LOCUS       sequence-200491-       14246 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  piggyBac vector expressing Dual-regulated Cas9 (Puro resistance).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14246)
  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 14246)
  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-ERiP (CRONUS-Puro)
FEATURES             Location/Qualifiers
     source          1..14246
                     /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)"
     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"
     polyA_signal    7411..7466
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     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    join(10016..10590,10591..10593,10594..10602)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     CDS             10603..11202
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(10603..10622)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     11099..11119
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    11455..11510
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     repeat_region   12213..12247
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(12340..12357)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(12511..13099)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(12591..12610)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(13270..14061,14062..14130))
                     /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     13893..13912
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(14131..14235)
                     /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 cgaattccgc ccccccccct
    10021 ctccctcccc cccccctaac gttactggcc gaagccgctt ggaataaggc cggtgtgcgt
    10081 ttgtctatat gttattttcc accatattgc cgtcttttgg caatgtgagg gcccggaaac
    10141 ctggccctgt cttcttgacg agcattccta ggggtctttc ccctctcgcc aaaggaatgc
    10201 aaggtctgtt gaatgtcgtg aaggaagcag ttcctctgga agcttcttga agacaaacaa
    10261 cgtctgtagc gaccctttgc aggcagcgga accccccacc tggcgacagg tgcctctgcg
    10321 gccaaaagcc acgtgtataa gatacacctg caaaggcggc acaaccccag tgccacgttg
    10381 tgagttggat agttgtggaa agagtcaaat ggctctcctc aagcgtattc aacaaggggc
    10441 tgaaggatgc ccagaaggta ccccattgta tgggatctga tctggggcct cggtgcacat
    10501 gctttacatg tgtttagtcg aggttaaaaa aacgtctagg ccccccgaac cacggggacg
    10561 tggttttcct ttgaaaaaca cgatgataat atggccacaa ccatgaccga gtacaagccc
    10621 acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct cgccgccgcg
    10681 ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat cgagcgggtc
    10741 accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc
    10801 gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg
    10861 gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag
    10921 caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc
    10981 accgtcggcg tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc
    11041 ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc
    11101 aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa
    11161 ggaccgcgca cctggtgcat gacccgcaag cccggtgcct gacgcccgcc ccacgacccg
    11221 cagcgcccga ccgaaaggag cgcacgaccc catgcatcga ttcgaatgta cagatctgga
    11281 tcctaggctt aagtatacct cgagttaaat tcactcctca ggtgcaggct gcctatcaga
    11341 aggtggtggc tggtgtggcc aatgccctgg ctcacaaata ccactgagat ctttttccct
    11401 ctgccaaaaa ttatggggac atcatgaagc cccttgagca tctgacttct ggctaataaa
    11461 ggaaatttat tttcattgca atagtgtgtt ggaatttttt gtgtctctca ctcggaagga
    11521 catatgggag ggcaaatcat ttaaaacatc agaatgagta tttggtttag agtttggcaa
    11581 catatgccca tatgctggct gccatgaaca aaggttggct ataaagaggt catcagtata
    11641 tgaaacagcc ccctgctgtc cattccttat tccatagaaa agccttgact tgaggttaga
    11701 ttttttttat attttgtttt gtgttatttt tttctttaac atccctaaaa ttttccttac
    11761 atgttttact agccagattt ttcctcctct cctgactact cccagtcata gctgtccctc
    11821 ttctcttatg gagatccctc gacttaatta accaacaagc tcgtcatcgc tttgcagaag
    11881 agcagagagg atatgctcat cgtctaaaga actacccatt ttattatata ttagtcacga
    11941 tatctataac aagaaaatat atatataata agttatcacg taagtagaac atgaaataac
    12001 aatataatta tcgtatgagt taaatcttaa aagtcacgta aaagataatc atgcgtcatt
    12061 ttgactcacg cggtcgttat agttcaaaat cagtgacact taccgcattg acaagcacgc
    12121 ctcacgggag ctccaagcgg cgactgagat gtcctaaatg cacagcgacg gattcgcgct
    12181 atttagaaag agagagcaat atttcaagaa tgcatgcgtc aattttacgc agactatctt
    12241 tctagggtta atacgtataa tacatatgat tcagctgcat taatgaatcg gccaacgcgc
    12301 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
    12361 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
    12421 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
    12481 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
    12541 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
    12601 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
    12661 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
    12721 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
    12781 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
    12841 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
    12901 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
    12961 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
    13021 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
    13081 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
    13141 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
    13201 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
    13261 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
    13321 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
    13381 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
    13441 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
    13501 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
    13561 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
    13621 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
    13681 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
    13741 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
    13801 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
    13861 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
    13921 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
    13981 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
    14041 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
    14101 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
    14161 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
    14221 aataggggtt ccgcgcacat ttcccc
//
