LOCUS       sequence-315174-       12349 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 12349)
  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 12349)
  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..12349
                     /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
                     IGGAAIDATGVPLPDEALEASKKVDAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVDPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     primer_bind     complement(3859..3878)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3978..4000
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4038..4056)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    4098..4601
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        4638..4742
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4743..4811,4812..5603)
                     /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(4961..4980)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5774..6362
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6263..6282
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6516..6533
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        join(6686..6691,6692..6709,6710..6716)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6724..6740
                     /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     6729..6751
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6748..6764
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6748..6764
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6783..6803
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6785..6803
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        6821..7219
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     primer_bind     7223..7239
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7223..7239
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             7251..11354
                     /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             7251..11354
                     /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             7251..11354
                     /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             11367..11387
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      11403..11650
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(11419..11437)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(11668..11687)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(11669..11687)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(11697..11714)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(11697..11713)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11706..11728)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      11858..12313
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11945..11964)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     12155..12176
                     /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 agaaggttga 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 ataaggttga 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 atagtaaagg aatacaggta
     3541 agcaaattga tactaatggc tcaacgtgat aaggaaaaag aattgcactt taacattaat
     3601 attgacaagg aggagggcac cacacaaaaa gttaggtgta acagaaaatc atgaaactac
     3661 gattcctaat ttgatattgg aggattttct ctaaaaaaaa aaaaatacaa caaataaaaa
     3721 acactcaatg acctgaccat ttgatggagt ttaagtcaat accttcttga accatttccc
     3781 ataatggtga aagttccctc aagaatttta ctctgtcaga aacggcctta cgacgtagtc
     3841 gatatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca
     3901 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     3961 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     4021 acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
     4081 aatggtttct taggacggat cgcttgcctg taacttacac gcgcctcgta tcttttaatg
     4141 atggaataat ttgggaattt actctgtgtt tatttatttt tatgttttgt atttggattt
     4201 tagaaagtaa ataaagaagg tagaagagtt acggaatgaa gaaaaaaaaa taaacaaagg
     4261 tttaaaaaat ttcaacaaaa agcgtacttt acatatatat ttattagaca agaaaagcag
     4321 attaaataga tatacattcg attaacgata agtaaaatgt aaaatcacag gattttcgtg
     4381 tgtggtcttc tacacagaca agatgaaaca attcggcatt aatacctgag agcaggaaga
     4441 aaaagataaa aggtagtatt tgttggcgat ccccctagag tcttttacat cttcggaaaa
     4501 caaaaactat tttttcttta atttcttttt ttactttcta tttttaattt atatatttat
     4561 attaaaaaat ttaaattata attattttta tagcacgtga tgaaaaggac ccaggtggca
