LOCUS       sequence-317362-       12652 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integrates CRISPRi effector and tet-responsive regulator.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12652)
  AUTHORS   McGlincy NJ, Meacham ZA, Reynaud KK, Muller R, Baum R, Ingolia NT
  TITLE     A genome-scale CRISPR interference guide library enables 
            comprehensive phenotypic profiling in yeast.
  JOURNAL   BMC Genomics. 2021 Mar 23;22(1):205. doi: 
            10.1186/s12864-021-07518-0.
  PUBMED    33757429
REFERENCE   2  (bases 1 to 12652)
  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: "BMC 
            Genomics."; date: "2021-03-23"; volume: "22(1):205. doi"; pages: " 
            10.1186/s12864-021-07518-0"
COMMENT     Sequence Label: pNTI647 dCas9-Mxi1 TetR KanMX
FEATURES             Location/Qualifiers
     source          1..12652
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(110..901,902..970))
                     /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     733..752
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(971..1062)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1129..1148
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    1355..1388
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     terminator      complement(2105..2352)
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     2318..2336
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     CDS             complement(2376..2498)
                     /codon_start=1
                     /product="transcriptional repressor domain from the mouse 
                     Max-interacting protein 1 (Schreiber-Agus et al., 1995)"
                     /label=Mxi1
                     /translation="MERVRMINVQRLLEAAEFLERRERECEHGYASSFPSMPSPR"
     CDS             complement(2532..2552)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(join(2574..4088,4089..4091,4092..4157,4158..4163,
                     4164..6647,6648..6650,6651..6677))
                     /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             complement(join(2574..4157,4158..4160,4161..6647,6648..6650,
                     6651..6677))
                     /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             complement(2574..6677)
                     /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             complement(6687..6707)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        complement(6730..7128)
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     CDS             7941..8564
                     /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(8014..8033)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     protein_bind    8973..9006
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     gene            9061..10417
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        9061..9404
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             9405..10214
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     terminator      10220..10417
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     protein_bind    10480..10513
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(10556..10575)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(10557..10575)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(10585..10601)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10594..10616)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     protein_bind    complement(11263..11296)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     primer_bind     complement(11766..11782)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11881..11898)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(12052..12640)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(12132..12151)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 atcttttcta cggggtctga cgctcagtgg aacgacgcgc gcgtaactca cgttaaggga
