LOCUS       sequence-323254-       13048 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of SpCas9 with 4 ERT2 fusion protein and empty gRNA 
            cassette. The activity of Cas9 can be switched on and off in human 
            cells with 4-hydroxytamoxifen (4-HT).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13048)
  AUTHORS   Liu KI, Ramli MN, Woo CW, Wang Y, Zhao T, Zhang X, Yim GR, Chong BY,
            Gowher A, Chua MZ, Jung J, Lee JH, Tan MH
  TITLE     A chemical-inducible CRISPR-Cas9 system for rapid control of genome 
            editing.
  JOURNAL   Nat Chem Biol. 2016 Sep 12. doi: 10.1038/nchembio.2179.
  PUBMED    27618190
REFERENCE   2  (bases 1 to 13048)
  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: "Nat Chem 
            Biol. 2016 Sep 12. doi: 10.1038/nchembio.2179."
COMMENT     Sequence Label: iCas
FEATURES             Location/Qualifiers
     source          1..13048
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        50..429
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        430..633
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     584..604
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     630..654
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      728..737
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             737..778
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             782..802
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             842..1777
                     /codon_start=1
                     /product="mutated ligand-binding domain of the human 
                     estrogen receptor (Feil et al., 1997)"
                     /label=ERT2
                     /translation="AGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPIL
                     YSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEI
                     LMIGLVWRSMEHPVKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEF
                     VCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRL
                     AQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEAADAHRLHAPTSRGGASVEET
                     DQSHLATAGSTSSHSLQKYYITGEAEGFPAT"
     CDS             1796..2731
                     /codon_start=1
                     /product="mutated ligand-binding domain of the human 
                     estrogen receptor (Feil et al., 1997)"
                     /label=ERT2
                     /translation="AGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPIL
                     YSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEI
                     LMIGLVWRSMEHPVKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEF
                     VCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRL
                     AQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEAADAHRLHAPTSRGGASVEET
                     DQSHLATAGSTSSHSLQKYYITGEAEGFPAT"
     CDS             2753..6853
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             2753..6853
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     RBS             6766..6774
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             6866..6886
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6893..7828
                     /codon_start=1
                     /product="mutated ligand-binding domain of the human 
                     estrogen receptor (Feil et al., 1997)"
                     /label=ERT2
                     /translation="AGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPIL
                     YSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEI
                     LMIGLVWRSMEHPVKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEF
                     VCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRL
                     AQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEAADAHRLHAPTSRGGASVEET
                     DQSHLATAGSTSSHSLQKYYITGEAEGFPAT"
     CDS             7850..8785
                     /codon_start=1
                     /product="mutated ligand-binding domain of the human 
                     estrogen receptor (Feil et al., 1997)"
                     /label=ERT2
                     /translation="AGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPIL
                     YSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEI
                     LMIGLVWRSMEHPVKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEF
                     VCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRL
                     AQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEAADAHRLHAPTSRGGASVEET
                     DQSHLATAGSTSSHSLQKYYITGEAEGFPAT"
     CDS             8804..8857
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             8858..9523
                     /codon_start=1
                     /product="orange fluorescent protein from Cerianthus (Ip et
                     al., 2007)"
                     /label=OFP
                     /translation="MNLSKNVSVSVYMKGNVNNHEFEYDGEGGGDPYTGKYSMKMTLRG
                     QNSLPFSYDIITTAFQYGFRVFTKYPEGIVDYFKDSLPDAFQWNRRIVFEDGGVLNMSS
                     DITYKDNVLHGDVKAEGVNFPPNGPVMKNEIVMEEPTEETFTPKNGVLVGFCPKAYLLK
                     DGSYYYGNMTTFYRSKKSGQAPPGYHFVKHRLVKTNVGHGFKTVEQTEYATAHVSDLPK
                     "
     primer_bind     complement(9563..9582)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    9607..9655
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      9857..10285
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9944..9963)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     10154..10175
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        10305..10553
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     10305..10325
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     10476..10495
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        10581..10656
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(10706..10722)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10706..10722)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10719..10741)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10730..10746
                     /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)."
