LOCUS       sequence-207339-       14238 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 14238)
  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 14238)
  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..14238
                     /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    485..503
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    522..540
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    559..577
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    596..614
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    633..651
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    670..688
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    707..725
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    744..762
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     784..808
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    888..912
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             938..5041
                     /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             938..5041
                     /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             4954..4962
                     /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(5864..5888)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    join(5930..6504,6505..6507,6508..6516)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     CDS             6517..7227
                     /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(6662..6680)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     6930..6949
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    7406..7461
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     promoter        7913..9091
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          8144..9082
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     9039..9059
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    9114..9138
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             join(9159..9776,9777..9782,9783..9818,9819..9821,9822..9857,
                     9858..9860,9861..9896,9897..9905)
                     /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(9942..9966)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    join(10008..10582,10583..10585,10586..10594)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     CDS             10595..11194
                     /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(10595..10614)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     11091..11111
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    11447..11502
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     repeat_region   12205..12239
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(12332..12349)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(12503..13091)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(12583..12602)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(13262..14053,14054..14122))
                     /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     13885..13904
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(14123..14227)
                     /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 acaaaaactc aaaatttctt ctataaagta acaaaacttt taaacattct ctcttttaca
      301 aaaataaact tattttgtac tttaaaaaca gtcatgttgt attataaaat aagtaattag
      361 cttaacctat acataataga aacaaattat acttattagt cagtcagaaa caactttggc
      421 acatatcaat attatgctct gctagcgttt aaacccctcg agggatccaa gctggccgca
      481 cgtctcccta tcagtgatag agaagtcgac acgtctcgag ctccctatca gtgatagaga
      541 aggtacgtct agaacgtctc cctatcagtg atagagaagt cgacacgtct cgagctccct
      601 atcagtgata gagaaggtac gtctagaacg tctccctatc agtgatagag aagtcgacac
