LOCUS       sequence-484407-        9833 bp    DNA     circular SYN 22-AUG-2025
DEFINITION  Expresses Cas9 protein, under the control of the TEF1 promoter, and 
            CYC terminator. KanMX resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9833)
  AUTHORS   Jessop-Fabre MM, Jakociunas T, Stovicek V, Dai Z, Jensen MK, 
            Keasling JD, Borodina I
  TITLE     EasyClone-MarkerFree: A vector toolkit for marker-less integration 
            of genes into Saccharomyces cerevisiae via CRISPR-Cas9.
  JOURNAL   Biotechnol J. 2016 Aug;11(8):1110-7. doi: 10.1002/biot.201600147. 
            Epub 2016 Jun 23.
  PUBMED    27166612
REFERENCE   2  (bases 1 to 9833)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 22, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1002/biot.201600147"; journalName: "Biotechnol J"; date: 
            "2016-08"; volume: "11"; issue: "8"; pages: "1110-7"
COMMENT     Sequence Label: pCfB2312 (TEF1p-Cas9-CYC1t_kanMX)
FEATURES             Location/Qualifiers
     source          1..9833
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    58..561
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        598..702
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(703..771,772..1563)
                     /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(921..940)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1734..2322
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2223..2242
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2476..2493
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2610..2631
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2646..2651,2652..2669,2670..2676)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2684..2700
                     /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     2689..2711
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2708..2724
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2708..2724
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2743..2763
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2745..2763
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        2781..3179
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     primer_bind     3183..3199
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3183..3199
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             3211..7314
                     /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             3211..7314
                     /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             3211..7314
                     /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             7327..7347
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      7363..7610
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(7379..7397)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(7628..7647)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7629..7647)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(7657..7674)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7657..7673)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7666..7688)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      7814..8194
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7901..7920)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8111..8132
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     protein_bind    complement(8246..8279)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     gene            complement(8342..9698)
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     terminator      complement(8342..8539)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(8545..9354)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     9268..9287
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(9355..9698)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     protein_bind    complement(9753..9786)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
ORIGIN
        1 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacggat
       61 cgcttgcctg taacttacac gcgcctcgta tcttttaatg atggaataat ttgggaattt
      121 actctgtgtt tatttatttt tatgttttgt atttggattt tagaaagtaa ataaagaagg
      181 tagaagagtt acggaatgaa gaaaaaaaaa taaacaaagg tttaaaaaat ttcaacaaaa
      241 agcgtacttt acatatatat ttattagaca agaaaagcag attaaataga tatacattcg
      301 attaacgata agtaaaatgt aaaatcacag gattttcgtg tgtggtcttc tacacagaca
      361 agatgaaaca attcggcatt aatacctgag agcaggaaga gcaagataaa aggtagtatt
      421 tgttggcgat ccccctagag tcttttacat cttcggaaaa caaaaactat tttttcttta
      481 atttcttttt ttactttcta tttttaattt atatatttat attaaaaaat ttaaattata
      541 attattttta tagcacgtga tgaaaaggac ccaggtggca cttttcgggg aaatgtgcgc
      601 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
      661 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
      721 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
      781 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
      841 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
      901 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
