LOCUS       sequence-200490-       14209 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  piggyBac vector expressing Dual-regulated Cas9 (Hygro resistance).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14209)
  AUTHORS   Ishida K, Xu H, Sasakawa N, Lung MSY, Kudryashev JA, Gee P, Hotta A
  TITLE     Site-specific randomization of the endogenous genome by a 
            regulatable CRISPR-Cas9 piggyBac system in human cells.
  JOURNAL   Sci Rep. 2018 Jan 10;8(1):310. doi: 10.1038/s41598-017-18568-4.
  PUBMED    29321585
REFERENCE   2  (bases 1 to 14209)
  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: "Sci Rep."; 
            date: "2018-01-10"; volume: "8(1):310. doi"; pages: " 
            10.1038/s41598-017-18568-4"
COMMENT     Sequence Label: pPV-TetO-SpCas9-GR-iC-ERiH (CRONUS-Hygro)
FEATURES             Location/Qualifiers
     source          1..14209
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   46..108
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    491..509
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    528..546
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    565..583
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    602..620
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    639..657
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    676..694
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    713..731
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    750..768
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     790..814
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    894..918
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             944..5047
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             944..5047
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     RBS             4960..4968
                     /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)"
     protein_bind    complement(5870..5894)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    join(5936..6509,6510..6512,6513..6521)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(6103..6120)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     6330..6349
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             6522..7232
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(6667..6685)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     6935..6954
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(7346..7365)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    7411..7466
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(7465..7484)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     promoter        7918..9100
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          8149..9091
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     9048..9068
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    9122..9146
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             join(9167..9784,9785..9790,9791..9826,9827..9829,9830..9865,
                     9866..9868,9869..9904,9905..9913)
                     /codon_start=1
                     /product="improved tetracycline-controlled transactivator"
                     /label=rtTA-Advanced
                     /note="In the Tet-On(R) system, rtTA-Advanced binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the presence of tetracycline or doxycycline."
                     /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPADALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     protein_bind    complement(9950..9974)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    10012..10456
                     /label=FMDV IRES
