LOCUS       sequence-313918-       12592 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 12592)
  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 12592)
  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..12592
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(10..28)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    76..579
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        609..713
                     /gene="bla"
                     /label=AmpR promoter
     CDS             800..1588
                     /codon_start=1
                     /gene="bla(M)"
                     /product="beta-lactamase lacking the signal sequence"
                     /label=bla(M)
                     /note="allows cytosolic expression of beta-lactamase"
                     /translation="PETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMST
                     FKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMS
                     DNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPVAMATT
                     LRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAA
                     LGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHW"
     primer_bind     complement(946..965)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1759..2347
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2248..2267
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    2742..2760
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        2833..2908
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      2921..2983
                     /label=RPR1 terminator
                     /note="transcription terminator for the S. cerevisiae RNase
                     P RNA gene"
     CDS             4118..4741
                     /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(4191..4210)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     5238..5255
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        join(5408..5413,5414..5431,5432..5438)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5446..5462
                     /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     5451..5473
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5470..5486
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5470..5486
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5505..5525
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5507..5525
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5543..5941
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     CDS             5964..5984
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(5994..6020,6021..6023,6024..8507,8508..8513,8514..8579,
                     8580..8582,8583..10097)
                     /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(5994..6020,6021..6023,6024..8510,8511..8513,
                     8514..10097)
                     /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             5994..10097
                     /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             10119..10139
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             10173..10295
                     /codon_start=1
                     /product="transcriptional repressor domain from the mouse 
                     Max-interacting protein 1 (Schreiber-Agus et al., 1995)"
                     /label=Mxi1
                     /translation="MERVRMINVQRLLEAAEFLERRERECEHGYASSFPSMPSPR"
     terminator      10319..10566
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(10335..10353)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(10584..10603)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(10585..10603)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(10613..10630)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(10613..10629)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10622..10644)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      10774..11229
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10861..10880)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     11071..11092
