LOCUS       sequence-318315-       12576 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pRS416-dCas9-Mxi1+TetR+pRPR1(TetO)-NotI-gRNA plasmid marked with 
            His, expressing dCas9-Mxi1 under Tef1 promoter, and a tet-inducible 
            promoter for gRNA expression..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12576)
  AUTHORS   Momen-Roknabadi A, Oikonomou P, Zegans M, Tavazoie S
  TITLE     An inducible CRISPR interference library for genetic interrogation 
            of Saccharomyces cerevisiae biology.
  JOURNAL   Commun Biol. 2020 Nov 27;3(1):723. doi: 10.1038/s42003-020-01452-9.
  PUBMED    33247197
REFERENCE   2  (bases 1 to 12576)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Commun 
            Biol."; date: "2020-11-27"; volume: "3(1):723. doi"; pages: " 
            10.1038/s42003-020-01452-9"
COMMENT     Sequence Label: ampl43
FEATURES             Location/Qualifiers
     source          1..12576
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    136..154
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        227..302
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      315..377
                     /label=RPR1 terminator
                     /note="transcription terminator for the S. cerevisiae RNase
                     P RNA gene"
     CDS             1512..2135
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(1585..1604)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     2632..2649
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2766..2787
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2802..2807,2808..2825,2826..2832)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2840..2856
                     /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     2845..2867
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2864..2880
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2864..2880
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2899..2919
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2901..2919
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        2937..3335
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     CDS             3358..3378
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(3388..3414,3415..3417,3418..5901,5902..5907,5908..5973,
                     5974..5976,5977..7491)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=Cas9m4
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the mutations D10A, D839A, 
                     H840A, and N863A (Mali et al., 2013)"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             join(3388..3414,3415..3417,3418..5904,5905..5907,
                     5908..7491)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             3388..7491
                     /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="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             7513..7533
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7567..7689
                     /codon_start=1
                     /product="transcriptional repressor domain from the mouse 
                     Max-interacting protein 1 (Schreiber-Agus et al., 1995)"
                     /label=Mxi1
                     /translation="MERVRMINVQRLLEAAEFLERRERECEHGYASSFPSMPSPR"
     terminator      7713..7960
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(7729..7747)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(7978..7997)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7979..7997)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8007..8024)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8007..8023)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8016..8038)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      8164..8619
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8251..8270)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8461..8482
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(8818..9480)
                     /codon_start=1
                     /gene="S. cerevisiae HIS3"
                     /product="imidazoleglycerol-phosphate dehydratase, required