      661 gtctcgagct ccctatcagt gatagagaag gtacgtctag aacgtctccc tatcagtgat
      721 agagaagtcg acacgtctcg agctccctat cagtgataga gaaggtaccc cctatataag
      781 cagagctcgt ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct
      841 ccatagaaga caccgggacc gatccagcct ccgcggtggc ggccatcaca agtttgtaca
      901 aaaaagcagg ctggcgccgg aaccaattca gtcgaccatg gataagaaat acagcattgg
      961 actggacatt gggacaaact ccgtgggatg ggccgtgatt acagacgaat acaaagtgcc
     1021 ttcaaagaag ttcaaggtgc tgggcaacac cgatagacac agcatcaaga aaaatctgat
     1081 tggagccctg ctgttcgact ccggcgagac agctgaagca actcggctga aaagaactgc
     1141 tcggagaagg tatacccgcc gaaagaatag gatctgctac ctgcaggaga ttttcagcaa
     1201 cgaaatggcc aaggtggacg atagtttctt tcaccgcctg gaggaatcat tcctggtcga
     1261 ggaagataag aaacacgagc ggcatcccat ctttggcaac attgtggacg aggtcgctta
     1321 tcacgaaaag taccctacca tctatcatct gaggaagaaa ctggtggact ccacagataa
     1381 agcagacctg cgcctgatct atctggccct ggctcacatg attaagttcc ggggccattt
     1441 tctgatcgag ggggatctga acccagacaa ttctgatgtg gacaagctgt tcatccagct
     1501 ggtccagaca tacaatcagc tgtttgagga aaaccccatt aatgcatctg gcgtggacgc
     1561 aaaagccatc ctgagtgcca gactgtctaa gagtcggaga ctggagaacc tgatcgctca
     1621 gctgccaggg gaaaagaaaa acggcctgtt tgggaatctg attgcactgt cactgggact
     1681 gactcccaac ttcaagagca attttgatct ggccgaggac gctaaactgc agctgtccaa
     1741 ggacacctat gacgatgacc tggataacct gctggctcag atcggggatc agtacgcaga
     1801 cctgttcctg gccgctaaga atctgtctga cgccatcctg ctgagtgata ttctgcgcgt
     1861 gaacaccgag attacaaaag cccccctgtc agctagcatg atcaagagat atgacgagca
     1921 ccatcaggat ctgaccctgc tgaaggctct ggtgaggcag cagctgcctg agaagtacaa
     1981 ggaaatcttc tttgatcagt ctaagaacgg atacgccggc tatattgacg gcggggctag
     2041 tcaggaggag ttctacaagt ttatcaaacc cattctggag aagatggatg gcacagagga
     2101 actgctggtg aaactgaatc gggaagacct gctgaggaag cagcgcactt ttgataacgg
     2161 aagcatccct caccagattc atctgggaga gctgcacgca atcctgaggc gccaggaaga
     2221 cttctaccca tttctgaagg ataacaggga gaagatcgaa aaaattctga cattccgcat
     2281 cccctactat gtgggccctc tggcaagagg caacagccgg tttgcctgga tgactcgcaa
     2341 atctgaggaa acaatcactc cctggaactt cgaggaagtg gtcgataagg gcgcttccgc
     2401 acagtctttc attgagcgga tgacaaactt cgacaagaac ctgccaaacg aaaaagtgct
     2461 gcccaagcac tctctgctgt acgagtattt cacagtctat aacgaactga ctaaggtgaa
     2521 atacgtcacc gaggggatga gaaagcctgc cttcctgagt ggagaacaga agaaagctat
     2581 cgtggacctg ctgtttaaaa ccaataggaa ggtgacagtc aagcagctga aagaggacta
     2641 tttcaagaaa attgaatgtt tcgattctgt ggagatcagt ggcgtcgaag acaggtttaa
     2701 cgcctccctg gggacctacc acgatctgct gaagatcatt aaggataaag acttcctgga
     2761 caacgaggaa aatgaggata tcctggaaga cattgtgctg accctgacac tgtttgagga
     2821 tagggaaatg atcgaggaac gcctgaagac ctatgcccat ctgttcgatg acaaagtgat
     2881 gaaacagctg aagcgacgga gatacacagg atggggccga ctgtctcgga agctgatcaa
     2941 tgggattcgc gacaaacaga gtggaaagac catcctggac tttctgaaat cagatggctt
     3001 cgccaaccgg aacttcatgc agctgattca cgatgacagc ctgacattca aagaggatat
     3061 ccagaaggca caggtgtccg ggcagggaga ctctctgcac gagcatatcg caaacctggc
     3121 cggcagccct gccatcaaga aagggattct gcagaccgtg aaggtggtgg acgagctggt
     3181 gaaagtcatg ggaagacata agccagaaaa catcgtgatt gagatggcca gggaaaatca
     3241 gaccacacag aaaggccaga agaactcaag ggagcgcatg aaaagaatcg aggaaggaat
     3301 taaggaactg ggcagccaga tcctgaaaga gcaccccgtg gaaaacacac agctgcagaa
     3361 tgagaagctg tatctgtact atctgcagaa tggacgcgat atgtacgtgg accaggagct
     3421 ggatattaac cgactgtccg attacgacgt ggatcatatc gtcccacagt cattcctgaa
     3481 agatgacagc attgacaata aggtgctgac ccgctctgac aaaaaccgag gcaagagtga
     3541 taatgtcccc tcagaggaag tggtcaagaa aatgaagaac tactggaggc agctgctgaa
