LOCUS       sequence-318661-       12346 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Pre-express Cas9 in yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12346)
  AUTHORS   Sankaran SM, Smith JD, Roy KR
  TITLE     CRISPR-Cas9 Gene Editing in Yeast: A Molecular Biology and 
            Bioinformatics Laboratory Module for Undergraduate and High School 
            Students.
  JOURNAL   J Microbiol Biol Educ. 2021 May 31;22(2):e00106-21. doi: 
            10.1128/jmbe.00106-21. eCollection 2021 Fall.
  PUBMED    34594460
REFERENCE   2  (bases 1 to 12346)
  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"; doi: 
            "10.1128/jmbe.00106-21"; journalName: "J Microbiol Biol Educ"; date:
            "2021-05-31- 31"; volume: "22"; issue: "2"; pages: "e00106-21"
COMMENT     Sequence Label: pCas9
FEATURES             Location/Qualifiers
     source          1..12346
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     gene            1..1576
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     promoter        1..344
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             345..1373
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             345..1373
                     /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      1379..1576
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     promoter        1864..2271
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             2272..3366
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     primer_bind     complement(3860..3879)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3979..4001
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4039..4057)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    4099..4602
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        4639..4743
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4744..4812,4813..5604)
                     /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(4962..4981)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5775..6363
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6264..6283
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6517..6534
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6651..6672
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6687..6692,6693..6710,6711..6717)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6725..6741
                     /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     6730..6752
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6749..6765
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6749..6765
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6784..6804
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6786..6804
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        6822..7220
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     primer_bind     7224..7240
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7224..7240
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             7252..11355
                     /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             7252..11355
                     /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             7252..11355
                     /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             11368..11388
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      11404..11651
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(11420..11438)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(11669..11688)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(11670..11688)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(11698..11715)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(11698..11714)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11707..11729)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      11855..12310