                     for histidine biosynthesis"
                     /label=HIS3
                     /note="yeast auxotrophic marker"
                     /translation="MTEQKALVKRITNETKIQIAISLKGGPLAIEHSIFPEKEAEAVAE
                     QATQSQVINVHTGIGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHTTEDCGIALGQAF
                     KEALGAVRGVKRFGSGFAPLDEALSRAVVDLSNRPYAVVELGLQREKVGDLSCEMIPHF
                     LESFAEASRITLHVDCLRGKNDHHRSESAFKALAVAIREATSPNGTNDVPSTKGVLM"
     promoter        complement(9481..9668)
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     primer_bind     complement(9815..9834)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     9934..9956
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(9994..10012)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    10060..10563
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        10593..10697
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(10698..10766,10767..11558)
                     /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(10916..10935)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      11729..12317
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     12218..12237
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ccttttaacc tgtaatcatt gtcaacagga tatgttaacg acccacattg ataaacgcta
       61 gtatttcttt ttcctcttct tattggccgg ctgtctctat actcccctat agtctgtttc
      121 ttttcgtttc gattgtccct atcagtgata gagatggcgc acatggtacg ctgtggtgct
      181 cgcggctggg aacgaaactc tgggagctgc gattggcagc ggccgcgttt tagagctaga
      241 aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt
      301 gcttttttct cgagccatat ccaacttcca atttaatctt tcttttttaa ttttcactta
      361 tttgcgatac agaaagaaaa aagcgatagt aactattgaa ttttgtttgg atttggttag
      421 attagatatg gtttctcttt atatttacat gctaaaaatg ggctacacca gagatacata
      481 attagatata tatacgccag tacaccttat cggcccaagc cttgtcccaa ggcagcgttt
      541 tgttcttgga aacgctgccc tacacgttcg ctatgcttca agaacttttc tgagcacttc
      601 atgatgcatg tttgttcctt attggttagc tttgatgttg tgaagtcatt gacacagtct
      661 gtgaaacatc tttctaccag attagagtac aaacgcatga aatccttcat ttgcttttgt
      721 tccactactt tttggaactc ttgttgttct ttcctgcagg tagtcgtgca atgtatgact
      781 ttaagatttg tgagcaggaa gaaaagggag aatcttctaa cgataaaccc ttgaaaaact
      841 gggtagacta cgctatgttg agttgctacg caggctgcac aattacacga gaatgctccc
      901 gcctaggatt taaggctaag ggacgtgcaa tgcagacgac agatctaaat gaccgtgtcg
      961 gtgaagtgtt cgccaaactt ttcggttaac acatgcagtg atgcacgcgc gatggtgcta
     1021 agttacatat atatatatat gtatagccat agtgatgtct aagtaacctt tatggtatat
     1081 ttcttaatgt ggaaagatac tagcgcgcgc acccacacac aagcttcgtc ttttcttgaa
     1141 gaaaagagga agctcgctaa atgggattcc actttccgtt ccctgccagc tgatggaaaa
     1201 aggttagtgg aacgatgaag aataaaaaga gagatccact gaggtgaaat ttcagctgac
     1261 agcgagtttc atgatcgtga tgaacaatgg taacgagttg tggctgttgc cagggagggt
     1321 ggttctcaac ttttaatgta tggccaaatc gctacttggg tttgttatat aacaaagaag
     1381 aaataatgaa ctgattctct tcctccttct tgtcctttct taattctgtt gtaattacct
     1441 tcctttgtaa ttttttttgt aattattctt cttaataatc caaacaaaca cacatattac
     1501 aatatattaa aatgtctaga ttagataaaa gtaaagtgat taacagcgca ttagagctgc
     1561 ttaatgaggt cggaatcgaa ggtttaacaa cccgtaaact cgcccagaag ctaggtgtag
     1621 agcagcctac attgtattgg catgtaaaaa ataagcgggc tttgctcgac gccttagcca
     1681 ttgagatgtt agataggcac catactcact tttgcccttt agaaggggaa agctggcaag
     1741 attttttacg taataacgct aaaagtttta gatgtgcttt actaagtcat cgcgatggag
     1801 caaaagtaca tttaggtaca cggcctacag aaaaacagta tgaaactctc gaaaatcaat
     1861 tagccttttt atgccaacaa ggtttttcac tagagaatgc attatatgca ctcagcgctg
     1921 tggggcattt tactttaggt tgcgtattgg aagatcaaga gcatcaagtc gctaaagaag
     1981 aaagggaaac acctactact gatagtatgc cgccattatt acgacaagct atcgaattat
     2041 ttgatcacca aggtgcagag ccagccttct tattcggcct tgaattgatc atatgcggat
     2101 tagaaaaaca acttaaatgt gaaagtgggt cttaagtctg aagaatgaat gatttgatga
     2161 tttctttttc cctccatttt tcttactgaa tatatcaatg atatagactt gtatagttta
     2221 ttatttcaaa ttaagtagct atatatagtc aagataacgt ttgtttgaca cgattacatt
     2281 attcgtcgac atcttttttc agcctgtcgt ggtagcaatt tgaggagtat tattaattga
