc aaggaactgc tgggcatcac
     7861 aatcatggag cgatcaagct tcgaaaaaaa ccccatcgac tttctcgagg cgaaaggata
     7921 taaagaggtc aaaaaagacc tcatcattaa gcttcccaag tactctctct ttgagcttga
     7981 aaacggccgg aaacgaatgc tcgctagtgc gggcgagctg cagaaaggta acgagctggc
     8041 actgccctct aaatacgtta atttcttgta tctggccagc cactatgaaa agctcaaagg
     8101 gtctcccgaa gataatgagc agaagcagct gttcgtggaa caacacaaac actaccttga
     8161 tgagatcatc gagcaaataa gcgaattctc caaaagagtg atcctcgccg acgctaacct
     8221 cgataaggtg ctttctgctt acaataagca cagggataag cccatcaggg agcaggcaga
     8281 aaacattatc cacttgttta ctctgaccaa cttgggcgcg cctgcagcct tcaagtactt
     8341 cgacaccacc atagacagaa agcggtacac ctctacaaag gaggtcctgg acgccacact
     8401 gattcatcag tcaattacgg ggctctatga aacaagaatc gacctctctc agctcggtgg
     8461 agacagcagg gctgacccca agaagaagag gaaggtgtga tctcttctcg agtcatgtaa
     8521 ttagttatgt cacgcttaca ttcacgccct ccccccacat ccgctctaac cgaaaaggaa
     8581 ggagttagac aacctgaagt ctaggtccct atttattttt ttatagttat gttagtatta
     8641 agaacgttat ttatatttca aatttttctt ttttttctgt acagacgcgt gtacgcatgt
     8701 aacattatac tgaaaacctt gcttgagaag gttttgggac gctcgaaggc tttaatttgc
     8761 ggccggtacc gcgggcccgc ttctttgaaa agataatgta tgattatgct ttcactcata
     8821 tttatacaga aacttgatgt tttctttcga gtatatacaa ggtgattaca tgtacgtttg
     8881 aagtacaact ctagattttg tagtgccctc ttgggctagc ggtaaaggtg cgcatttttt
     8941 cacaccctac aatgttctgt tcaaaagatt ttggtcaaac gctgtagaag tgaaagttgg
     9001 tgcgcatgtt tcggcgttcg aaacttctcc gcagtgaaag ataaatgatc catgtcgaaa
     9061 gctacatata gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac
     9121 ttgaaaaagt ggcaccgagt cggtggtgct ttttttgttt tttatgtctg cggccgcggt
     9181 acccaattcg ctctttgaaa agataatgta tgattatgct ttcactcata tttatacaga
     9241 aacttgatgt tttctttcga gtatatacaa ggtgattaca tgtacgtttg aagtacaact
     9301 ctagattttg tagtgccctc ttgggctagc ggtaaaggtg cgcatttttt cacaccctac
     9361 aatgttctgt tcaaaagatt ttggtcaaac gctgtagaag tgaaagttgg tgcgcatgtt
     9421 tcggcgttcg aaacttctcc gcagtgaaag ataaatgatc gcaaaactaa aaaactgtat
     9481 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt
     9541 ggcaccgagt cggtggtgct ttttttgttt tttatgtctg cggccgcggt acccaattcg
     9601 ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg
     9661 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccagatc tgtttagctt
     9721 gcctcgtccc cgccgggtca cccggccagc gacatggagg cccagaatac cctccttgac
     9781 agtcttgacg tgcgcagctc aggggcatga tgtgactgtc gcccgtacat ttagcccata
     9841 catccccatg tataatcatt tgcatccata cattttgatg gccgcacggc gcgaagcaaa
     9901 aattacggct cctcgctgcg gacctgcgag cagggaaacg ctcccctcac agacgcgttg
     9961 aattgtcccc acgccgcgcc cctgtagaga aatataaaag gttaggattt gccactgagg
    10021 ttcttctttc atatacttcc ttttaaaatc ttgctaggat acagttctca catcacatcc
    10081 gaacataaac aaccatgggt aaaaagcctg aactcaccgc gacgtctgtc gagaagtttc
    10141 tgatcgaaaa gttcgacagc gtctccgacc tgatgcagct ctcggagggc gaagaatctc
    10201 gtgctttcag cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat agctgcgccg
    10261 atggtttcta caaagatcgt tatgtttatc ggcactttgc atcggccgcg ctcccgattc
    10321 cggaagtgct tgacattggg gaattcagcg agagcctgac ctattgcatc tcccgccgtg
    10381 cacagggtgt cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt ctgcagccgg
    10441 tcgcggaggc catggatgcg atcgctgcgg ccgatcttag ccagacgagc gggttcggcc
    10501 cattcggacc gcaaggaatc ggtcaataca ctacatggcg tgatttcata tgcgcgattg
    10561 ctgatcccca tgtgtatcac tggcaaactg tgatggacga caccgtcagt gcgtccgtcg
    10621 cgcaggctct cgatgagctg atgctttggg ccgaggactg ccccgaagtc cggcacctcg
    10681 tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa tggccgcata acagcggtca
    10741 ttgactggag cgaggcgatg ttcggggatt cccaatacga ggtcgccaac atcttcttct
    10801 ggaggccgtg gttggcttgt atggagcagc agacgcgcta cttcgagcgg aggcatccgg
    10861 agcttgcagg atcgccgcgg ctccgggcgt atatgctccg cattggtctt gaccaactct
    10921 atcagagctt ggttgacggc aatttcgatg atgcagcttg ggcgcagggt cgatgcgacg
    10981 caatcgtccg atccggagcc gggactgtcg ggcgtacaca aatcgcccgc agaagcgcgg
    11041 ccgtctggac cgatggctgt gtagaagtac tcgccgatag tggaaaccga cgccccagca
    11101 ctcgtccgag ggcaaaggaa taatcagtac tgacaataaa aagattcttg ttttcaagaa
    11161 cttgtcattt gtatagtttt tttatattgt agttgttcta ttttaatcaa atgttagcgt
    11221 gatttatatt ttttttcgcc tcgacatcat ctgcccagat gcgaagttaa gtgc
//