     promoter        complement(join(10754..10760,10761..10778,10779..10784))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10799..10820
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10937..10954)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11108..11696)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11188..11207)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11867..12658,12659..12727))
                     /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     12490..12509
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12728..12832)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(12907..12926)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta
       61 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
      121 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
      181 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
      241 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
      301 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
      361 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
      421 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
      481 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
      541 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
      601 cgtgtacggt gggaggtcta tataagcaga gctcgtttag tgaaccgtca gatcgcctgg
      661 agacgccatc cacgctgttt tgacctccat agaagacacc gggaccgatc cagcctccgg
      721 agcggccgcc accatgggca agcccatccc taaccccctg ttggggctgg acagcaccgc
      781 tcccaaaaag aaaaggaagg tgggcattca cggcgtgcct gcggccaccg gtggaggctc
      841 tgctggagac atgagagctg ccaacctttg gccaagcccg ctcatgatca aacgctctaa
      901 gaagaacagc ctggccttgt ccctgacggc cgaccagatg gtcagtgcct tgttggatgc
      961 tgagcccccc atactctatt ccgagtatga tcctaccaga cccttcagtg aagcttcgat
     1021 gatgggctta ctgaccaacc tggcagacag ggagctggtt cacatgatca actgggcgaa
     1081 gagggtgcca ggctttgtgg atttgaccct ccatgatcag gtccaccttc ttgaatgtgc
     1141 ctggctagag atcctgatga ttggtctcgt ctggcgctcc atggagcacc cagtgaagct
     1201 actgtttgct cctaacttgc tcttggacag gaaccaggga aaatgtgtag agggcatggt
     1261 ggagatcttc gacatgctgc tggctacatc atctcggttc cgcatgatga atctgcaggg
     1321 agaggagttt gtgtgcctca aatctattat tttgcttaat tctggagtgt acacatttct
     1381 gtccagcacc ctgaagtctc tggaagagaa ggaccatatc caccgagtcc tggacaagat
     1441 cacagacact ttgatccacc tgatggccaa ggcaggcctg accctgcagc agcagcacca
     1501 gcggctggcc cagctcctcc tcatcctctc ccacatcagg cacatgagta acaaaggcat
     1561 ggagcatctg tacagcatga agtgcaagaa cgtggtgccc ctctatgacc tgctgctgga
     1621 ggcggcggac gcccaccgcc tacatgcgcc cactagccgt ggaggggcat ccgtggagga
     1681 gacggaccaa agccacttgg ccactgcggg ctctacttca tcgcattcct tgcaaaagta
     1741 ttacatcacg ggggaggcag agggtttccc tgccacagct cctaggggag gctctgctgg
     1801 agacatgaga gctgccaacc tttggccaag cccgctcatg atcaaacgct ctaagaagaa
     1861 cagcctggcc ttgtccctga cggccgacca gatggtcagt gccttgttgg atgctgagcc
     1921 ccccatactc tattccgagt atgatcctac cagacccttc agtgaagctt cgatgatggg
     1981 cttactgacc aacctggcag acagggagct ggttcacatg atcaactggg cgaagagggt
     2041 gccaggcttt gtggatttga ccctccatga tcaggtccac cttcttgaat gtgcctggct
     2101 agagatcctg atgattggtc tcgtctggcg ctccatggag cacccagtga agctactgtt
     2161 tgctcctaac ttgctcttgg acaggaacca gggaaaatgt gtagagggca tggtggagat