     2341 ataggttcat tttgcgctcg cataaacagt tttcgtcagg gacagtatgt tggaatgagt
     2401 ggtaattaat ggtgacatga catgttatag caataacctt gatgtttaca tcgtagttta
     2461 atgtacaccc cgcgaattcg ttcaagtagg agtgcaccaa ttgcaaaggg aaaagctgaa
     2521 tgggcagttc gaaccgcggt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     2581 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     2641 gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg
     2701 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac
     2761 gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg
     2821 gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac
     2881 catgattacg ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctcata
     2941 gcttcaaaat gtttctactc cttttttact cttccagatt ttctcggact ccgcgcatcg
     3001 ccgtaccact tcaaaacacc caagcacagc atactaaatt tcccctcttt cttcctctag
     3061 ggtgtcgtta attacccgta ctaaaggttt ggaaaagaaa aaagagaccg cctcgtttct
     3121 ttttcttcgt cgaaaaaggc aataaaaatt tttatcacgt ttctttttct tgaaaatttt
     3181 ttttttgatt tttttctctt tcgatgacct cccattgata tttaagttaa taaacggtct
     3241 tcaatttctc aagtttcagt ttcatttttc ttgttctatt acaacttttt ttacttcttg
     3301 ctcattagaa agaaagcata gcaatctaat ctaagttttc tagagatgtc tagagcccca
     3361 aagaagaaga gaaaagttag acccgggatg gacaagaagt actccattgg gctcgctatc
     3421 ggcacaaaca gcgtcggctg ggccgtcatt acggacgagt acaaggtgcc gagcaaaaaa
     3481 ttcaaagttc tgggcaatac cgatcgccac agcataaaga agaacctcat tggcgccctc
     3541 ctgttcgact ccggggagac ggccgaagcc acgcggctca aaagaacagc acggcgcaga
     3601 tatacccgca gaaagaatcg gatctgctac ctgcaggaga tctttagtaa tgagatggct
     3661 aaggtggatg actctttctt ccataggctg gaggagtcct ttttggtgga ggaggataaa
     3721 aagcacgagc gccacccaat ctttggcaat atcgtggacg aggtggcgta ccatgaaaag
     3781 tacccaacca tatatcatct gaggaagaag cttgtagaca gtactgataa ggctgacttg
     3841 cggttgatct atctcgcgct ggcgcatatg atcaaatttc ggggacactt cctcatcgag
     3901 ggggacctga acccagacaa cagcgatgtc gacaaactct ttatccaact ggttcagact
     3961 tacaatcagc ttttcgaaga gaacccgatc aacgcatccg gagttgacgc caaagcaatc
     4021 ctgagcgcta ggctgtccaa atcccggcgg ctcgaaaacc tcatcgcaca gctccctggg
     4081 gagaagaaga acggcctgtt tggtaatctt atcgccctgt cactcgggct gacccccaac
     4141 tttaaatcta acttcgacct ggccgaagat gccaagcttc aactgagcaa agacacctac
     4201 gatgatgatc tcgacaatct gctggcccag atcggcgacc agtacgcaga cctttttttg
     4261 gcggcaaaga acctgtcaga cgccattctg ctgagtgata ttctgcgagt gaacacggag
     4321 atcaccaaag ctccgctgag cgctagtatg atcaagcgct atgatgagca ccaccaagac
     4381 ttgactttgc tgaaggccct tgtcagacag caactgcctg agaagtacaa ggaaattttc
     4441 ttcgatcagt ctaaaaatgg ctacgccgga tacattgacg gcggagcaag ccaggaggaa
     4501 ttttacaaat ttattaagcc catcttggaa aaaatggacg gcaccgagga gctgctggta
     4561 aagcttaaca gagaagatct gttgcgcaaa cagcgcactt tcgacaatgg aagcatcccc
     4621 caccagattc acctgggcga actgcacgct atcctcaggc ggcaagagga tttctacccc
     4681 tttttgaaag ataacaggga aaagattgag aaaatcctca catttcggat accctactat
     4741 gtaggccccc tcgcccgggg aaattccaga ttcgcgtgga tgactcgcaa atcagaagag
     4801 accatcactc cctggaactt cgaggaagtc gtggataagg gggcctctgc ccagtccttc
     4861 atcgaaagga tgactaactt tgataaaaat ctgcctaacg aaaaggtgct tcctaaacac
     4921 tctctgctgt acgagtactt cacagtttat aacgagctca ccaaggtcaa atacgtcaca
     4981 gaagggatga gaaagccagc attcctgtct ggagagcaga agaaagctat cgtggacctc
     5041 ctcttcaaga cgaaccggaa agttaccgtg aaacagctca aagaagacta tttcaaaaag
     5101 attgaatgtt tcgactctgt tgaaatcagc ggagtggagg atcgcttcaa cgcatccctg
     5161 ggaacgtatc acgatctcct gaaaatcatt aaagacaagg acttcctgga caatgaggag
     5221 aacgaggaca ttcttgagga cattgtcctc acccttacgt tgtttgaaga tagggagatg
     5281 attgaagaac gcttgaaaac ttacgctcat ctcttcgacg acaaagtcat gaaacagctc
     5341 aagaggcgcc gatatacagg atgggggcgg ctgtcaagaa aactgatcaa tgggatccga
     5401 gacaagcaga gtggaaagac aatcctggat tttcttaagt ccgatggatt tgccaaccgg
     5461 aacttcatgc agttgatcca tgatgactct ctcaccttta aggaggacat ccagaaagca
     5521 caagtttctg gccaggggga cagtcttcac gagcacatcg ctaatcttgc aggtagccca
     5581 gctatcaaaa agggaatact gcagaccgtt aaggtcgtgg atgaactcgt caaagtaatg