                     /note="internal ribosome entry site (IRES) of the 
                     foot-and-mouth disease virus"
     promoter        10490..10536
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        10528..10546
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             10556..11581
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     repeat_region   12176..12210
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(12303..12320)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(12474..13062)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(12554..12573)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(13233..14024,14025..14093))
                     /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     13856..13875
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(14094..14198)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gaaaagtgcc acctgacgtc atctgttaac attatacgcg tttaacccta gaaagataat
       61 catattgtga cgtacgttaa agataatcat gcgtaaaatt gacgcatgtg ttttatcggt
      121 ctgtatatcg aggtttattt attaatttga atagatatta agttttatta tatttacact
      181 tacatactaa taataaattc aacaaacaat ttatttatgt ttatttattt attaaaaaaa
      241 aacaaaaact caaaatttct tctataaagt aacaaaactt ttaaacattc tctcttttac
      301 aaaaataaac ttattttgta ctttaaaaac agtcatgttg tattataaaa taagtaatta
      361 gcttaaccta tacataatag aaacaaatta tacttattag tcagtcagaa acaactttgg
      421 cacatatcaa tattatgctc tgctagcgtt taaacccatt aactcgaggg atccaagctg
      481 gccgcacgtc tccctatcag tgatagagaa gtcgacacgt ctcgagctcc ctatcagtga
      541 tagagaaggt acgtctagaa cgtctcccta tcagtgatag agaagtcgac acgtctcgag
      601 ctccctatca gtgatagaga aggtacgtct agaacgtctc cctatcagtg atagagaagt
      661 cgacacgtct cgagctccct atcagtgata gagaaggtac gtctagaacg tctccctatc
      721 agtgatagag aagtcgacac gtctcgagct ccctatcagt gatagagaag gtacccccta
      781 tataagcaga gctcgtttag tgaaccgtca gatcgcctgg agacgccatc cacgctgttt
      841 tgacctccat agaagacacc gggaccgatc cagcctccgc ggtggcggcc atcacaagtt
      901 tgtacaaaaa agcaggctgg cgccggaacc aattcagtcg accatggata agaaatacag
      961 cattggactg gacattggga caaactccgt gggatgggcc gtgattacag acgaatacaa
     1021 agtgccttca aagaagttca aggtgctggg caacaccgat agacacagca tcaagaaaaa
     1081 tctgattgga gccctgctgt tcgactccgg cgagacagct gaagcaactc ggctgaaaag
     1141 aactgctcgg agaaggtata cccgccgaaa gaataggatc tgctacctgc aggagatttt
     1201 cagcaacgaa atggccaagg tggacgatag tttctttcac cgcctggagg aatcattcct
     1261 ggtcgaggaa gataagaaac acgagcggca tcccatcttt ggcaacattg tggacgaggt
     1321 cgcttatcac gaaaagtacc ctaccatcta tcatctgagg aagaaactgg tggactccac
     1381 agataaagca gacctgcgcc tgatctatct ggccctggct cacatgatta agttccgggg
     1441 ccattttctg atcgaggggg atctgaaccc agacaattct gatgtggaca agctgttcat
     1501 ccagctggtc cagacataca atcagctgtt tgaggaaaac cccattaatg catctggcgt
     1561 ggacgcaaaa gccatcctga gtgccagact gtctaagagt cggagactgg agaacctgat
     1621 cgctcagctg ccaggggaaa agaaaaacgg cctgtttggg aatctgattg cactgtcact
     1681 gggactgact cccaacttca agagcaattt tgatctggcc gaggacgcta aactgcagct
     1741 gtccaaggac acctatgacg atgacctgga taacctgctg gctcagatcg gggatcagta
     1801 cgcagacctg ttcctggccg ctaagaatct gtctgacgcc atcctgctga gtgatattct
     1861 gcgcgtgaac accgagatta caaaagcccc cctgtcagct agcatgatca agagatatga
     1921 cgagcaccat caggatctga ccctgctgaa ggctctggtg aggcagcagc tgcctgagaa
     1981 gtacaaggaa atcttctttg atcagtctaa gaacggatac gccggctata ttgacggcgg
     2041 ggctagtcag gaggagttct acaagtttat caaacccatt ctggagaaga tggatggcac
     2101 agaggaactg ctggtgaaac tgaatcggga agacctgctg aggaagcagc gcacttttga
     2161 taacggaagc atccctcacc agattcatct gggagagctg cacgcaatcc tgaggcgcca