       61 ttttggtcat gagcttgcgc cgtcccgtca agtcagcgta atgctctgct taccaatgct
      121 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
      181 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agcgctgcga
      241 tgataccgcg agaaccacgc tcaccggctc cggatttatc agcaataaac cagccagccg
      301 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
      361 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
      421 tcgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
      481 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
      541 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
      601 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
      661 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
      721 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
      781 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
      841 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
      901 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
      961 gaatactcat attcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     1021 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtc agtgttacaa
     1081 ccaattaacc aattctgaac attatcgcga gcccatttat acctgaatat ggctcataac
     1141 accccttgtt tgcctggcgg cagtagcgcg gtggtcccac ctgaccccat gccgaactca
     1201 gaagtgaaac gccgtagcgc cgatggtagt gtggggactc cccatgcgag agtagggaac
     1261 tgccaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc gcccgggcta
     1321 attatggggt gtcgccctta ttcgactcta tagtgaagtt cctattctct agaaagtata
     1381 ggaacttctg aagtggggat ttaaatgcgg ccgcgctgag ggtttaatcg gcatgcaaac
     1441 atctacacaa ttagcaaggg caatccatat tttgtctttt cgcgccctgg aaaggcctaa
     1501 gtaatgtcgt aaacgcattc tatctgtact tcaactctcc tctgtgcatt ggtttgtgca
     1561 aatcacattt tacgatactg ccagatatat gcaaaaagag aaaaccaagg gaccagaaca
     1621 aagcaaaatt acgatattct tcgaattcct tcgtgcttga ctaagacaaa gggatggacg
     1681 tagcgatttt tagcgggcca agaactggtt ccgaaaaagc acaggtacac cgaaccctca
     1741 gctaaggagg gacagcaccg atgcggaagg acaaactttc tttttgccta tcacagtatc
     1801 ttatcgagct aactattttc gacacacatg aaaaagcaga aatattaacg aaaaagaaaa
     1861 gaaagaccat gtcatgtacg ggcaatcaga atctgtaaca agcgccattt ttttttctgt
     1921 atcgggccct ccttactgct ctccttccgt gtaacgcgtt atgattaagt cctcagcgag
     1981 ctcgcatgga atgcgtgcga tgagcgacct catgctatac ctgagaaagc aacctgacct
     2041 acaggaaaga gttactcaag aataagaatt ttcgttttaa aacctaagag tcactttaaa
     2101 atttgtaaat taaagccttc gagcgtccca aaaccttctc aagcaaggtt ttcagtataa
     2161 tgttacatgc gtacacgcgt ctgtacagaa aaaaaagaaa aatttgaaat ataaataacg
     2221 ttcttaatac taacataact ataaaaaaat aaatagggac ctagacttca ggttgtctaa
     2281 ctccttcctt ttcggttaga gcggatgtgg ggggagggcg tgaatgtaag cgtgacataa
     2341 ctaattacat gactcgagaa gagctcgagt tagcctctgg gagagggcat gctagggaaa
     2401 gaactggcat acccgtgttc gcattctctt tctcttcttt ctaaaaactc tgcggcttct
     2461 aacagccttt gcacattaat cattctcaca cgttccatgc tgccgccgct gccgccaagc
     2521 ttggaactac ctaccttgcg ctttttcttg ggagctccct cgtcagccct gctgtctcca
     2581 ccgagctgag agaggtcgat tcttgtttca tagagccccg taattgactg atgaatcagt
     2641 gtggcgtcca ggacctcctt tgtagaggtg taccgctttc tgtctatggt ggtgtcgaag
     2701 tacttgaagg ctgcaggcgc gcccaagttg gtcagagtaa acaagtggat aatgttttct
     2761 gcctgctccc tgatgggctt atccctgtgc ttattgtaag cagaaagcac cttatcgagg
     2821 ttagcgtcgg cgaggatcac tcttttggag aattcgctta tttgctcgat gatctcatca
     2881 aggtagtgtt tgtgttgttc cacgaacagc tgcttctgct cattatcttc gggagaccct
     2941 ttgagctttt catagtggct ggccagatac aagaaattaa cgtatttaga gggcagtgcc
     3001 agctcgttac ctttctgcag ctcgcccgca ctagcgagca ttcgtttccg gccgttttca
     3061 agctcaaaga gagagtactt gggaagctta atgatgaggt cttttttgac ctctttatat
     3121 cctttcgcct cgagaaagtc gatggggttt ttttcgaagc ttgatcgctc catgattgtg
     3181 atgcccagca gttccttgac gcttttgagt tttttagact tccctttctc cactttggcc