     5641 ggaaggcata agcccgagaa tatcgttatc gagatggccc gagagaacca aactacccag
     5701 aagggacaga agaacagtag ggaaaggatg aagaggattg aagagggtat aaaagaactg
     5761 gggtcccaaa tccttaagga acacccagtt gaaaacaccc agcttcagaa tgagaagctc
     5821 tacctgtact acctgcagaa cggcagggac atgtacgtgg atcaggaact ggacatcaat
     5881 cggctctccg actacgacgt ggatgctatc gtgccccagt cttttctcaa agatgattct
     5941 attgataata aagtgttgac aagatccgat aaaaatagag ggaagagtga taacgtcccc
     6001 tcagaagaag ttgtcaagaa aatgaaaaat tattggcggc agctgctgaa cgccaaactg
     6061 atcacacaac ggaagttcga taatctgact aaggctgaac gaggtggcct gtctgagttg
     6121 gataaagccg gcttcatcaa aaggcagctt gttgagacac gccagatcac caagcacgtg
     6181 gcccaaattc tcgattcacg catgaacacc aagtacgatg aaaatgacaa actgattcga
     6241 gaggtgaaag ttattactct gaagtctaag ctggtctcag atttcagaaa ggactttcag
     6301 ttttataagg tgagagagat caacaattac caccatgcgc atgatgccta cctgaatgca
     6361 gtggtaggca ctgcacttat caaaaaatat cccaagcttg aatctgaatt tgtttacgga
     6421 gactataaag tgtacgatgt taggaaaatg atcgcaaagt ctgagcagga aataggcaag
     6481 gccaccgcta agtacttctt ttacagcaat attatgaatt ttttcaagac cgagattaca
     6541 ctggccaatg gagagattcg gaagcgacca cttatcgaaa caaacggaga aacaggagaa
     6601 atcgtgtggg acaagggtag ggatttcgcg acagtccgga aggtcctgtc catgccgcag
     6661 gtgaacatcg ttaaaaagac cgaagtacag accggaggct tctccaagga aagtatcctc
     6721 ccgaaaagga acagcgacaa gctgatcgca cgcaaaaaag attgggaccc caagaaatac
     6781 ggcggattcg attctcctac agtcgcttac agtgtactgg ttgtggccaa agtggagaaa
     6841 gggaagtcta aaaaactcaa aagcgtcaag gaactgctgg gcatcacaat catggagcga
     6901 tcaagcttcg aaaaaaaccc catcgacttt ctcgaggcga aaggatataa agaggtcaaa
     6961 aaagacctca tcattaagct tcccaagtac tctctctttg agcttgaaaa cggccggaaa
     7021 cgaatgctcg ctagtgcggg cgagctgcag aaaggtaacg agctggcact gccctctaaa
     7081 tacgttaatt tcttgtatct ggccagccac tatgaaaagc tcaaagggtc tcccgaagat
     7141 aatgagcaga agcagctgtt cgtggaacaa cacaaacact accttgatga gatcatcgag
     7201 caaataagcg aattctccaa aagagtgatc ctcgccgacg ctaacctcga taaggtgctt
     7261 tctgcttaca ataagcacag ggataagccc atcagggagc aggcagaaaa cattatccac
     7321 ttgtttactc tgaccaactt gggcgcgcct gcagccttca agtacttcga caccaccata
     7381 gacagaaagc ggtacacctc tacaaaggag gtcctggacg ccacactgat tcatcagtca
     7441 attacggggc tctatgaaac aagaatcgac ctctctcagc tcggtggaga cagcagggct
     7501 gacgagggag ctcccaagaa aaagcgcaag gtaggtagtt ccaagcttgg cggcagcggc
     7561 ggcagcatgg aacgtgtgag aatgattaat gtgcaaaggc tgttagaagc cgcagagttt
     7621 ttagaaagaa gagaaagaga atgcgaacac gggtatgcca gttctttccc tagcatgccc
     7681 tctcccagag gctaactcga gctcttctcg agtcatgtaa ttagttatgt cacgcttaca
     7741 ttcacgccct ccccccacat ccgctctaac cgaaaaggaa ggagttagac aacctgaagt
     7801 ctaggtccct atttattttt ttatagttat gttagtatta agaacgttat ttatatttca
     7861 aatttttctt ttttttctgt acagacgcgt gtacgcatgt aacattatac tgaaaacctt
     7921 gcttgagaag gttttgggac gctcgaaggc tttaatttgc ggccggtacc caattcgccc
     7981 tatagtgagt cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa
     8041 aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt
     8101 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa
     8161 tggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     8221 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     8281 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     8341 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     8401 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     8461 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     8521 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     8581 ttaacgcgaa ttttaacaaa atattaacgt ttacaatttc ctgatgcaaa ttgaagctct
     8641 aatttgtgag tttagtaaaa gaagcgcgcc tcgttcagaa tgacacgtat agaatgatgc
     8701 attaccttgt catcttcagt atcatactgt tcgtatacat acttactgac attcataggt
     8761 atacatatat acacatgtat atatatcgta tgctgcagct ttaaataatc ggtgtcacta
     8821 cataagaaca cctttggtgg agggaacatc gttggtacca ttgggcgagg tggcttctct
     8881 tatggcaacc gcaagagcct tgaacgcact ctcactacgg tgatgatcat tcttgcctcg
     8941 cagacaatca acgtggaggg taattctgct agcctctgca aagctttcaa gaaaatgcgg