      961 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     1021 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     1081 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     1141 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     1201 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     1261 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     1321 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     1381 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     1441 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     1501 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     1561 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     1621 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     1681 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     1741 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     1801 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     1861 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     1921 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     1981 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     2041 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     2101 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     2161 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     2221 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     2281 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     2341 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     2401 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     2461 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa
     2521 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
     2581 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc
     2641 caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa
     2701 tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa ccctcactaa
     2761 agggaacaaa agctggagct catagcttca aaatgtttct actccttttt tactcttcca
     2821 gattttctcg gactccgcgc atcgccgtac cacttcaaaa cacccaagca cagcatacta
     2881 aatttcccct ctttcttcct ctagggtgtc gttaattacc cgtactaaag gtttggaaaa
     2941 gaaaaaagag accgcctcgt ttctttttct tcgtcgaaaa aggcaataaa aatttttatc
     3001 acgtttcttt ttcttgaaaa tttttttttt gatttttttc tctttcgatg acctcccatt
     3061 gatatttaag ttaataaacg gtcttcaatt tctcaagttt cagtttcatt tttcttgttc
     3121 tattacaact ttttttactt cttgctcatt agaaagaaag catagcaatc taatctaagt
     3181 tttctagaac tagtggatcc cccgggaaaa atggacaaga agtactccat tgggctcgat
     3241 atcggcacaa acagcgtcgg ttgggccgtc attacggacg agtacaaggt gccgagcaaa
     3301 aaattcaaag ttctgggcaa taccgatcgc cacagcataa agaagaacct cattggcgcc
     3361 ctcctgttcg actccgggga gacggccgaa gccacgcggc tcaaaagaac agcacggcgc
     3421 agatataccc gcagaaagaa tcggatctgc tacctgcagg agatctttag taatgagatg
     3481 gctaaggtgg atgactcttt cttccatagg ctggaggagt cctttttggt ggaggaggat
     3541 aaaaagcacg agcgccaccc aatctttggc aatatcgtgg acgaggtggc gtaccatgaa
     3601 aagtacccaa ccatatatca tctgaggaag aagcttgtag acagtactga taaggctgac
     3661 ttgcggttga tctatctcgc gctggcgcat atgatcaaat ttcggggaca cttcctcatc
     3721 gagggggacc tgaacccaga caacagcgat gtcgacaaac tctttatcca actggttcag
     3781 acttacaatc agcttttcga agagaacccg atcaacgcat ccggagttga cgccaaagca
     3841 atcctgagcg ctaggctgtc caaatcccgg cggctcgaaa acctcatcgc acagctccct
     3901 ggggagaaga agaacggcct gtttggtaat cttatcgccc tgtcactcgg gctgaccccc
     3961 aactttaaat ctaacttcga cctggccgaa gatgccaagc ttcaactgag caaagacacc
     4021 tacgatgatg atctcgacaa tctgctggcc cagatcggcg accagtacgc agaccttttt
     4081 ttggcggcaa agaacctgtc agacgccatt ctgctgagtg atattctgcg agtgaacacg
     4141 gagatcacca aagctccgct gagcgctagt atgatcaagc gctatgatga gcaccaccaa
     4201 gacttgactt tgctgaaggc ccttgtcaga cagcaactgc ctgagaagta caaggaaatt
     4261 ttcttcgatc agtctaaaaa tggctacgcc ggatacattg acggcggagc aagccaggag
     4321 gaattttaca aatttattaa gcccatcttg gaaaaaatgg acggcaccga ggagctgctg
     4381 gtaaagctta acagagaaga tctgttgcgc aaacagcgca ctttcgacaa tggaagcatc
     4441 ccccaccaga ttcacctggg cgaactgcac gctatcctca ggcggcaaga ggatttctac
     4501 ccctttttga aagataacag ggaaaagatt gagaaaatcc tcacatttcg gataccctac
     4561 tatgtaggcc ccctcgcccg gggaaattcc agattcgcgt ggatgactcg caaatcagaa
     4621 gagaccatca ctccctggaa cttcgaggaa gtcgtggata agggggcctc tgcccagtcc
     4681 ttcatcgaaa ggatgactaa ctttgataaa aatctgccta acgaaaaggt gcttcctaaa
     4741 cactctctgc tgtacgagta cttcacagtt tataacgagc tcaccaaggt caaatacgtc
     4801 acagaaggga tgagaaagcc agcattcctg tctggagagc agaagaaagc tatcgtggac
     4861 ctcctcttca agacgaaccg gaaagttacc gtgaaacagc tcaaagaaga ctatttcaaa
     4921 aagattgaat gtttcgactc tgttgaaatc agcggagtgg aggatcgctt caacgcatcc
     4981 ctgggaacgt atcacgatct cctgaaaatc attaaagaca aggacttcct ggacaatgag
     5041 gagaacgagg acattcttga ggacattgtc ctcaccctta cgttgtttga agatagggag
     5101 atgattgaag aacgcttgaa aacttacgct catctcttcg acgacaaagt catgaaacag
     5161 ctcaagaggc gccgatatac aggatggggg cggctgtcaa gaaaactgat caatgggatc
     5221 cgagacaagc agagtggaaa gacaatcctg gattttctta agtccgatgg atttgccaac
     5281 cggaacttca tgcagttgat ccatgatgac tctctcacct ttaaggagga catccagaaa
     5341 gcacaagttt ctggccaggg ggacagtctt cacgagcaca tcgctaatct tgcaggtagc
     5401 ccagctatca aaaagggaat actgcagacc gttaaggtcg tggatgaact cgtcaaagta
     5461 atgggaagac ataagcccga gaatatcgtt atcgagatgg cccgagagaa ccaaactacc
     5521 cagaagggac agaagaacag tagggaaagg atgaagagga ttgaagaggg tataaaagaa