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(11430..12089)
                     /codon_start=1
                     /gene="S. cerevisiae HIS3"
                     /product="imidazoleglycerol-phosphate dehydratase, required
                     for histidine biosynthesis"
                     /label=HIS3
                     /note="yeast auxotrophic marker"
                     /translation="MTEQKALVKRITNETKIQIAISLKGGPLAIEHSIFPEKEAEAVAE
                     QATQSQVINVHTGIGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHTTEDCGIALGQAF
                     KEALLARGVKRFGSGFAPLDEALSRAVVDLSNRPYAVVELGLQREKVGDLSCEMIPHFL
                     ESFAEASRITLHVDCLRGKNDHHRSESAFKALAVAIREATSPNGTNDVPSTKGVLM"
     promoter        complement(12090..12276)
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     primer_bind     complement(12423..12442)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     12542..12564
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttagggtcc ttttcatcac gtgctataaa aataattata atttaaattt tttaatataa
      121 atatataaat taaaaataga aagtaaaaaa agaaattaaa gaaaaaatag tttttgtttt
      181 ccgaagatgt aaaagactct agggggatcg ccaacaaata ctacctttta tcttgctctt
      241 cctgctctca ggtattaatg ccgaattgtt tcatcttgtc tgtgtagaag accacacacg
      301 aaaatcctgt gattttacat tttacttatc gttaatcgaa tgtatatcta tttaatctgc
      361 ttttcttgtc taataaatat atatgtaaag tacgcttttt gttgaaattt tttaaacctt
      421 tgtttatttt tttttcttca ttccgtaact cttctacctt ctttatttac tttctaaaat
      481 ccaaatacaa aacataaaaa taaataaaca cagagtaaat tcccaaatta ttccatcatt
      541 aaaagatacg aggcgcgtgt aagttacagg caagcgatcc gtcaggtggc acttttcggg
      601 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
      661 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
      721 ttcaacattt ccgtgtcaac tctgggagct ggcccttatt cccttttttg cggcattttg
      781 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
      841 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
      901 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
      961 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     1021 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     1081 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     1141 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     1201 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     1261 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     1321 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     1381 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     1441 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     1501 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     1561 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     1621 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     1681 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     1741 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     1801 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     1861 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     1921 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     1981 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     2041 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     2101 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     2161 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     2221 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     2281 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     2341 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     2401 tggccttttg ctcgggggat ctgccaattg aacataacat ggtagttaca tatactagta
     2461 atatggttcg gcacacatta aaagtataaa aactatctga attacgaatt acatatattg
     2521 gtcataaaaa tcaatcaatc atcgtgtgtt ttatatgtct cttatctaag tataagaata
     2581 tccatagtta atattcactt acgctacctt ttaacctgta atcattgtca acaggatatg
     2641 ttaacgaccc acattgataa acgctagtat ttctttttcc tcttcttatt ggccggctgt
     2701 ctctatactc ccctatagtc tgtttctttt cgtttcgatt gtccctatca gtgatagaga
     2761 tggcgcacat ggtacgctgt ggtgctcgcg gctgggaacg aaactctggg agctgcgatt