     2221 ggaagacttc tacccatttc tgaaggataa cagggagaag atcgaaaaaa ttctgacatt
     2281 ccgcatcccc tactatgtgg gccctctggc aagaggcaac agccggtttg cctggatgac
     2341 tcgcaaatct gaggaaacaa tcactccctg gaacttcgag gaagtggtcg ataagggcgc
     2401 ttccgcacag tctttcattg agcggatgac aaacttcgac aagaacctgc caaacgaaaa
     2461 agtgctgccc aagcactctc tgctgtacga gtatttcaca gtctataacg aactgactaa
     2521 ggtgaaatac gtcaccgagg ggatgagaaa gcctgccttc ctgagtggag aacagaagaa
     2581 agctatcgtg gacctgctgt ttaaaaccaa taggaaggtg acagtcaagc agctgaaaga
     2641 ggactatttc aagaaaattg aatgtttcga ttctgtggag atcagtggcg tcgaagacag
     2701 gtttaacgcc tccctgggga cctaccacga tctgctgaag atcattaagg ataaagactt
     2761 cctggacaac gaggaaaatg aggatatcct ggaagacatt gtgctgaccc tgacactgtt
     2821 tgaggatagg gaaatgatcg aggaacgcct gaagacctat gcccatctgt tcgatgacaa
     2881 agtgatgaaa cagctgaagc gacggagata cacaggatgg ggccgactgt ctcggaagct
     2941 gatcaatggg attcgcgaca aacagagtgg aaagaccatc ctggactttc tgaaatcaga
     3001 tggcttcgcc aaccggaact tcatgcagct gattcacgat gacagcctga cattcaaaga
     3061 ggatatccag aaggcacagg tgtccgggca gggagactct ctgcacgagc atatcgcaaa
     3121 cctggccggc agccctgcca tcaagaaagg gattctgcag accgtgaagg tggtggacga
     3181 gctggtgaaa gtcatgggaa gacataagcc agaaaacatc gtgattgaga tggccaggga
     3241 aaatcagacc acacagaaag gccagaagaa ctcaagggag cgcatgaaaa gaatcgagga
     3301 aggaattaag gaactgggca gccagatcct gaaagagcac cccgtggaaa acacacagct
     3361 gcagaatgag aagctgtatc tgtactatct gcagaatgga cgcgatatgt acgtggacca
     3421 ggagctggat attaaccgac tgtccgatta cgacgtggat catatcgtcc cacagtcatt
     3481 cctgaaagat gacagcattg acaataaggt gctgacccgc tctgacaaaa accgaggcaa
     3541 gagtgataat gtcccctcag aggaagtggt caagaaaatg aagaactact ggaggcagct
     3601 gctgaatgcc aaactgatca cacagcgaaa gtttgataac ctgactaaag ctgagcgggg
     3661 aggcctgagt gaactggaca aagcaggctt cattaagcga cagctggtgg agacacggca
     3721 gatcacaaag cacgtcgccc agattctgga ttcaagaatg aacactaagt acgatgagaa
     3781 tgacaaactg atcagagaag tgaaggtcat taccctgaag tcaaaactgg tgagcgactt
     3841 tcggaaagat ttccagtttt ataaggtcag agagatcaac aactaccacc atgctcatga
     3901 cgcatacctg aacgcagtgg tcggcacagc cctgattaag aaatacccta aactggagtc
     3961 cgagttcgtg tacggggact ataaggtgta cgatgtcaga aaaatgatcg ccaagtctga
     4021 gcaggaaatt ggcaaagcca ctgctaagta tttcttttac agtaacatca tgaatttctt
     4081 taagactgag atcaccctgg caaatgggga aatccgaaag cggccactga ttgagactaa
     4141 cggcgagaca ggagaaatcg tgtgggacaa aggaagagat tttgctaccg tgaggaaggt
     4201 cctgagcatg ccccaagtga atattgtcaa gaaaacagag gtgcagactg ggggattcag
     4261 taaggaatca attctgccta aacgcaactc cgataagctg atcgcccgaa agaaagactg
     4321 ggaccccaag aagtatggcg ggttcgactc cccaactgtg gcttactctg tcctggtggt
     4381 cgcaaaggtg gagaagggaa aaagcaagaa actgaaatcc gtcaaggaac tgctgggcat
     4441 caccattatg gagcgcagct ccttcgaaaa gaatcctatc gattttctgg aggccaaagg
     4501 ctataaggaa gtgaagaaag acctgatcat caagctgcca aagtactcac tgtttgagct
     4561 ggaaaacggg agaaagagga tgctggcaag cgccggggag ctgcagaaag gaaatgaact
     4621 ggccctgccc tccaagtacg tgaacttcct gtatctggct agccactacg agaagctgaa
     4681 agggtcccct gaggataacg aacagaaaca gctgtttgtg gagcagcaca agcattatct
     4741 ggacgagatc attgaacaga ttagcgagtt ctccaaaaga gtgatcctgg ctgacgcaaa
     4801 tctggataag gtcctgagcg catacaacaa acaccgggat aagccaatca gagagcaggc
     4861 cgaaaatatc attcatctgt tcactctgac caacctggga gcccccgcag ccttcaagta
     4921 ttttgacact accatcgatc gcaaacgata cacaagcact aaggaggtgc tggacgctac
     4981 cctgattcat cagagcatta ctggcctgta tgaaacaagg attgacctgt ctcagctggg
     5041 cggcgactcc ggagctgacg gaagcgaaaa tcctggtaac aaaacaatag ttcctgcaac
     5101 gttaccacaa ctcaccccta ccctggtgtc actgttggag gttattgaac ctgaagtgtt