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11942..11961)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     12152..12173
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 gacatggagg cccagaatac cctccttgac agtcttgacg tgcgcagctc aggggcatga
       61 tgtgactgtc gcccgtacat ttagcccata catccccatg tataatcatt tgcatccata
      121 cattttgatg gccgcacggc gcgaagcaaa aattacggct cctcgctgcg gacctgcgag
      181 cagggaaacg ctcccctcac agacgcgttg aattgtcccc acgccgcgcc cctgtagaga
      241 aatataaaag gttaggattt gccactgagg ttcttctttc atatacttcc ttttaaaatc
      301 ttgctaggat acagttctca catcacatcc gaacataaac aaccatgggt aaaaagcctg
      361 aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc
      421 tgatgcagct ctcggagggc gaagaatctc gtgctttcag cttcgatgta ggagggcgtg
      481 gatatgtcct gcgggtaaat agctgcgccg atggtttcta caaagatcgt tatgtttatc
      541 ggcactttgc atcggccgcg ctcccgattc cggaagtgct tgacattggg gaattcagcg
      601 agagcctgac ctattgcatc tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg
      661 aaaccgaact gcccgctgtt ctgcagccgg tcgcggaggc catggatgcg atcgctgcgg
      721 ccgatcttag ccagacgagc gggttcggcc cattcggacc gcaaggaatc ggtcaataca
      781 ctacatggcg tgatttcata tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg
      841 tgatggacga caccgtcagt gcgtccgtcg cgcaggctct cgatgagctg atgctttggg
      901 ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc
      961 tgacggacaa tggccgcata acagcggtca ttgactggag cgaggcgatg ttcggggatt
     1021 cccaatacga ggtcgccaac atcttcttct ggaggccgtg gttggcttgt atggagcagc
     1081 agacgcgcta cttcgagcgg aggcatccgg agcttgcagg atcgccgcgg ctccgggcgt
     1141 atatgctccg cattggtctt gaccaactct atcagagctt ggttgacggc aatttcgatg
     1201 atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg atccggagcc gggactgtcg
     1261 ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac cgatggctgt gtagaagtac
     1321 tcgccgatag tggaaaccga cgccccagca ctcgtccgag ggcaaaggaa taatcagtac
     1381 tgacaataaa aagattcttg ttttcaagaa cttgtcattt gtatagtttt tttatattgt
     1441 agttgttcta ttttaatcaa atgttagcgt gatttatatt ttttttcgcc tcgacatcat
     1501 ctgcccagat gcgaagttaa gtgcgcagaa agtaatatca tgcgtcaatc gtatgtgaat
     1561 gctggtcgct atactgctgt cgattcgata ctaacgccgc catccagtgt taattaacga
     1621 cggtcgagga gaacttctag tatatctaca tacctaatat tattgcctta ttaaaaatgg
     1681 aatcccaaca attacatcaa aatccacatt ctcttcaaaa tcaattgtcc tgtacttcct
     1741 tgttcatgtg tgttcaaaaa cgttatattt ataggataat tatactctat ttctcaacaa
     1801 gtaattggtt gtttggccga gcggtctaag gcgcctgatt caagaaatat cttgaccgca
     1861 gttaactgtg ggaatactca ggtatcgtaa gatgcaagag ttcgaatctc ttagcaacca
     1921 ttattttttt cctcaacata acgagaacac acaggggcgc tatcgcacag aatcaaattc
     1981 gatgactgga aattttttgt taatttcaga ggtcgcctga cgcatatacc tttttcaact
     2041 gaaaaattgg gagaaaaagg aaaggtgaga gcgccggaac cggcttttca tatagaatag
     2101 agaagcgttc atgactaaat gcttgcatca caatacttga agttgacaat attatttaag
     2161 gacctattgt tttttccaat aggtggttag caatcgtctt actttctaac ttttcttacc
     2221 ttttacattt cagcaatata tatatatata tttcaaggat ataccattct aatgtctgcc
     2281 cctaagaaga tcgtcgtttt gccaggtgac cacgttggtc aagaaatcac agccgaagcc
     2341 attaaggttc ttaaagctat ttctgatgtt cgttccaatg tcaagttcga tttcgaaaat
     2401 catttaattg gtggtgctgc tatcgatgct acaggtgttc cacttccaga tgaggcgctg
     2461 gaagcctcca agaaggctga tgccgttttg ttaggtgctg tgggtggtcc taaatggggt
     2521 accggtagtg ttagacctga acaaggttta ctaaaaatcc gtaaagaact tcaattgtac
     2581 gccaacttaa gaccatgtaa ctttgcatcc gactctcttt tagacttatc tccaatcaag
     2641 ccacaatttg ctaaaggtac tgacttcgtt gttgtcagag aattagtggg aggtatttac
     2701 tttggtaaga gaaaggaaga cgatggtgat ggtgtcgctt gggatagtga acaatacacc
     2761 gttccagaag tgcaaagaat cacaagaatg gccgctttca tggccctaca acatgagcca