     2221 cttcgacatg ctgctggcta catcatctcg gttccgcatg atgaatctgc agggagagga
     2281 gtttgtgtgc ctcaaatcta ttattttgct taattctgga gtgtacacat ttctgtccag
     2341 caccctgaag tctctggaag agaaggacca tatccaccga gtcctggaca agatcacaga
     2401 cactttgatc cacctgatgg ccaaggcagg cctgaccctg cagcagcagc accagcggct
     2461 ggcccagctc ctcctcatcc tctcccacat caggcacatg agtaacaaag gcatggagca
     2521 tctgtacagc atgaagtgca agaacgtggt gcccctctat gacctgctgc tggaggcggc
     2581 ggacgcccac cgcctacatg cgcccactag ccgtggaggg gcatccgtgg aggagacgga
     2641 ccaaagccac ttggccactg cgggctctac ttcatcgcat tccttgcaaa agtattacat
     2701 cacgggggag gcagagggtt tccctgccac agctacacca gaatcgggcg ccgacaaaaa
     2761 gtacagcatc ggccttgata tcggcaccaa tagcgtgggc tgggccgtta tcacagacga
     2821 atacaaggta cccagcaaga agttcaaggt gctggggaat acagacaggc actctatcaa
     2881 gaaaaacctt atcggggctc tgctgtttga ctcaggcgag accgccgagg ccaccaggtt
     2941 gaagaggacc gcaaggcgaa ggtacacccg gaggaagaac aggatctgct atctgcagga
     3001 gatcttcagc aacgagatgg ccaaggtgga cgacagcttc ttccacaggc tggaggagag
     3061 cttccttgtc gaggaggata agaagcacga acgacacccc atcttcggca acatagtcga
     3121 cgaggtcgct tatcacgaga agtaccccac catctaccac ctgcgaaaga aattggtgga
     3181 tagcaccgat aaagccgact tgcgacttat ctacttggct ctggcgcaca tgattaagtt
     3241 caggggccac ttcctgatcg agggcgacct taaccccgac aacagtgacg tagacaaatt
     3301 gttcatccag cttgtacaga cctataacca gctgttcgag gaaaacccta ttaacgccag
     3361 cggggtggat gcgaaggcca tacttagcgc caggctgagc aaaagcaggc gcttggagaa
     3421 cctgatagcc cagctgcccg gtgaaaagaa gaacggcctc ttcggtaatc tgattgccct
     3481 gagcctgggc ctgaccccca acttcaagag caacttcgac ctggcagaag atgccaagct
     3541 gcagttgagt aaggacacct atgacgacga cttggacaat ctgctcgccc aaatcggcga
     3601 ccagtacgct gacctgttcc tcgccgccaa gaacctttct gacgcaatcc tgcttagcga
     3661 tatccttagg gtgaacacag agatcaccaa ggcccccctg agcgccagca tgatcaagag
     3721 gtacgacgag caccatcagg acctgaccct tctgaaggcc ctggtgaggc agcaactgcc
     3781 cgagaagtac aaggagatct ttttcgacca gagcaagaac ggctacgccg gctacatcga
     3841 cggcggagcc agccaagagg agttctacaa gttcatcaag cccatcctgg agaagatgga
     3901 tggcaccgag gagctgctgg tgaagctgaa cagggaagat ttgctccgga agcagaggac
     3961 ctttgacaac ggtagcatcc cccaccagat ccacctgggc gagctgcacg caatactgag
     4021 gcgacaggag gatttctacc ccttcctcaa ggacaatagg gagaaaatcg aaaagattct
     4081 gaccttcagg atcccctact acgtgggccc tcttgccagg ggcaacagcc gattcgcttg
     4141 gatgacaaga aagagcgagg agaccatcac cccctggaac ttcgaggaag tggtggacaa
     4201 aggagcaagc gcgcagtctt tcatcgaacg gatgaccaat ttcgacaaaa acctgcctaa
     4261 cgagaaggtg ctgcccaagc acagcctgct ttacgagtac ttcaccgtgt acaacgagct
     4321 caccaaggtg aaatatgtga ccgagggcat gcgaaaaccc gctttcctga gcggcgagca
     4381 gaagaaggcc atcgtggacc tgctgttcaa gaccaacagg aaggtgaccg tgaagcagct
     4441 gaaggaggac tacttcaaga agatcgagtg ctttgatagc gtggaaataa gcggcgtgga
     4501 ggacaggttc aacgccagcc tgggcaccta ccacgacttg ttgaagataa tcaaagacaa
     4561 ggatttcctg gataatgagg agaacgagga tatactcgag gacatcgtgc tgactttgac
     4621 cctgtttgag gaccgagaga tgattgaaga aaggctcaaa acctacgccc acctgttcga
     4681 cgacaaagtg atgaaacaac tgaagagacg aagatacacc ggctggggca gactgtccag
     4741 gaagctcatc aacggcatta gggacaagca gagcggcaag accatcctgg atttcctgaa