     3601 tgccaaactg atcacacagc gaaagtttga taacctgact aaagctgagc ggggaggcct
     3661 gagtgaactg gacaaagcag gcttcattaa gcgacagctg gtggagacac ggcagatcac
     3721 aaagcacgtc gcccagattc tggattcaag aatgaacact aagtacgatg agaatgacaa
     3781 actgatcaga gaagtgaagg tcattaccct gaagtcaaaa ctggtgagcg actttcggaa
     3841 agatttccag ttttataagg tcagagagat caacaactac caccatgctc atgacgcata
     3901 cctgaacgca gtggtcggca cagccctgat taagaaatac cctaaactgg agtccgagtt
     3961 cgtgtacggg gactataagg tgtacgatgt cagaaaaatg atcgccaagt ctgagcagga
     4021 aattggcaaa gccactgcta agtatttctt ttacagtaac atcatgaatt tctttaagac
     4081 tgagatcacc ctggcaaatg gggaaatccg aaagcggcca ctgattgaga ctaacggcga
     4141 gacaggagaa atcgtgtggg acaaaggaag agattttgct accgtgagga aggtcctgag
     4201 catgccccaa gtgaatattg tcaagaaaac agaggtgcag actgggggat tcagtaagga
     4261 atcaattctg cctaaacgca actccgataa gctgatcgcc cgaaagaaag actgggaccc
     4321 caagaagtat ggcgggttcg actccccaac tgtggcttac tctgtcctgg tggtcgcaaa
     4381 ggtggagaag ggaaaaagca agaaactgaa atccgtcaag gaactgctgg gcatcaccat
     4441 tatggagcgc agctccttcg aaaagaatcc tatcgatttt ctggaggcca aaggctataa
     4501 ggaagtgaag aaagacctga tcatcaagct gccaaagtac tcactgtttg agctggaaaa
     4561 cgggagaaag aggatgctgg caagcgccgg ggagctgcag aaaggaaatg aactggccct
     4621 gccctccaag tacgtgaact tcctgtatct ggctagccac tacgagaagc tgaaagggtc
     4681 ccctgaggat aacgaacaga aacagctgtt tgtggagcag cacaagcatt atctggacga
     4741 gatcattgaa cagattagcg agttctccaa aagagtgatc ctggctgacg caaatctgga
     4801 taaggtcctg agcgcataca acaaacaccg ggataagcca atcagagagc aggccgaaaa
     4861 tatcattcat ctgttcactc tgaccaacct gggagccccc gcagccttca agtattttga
     4921 cactaccatc gatcgcaaac gatacacaag cactaaggag gtgctggacg ctaccctgat
     4981 tcatcagagc attactggcc tgtatgaaac aaggattgac ctgtctcagc tgggcggcga
     5041 ctccggagct gacggaagcg aaaatcctgg taacaaaaca atagttcctg caacgttacc
     5101 acaactcacc cctaccctgg tgtcactgtt ggaggttatt gaacctgaag tgttatatgc
     5161 aggatatgat agctctgttc cagactcaac ttggaggatc atgactacgc tcaacatgtt
     5221 aggagggcgg caagtgattg cagcagtgaa atgggcaaag gcaataccag gtttcaggaa
     5281 cttacacctg gatgaccaaa tgaccctact gcagtactcc tggatgtttc ttatggcatt
     5341 tgctctgggg tggagatcat atagacaatc aagtgcaaac ctgctgtgtt ttgctcctga
     5401 tctgattatt aatgagcaga gaatgactct accctgcatg tacgaccaat gtaaacacat
     5461 gctgtatgtt tcctctgagt tacacaggct tcaggtatct tatgaagagt atctctgtat
     5521 gaaaacctta ctgcttctct cttcagttcc taaggacggt ctgaagagcc aagagctatt
     5581 tgatgaaatt agaatgacct acatcaaaga gctaggaaaa gccattgtca agagggaagg
     5641 aaactccagc cagaactggc agcggtttta tcaactgaca aaactcttgg attctatgca
     5701 tgaagtggtt gaaaatctcc ttaactattg cttccaaaca tttttggata agaccatgtc
     5761 catcgagttc cccgagatgt tagctgaaat catcaccaat cagataccaa aatattcaaa
     5821 tggaaatatc aaaaaacttc tgtttcatca aaagtgagtc tagacccagc tttcttgtac
     5881 aaagtggtga tctagctaaa acgcggcctc gaatcgaatt ccgccccccc ccctctccct
     5941 cccccccccc taacgttact ggccgaagcc gcttggaata aggccggtgt gcgtttgtct
     6001 atatgttatt ttccaccata ttgccgtctt ttggcaatgt gagggcccgg aaacctggcc
     6061 ctgtcttctt gacgagcatt cctaggggtc tttcccctct cgccaaagga atgcaaggtc
     6121 tgttgaatgt cgtgaaggaa gcagttcctc tggaagcttc ttgaagacaa acaacgtctg
     6181 tagcgaccct ttgcaggcag cggaaccccc cacctggcga caggtgcctc tgcggccaaa
     6241 agccacgtgt ataagataca cctgcaaagg cggcacaacc ccagtgccac gttgtgagtt
     6301 ggatagttgt ggaaagagtc aaatggctct cctcaagcgt attcaacaag gggctgaagg
     6361 atgcccagaa ggtaccccat tgtatgggat ctgatctggg gcctcggtgc acatgcttta
     6421 catgtgttta gtcgaggtta aaaaaacgtc taggcccccc gaaccacggg gacgtggttt
     6481 tcctttgaaa aacacgatga taatatggcc acaaccatgg tgagcaaggg cgaggaggat
     6541 aacatggcca tcatcaagga gttcatgcgc ttcaaggtgc acatggaggg ctccgtgaac