     2821 ccattgccta tttggtcctt ggataaagct aatgttttgg cctcttcaag attatggaga
     2881 aaaactgtgg aggaaaccat caagaacgaa ttccctacat tgaaggttca acatcaattg
     2941 attgattctg ccgccatgat cctagttaag aacccaaccc acctaaatgg tattataatc
     3001 accagcaata tgtttggtga tatcatctcc gatgaagcct ccgttatccc aggttccttg
     3061 ggtttgttgc catctgcgtc cttggcctct ttgccagaca agaacaccgc atttggtttg
     3121 tacgaaccat gccacggttc tgctccagat ttgccaaaga ataaggtcaa ccctatcgcc
     3181 actatcttgt ctgctgcaat gatgttgaaa ttgtcattga acttgcctga agaaggtaag
     3241 gccattgaag atgcagttaa aaaggttttg gatgcaggta tcagaactgg tgatttaggt
     3301 ggttccaaca gtaccaccga agtcggtgat gctgtcgccg aagaagttaa gaaaatcctt
     3361 gcttaaaaag attctctttt tttatgatat ttgtacataa actttataaa tgaaattcat
     3421 aatagaaacg acacgaaatt acaaaatgga atatgttcat agggtagacg aaactatata
     3481 cgcaatctac atacatttat caagaaggag aaaaaggagg atgtaaagga atacaggtaa
     3541 gcaaattgat actaatggct caacgtgata aggaaaaaga attgcacttt aacattaata
     3601 ttgacaagga ggagggcacc acacaaaaag ttaggtgtaa cagaaaatca tgaaactatg
     3661 attcctaatt tatatattgg aggattttct ctaaaaaaaa aaaaatacaa caaataaaaa
     3721 acactcaatg acctgaccat ttgatggagt ttaagtcaat accttcttga accatttccc
     3781 ataatggtga aagttccctc aagaatttta ctctgtcaga aacggcctta acgacgtagt
     3841 cgatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
     3901 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     3961 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     4021 aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
     4081 taatggtttc ttaggacgga tcgcttgcct gtaacttaca cgcgcctcgt atcttttaat
     4141 gatggaataa tttgggaatt tactctgtgt ttatttattt ttatgttttg tatttggatt
     4201 ttagaaagta aataaagaag gtagaagagt tacggaatga agaaaaaaaa ataaacaaag
     4261 gtttaaaaaa tttcaacaaa aagcgtactt tacatatata tttattagac aagaaaagca
     4321 gattaaatag atatacattc gattaacgat aagtaaaatg taaaatcaca ggattttcgt
     4381 gtgtggtctt ctacacagac aagatgaaac aattcggcat taatacctga gagcaggaag
     4441 aaaaagataa aaggtagtat ttgttggcga tccccctaga gtcttttaca tcttcggaaa
     4501 acaaaaacta ttttttcttt aatttctttt tttactttct atttttaatt tatatattta
     4561 tattaaaaaa tttaaattat aattattttt atagcacgtg atgaaaagga cccaggtggc
     4621 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     4681 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     4741 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     4801 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     4861 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     4921 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     4981 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     5041 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     5101 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     5161 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     5221 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     5281 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     5341 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     5401 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     5461 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     5521 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     5581 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     5641 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     5701 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     5761 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     5821 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     5881 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     5941 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     6001 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     6061 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     6121 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     6181 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     6241 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     6301 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     6361 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     6421 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     6481 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     6541 gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