     4621 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     4681 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     4741 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     4801 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     4861 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     4921 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     4981 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     5041 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     5101 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     5161 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     5221 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     5281 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     5341 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     5401 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     5461 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     5521 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     5581 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     5641 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     5701 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     5761 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     5821 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     5881 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     5941 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     6001 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     6061 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     6121 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     6181 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     6241 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     6301 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     6361 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     6421 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     6481 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     6541 aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     6601 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt
     6661 acctcactca ttaggcaccc caggctttac actttatgct tccggctcct atgttgtgtg
     6721 gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc
     6781 gcgcaattaa ccctcactaa agggaacaaa agctggagct catagcttca aaatgtttct
     6841 actccttttt tactcttcca gattttctcg gactccgcgc atcgccgtac cacttcaaaa
     6901 cacccaagca cagcatacta aatttcccct ctttcttcct ctagggtgtc gttaattacc
     6961 cgtactaaag gtttggaaaa gaaaaaagag accgcctcgt ttctttttct tcgtcgaaaa
     7021 aggcaataaa aatttttatc acgtttcttt ttcttgaaaa tttttttttt gatttttttc
     7081 tctttcgatg acctcccatt gatatttaag ttaataaacg gtcttcaatt tctcaagttt
     7141 cagtttcatt tttcttgttc tattacaact ttttttactt cttgctcatt agaaagaaag
     7201 catagcaatc taatctaagt tttctagaac tagtggatcc cccgggaaaa atggacaaga
     7261 agtactccat tgggctcgat atcggcacaa acagcgtcgg ttgggccgtc attacggacg
     7321 agtacaaggt gccgagcaaa aaattcaaag ttctgggcaa taccgatcgc cacagcataa
     7381 agaagaacct cattggcgcc ctcctgttcg actccgggga gacggccgaa gccacgcggc
     7441 tcaaaagaac agcacggcgc agatataccc gcagaaagaa tcggatctgc tacctgcagg
     7501 agatctttag taatgagatg gctaaggtgg atgactcttt cttccatagg ctggaggagt
     7561 cctttttggt ggaggaggat aaaaagcacg agcgccaccc aatctttggc aatatcgtgg
     7621 acgaggtggc gtaccatgaa aagtacccaa ccatatatca tctgaggaag aagcttgtag
     7681 acagtactga taaggctgac ttgcggttga tctatctcgc gctggcgcat atgatcaaat
     7741 ttcggggaca cttcctcatc gagggggacc tgaacccaga caacagcgat gtcgacaaac
     7801 tctttatcca actggttcag acttacaatc agcttttcga agagaacccg atcaacgcat
     7861 ccggagttga cgccaaagca atcctgagcg ctaggctgtc caaatcccgg cggctcgaaa
     7921 acctcatcgc acagctccct ggggagaaga agaacggcct gtttggtaat cttatcgccc
     7981 tgtcactcgg gctgaccccc aactttaaat ctaacttcga cctggccgaa gatgccaagc
     8041 ttcaactgag caaagacacc tacgatgatg atctcgacaa tctgctggcc cagatcggcg
     8101 accagtacgc agaccttttt ttggcggcaa agaacctgtc agacgccatt ctgctgagtg
     8161 atattctgcg agtgaacacg gagatcacca aagctccgct gagcgctagt atgatcaagc
     8221 gctatgatga gcaccaccaa gacttgactt tgctgaaggc ccttgtcaga cagcaactgc
     8281 ctgagaagta caaggaaatt ttcttcgatc agtctaaaaa tggctacgcc ggatacattg
     8341 acggcggagc aagccaggag gaattttaca aatttattaa gcccatcttg gaaaaaatgg
     8401 acggcaccga ggagctgctg gtaaagctta acagagaaga tctgttgcgc aaacagcgca
     8461 ctttcgacaa tggaagcatc ccccaccaga ttcacctggg cgaactgcac gctatcctca
     8521 ggcggcaaga ggatttctac ccctttttga aagataacag ggaaaagatt gagaaaatcc
     8581 tcacatttcg gataccctac tatgtaggcc ccctcgcccg gggaaattcc agattcgcgt