     5581 ctggggtccc aaatccttaa ggaacaccca gttgaaaaca cccagcttca gaatgagaag
     5641 ctctacctgt actacctgca gaacggcagg gacatgtacg tggatcagga actggacatc
     5701 aatcggctct ccgactacga cgtggatcat atcgtgcccc agtcttttct caaagatgat
     5761 tctattgata ataaagtgtt gacaagatcc gataaaaata gagggaagag tgataacgtc
     5821 ccctcagaag aagttgtcaa gaaaatgaaa aattattggc ggcagctgct gaacgccaaa
     5881 ctgatcacac aacggaagtt cgataatctg actaaggctg aacgaggtgg cctgtctgag
     5941 ttggataaag ccggcttcat caaaaggcag cttgttgaga cacgccagat caccaagcac
     6001 gtggcccaaa ttctcgattc acgcatgaac accaagtacg atgaaaatga caaactgatt
     6061 cgagaggtga aagttattac tctgaagtct aagctggtct cagatttcag aaaggacttt
     6121 cagttttata aggtgagaga gatcaacaat taccaccatg cgcatgatgc ctacctgaat
     6181 gcagtggtag gcactgcact tatcaaaaaa tatcccaagc ttgaatctga atttgtttac
     6241 ggagactata aagtgtacga tgttaggaaa atgatcgcaa agtctgagca ggaaataggc
     6301 aaggccaccg ctaagtactt cttttacagc aatattatga attttttcaa gaccgagatt
     6361 acactggcca atggagagat tcggaagcga ccacttatcg aaacaaacgg agaaacagga
     6421 gaaatcgtgt gggacaaggg tagggatttc gcgacagtcc ggaaggtcct gtccatgccg
     6481 caggtgaaca tcgttaaaaa gaccgaagta cagaccggag gcttctccaa ggaaagtatc
     6541 ctcccgaaaa ggaacagcga caagctgatc gcacgcaaaa aagattggga ccccaagaaa
     6601 tacggcggat tcgattctcc tacagtcgct tacagtgtac tggttgtggc caaagtggag
     6661 aaagggaagt ctaaaaaact caaaagcgtc aaggaactgc tgggcatcac aatcatggag
     6721 cgatcaagct tcgaaaaaaa ccccatcgac tttctcgagg cgaaaggata taaagaggtc
     6781 aaaaaagacc tcatcattaa gcttcccaag tactctctct ttgagcttga aaacggccgg
     6841 aaacgaatgc tcgctagtgc gggcgagctg cagaaaggta acgagctggc actgccctct
     6901 aaatacgtta atttcttgta tctggccagc cactatgaaa agctcaaagg gtctcccgaa
     6961 gataatgagc agaagcagct gttcgtggaa caacacaaac actaccttga tgagatcatc
     7021 gagcaaataa gcgaattctc caaaagagtg atcctcgccg acgctaacct cgataaggtg
     7081 ctttctgctt acaataagca cagggataag cccatcaggg agcaggcaga aaacattatc
     7141 cacttgttta ctctgaccaa cttgggcgcg cctgcagcct tcaagtactt cgacaccacc
     7201 atagacagaa agcggtacac ctctacaaag gaggtcctgg acgccacact gattcatcag
     7261 tcaattacgg ggctctatga aacaagaatc gacctctctc agctcggtgg agacagcagg
     7321 gctgacccca agaagaagag gaaggtgtga tctcttctcg agtcatgtaa ttagttatgt
     7381 cacgcttaca ttcacgccct ccccccacat ccgctctaac cgaaaaggaa ggagttagac
     7441 aacctgaagt ctaggtccct atttattttt ttatagttat gttagtatta agaacgttat
     7501 ttatatttca aatttttctt ttttttctgt acagacgcgt gtacgcatgt aacattatac
     7561 tgaaaacctt gcttgagaag gttttgggac gctcgaaggc tttaatttgc ggccggtacc
     7621 caattcgccc tatagtgagt cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg
     7681 tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc
     7741 cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct
     7801 gaatggcgaa tggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     7861 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     7921 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     7981 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     8041 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     8101 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     8161 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaattg ggtgcatagg
     8221 ccactagtgg atctgatatc acctaataac ttcgtatagc atacattata cgaagttata
     8281 ttaagggttc tcgagagctc gttttcgaca ctggatggcg gcgttagtat cgaatcgaca
     8341 gcagtatagc gaccagcatt cacatacgat tgacgcatga tattactttc tgcgcactta
     8401 acttcgcatc tgggcagatg atgtcgaggc gaaaaaaaat ataaatcacg ctaacatttg
     8461 attaaaatag aacaactaca atataaaaaa actatacaaa tgacaagttc ttgaaaacaa
     8521 gaatcttttt attgtcagta ctgattagaa aaactcatcg agcatcaaat gaaactgcaa
     8581 tttattcata tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg
     8641 agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc
     8701 gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag
     8761 tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagct tatgcatttc
     8821 tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac
     8881 caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat cgctgttaaa
     8941 aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac
     9001 aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt tgccggggat
     9061 cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag
     9121 aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac
     9181 gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata
     9241 gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc
     9301 atccatgttg gaatttaatc gcggcctcga aacgtgagtc ttttccttac ccatggttgt
     9361 ttatgttcgg atgtgatgtg agaactgtat cctagcaaga ttttaaaagg aagtatatga
     9421 aagaagaacc tcagtggcaa atcctaacct tttatatttc tctacagggg cgcggcgtgg
     9481 ggacaattca acgcgtctgt gaggggagcg tttccctgct cgcaggtctg cagcgaggag
     9541 ccgtaatttt tgcttcgcgc cgtgcggcca tcaaaatgta tggatgcaaa tgattataca
     9601 tggggatgta tgggctaaat gtacgggcga cagtcacatc atgcccctga gctgcgcacg
     9661 tcaagactgt caaggagggt attctgggcc tccatgtcgc tggccgggtg acccggcggg
     9721 gacgaggcaa gctaaacaga tctctagacc taataacttc gtatagcata cattatacga
     9781 agttatatta agggttgtcg acctgcagcg tacgaagctt cagctgacgc gat
//