     6601 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt
     6661 tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt
     6721 ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag
     6781 cgcgcaatta accctcacta aagggaacaa aagctggagc tcatagcttc aaaatgtttc
     6841 tactcctttt ttactcttcc agattttctc ggactccgcg catcgccgta ccacttcaaa
     6901 acacccaagc acagcatact aaatttcccc tctttcttcc tctagggtgt cgttaattac
     6961 ccgtactaaa ggtttggaaa agaaaaaaga gaccgcctcg tttctttttc ttcgtcgaaa
     7021 aaggcaataa aaatttttat cacgtttctt tttcttgaaa attttttttt tgattttttt
     7081 ctctttcgat gacctcccat tgatatttaa gttaataaac ggtcttcaat ttctcaagtt
     7141 tcagtttcat ttttcttgtt ctattacaac tttttttact tcttgctcat tagaaagaaa
     7201 gcatagcaat ctaatctaag ttttctagaa ctagtggatc ccccgggaaa aatggacaag
     7261 aagtactcca ttgggctcga tatcggcaca aacagcgtcg gttgggccgt cattacggac
     7321 gagtacaagg tgccgagcaa aaaattcaaa gttctgggca ataccgatcg ccacagcata
     7381 aagaagaacc tcattggcgc cctcctgttc gactccgggg agacggccga agccacgcgg
     7441 ctcaaaagaa cagcacggcg cagatatacc cgcagaaaga atcggatctg ctacctgcag
     7501 gagatcttta gtaatgagat ggctaaggtg gatgactctt tcttccatag gctggaggag
     7561 tcctttttgg tggaggagga taaaaagcac gagcgccacc caatctttgg caatatcgtg
     7621 gacgaggtgg cgtaccatga aaagtaccca accatatatc atctgaggaa gaagcttgta
     7681 gacagtactg ataaggctga cttgcggttg atctatctcg cgctggcgca tatgatcaaa
     7741 tttcggggac acttcctcat cgagggggac ctgaacccag acaacagcga tgtcgacaaa
     7801 ctctttatcc aactggttca gacttacaat cagcttttcg aagagaaccc gatcaacgca
     7861 tccggagttg acgccaaagc aatcctgagc gctaggctgt ccaaatcccg gcggctcgaa
     7921 aacctcatcg cacagctccc tggggagaag aagaacggcc tgtttggtaa tcttatcgcc
     7981 ctgtcactcg ggctgacccc caactttaaa tctaacttcg acctggccga agatgccaag
     8041 cttcaactga gcaaagacac ctacgatgat gatctcgaca atctgctggc ccagatcggc
     8101 gaccagtacg cagacctttt tttggcggca aagaacctgt cagacgccat tctgctgagt
     8161 gatattctgc gagtgaacac ggagatcacc aaagctccgc tgagcgctag tatgatcaag
     8221 cgctatgatg agcaccacca agacttgact ttgctgaagg cccttgtcag acagcaactg
     8281 cctgagaagt acaaggaaat tttcttcgat cagtctaaaa atggctacgc cggatacatt
     8341 gacggcggag caagccagga ggaattttac aaatttatta agcccatctt ggaaaaaatg
     8401 gacggcaccg aggagctgct ggtaaagctt aacagagaag atctgttgcg caaacagcgc
     8461 actttcgaca atggaagcat cccccaccag attcacctgg gcgaactgca cgctatcctc
     8521 aggcggcaag aggatttcta cccctttttg aaagataaca gggaaaagat tgagaaaatc
     8581 ctcacatttc ggatacccta ctatgtaggc cccctcgccc ggggaaattc cagattcgcg
     8641 tggatgactc gcaaatcaga agagaccatc actccctgga acttcgagga agtcgtggat
     8701 aagggggcct ctgcccagtc cttcatcgaa aggatgacta actttgataa aaatctgcct
     8761 aacgaaaagg tgcttcctaa acactctctg ctgtacgagt acttcacagt ttataacgag
     8821 ctcaccaagg tcaaatacgt cacagaaggg atgagaaagc cagcattcct gtctggagag
     8881 cagaagaaag ctatcgtgga cctcctcttc aagacgaacc ggaaagttac cgtgaaacag
     8941 ctcaaagaag actatttcaa aaagattgaa tgtttcgact ctgttgaaat cagcggagtg
     9001 gaggatcgct tcaacgcatc cctgggaacg tatcacgatc tcctgaaaat cattaaagac
     9061 aaggacttcc tggacaatga ggagaacgag gacattcttg aggacattgt cctcaccctt
     9121 acgttgtttg aagataggga gatgattgaa gaacgcttga aaacttacgc tcatctcttc
     9181 gacgacaaag tcatgaaaca gctcaagagg cgccgatata caggatgggg gcggctgtca