     3241 acaaccagta cactgtaagc gactgtagga gaatcgaatc cgccgtattt cttggggtcc
     3301 caatcttttt tgcgtgcgat cagcttgtcg ctgttccttt tcgggaggat actttccttg
     3361 gagaagcctc cggtctgtac ttcggtcttt ttaacgatgt tcacctgcgg catggacagg
     3421 accttccgga ctgtcgcgaa atccctaccc ttgtcccaca cgatttctcc tgtttctccg
     3481 tttgtttcga taagtggtcg cttccgaatc tctccattgg ccagtgtaat ctcggtcttg
     3541 aaaaaattca taatattgct gtaaaagaag tacttagcgg tggccttgcc tatttcctgc
     3601 tcagactttg cgatcatttt cctaacatcg tacactttat agtctccgta aacaaattca
     3661 gattcaagct tgggatattt tttgataagt gcagtgccta ccactgcatt caggtaggca
     3721 tcatgcgcat ggtggtaatt gttgatctct ctcaccttat aaaactgaaa gtcctttctg
     3781 aaatctgaga ccagcttaga cttcagagta ataactttca cctctcgaat cagtttgtca
     3841 ttttcatcgt acttggtgtt catgcgtgaa tcgagaattt gggccacgtg cttggtgatc
     3901 tggcgtgtct caacaagctg ccttttgatg aagccggctt tatccaactc agacaggcca
     3961 cctcgttcag ccttagtcag attatcgaac ttccgttgtg tgatcagttt ggcgttcagc
     4021 agctgccgcc aataattttt cattttcttg acaacttctt ctgaggggac gttatcactc
     4081 ttccctctat ttttatcgga tcttgtcaac actttattat caatagaatc atctttgaga
     4141 aaagactggg gcacgatagc atccacgtcg tagtcggaga gccgattgat gtccagttcc
     4201 tgatccacgt acatgtccct gccgttctgc aggtagtaca ggtagagctt ctcattctga
     4261 agctgggtgt tttcaactgg gtgttcctta aggatttggg accccagttc ttttataccc
     4321 tcttcaatcc tcttcatcct ttccctactg ttcttctgtc ccttctgggt agtttggttc
     4381 tctcgggcca tctcgataac gatattctcg ggcttatgcc ttcccattac tttgacgagt
     4441 tcatccacga ccttaacggt ctgcagtatt ccctttttga tagctgggct acctgcaaga
     4501 ttagcgatgt gctcgtgaag actgtccccc tggccagaaa cttgtgcttt ctggatgtcc
     4561 tccttaaagg tgagagagtc atcatggatc aactgcatga agttccggtt ggcaaatcca
     4621 tcggacttaa gaaaatccag gattgtcttt ccactctgct tgtctcggat cccattgatc
     4681 agttttcttg acagccgccc ccatcctgta tatcggcgcc tcttgagctg tttcatgact
     4741 ttgtcgtcga agagatgagc gtaagttttc aagcgttctt caatcatctc cctatcttca
     4801 aacaacgtaa gggtgaggac aatgtcctca agaatgtcct cgttctcctc attgtccagg
     4861 aagtccttgt ctttaatgat tttcaggaga tcgtgatacg ttcccaggga tgcgttgaag
     4921 cgatcctcca ctccgctgat ttcaacagag tcgaaacatt caatcttttt gaaatagtct
     4981 tctttgagct gtttcacggt aactttccgg ttcgtcttga agaggaggtc cacgatagct
     5041 ttcttctgct ctccagacag gaatgctggc tttctcatcc cttctgtgac gtatttgacc
     5101 ttggtgagct cgttataaac tgtgaagtac tcgtacagca gagagtgttt aggaagcacc
     5161 ttttcgttag gcagattttt atcaaagtta gtcatccttt cgatgaagga ctgggcagag
     5221 gcccccttat ccacgacttc ctcgaagttc cagggagtga tggtctcttc tgatttgcga
     5281 gtcatccacg cgaatctgga atttccccgg gcgagggggc ctacatagta gggtatccga
     5341 aatgtgagga ttttctcaat cttttccctg ttatctttca aaaaggggta gaaatcctct
     5401 tgccgcctga ggatagcgtg cagttcgccc aggtgaatct ggtgggggat gcttccattg
     5461 tcgaaagtgc gctgtttgcg caacagatct tctctgttaa gctttaccag cagctcctcg
     5521 gtgccgtcca ttttttccaa gatgggctta ataaatttgt aaaattcctc ctggcttgct
     5581 ccgccgtcaa tgtatccggc gtagccattt ttagactgat cgaagaaaat ttccttgtac
     5641 ttctcaggca gttgctgtct gacaagggcc ttcagcaaag tcaagtcttg gtggtgctca
     5701 tcatagcgct tgatcatact agcgctcagc ggagctttgg tgatctccgt gttcactcgc
     5761 agaatatcac tcagcagaat ggcgtctgac aggttctttg ccgccaaaaa aaggtctgcg
     5821 tactggtcgc cgatctgggc cagcagattg tcgagatcat catcgtaggt gtctttgctc
     5881 agttgaagct tggcatcttc ggccaggtcg aagttagatt taaagttggg ggtcagcccg
     5941 agtgacaggg cgataagatt accaaacagg ccgttcttct tctccccagg gagctgtgcg
     6001 atgaggtttt cgagccgccg ggatttggac agcctagcgc tcaggattgc tttggcgtca
     6061 actccggatg cgttgatcgg gttctcttcg aaaagctgat tgtaagtctg aaccagttgg
     6121 ataaagagtt tgtcgacatc gctgttgtct gggttcaggt ccccctcgat gaggaagtgt
     6181 ccccgaaatt tgatcatatg cgccagcgcg agatagatca accgcaagtc agccttatca
     6241 gtactgtcta caagcttctt cctcagatga tatatggttg ggtacttttc atggtacgcc
     6301 acctcgtcca cgatattgcc aaagattggg tggcgctcgt gctttttatc ctcctccacc