     4801 gtccgacggc ttcgccaacc gaaacttcat gcagctgatt cacgatgaca gcttgacctt
     4861 caaggaggac atccagaagg cccaggttag cggccagggc gactccctgc acgaacatat
     4921 tgcaaacctg gcaggctccc ctgcgatcaa gaagggcata ctgcagaccg ttaaggttgt
     4981 ggacgaattg gtcaaggtca tgggcaggca caagcccgaa aacatagtta tagagatggc
     5041 cagagagaac cagaccaccc aaaagggcca gaagaacagc cgggagcgca tgaaaaggat
     5101 cgaggagggt atcaaggaac tcggaagcca gatcctcaaa gagcaccccg tggagaatac
     5161 ccagctccag aacgagaagc tgtacctgta ctacctgcag aacggtaggg acatgtacgt
     5221 tgaccaggag ttggacatca acaggctttc agactatgac gtggatcaca tagtgcccca
     5281 gagctttctt aaagacgata gcatcgacaa caaggtcctg acccgctccg acaaaaacag
     5341 gggcaaaagc gacaacgtgc caagcgaaga ggtggttaaa aagatgaaga actactggag
     5401 gcaactgctc aacgcgaaat tgatcaccca gagaaagttc gataacctga ccaaggccga
     5461 gaggggcgga ctctccgaac ttgacaaagc gggcttcata aagaggcagc tggtcgagac
     5521 ccgacagatc acgaagcacg tggcccaaat cctcgacagc agaatgaata ccaagtacga
     5581 tgagaatgac aaactcatca gggaagtgaa agtgattacc ctgaagagca agttggtgtc
     5641 cgactttcgc aaagatttcc agttctacaa ggtgagggag atcaacaact accaccatgc
     5701 ccacgacgca tacctgaacg ccgtggtcgg caccgccctg attaagaagt atccaaagct
     5761 ggagtccgaa tttgtctacg gcgactacaa agtttacgat gtgaggaaga tgatcgctaa
     5821 gagcgaacag gagatcggca aggccaccgc taagtatttc ttctacagca acatcatgaa
     5881 ctttttcaag accgagatca cacttgccaa cggcgaaatc aggaagaggc cgcttatcga
     5941 gaccaacggt gagaccggcg agatcgtgtg ggacaagggc agggacttcg ccaccgtgag
     6001 gaaagtcctg agcatgcccc aggtgaatat tgtgaaaaaa actgaggtgc agacaggcgg
     6061 ctttagcaag gaatccatcc tgcccaagag gaacagcgac aagctgatcg cccggaagaa
     6121 ggactgggac cctaagaagt atggaggctt cgacagcccc accgtagcct acagcgtgct
     6181 ggtggtcgcg aaggtagaga aggggaagag caagaaactg aagagcgtga aggagctgct
     6241 cggcataacc atcatggaga ggtccagctt tgagaagaac cccattgact ttttggaagc
     6301 caagggctac aaagaggtca aaaaggacct gatcatcaaa ctccccaagt actccctgtt
     6361 tgaattggag aacggcagaa agaggatgct ggcgagcgct ggggaactgc aaaagggcaa
     6421 cgaactggcg ctgcccagca agtacgtgaa ttttctgtac ctggcgtccc actacgaaaa
     6481 gctgaaaggc agccccgagg acaacgagca gaagcagctg ttcgtggagc agcacaagca
     6541 ttacctggac gagataatcg agcaaatcag cgagttcagc aagagggtga ttctggccga
     6601 cgcgaacctg gataaggtcc tcagcgccta caacaagcac cgagacaaac ccatcaggga
     6661 gcaggccgag aatatcatac acctgttcac cctgacaaat ctgggcgcac ctgcggcatt
     6721 caaatacttc gataccacca tcgacaggaa aaggtacact agcactaagg aggtgctgga
     6781 tgccaccttg atccaccagt ccattaccgg cctgtatgag accaggatcg acctgagcca
     6841 gcttggaggc gactctaggg cggacccaaa aaagaaaagg aaggtggaat tcgctggaga
     6901 catgagagct gccaaccttt ggccaagccc gctcatgatc aaacgctcta agaagaacag
     6961 cctggccttg tccctgacgg ccgaccagat ggtcagtgcc ttgttggatg ctgagccccc
     7021 catactctat tccgagtatg atcctaccag acccttcagt gaagcttcga tgatgggctt
     7081 actgaccaac ctggcagaca gggagctggt tcacatgatc aactgggcga agagggtgcc
     7141 aggctttgtg gatttgaccc tccatgatca ggtccacctt cttgaatgtg cctggctaga
     7201 gatcctgatg attggtctcg tctggcgctc catggagcac ccagtgaagc tactgtttgc
     7261 tcctaacttg ctcttggaca ggaaccaggg aaaatgtgta gagggcatgg tggagatctt
     7321 cgacatgctg ctggctacat catctcggtt ccgcatgatg aatctgcagg gagaggagtt
     7381 tgtgtgcctc aaatctatta ttttgcttaa ttctggagtg tacacatttc tgtccagcac