     2821 ggcagcggcc gcgttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca
     2881 acttgaaaaa gtggcaccga gtcggtgctt ttttctcgag ccatatccaa cttccaattt
     2941 aatctttctt ttttaatttt cacttatttg cgatacagaa agaaaaaagc gatagtaact
     3001 attgaatttt gtttggattt ggttagatta gatatggttt ctctttatat ttacatgcta
     3061 aaaatgggct acaccagaga tacataatta gatatatata cgccagtaca ccttatcggc
     3121 ccaagccttg tcccaaggca gcgttttgtt cttggaaacg ctgccctaca cgttcgctat
     3181 gcttcaagaa cttttctgag cacttcatga tgcatgtttg ttccttattg gttagctttg
     3241 atgttgtgaa gtcattgaca cagtctgtga aacatctttc taccagatta gagtacaaac
     3301 gcatgaaatc cttcatttgc ttttgttcca ctactttttg gaactcttgt tgttctttcc
     3361 tgcaggtagt cgtgcaatgt atgactttaa gatttgtgag caggaagaaa agggagaatc
     3421 ttctaacgat aaacccttga aaaactgggt agactacgct atgttgagtt gctacgcagg
     3481 ctgcacaatt acacgagaat gctcccgcct aggatttaag gctaagggac gtgcaatgca
     3541 gacgacagat ctaaatgacc gtgtcggtga agtgttcgcc aaacttttcg gttaacacat
     3601 gcagtgatgc acgcgcgatg gtgctaagtt acatatatat atatatgtat agccatagtg
     3661 atgtctaagt aacctttatg gtatatttct taatgtggaa agatactagc gcgcgcaccc
     3721 acacacaagc ttcgtctttt cttgaagaaa agaggaagct cgctaaatgg gattccactt
     3781 tccgttccct gccagctgat ggaaaaaggt tagtggaacg atgaagaata aaaagagaga
     3841 tccactgagg tgaaatttca gctgacagcg agtttcatga tcgtgatgaa caatggtaac
     3901 gagttgtggc tgttgccagg gagggtggtt ctcaactttt aatgtatggc caaatcgcta
     3961 cttgggtttg ttatataaca aagaagaaat aatgaactga ttctcttcct ccttcttgtc
     4021 ctttcttaat tctgttgtaa ttaccttcct ttgtaatttt ttttgtaatt attcttctta
     4081 ataatccaaa caaacacaca tattacaata tattaaaatg tctagattag ataaaagtaa
     4141 agtgattaac agcgcattag agctgcttaa tgaggtcgga atcgaaggtt taacaacccg
     4201 taaactcgcc cagaagctag gtgtagagca gcctacattg tattggcatg taaaaaataa
     4261 gcgggctttg ctcgacgcct tagccattga gatgttagat aggcaccata ctcacttttg
     4321 ccctttagaa ggggaaagct ggcaagattt tttacgtaat aacgctaaaa gttttagatg
     4381 tgctttacta agtcatcgcg atggagcaaa agtacattta ggtacacggc ctacagaaaa
     4441 acagtatgaa actctcgaaa atcaattagc ctttttatgc caacaaggtt tttcactaga
     4501 gaatgcatta tatgcactca gcgctgtggg gcattttact ttaggttgcg tattggaaga
     4561 tcaagagcat caagtcgcta aagaagaaag ggaaacacct actactgata gtatgccgcc
     4621 attattacga caagctatcg aattatttga tcaccaaggt gcagagccag ccttcttatt
     4681 cggccttgaa ttgatcatat gcggattaga aaaacaactt aaatgtgaaa gtgggtctta
     4741 agtctgaaga atgaatgatt tgatgatttc tttttccctc catttttctt actgaatata
     4801 tcaatgatat agacttgtat agtttattat ttcaaattaa gtagctatat atagtcaaga
     4861 taacgtttgt ttgacacgat tacattattc gtcgacatct tttttcagcc tgtcgtggta
     4921 gcaatttgag gagtattatt aattgaatag gttcattttg cgctcgcata aacagttttc
     4981 gtcagggaca gtatgttgga atgagtggta attaatggtg acatgacatg ttatagcaat
     5041 aaccttgatg tttacatcgt agtttaatgt acaccccgcg aattcgttca agtaggagtg
     5101 caccaattgc aaagggaaaa gctgaatggg cagttcgaac cgcggtcttt cctgcgttat
     5161 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     5221 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca
     5281 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
     5341 actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttacctcact cattaggcac
     5401 cccaggcttt acactttatg cttccggctc ctatgttgtg tggaattgtg agcggataac
     5461 aatttcacac aggaaacagc tatgaccatg attacgccaa gcgcgcaatt aaccctcact
     5521 aaagggaaca aaagctggag ctcatagctt caaaatgttt ctactccttt tttactcttc
     5581 cagattttct cggactccgc gcatcgccgt accacttcaa aacacccaag cacagcatac
     5641 taaatttccc ctctttcttc ctctagggtg tcgttaatta cccgtactaa aggtttggaa
     5701 aagaaaaaag agaccgcctc gtttcttttt cttcgtcgaa aaaggcaata aaaattttta
     5761 tcacgtttct ttttcttgaa aatttttttt ttgatttttt tctctttcga tgacctccca
     5821 ttgatattta agttaataaa cggtcttcaa tttctcaagt ttcagtttca tttttcttgt
     5881 tctattacaa ctttttttac ttcttgctca ttagaaagaa agcatagcaa tctaatctaa
     5941 gttttctaga gatgtctaga gccccaaaga agaagagaaa agttagaccc gggatggaca
     6001 agaagtactc cattgggctc gctatcggca caaacagcgt cggctgggcc gtcattacgg