     6361 aaaaaggact cctccagcct atggaagaaa gagtcatcca ccttagccat ctcattacta
     6421 aagatctcct gcaggtagca gatccgattc tttctgcggg tatatctgcg ccgtgctgtt
     6481 cttttgagcc gcgtggcttc ggccgtctcc ccggagtcga acaggagggc gccaatgagg
     6541 ttcttcttta tgctgtggcg atcggtattg cccagaactt tgaatttttt gctcggcacc
     6601 ttgtactcgt ccgtaatgac ggcccagccg acgctgtttg tgccgatagc gagcccaatg
     6661 gagtacttct tgtccatccc gggtctaact tttctcttct tctttggggc tctagacatc
     6721 tctagaaaac ttagattaga ttgctatgct ttctttctaa tgagcaagaa gtaaaaaaag
     6781 ttgtaataga acaagaaaaa tgaaactgaa acttgagaaa ttgaagaccg tttattaact
     6841 taaatatcaa tgggaggtca tcgaaagaga aaaaaatcaa aaaaaaaatt ttcaagaaaa
     6901 agaaacgtga taaaaatttt tattgccttt ttcgacgaag aaaaagaaac gaggcggtct
     6961 cttttttctt ttccaaacct ttagtacggg taattaacga caccctagag gaagaaagag
     7021 gggaaattta gtatgctgtg cttgggtgtt ttgaagtggt acggcgatgc gcggagtccg
     7081 agaaaatctg gaagagtaaa aaaggagtag aaacattttg aagctatgcg cttatttaga
     7141 agtgtcaaca acgtatctac caacggaatg cgtgcgatcg cgtgcattct agtcgtgcaa
     7201 tgtatgactt taagatttgt gagcaggaag aaaagggaga atcttctaac gataaaccct
     7261 tgaaaaactg ggtagactac gctatgttga gttgctacgc aggctgcaca attacacgag
     7321 aatgctcccg cctaggattt aaggctaagg gacgtgcaat gcagacgaca gatctaaatg
     7381 accgtgtcgg tgaagtgttc gccaaacttt tcggttaaca catgcagtga tgcacgcgcg
     7441 atggtgctaa gttacatata tatatatatg tatagccata gtgatgtcta agtaaccttt
     7501 atggtatatt tcttaatgtg gaaagatact agcgcgcgca cccacacaca agcttcgtct
     7561 tttcttgaag aaaagaggaa gctcgctaaa tgggattcca ctttccgttc cctgccagct
     7621 gatggaaaaa ggttagtgga acgatgaaga ataaaaagag agatccactg aggtgaaatt
     7681 tcagctgaca gcgagtttca tgatcgtgat gaacaatggt aacgagttgt ggctgttgcc
     7741 agggagggtg gttctcaact tttaatgtat ggccaaatcg ctacttgggt ttgttatata
     7801 acaaagaaga aataatgaac tgattctctt cctccttctt gtcctttctt aattctgttg
     7861 taattacctt cctttgtaat tttttttgta attattcttc ttaataatcc aaacaaacac
     7921 acatattaca atatattaaa atgtctagat tagataaaag taaagtgatt aacagcgcat
     7981 tagagctgct taatgaggtc ggaatcgaag gtttaacaac ccgtaaactc gcccagaagc
     8041 taggtgtaga gcagcctaca ttgtattggc atgtaaaaaa taagcgggct ttgctcgacg
     8101 ccttagccat tgagatgtta gataggcacc atactcactt ttgcccttta gaaggggaaa
     8161 gctggcaaga ttttttacgt aataacgcta aaagttttag atgtgcttta ctaagtcatc
     8221 gcgatggagc aaaagtacat ttaggtacac ggcctacaga aaaacagtat gaaactctcg
     8281 aaaatcaatt agccttttta tgccaacaag gtttttcact agagaatgca ttatatgcac
     8341 tcagcgctgt ggggcatttt actttaggtt gcgtattgga agatcaagag catcaagtcg
     8401 ctaaagaaga aagggaaaca cctactactg atagtatgcc gccattatta cgacaagcta
     8461 tcgaattatt tgatcaccaa ggtgcagagc cagccttctt attcggcctt gaattgatca
     8521 tatgcggatt agaaaaacaa cttaaatgtg aaagtgggtc ttaagtctga agaatgaatg
     8581 atttgatgat ttctttttcc ctccattttt cttactgaat atatcaatga tatagacttg
     8641 tatagtttat tatttcaaat taagtagcta tatatagtca agataacgtt tgtttgacac
     8701 gattacatta ttcgtcgaca tcttttttca gcctgtcgtg gtagcaattt gaggagtatt
     8761 attaattgaa taggttcatt ttgcgctcgc ataaacagtt ttcgtcaggg acagtatgtt
     8821 ggaatgagtg gtaattaatg gtgacatgac atgttatagc aataaccttg atgtttacat
     8881 cgtagtttaa tgtacacccc gcgaattcgt tcaagtagga gtgcaccaat tgcaaaggga
     8941 aaagctgaat gggcagttcg aaaaccctta atataacttc gtataatgta tgctatacga
     9001 agttattagg tctagagatc tgtttagctt gcctcgtccc cgccgggtca cccggccagc
     9061 gacatggagg cccagaatac cctccttgac agtcttgacg tgcgcagctc aggggcatga
     9121 tgtgactgtc gcccgtacat ttagcccata catccccatg tataatcatt tgcatccata
     9181 cattttgatg gccgcacggc gcgaagcaaa aattacggct cctcgctgca gacctgcgag
     9241 cagggaaacg ctcccctcac agacgcgttg aattgtcccc acgccgcgcc cctgtagaga
     9301 aatataaaag gttaggattt gccactgagg ttcttctttc atatacttcc ttttaaaatc
     9361 ttgctaggat acagttctca catcacatcc gaacataaac aaccatgggt aaggaaaaga
     9421 ctcacgtttc gaggccgcga ttaaattcca acatggatgc tgatttatat gggtataaat