     7441 cctgaagtct ctggaagaga aggaccatat ccaccgagtc ctggacaaga tcacagacac
     7501 tttgatccac ctgatggcca aggcaggcct gaccctgcag cagcagcacc agcggctggc
     7561 ccagctcctc ctcatcctct cccacatcag gcacatgagt aacaaaggca tggagcatct
     7621 gtacagcatg aagtgcaaga acgtggtgcc cctctatgac ctgctgctgg aggcggcgga
     7681 cgcccaccgc ctacatgcgc ccactagccg tggaggggca tccgtggagg agacggacca
     7741 aagccacttg gccactgcgg gctctacttc atcgcattcc ttgcaaaagt attacatcac
     7801 gggggaggca gagggtttcc ctgccacagc tcctgcaggt ggaggctctg ctggagacat
     7861 gagagctgcc aacctttggc caagcccgct catgatcaaa cgctctaaga agaacagcct
     7921 ggccttgtcc ctgacggccg accagatggt cagtgccttg ttggatgctg agccccccat
     7981 actctattcc gagtatgatc ctaccagacc cttcagtgaa gcttcgatga tgggcttact
     8041 gaccaacctg gcagacaggg agctggttca catgatcaac tgggcgaaga gggtgccagg
     8101 ctttgtggat ttgaccctcc atgatcaggt ccaccttctt gaatgtgcct ggctagagat
     8161 cctgatgatt ggtctcgtct ggcgctccat ggagcaccca gtgaagctac tgtttgctcc
     8221 taacttgctc ttggacagga accagggaaa atgtgtagag ggcatggtgg agatcttcga
     8281 catgctgctg gctacatcat ctcggttccg catgatgaat ctgcagggag aggagtttgt
     8341 gtgcctcaaa tctattattt tgcttaattc tggagtgtac acatttctgt ccagcaccct
     8401 gaagtctctg gaagagaagg accatatcca ccgagtcctg gacaagatca cagacacttt
     8461 gatccacctg atggccaagg caggcctgac cctgcagcag cagcaccagc ggctggccca
     8521 gctcctcctc atcctctccc acatcaggca catgagtaac aaaggcatgg agcatctgta
     8581 cagcatgaag tgcaagaacg tggtgcccct ctatgacctg ctgctggagg cggcggacgc
     8641 ccaccgccta catgcgccca ctagccgtgg aggggcatcc gtggaggaga cggaccaaag
     8701 ccacttggcc actgcgggct ctacttcatc gcattccttg caaaagtatt acatcacggg
     8761 ggaggcagag ggtttccctg ccacagcttc tagaggcagt ggagagggca gaggaagtct
     8821 gctaacatgc ggtgacgtcg aggagaatcc tggcccaatg aacctgagca aaaacgtgag
     8881 cgtgagcgtg tatatgaagg ggaacgtcaa caatcatgag tttgagtacg acggggaagg
     8941 tggtggtgat ccttatacag gtaaatattc catgaagatg acgctacgtg gtcaaaattc
     9001 cctacccttt tcctatgata tcattaccac ggcatttcag tatggtttcc gcgtatttac
     9061 aaaataccct gagggaattg ttgactattt taaggactcg cttcccgacg cattccagtg
     9121 gaacagacga attgtgtttg aagatggtgg agtactaaac atgagcagtg atatcacata
     9181 taaagataat gttctgcatg gtgacgtcaa ggctgaggga gtgaacttcc cgccgaatgg
     9241 gccagtgatg aagaatgaaa ttgtgatgga ggaaccgact gaagaaacat ttactccaaa
     9301 aaacggggtt cttgttggct tttgtcccaa agcgtactta cttaaagacg gttcctatta
     9361 ctatggaaat atgacaacat tttacagatc caagaaatct ggccaggcac ctcctgggta
     9421 tcactttgtt aagcatcgtc tcgtcaagac caatgtggga catggattta agacggttga
     9481 gcagactgaa tatgccactg ctcatgtcag tgatcttccc aagttcgaag cttgataatg
     9541 agtttaaacg ggggaggcta actgaaacac ggaaggagac aataccggaa ggaacccgcg
     9601 ctatgacggc aataaaaaga cagaataaaa cgcacgggtg ttgggtcgtt tgttcataaa
     9661 cgcggggttc ggtcccaggg ctggcactct gtcgataccc caccgagacc ccattggggc
     9721 caatacgccc gcgtttcttc cttttcccca ccccaccccc caagttcggg tgaaggccca
     9781 gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag cagatctgcg cagctggggc
     9841 tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     9901 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     9961 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
    10021 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