     6061 acgagtacaa ggtgccgagc aaaaaattca aagttctggg caataccgat cgccacagca
     6121 taaagaagaa cctcattggc gccctcctgt tcgactccgg ggagacggcc gaagccacgc
     6181 ggctcaaaag aacagcacgg cgcagatata cccgcagaaa gaatcggatc tgctacctgc
     6241 aggagatctt tagtaatgag atggctaagg tggatgactc tttcttccat aggctggagg
     6301 agtccttttt ggtggaggag gataaaaagc acgagcgcca cccaatcttt ggcaatatcg
     6361 tggacgaggt ggcgtaccat gaaaagtacc caaccatata tcatctgagg aagaagcttg
     6421 tagacagtac tgataaggct gacttgcggt tgatctatct cgcgctggcg catatgatca
     6481 aatttcgggg acacttcctc atcgaggggg acctgaaccc agacaacagc gatgtcgaca
     6541 aactctttat ccaactggtt cagacttaca atcagctttt cgaagagaac ccgatcaacg
     6601 catccggagt tgacgccaaa gcaatcctga gcgctaggct gtccaaatcc cggcggctcg
     6661 aaaacctcat cgcacagctc cctggggaga agaagaacgg cctgtttggt aatcttatcg
     6721 ccctgtcact cgggctgacc cccaacttta aatctaactt cgacctggcc gaagatgcca
     6781 agcttcaact gagcaaagac acctacgatg atgatctcga caatctgctg gcccagatcg
     6841 gcgaccagta cgcagacctt tttttggcgg caaagaacct gtcagacgcc attctgctga
     6901 gtgatattct gcgagtgaac acggagatca ccaaagctcc gctgagcgct agtatgatca
     6961 agcgctatga tgagcaccac caagacttga ctttgctgaa ggcccttgtc agacagcaac
     7021 tgcctgagaa gtacaaggaa attttcttcg atcagtctaa aaatggctac gccggataca
     7081 ttgacggcgg agcaagccag gaggaatttt acaaatttat taagcccatc ttggaaaaaa
     7141 tggacggcac cgaggagctg ctggtaaagc ttaacagaga agatctgttg cgcaaacagc
     7201 gcactttcga caatggaagc atcccccacc agattcacct gggcgaactg cacgctatcc
     7261 tcaggcggca agaggatttc tacccctttt tgaaagataa cagggaaaag attgagaaaa
     7321 tcctcacatt tcggataccc tactatgtag gccccctcgc ccggggaaat tccagattcg
     7381 cgtggatgac tcgcaaatca gaagagacca tcactccctg gaacttcgag gaagtcgtgg
     7441 ataagggggc ctctgcccag tccttcatcg aaaggatgac taactttgat aaaaatctgc
     7501 ctaacgaaaa ggtgcttcct aaacactctc tgctgtacga gtacttcaca gtttataacg
     7561 agctcaccaa ggtcaaatac gtcacagaag ggatgagaaa gccagcattc ctgtctggag
     7621 agcagaagaa agctatcgtg gacctcctct tcaagacgaa ccggaaagtt accgtgaaac
     7681 agctcaaaga agactatttc aaaaagattg aatgtttcga ctctgttgaa atcagcggag
     7741 tggaggatcg cttcaacgca tccctgggaa cgtatcacga tctcctgaaa atcattaaag
     7801 acaaggactt cctggacaat gaggagaacg aggacattct tgaggacatt gtcctcaccc
     7861 ttacgttgtt tgaagatagg gagatgattg aagaacgctt gaaaacttac gctcatctct
     7921 tcgacgacaa agtcatgaaa cagctcaaga ggcgccgata tacaggatgg gggcggctgt
     7981 caagaaaact gatcaatggg atccgagaca agcagagtgg aaagacaatc ctggattttc
     8041 ttaagtccga tggatttgcc aaccggaact tcatgcagtt gatccatgat gactctctca
     8101 cctttaagga ggacatccag aaagcacaag tttctggcca gggggacagt cttcacgagc
     8161 acatcgctaa tcttgcaggt agcccagcta tcaaaaaggg aatactgcag accgttaagg
     8221 tcgtggatga actcgtcaaa gtaatgggaa ggcataagcc cgagaatatc gttatcgaga
     8281 tggcccgaga gaaccaaact acccagaagg gacagaagaa cagtagggaa aggatgaaga
     8341 ggattgaaga gggtataaaa gaactggggt cccaaatcct taaggaacac ccagttgaaa
     8401 acacccagct tcagaatgag aagctctacc tgtactacct gcagaacggc agggacatgt
     8461 acgtggatca ggaactggac atcaatcggc tctccgacta cgacgtggat gctatcgtgc
     8521 cccagtcttt tctcaaagat gattctattg ataataaagt gttgacaaga tccgataaaa
     8581 atagagggaa gagtgataac gtcccctcag aagaagttgt caagaaaatg aaaaattatt
     8641 ggcggcagct gctgaacgcc aaactgatca cacaacggaa gttcgataat ctgactaagg
     8701 ctgaacgagg tggcctgtct gagttggata aagccggctt catcaaaagg cagcttgttg
     8761 agacacgcca gatcaccaag cacgtggccc aaattctcga ttcacgcatg aacaccaagt
     8821 acgatgaaaa tgacaaactg attcgagagg tgaaagttat tactctgaag tctaagctgg
     8881 tctcagattt cagaaaggac tttcagtttt ataaggtgag agagatcaac aattaccacc
     8941 atgcgcatga tgcctacctg aatgcagtgg taggcactgc acttatcaaa aaatatccca
     9001 agcttgaatc tgaatttgtt tacggagact ataaagtgta cgatgttagg aaaatgatcg
     9061 caaagtctga gcaggaaata ggcaaggcca ccgctaagta cttcttttac agcaatatta
     9121 tgaatttttt caagaccgag attacactgg ccaatggaga gattcggaag cgaccactta
     9181 tcgaaacaaa cggagaaaca ggagaaatcg tgtgggacaa gggtagggat ttcgcgacag