     9481 gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgattgtat gggaagcccg
     9541 atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg
     9601 agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc aagcatttta
     9661 tccgtactcc tgatgatgca tggttactca ccactgcgat ccccggcaaa acagcattcc
     9721 aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc
     9781 tgcgccggtt acattcgatt cctgtttgta attgtccttt taacagcgac cgtgtatttc
     9841 gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt gattttgatg
     9901 acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataag cttttgccat
     9961 tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt atttttgacg
    10021 aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac cgataccagg
    10081 atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag aaacggcttt
    10141 ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat ttgatgctcg
    10201 atgagttttt ctaatcagta ctgacaataa aaagattctt gttttcaaga acttgtcatt
    10261 tgtatagttt ttttatattg tagttgttct attttaatca aatgttagcg tgatttatat
    10321 tttttttcgc ctcgacatca tctgcccaga tgcgaagtta agtgcgcaga aagtaatatc
    10381 atgcgtcaat cgtatgtgaa tgctggtcgc tatactgctg tcgattcgat actaacgccg
    10441 ccatccagtg tcgaaaacga gctctcgaga acccttaata taacttcgta taatgtatgc
    10501 tatacgaagt tattaggtga tatcagatcc actagtggcc tatgcaccca attcgcccta
    10561 tagtgagtcg tattacgcgc gctcactggc cgtcgtttta caacgtcgtg actgggaaaa
    10621 ccctggcgtt acccctgcag gactagtgct gaggcattaa tctactatcg gcgactctct
    10681 cgaaattttt cttaacgcgt ccttgtactg cgtctaacgc ttttgccact tggatttcta
    10741 ttataggaaa tagtctcact tactgggcga cgaattttcg cgttttgatg aagcacagga
    10801 agaatttctt ttttttttgg cttcttctgg ttccgttttt tacgcgcaca aatctaaaaa
    10861 aagaaataat tataacctag tctcgaaaat tttcatcgat ccattcgttc ctttttttcg
    10921 attttttcag atcaaaattc ttgtttcttt ctttgtctta gtttatatta aaagatattt
    10981 tgattttact cctgaactat ttattctttc taagaaggcc agaacactac agctgtttta
    11041 accgactacg aagttctcca ttctcgaaca ctagccttca tttaccaaac aggaactagc
    11101 gtatatcatt agtccttatt cgaaaagaga ttggtagata tttattgtag tttgtgagaa
    11161 ggagaaaata ctgtcattgg actgatagtt agaggacatt aacctctctt acgttcgctc
    11221 attaagacct cagcgcggcc gcaaatttaa ataaaatgaa gtgaagttcc tatactttct
    11281 agagaatagg aacttctata gtgagtcgaa taagggcgac acaaaattta ttctaaatgc
    11341 ataataaata ctgataacat cttatagttt gtattatatt ttgtattatc gttgacatgt
    11401 ataattttga tatcaaaaac tgattttccc tttattattt tcgagattta ttttcttaat
    11461 tctctttaac aaactagaaa tattgtatat acaaaaaatc ataaataata gatgaatagt
    11521 ttaattatag gtgttcatca atcgaaaaag caacgtatct tatttaaagt gcgttgcttt
    11581 tttctcattt ataaggttaa ataattctca tatatcaagc aaagtgacag gcgcccttaa
    11641 atattctgac aaatgctctt tccctaaact ccccccataa aaaaacccgc cgaagcgggt
    11701 ttttacgtta tttgcggatt aacgattact cgttatcaga accgcccagg gggcccgagc
    11761 ttaagactgg ccgtcgtttt acaacacaga aagagtttgt agaaacgcaa aaaggccatc
    11821 cgtcaggggc cttctgctta gtttgatgcc tggcagttcc ctactctcgc cttccgcttc
    11881 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
    11941 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
    12001 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
    12061 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
    12121 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
    12181 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
    12241 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
    12301 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
    12361 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
    12421 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggg ctaactacgg
    12481 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
    12541 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
    12601 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tg
//