     8641 ggatgactcg caaatcagaa gagaccatca ctccctggaa cttcgaggaa gtcgtggata
     8701 agggggcctc tgcccagtcc ttcatcgaaa ggatgactaa ctttgataaa aatctgccta
     8761 acgaaaaggt gcttcctaaa cactctctgc tgtacgagta cttcacagtt tataacgagc
     8821 tcaccaaggt caaatacgtc acagaaggga tgagaaagcc agcattcctg tctggagagc
     8881 agaagaaagc tatcgtggac ctcctcttca agacgaaccg gaaagttacc gtgaaacagc
     8941 tcaaagaaga ctatttcaaa aagattgaat gtttcgactc tgttgaaatc agcggagtgg
     9001 aggatcgctt caacgcatcc ctgggaacgt atcacgatct cctgaaaatc attaaagaca
     9061 aggacttcct ggacaatgag gagaacgagg acattcttga ggacattgtc ctcaccctta
     9121 cgttgtttga agatagggag atgattgaag aacgcttgaa aacttacgct catctcttcg
     9181 acgacaaagt catgaaacag ctcaagaggc gccgatatac aggatggggg cggctgtcaa
     9241 gaaaactgat caatgggatc cgagacaagc agagtggaaa gacaatcctg gattttctta
     9301 agtccgatgg atttgccaac cggaacttca tgcagttgat ccatgatgac tctctcacct
     9361 ttaaggagga catccagaaa gcacaagttt ctggccaggg ggacagtctt cacgagcaca
     9421 tcgctaatct tgcaggtagc ccagctatca aaaagggaat actgcagacc gttaaggtcg
     9481 tggatgaact cgtcaaagta atgggaaggc ataagcccga gaatatcgtt atcgagatgg
     9541 cccgagagaa ccaaactacc cagaagggac agaagaacag tagggaaagg atgaagagga
     9601 ttgaagaggg tataaaagaa ctggggtccc aaatccttaa ggaacaccca gttgaaaaca
     9661 cccagcttca gaatgagaag ctctacctgt actacctgca gaacggcagg gacatgtacg
     9721 tggatcagga actggacatc aatcggctct ccgactacga cgtggatcat atcgtgcccc
     9781 agtcttttct caaagatgat tctattgata ataaagtgtt gacaagatcc gataaaaata
     9841 gagggaagag tgataacgtc ccctcagaag aagttgtcaa gaaaatgaaa aattattggc
     9901 ggcagctgct gaacgccaaa ctgatcacac aacggaagtt cgataatctg actaaggctg
     9961 aacgaggtgg cctgtctgag ttggataaag ccggcttcat caaaaggcag cttgttgaga
    10021 cacgccagat caccaagcac gtggcccaaa ttctcgattc acgcatgaac accaagtacg
    10081 atgaaaatga caaactgatt cgagaggtga aagttattac tctgaagtct aagctggtct
    10141 cagatttcag aaaggacttt cagttttata aggtgagaga gatcaacaat taccaccatg
    10201 cgcatgatgc ctacctgaat gcagtggtag gcactgcact tatcaaaaaa tatcccaagc
    10261 ttgaatctga atttgtttac ggagactata aagtgtacga tgttaggaaa atgatcgcaa
    10321 agtctgagca ggaaataggc aaggccaccg ctaagtactt cttttacagc aatattatga
    10381 attttttcaa gaccgagatt acactggcca atggagagat tcggaagcga ccacttatcg
    10441 aaacaaacgg agaaacagga gaaatcgtgt gggacaaggg tagggatttc gcgacagtcc
    10501 ggaaggtcct gtccatgccg caggtgaaca tcgttaaaaa gaccgaagta cagaccggag
    10561 gcttctccaa ggaaagtatc ctcccgaaaa ggaacagcga caagctgatc gcacgcaaaa
    10621 aagattggga ccccaagaaa tacggcggat tcgattctcc tacagtcgct tacagtgtac
    10681 tggttgtggc caaagtggag aaagggaagt ctaaaaaact caaaagcgtc aaggaactgc
    10741 tgggcatcac aatcatggag cgatcaagct tcgaaaaaaa ccccatcgac tttctcgagg
    10801 cgaaaggata taaagaggtc aaaaaagacc tcatcattaa gcttcccaag tactctctct
    10861 ttgagcttga aaacggccgg aaacgaatgc tcgctagtgc gggcgagctg cagaaaggta
    10921 acgagctggc actgccctct aaatacgtta atttcttgta tctggccagc cactatgaaa
    10981 agctcaaagg gtctcccgaa gataatgagc agaagcagct gttcgtggaa caacacaaac
    11041 actaccttga tgagatcatc gagcaaataa gcgaattctc caaaagagtg atcctcgccg
    11101 acgctaacct cgataaggtg ctttctgctt acaataagca cagggataag cccatcaggg
    11161 agcaggcaga aaacattatc cacttgttta ctctgaccaa cttgggcgcg cctgcagcct
    11221 tcaagtactt cgacaccacc atagacagaa agcggtacac ctctacaaag gaggtcctgg
    11281 acgccacact gattcatcag tcaattacgg ggctctatga aacaagaatc gacctctctc
    11341 agctcggtgg agacagcagg gctgacccca agaagaagag gaaggtgtga tctcttctcg
    11401 agtcatgtaa ttagttatgt cacgcttaca ttcacgccct ccccccacat ccgctctaac
    11461 cgaaaaggaa ggagttagac aacctgaagt ctaggtccct atttattttt ttatagttat
    11521 gttagtatta agaacgttat ttatatttca aatttttctt ttttttctgt acagacgcgt
    11581 gtacgcatgt aacattatac tgaaaacctt gcttgagaag gttttgggac gctcgaaggc
    11641 tttaatttgc ggccggtacc caattcgccc tatagtgagt cgtattacgc gcgctcactg
    11701 gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt
    11761 gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct
    11821 tcccaacagt tgcgcagcct gaatggcgaa tggcgcgacg cgccctgtag cggcgcatta
    11881 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
    11941 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
    12001 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
    12061 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
    12121 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
    12181 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
    12241 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
    12301 acgtttacaa tttcctgatg cggtattttc tccttacgca tctgtgcgg
//