     5161 atatgcagga tatgatagct ctgttccaga ctcaacttgg aggatcatga ctacgctcaa
     5221 catgttagga gggcggcaag tgattgcagc agtgaaatgg gcaaaggcaa taccaggttt
     5281 caggaactta cacctggatg accaaatgac cctactgcag tactcctgga tgtttcttat
     5341 ggcatttgct ctggggtgga gatcatatag acaatcaagt gcaaacctgc tgtgttttgc
     5401 tcctgatctg attattaatg agcagagaat gactctaccc tgcatgtacg accaatgtaa
     5461 acacatgctg tatgtttcct ctgagttaca caggcttcag gtatcttatg aagagtatct
     5521 ctgtatgaaa accttactgc ttctctcttc agttcctaag gacggtctga agagccaaga
     5581 gctatttgat gaaattagaa tgacctacat caaagagcta ggaaaagcca ttgtcaagag
     5641 ggaaggaaac tccagccaga actggcagcg gttttatcaa ctgacaaaac tcttggattc
     5701 tatgcatgaa gtggttgaaa atctccttaa ctattgcttc caaacatttt tggataagac
     5761 catgtccatc gagttccccg agatgttagc tgaaatcatc accaatcaga taccaaaata
     5821 ttcaaatgga aatatcaaaa aacttctgtt tcatcaaaag tgagtctaga cccagctttc
     5881 ttgtacaaag tggtgatcta gctaaaacgc ggcctcgaat cgaattccgc ccccccccct
     5941 ctccctcccc cccccctaac gttactggcc gaagccgctt ggaataaggc cggtgtgcgt
     6001 ttgtctatat gttattttcc accatattgc cgtcttttgg caatgtgagg gcccggaaac
     6061 ctggccctgt cttcttgacg agcattccta ggggtctttc ccctctcgcc aaaggaatgc
     6121 aaggtctgtt gaatgtcgtg aaggaagcag ttcctctgga agcttcttga agacaaacaa
     6181 cgtctgtagc gaccctttgc aggcagcgga accccccacc tggcgacagg tgcctctgcg
     6241 gccaaaagcc acgtgtataa gatacacctg caaaggcggc acaaccccag tgccacgttg
     6301 tgagttggat agttgtggaa agagtcaaat ggctctcctc aagcgtattc aacaaggggc
     6361 tgaaggatgc ccagaaggta ccccattgta tgggatctga tctggggcct cggtgcacat
     6421 gctttacatg tgtttagtcg aggttaaaaa acgtctaggc cccccgaacc acggggacgt
     6481 ggttttcctt tgaaaaacac gatgataata tggccacaac catggtgagc aagggcgagg
     6541 aggataacat ggccatcatc aaggagttca tgcgcttcaa ggtgcacatg gagggctccg
     6601 tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac gagggcaccc
     6661 agaccgccaa gctgaaggtg accaagggtg gccccctgcc cttcgcctgg gacatcctgt
     6721 cccctcagtt catgtacggc tccaaggcct acgtgaagca ccccgccgac atccccgact
     6781 acttgaagct gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggacg
     6841 gcggcgtggt gaccgtgacc caggactcct ccctgcagga cggcgagttc atctacaagg
     6901 tgaagctgcg cggcaccaac ttcccctccg acggccccgt aatgcagaag aagaccatgg
     6961 gctgggaggc ctcctccgag cggatgtacc ccgaggacgg cgccctgaag ggcgagatca
     7021 agcagaggct gaagctgaag gacggcggcc actacgacgc tgaggtcaag accacctaca
     7081 aggccaagaa gcccgtgcag ctgcccggcg cctacaacgt caacatcaag ttggacatca
     7141 cctcccacaa cgaggactac accatcgtgg aacagtacga acgcgccgag ggccgccact
     7201 ccaccggcgg catggacgag ctgtacaagt aaagcgctct aggcttaagt atacctcgag
     7261 ttaaattcac tcctcaggtg caggctgcct atcagaaggt ggtggctggt gtggccaatg
     7321 ccctggctca caaataccac tgagatcttt ttccctctgc caaaaattat ggggacatca
     7381 tgaagcccct tgagcatctg acttctggct aataaaggaa atttattttc attgcaatag
     7441 tgtgttggaa ttttttgtgt ctctcactcg gaaggacata tgggagggca aatcatttaa
     7501 aacatcagaa tgagtatttg gtttagagtt tggcaacata tgcccatatg ctggctgcca
     7561 tgaacaaagg ttggctataa agaggtcatc agtatatgaa acagccccct gctgtccatt
     7621 ccttattcca tagaaaagcc ttgacttgag gttagatttt ttttatattt tgttttgtgt
     7681 tatttttttc tttaacatcc ctaaaatttt ccttacatgt tttactagcc agatttttcc
     7741 tcctctcctg actactccca gtcatagctg tccctcttct cttatggaga tccctcgact
     7801 taatcgctct agactagtgc ggccgctgca gaagctttgc aaagatggat aaagttttaa
     7861 acagagagga atctttgcag ctaatggacc ttctaggtct tgaaaggagt gggaattggc
     7921 tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa gttgggggga
     7981 ggggtcggca attgaaccgg tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat
     8041 gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat aagtgcagta
     8101 gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacaggt aagtgccgtg
     8161 tgtggttccc gcgggcctgg cctctttacg ggttatggcc cttgcgtgcc ttgaattact
     8221 tccacgcccc tggctgcagt acgtgattct tgatcccgag cttcgggttg gaagtgggtg
     8281 ggagagttcg aggccttgcg cttaaggagc cccttcgcct cgtgcttgag ttgaggcctg
     8341 gcctgggcgc tggggccgcc gcgtgcgaat ctggtggcac cttcgcgcct gtctcgctgc
     8401 tttcgataag tctctagcca tttaaaattt ttgatgacct gctgcgacgc tttttttctg
     8461 gcaagatagt cttgtaaatg cgggccaaga tctgcacact ggtatttcgg tttttggggc
     8521 cgcgggcggc gacggggccc gtgcgtccca gcgcacatgt tcggcgaggc ggggcctgcg
     8581 agcgcggcca ccgagaatcg gacgggggta gtctcaagct ggccggcctg ctctggtgcc
     8641 tggcctcgcg ccgccgtgta tcgccccgcc ctgggcggca aggctggccc ggtcggcacc
     8701 agttgcgtga gcggaaagat ggccgcttcc cggccctgct gcagggagct caaaatggag
     8761 gacgcggcgc tcgggagagc gggcgggtga gtcacccaca caaaggaaaa gggcctttcc
     8821 gtcctcagcc gtcgcttcat gtgactccac ggagtaccgg gcgccgtcca ggcacctcga
     8881 ttagttctcg agcttttgga gtacgtcgtc tttaggttgg ggggaggggt tttatgcgat
     8941 ggagtttccc cacactgagt gggtggagac tgaagttagg ccagcttggc acttgatgta
     9001 attctccttg gaatttgccc tttttgagtt tggatcttgg ttcattctca agcctcagac
     9061 agtggttcaa agtttttttc ttccatttca ggtgtcgtga gaattagcgg tggcggccat
     9121 cacaagtttg tacaaaaaag caggctccgc ggccgccccc ttcaccatgt ctagactgga
     9181 caagagcaaa gtcataaacg gcgctctgga attactcaat ggagtcggta tcgaaggcct
     9241 gacgacaagg aaactcgctc aaaagctggg agttgagcag cctaccctgt actggcacgt
     9301 gaagaacaag cgggccctgc tcgatgccct gccaatcgag atgctggaca ggcatcatac
     9361 ccacttctgc cccctggaag gcgagtcatg gcaagacttt ctgcggaaca acgccaagtc
     9421 attccgctgt gctctcctct cacatcgcga cggggctaaa gtgcatctcg gcacccgccc
     9481 aacagagaaa cagtacgaaa ccctggaaaa tcagctcgcg ttcctgtgtc agcaaggctt
     9541 ctccctggag aacgcactgt acgctctgtc cgccgtgggc cactttacac tgggctgcgt
     9601 attggaggaa caggagcatc aagtagcaaa agaggaaaga gagacaccta ccaccgattc
     9661 tatgccccca cttctgagac aagcaattga gctgttcgac cggcagggag ccgaacctgc
     9721 cttccttttc ggcctggaac taatcatatg tggcctggag aaacagctaa agtgcgaaag
     9781 cggcgggccg gccgacgccc ttgacgattt tgacttagac atgctcccag ccgatgccct
     9841 tgacgacttt gaccttgata tgctgcctgc tgacgctctt gacgattttg accttgacat
     9901 gctccccggg taactaagta aggatcccgt acgaagggtg ggcgcgccga cccagctttc
     9961 ttgtacaaag tggtgatcta gctaaaacgc ggcctcgaat cgaattctag gagcaggttt
    10021 ccccaatgac acaaaacgtg caacttgaaa ctccgcctgg tctttccagg tctagagggg
    10081 taacactttg tactgcgttt ggctccacgc tcgatccact ggcgagtgtt agtaacagca
    10141 ctgttgcttc gtagcggagc atgacggccg tgggaactcc tccttggtaa caaggaccca
    10201 cggggccaaa agccacgccc acacgggccc gtcatgtgtg caaccccagc acggcgactt
    10261 tactgcgaaa cccactttaa agtgacattg aaactggtac ccacacactg gtgacaggct
    10321 aaggatgccc ttcaggtacc ccgaggtaac acgcgacact cgggatctga gaaggggact
    10381 ggggcttcta taaaagcgct cggtttaaaa agcttctatg cctgaatagg tgaccggagg
    10441 tcggcacctt tcctttgcaa ttactgaccc tatgaataca actgactgtt tgacaattaa
    10501 tcatcggcat agtatatcgg catagtataa tacgactcac tataggaggg ccaccatgaa
    10561 gaaacctgaa ctgacagcaa cttctgttga gaagtttctc attgaaaaat ttgattctgt
    10621 ttctgatctc atgcagctgt ctgaaggtga agaaagcaga gccttttctt ttgatgttgg
    10681 aggaagaggt tatgttctga gggtcaattc ttgtgctgat ggtttttaca aagacagata
    10741 tgtttacaga cactttgcct ctgctgctct gccaattcca gaagttctgg acattggaga
    10801 attttctgaa tctctcacct actgcatcag cagaagagca caaggagtca ctctccagga
    10861 tctccctgaa actgagctgc cagctgttct gcaacctgtt gctgaagcaa tggatgccat
    10921 tgcagcagct gatctgagcc aaacctctgg atttggtcct tttggtcccc aaggcattgg