     9241 agaaaactga tcaatgggat ccgagacaag cagagtggaa agacaatcct ggattttctt
     9301 aagtccgatg gatttgccaa ccggaacttc atgcagttga tccatgatga ctctctcacc
     9361 tttaaggagg acatccagaa agcacaagtt tctggccagg gggacagtct tcacgagcac
     9421 atcgctaatc ttgcaggtag cccagctatc aaaaagggaa tactgcagac cgttaaggtc
     9481 gtggatgaac tcgtcaaagt aatgggaagg cataagcccg agaatatcgt tatcgagatg
     9541 gcccgagaga accaaactac ccagaaggga cagaagaaca gtagggaaag gatgaagagg
     9601 attgaagagg gtataaaaga actggggtcc caaatcctta aggaacaccc agttgaaaac
     9661 acccagcttc agaatgagaa gctctacctg tactacctgc agaacggcag ggacatgtac
     9721 gtggatcagg aactggacat caatcggctc tccgactacg acgtggatca tatcgtgccc
     9781 cagtcttttc tcaaagatga ttctattgat aataaagtgt tgacaagatc cgataaaaat
     9841 agagggaaga gtgataacgt cccctcagaa gaagttgtca agaaaatgaa aaattattgg
     9901 cggcagctgc tgaacgccaa actgatcaca caacggaagt tcgataatct gactaaggct
     9961 gaacgaggtg gcctgtctga gttggataaa gccggcttca tcaaaaggca gcttgttgag
    10021 acacgccaga tcaccaagca cgtggcccaa attctcgatt cacgcatgaa caccaagtac
    10081 gatgaaaatg acaaactgat tcgagaggtg aaagttatta ctctgaagtc taagctggtc
    10141 tcagatttca gaaaggactt tcagttttat aaggtgagag agatcaacaa ttaccaccat
    10201 gcgcatgatg cctacctgaa tgcagtggta ggcactgcac ttatcaaaaa atatcccaag
    10261 cttgaatctg aatttgttta cggagactat aaagtgtacg atgttaggaa aatgatcgca
    10321 aagtctgagc aggaaatagg caaggccacc gctaagtact tcttttacag caatattatg
    10381 aattttttca agaccgagat tacactggcc aatggagaga ttcggaagcg accacttatc
    10441 gaaacaaacg gagaaacagg agaaatcgtg tgggacaagg gtagggattt cgcgacagtc
    10501 cggaaggtcc tgtccatgcc gcaggtgaac atcgttaaaa agaccgaagt acagaccgga
    10561 ggcttctcca aggaaagtat cctcccgaaa aggaacagcg acaagctgat cgcacgcaaa
    10621 aaagattggg accccaagaa atacggcgga ttcgattctc ctacagtcgc ttacagtgta
    10681 ctggttgtgg ccaaagtgga gaaagggaag tctaaaaaac tcaaaagcgt caaggaactg
    10741 ctgggcatca caatcatgga gcgatcaagc ttcgaaaaaa accccatcga ctttctcgag
    10801 gcgaaaggat ataaagaggt caaaaaagac ctcatcatta agcttcccaa gtactctctc
    10861 tttgagcttg aaaacggccg gaaacgaatg ctcgctagtg cgggcgagct gcagaaaggt
    10921 aacgagctgg cactgccctc taaatacgtt aatttcttgt atctggccag ccactatgaa
    10981 aagctcaaag ggtctcccga agataatgag cagaagcagc tgttcgtgga acaacacaaa
    11041 cactaccttg atgagatcat cgagcaaata agcgaattct ccaaaagagt gatcctcgcc
    11101 gacgctaacc tcgataaggt gctttctgct tacaataagc acagggataa gcccatcagg
    11161 gagcaggcag aaaacattat ccacttgttt actctgacca acttgggcgc gcctgcagcc
    11221 ttcaagtact tcgacaccac catagacaga aagcggtaca cctctacaaa ggaggtcctg
    11281 gacgccacac tgattcatca gtcaattacg gggctctatg aaacaagaat cgacctctct
    11341 cagctcggtg gagacagcag ggctgacccc aagaagaaga ggaaggtgtg atctcttctc
    11401 gagtcatgta attagttatg tcacgcttac attcacgccc tccccccaca tccgctctaa
    11461 ccgaaaagga aggagttaga caacctgaag tctaggtccc tatttatttt tttatagtta
    11521 tgttagtatt aagaacgtta tttatatttc aaatttttct tttttttctg tacagacgcg
    11581 tgtacgcatg taacattata ctgaaaacct tgcttgagaa ggttttggga cgctcgaagg
    11641 ctttaatttg cggccggtac ccaattcgcc ctatagtgag tcgtattacg cgcgctcact
    11701 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
    11761 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
    11821 ttcccaacag ttgcgcagcc tgaatggcga atggacgcgc cctgtagcgg cgcattaagc
    11881 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
    11941 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
    12001 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
    12061 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
    12121 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
    12181 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
    12241 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
    12301 cttacaattt cctgatgcgg tattttctcc ttacgcatct gtgcgg
//