     9241 tccggaaggt cctgtccatg ccgcaggtga acatcgttaa aaagaccgaa gtacagaccg
     9301 gaggcttctc caaggaaagt atcctcccga aaaggaacag cgacaagctg atcgcacgca
     9361 aaaaagattg ggaccccaag aaatacggcg gattcgattc tcctacagtc gcttacagtg
     9421 tactggttgt ggccaaagtg gagaaaggga agtctaaaaa actcaaaagc gtcaaggaac
     9481 tgctgggcat cacaatcatg gagcgatcaa gcttcgaaaa aaaccccatc gactttctcg
     9541 aggcgaaagg atataaagag gtcaaaaaag acctcatcat taagcttccc aagtactctc
     9601 tctttgagct tgaaaacggc cggaaacgaa tgctcgctag tgcgggcgag ctgcagaaag
     9661 gtaacgagct ggcactgccc tctaaatacg ttaatttctt gtatctggcc agccactatg
     9721 aaaagctcaa agggtctccc gaagataatg agcagaagca gctgttcgtg gaacaacaca
     9781 aacactacct tgatgagatc atcgagcaaa taagcgaatt ctccaaaaga gtgatcctcg
     9841 ccgacgctaa cctcgataag gtgctttctg cttacaataa gcacagggat aagcccatca
     9901 gggagcaggc agaaaacatt atccacttgt ttactctgac caacttgggc gcgcctgcag
     9961 ccttcaagta cttcgacacc accatagaca gaaagcggta cacctctaca aaggaggtcc
    10021 tggacgccac actgattcat cagtcaatta cggggctcta tgaaacaaga atcgacctct
    10081 ctcagctcgg tggagacagc agggctgacg agggagctcc caagaaaaag cgcaaggtag
    10141 gtagttccaa gcttggcggc agcggcggca gcatggaacg tgtgagaatg attaatgtgc
    10201 aaaggctgtt agaagccgca gagtttttag aaagaagaga aagagaatgc gaacacgggt
    10261 atgccagttc tttccctagc atgccctctc ccagaggcta actcgagctc ttctcgagtc
    10321 atgtaattag ttatgtcacg cttacattca cgccctcccc ccacatccgc tctaaccgaa
    10381 aaggaaggag ttagacaacc tgaagtctag gtccctattt atttttttat agttatgtta
    10441 gtattaagaa cgttatttat atttcaaatt tttctttttt ttctgtacag acgcgtgtac
    10501 gcatgtaaca ttatactgaa aaccttgctt gagaaggttt tgggacgctc gaaggcttta
    10561 atttgcggcc ggtacccaat tcgccctata gtgagtcgta ttacgcgcgc tcactggccg
    10621 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag
    10681 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
    10741 aacagttgcg cagcctgaat ggcgaatggc gcgacgcgcc ctgtagcggc gcattaagcg
    10801 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
    10861 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
    10921 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
    10981 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
    11041 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
    11101 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
    11161 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt
    11221 ttacaatttc ctgatgcaaa ttgaagctct aatttgtgag tttagtaaaa ggaaagcgcg
    11281 cctcgttcag aatgacacgt atagaatgat gcattacctt gtcatcttca gtatcatact
    11341 gttcgtatac atacttactg acattcatag gtatacatat atacacatgt atatatatcg
    11401 tatgctgcag ctttaaataa tcggtgtcac tacataagaa cacctttggt ggagggaaca
    11461 tcgttggtac cattgggcga ggtggcttct cttatggcaa ccgcaagagc cttgaacgca
    11521 ctctcactac ggtgatgatc attcttgcct cgcagacaat caacgtggag ggtaattctg
    11581 ctagcctctg caaagctttc aagaaaatgc gggatcatct cgcaagagag atctcctact
    11641 ttctcccttt gcaaaccaag ttcgacaact gcgtacggcc tgttcgaaag atctaccacc
    11701 gctctggaaa gtgcctcatc caaaggcgca aatcctgatc caaacctttt tactccacgc
    11761 gccagtaggg cctctttaaa agcttgaccg agagcaatcc cgcagtcttc agtggtgtga
    11821 tggtcgtcta tgtgtaagtc accaatgcac tcaacgatta gcgaccagcc ggaatgcttg
    11881 gccagagcat gtatcatatg gtccagaaac cctatacctg tgtggacgtt aatcacttgc
    11941 gattgtgtgg cctgttctgc tactgcttct gcctcttttt ctgggaagat cgagtgctct
    12001 atcgctaggg gaccaccctt taaagagatc gcaatctgaa tcttggtttc atttgtaata
    12061 cgctttacta gggctttctg ctctgtcatc tttgccttcg tttatcttgc ctgctcattt
    12121 tttagtatat tcttcgaaga aatcacatta ctttatataa tgtataattc attatgtgat
    12181 aatgccaatc gctaagaaaa aaaaagagtc atccgctagg ggaaaaaaaa aaatgaaaat
    12241 cattaccgag gcataaaaaa atatagagtg tactagagga ggccaagagt aatagaaaaa
    12301 gaaaattgcg ggaaaggact gtgttatgac ttccctgact aatgccgtgt tcaaacgata
    12361 cctggcagtg actcctagcg ctcaccaagc tcttaaaacg ggaatttatg gtgcactctc
    12421 agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacgcgct
    12481 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
    12541 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc ga
//