    10981 tcagtacacc acttggaggg atttcatttg tgccattgct gatcctcatg tctatcactg
    11041 gcagactgtg atggatgaca cagtttctgc ttctgttgct caggcactgg atgaactcat
    11101 gctgtgggca gaagattgtc ctgaagtcag acacctggtc catgctgatt ttggaagcaa
    11161 caatgttctg acagacaatg gcagaatcac tgcagtcatt gactggtctg aagccatgtt
    11221 tggagattct caatatgagg ttgccaacat ttttttttgg agaccttggc tggcttgcat
    11281 ggaacaacaa acaagatatt ttgaaagaag acacccagaa ctggctggtt cccccagact
    11341 gagagcctac atgctcagaa ttggcctgga ccaactgtat caatctctgg ttgatggaaa
    11401 ctttgatgat gctgcttggg cacaaggaag atgtgatgcc attgtgaggt ctggtgctgg
    11461 aactgttgga agaactcaaa ttgcaagaag gtctgctgct gtttggactg atggatgtgt
    11521 tgaagttctg gctgactctg gaaacaggag accctccaca agacccagag ccaaggaatg
    11581 aatattagct agattatccc taatacctgc caccccactc ttaatcagtg gtggaagaac
    11641 ggtctcagaa ctgtttgttt caattggcca tttaagttta gtagtaaaag actggttaat
    11701 gataacaatg catcgtaaaa ccttcagaag gaaaggagaa tgttttgtgg accactttgg
    11761 ttttcttttt tgcgtgtggc agttttaagt tattagtttt taaaatcagt actttttaat
    11821 ggaaacaact tgaccaaaaa tttgtcacag aattttgaga cccattaaaa aagttaaatg
    11881 agaaacctgt gtgttccttt ggtcatctat aacaagaaaa tatatatata ataagttatc
    11941 acgtaagtag aacatgaaat aacaatataa ttatcgtatg agttaaatct taaaagtcac
    12001 gtaaaagata atcatgcgtc attttgactc acgcggtcgt tatagttcaa aatcagtgac
    12061 acttaccgca ttgacaagca cgcctcacgg gagctccaag cggcgactga gatgtcctaa
    12121 atgcacagcg acggattcgc gctatttaga aagagagagc aatatttcaa gaatgcatgc
    12181 gtcaatttta cgcagactat ctttctaggg ttaatacgta taatacatat gattcagctg
    12241 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
    12301 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
    12361 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
    12421 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
    12481 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
    12541 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
    12601 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
    12661 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
    12721 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
    12781 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
    12841 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
    12901 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
    12961 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
    13021 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
    13081 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
    13141 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
    13201 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
    13261 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
    13321 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
    13381 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
    13441 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
    13501 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
    13561 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
    13621 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
    13681 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
    13741 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
    13801 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
    13861 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
    13921 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
    13981 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
    14041 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
    14101 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
    14161 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttcccc
//