    10081 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
    10141 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
    10201 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
    10261 atttaacaaa aatttaacgc gaattaatta aggtcgggca ggaagagggc ctatttccca
    10321 tgattccttc atatttgcat atacgataca aggctgttag agagataatt agaattaatt
    10381 tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg
    10441 ggtagtttgc agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt
    10501 gaaagtattt cgatttcttg gctttatata tcttgtggaa aggacgaaac accgggtctt
    10561 cgagctagcc tcgaagacct gttttagagc tagaaatagc aagttaaaat aaggctagtc
    10621 cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttctagtata ccgtcgacct
    10681 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
    10741 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
    10801 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
    10861 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
    10921 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
    10981 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
    11041 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
    11101 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
    11161 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
    11221 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
    11281 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
    11341 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
    11401 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
    11461 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
    11521 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
    11581 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
    11641 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
    11701 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
    11761 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
    11821 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
    11881 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
    11941 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
    12001 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
    12061 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
    12121 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
    12181 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
    12241 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
    12301 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
    12361 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
    12421 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
    12481 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
    12541 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
    12601 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
    12661 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
    12721 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
    12781 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
    12841 cccgaaaagt gccacctgac gtcgacggat cgggagatct cccgatcccc tatggtgcac
    12901 tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc ctgcttgtgt
    12961 gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac
    13021 cgacaattgc atgaagaatc tgcttagg
//