     9001 gatcatctcg caagagagat ctcctacttt ctccctttgc aaaccaagtt cgacaactgc
     9061 gtacggcctg ttcgaaagat ctaccaccgc tctggaaagt gcctcatcca aaggcgcaaa
     9121 tcctgatcca aaccttttta ctccacgcac ggcccctagg gcctctttaa aagcttgacc
     9181 gagagcaatc ccgcagtctt cagtggtgtg atggtcgtct atgtgtaagt caccaatgca
     9241 ctcaacgatt agcgaccagc cggaatgctt ggccagagca tgtatcatat ggtccagaaa
     9301 ccctatacct gtgtggacgt taatcacttg cgattgtgtg gcctgttctg ctactgcttc
     9361 tgcctctttt tctgggaaga tcgagtgctc tatcgctagg ggaccaccct ttaaagagat
     9421 cgcaatctga atcttggttt catttgtaat acgctttact agggctttct gctctgtcat
     9481 ctttgccttc gtttatcttg cctgctcatt ttttagtata ttcttcgaag aaatcacatt
     9541 actttatata atgtataatt cattatgtga taatgccaat cgctaagaaa aaaaaagagt
     9601 catccgctag gtggaaaaaa aaaaatgaaa atcattaccg aggcataaaa aaatatagag
     9661 tgtactagag gaggccaaga gtaatagaaa aagaaaattg cgggaaagga ctgtgttatg
     9721 acttccctga ctaatgccgt gttcaaacga tacctggcag tgactcctag cgctcaccaa
     9781 gctcttaaaa cgggaattta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     9841 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     9901 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     9961 caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg
    10021 ttaatgtcat gataataatg gtttcttagg gtccttttca tcacgtgcta taaaaataat
    10081 tataatttaa attttttaat ataaatatat aaattaaaaa tagaaagtaa aaaaagaaat
    10141 taaagaaaaa atagtttttg ttttccgaag atgtaaaaga ctctaggggg atcgccaaca
    10201 aatactacct tttatcttgc tcttcctgct ctcaggtatt aatgccgaat tgtttcatct
    10261 tgtctgtgta gaagaccaca cacgaaaatc ctgtgatttt acattttact tatcgttaat
    10321 cgaatgtata tctatttaat ctgcttttct tgtctaataa atatatatgt aaagtacgct
    10381 ttttgttgaa attttttaaa cctttgttta tttttttttc ttcattccgt aactcttcta
    10441 ccttctttat ttactttcta aaatccaaat acaaaacata aaaataaata aacacagagt
    10501 aaattcccaa attattccat cattaaaaga tacgaggcgc gtgtaagtta caggcaagcg
    10561 atccgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
    10621 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
    10681 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
    10741 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
    10801 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
    10861 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
    10921 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
    10981 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
    11041 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
    11101 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
    11161 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
    11221 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
    11281 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
    11341 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
    11401 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
    11461 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
    11521 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
    11581 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
    11641 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
    11701 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
    11761 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
    11821 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
    11881 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
    11941 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
    12001 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
    12061 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
    12121 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
    12181 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
    12241 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
    12301 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
    12361 ggccttttgc tggccttttg ctcgggggat ctgccaattg aacataacat ggtagttaca
    12421 tatactagta atatggttcg gcacacatta aaagtataaa aactatctga attacgaatt
    12481 acatatattg gtcataaaaa tcaatcaatc atcgtgtgtt ttatatgtct cttatctaag
    12541 tataagaata tccatagtta atattcactt acgcta
//