     6601 ggccacgagt tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc
     6661 gccaagctga aggtgaccaa gggtggcccc ctgcccttcg cctgggacat cctgtcccct
     6721 cagttcatgt acggctccaa ggcctacgtg aagcaccccg ccgacatccc cgactacttg
     6781 aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc
     6841 gtggtgaccg tgacccagga ctcctccctg caggacggcg agttcatcta caaggtgaag
     6901 ctgcgcggca ccaacttccc ctccgacggc cccgtaatgc agaagaagac catgggctgg
     6961 gaggcctcct ccgagcggat gtaccccgag gacggcgccc tgaagggcga gatcaagcag
     7021 aggctgaagc tgaaggacgg cggccactac gacgctgagg tcaagaccac ctacaaggcc
     7081 aagaagcccg tgcagctgcc cggcgcctac aacgtcaaca tcaagttgga catcacctcc
     7141 cacaacgagg actacaccat cgtggaacag tacgaacgcg ccgagggccg ccactccacc
     7201 ggcggcatgg acgagctgta caagtaaagc gctctaggct taagtatacc tcgagttaaa
     7261 ttcactcctc aggtgcaggc tgcctatcag aaggtggtgg ctggtgtggc caatgccctg
     7321 gctcacaaat accactgaga tctttttccc tctgccaaaa attatgggga catcatgaag
     7381 ccccttgagc atctgacttc tggctaataa aggaaattta ttttcattgc aatagtgtgt
     7441 tggaattttt tgtgtctctc actcggaagg acatatggga gggcaaatca tttaaaacat
     7501 cagaatgagt atttggttta gagtttggca acatatgccc atatgctggc tgccatgaac
     7561 aaaggttggc tataaagagg tcatcagtat atgaaacagc cccctgctgt ccattcctta
     7621 ttccatagaa aagccttgac ttgaggttag atttttttta tattttgttt tgtgttattt
     7681 ttttctttaa catccctaaa attttcctta catgttttac tagccagatt tttcctcctc
     7741 tcctgactac tcccagtcat agctgtccct cttctcttat ggagatccct cgacttaatc
     7801 gctctagact agtgcggccg ctgcagaagc tttgcaaaga tggataaagt tttaaacaga
     7861 gaggaatctt tgcagctaat ggaccttcta ggtcttgaaa ggagtgggaa ttggctccgg
     7921 tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc gagaagttgg ggggaggggt
     7981 cggcaattga accggtgcct agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt
     8041 gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg cagtagtcgc
     8101 cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggtaagtg ccgtgtgtgg
     8161 ttcccgcggg cctggcctct ttacgggtta tggcccttgc gtgccttgaa ttacttccac
     8221 ctggctgcag tacgtgattc ttgatcccga gcttcgggtt ggaagtgggt gggagagttc
     8281 gaggccttgc gcttaaggag ccccttcgcc tcgtgcttga gttgaggcct ggcctgggcg
     8341 ctggggccgc cgcgtgcgaa tctggtggca ccttcgcgcc tgtctcgctg ctttcgataa
     8401 gtctctagcc atttaaaatt tttgatgacc tgctgcgacg ctttttttct ggcaagatag
     8461 tcttgtaaat gcgggccaag atctgcacac tggtatttcg gtttttgggg ccgcgggcgg
     8521 cgacggggcc cgtgcgtccc agcgcacatg ttcggcgagg cggggcctgc gagcgcggcc
     8581 accgagaatc ggacgggggt agtctcaagc tggccggcct gctctggtgc ctggcctcgc
     8641 gccgccgtgt atcgccccgc cctgggcggc aaggctggcc cggtcggcac cagttgcgtg
     8701 agcggaaaga tggccgcttc ccggccctgc tgcagggagc tcaaaatgga ggacgcggcg
     8761 ctcgggagag cgggcgggtg agtcacccac acaaaggaaa agggcctttc cgtcctcagc
     8821 cgtcgcttca tgtgactcca cggagtaccg ggcgccgtcc aggcacctcg attagttctc
     8881 gagcttttgg agtacgtcgt ctttaggttg gggggagggg ttttatgcga tggagtttcc
     8941 ccacactgag tgggtggaga ctgaagttag gccagcttgg cacttgatgt aattctcctt
     9001 ggaatttgcc ctttttgagt ttggatcttg gttcattctc aagcctcaga cagtggttca
     9061 aagttttttt cttccatttc aggtgtcgtg aggaattagc ggtggcggcc atcacaagtt
     9121 tgtacaaaaa agcaggctcc gcggccgccc ccttcaccat gtctagactg gacaagagca
     9181 aagtcataaa cggcgctctg gaattactca atggagtcgg tatcgaaggc ctgacgacaa
     9241 ggaaactcgc tcaaaagctg ggagttgagc agcctaccct gtactggcac gtgaagaaca
     9301 agcgggccct gctcgatgcc ctgccaatcg agatgctgga caggcatcat acccacttct
     9361 gccccctgga aggcgagtca tggcaagact ttctgcggaa caacgccaag tcattccgct
     9421 gtgctctcct ctcacatcgc gacggggcta aagtgcatct cggcacccgc ccaacagaga
     9481 aacagtacga aaccctggaa aatcagctcg cgttcctgtg tcagcaaggc ttctccctgg
     9541 agaacgcact gtacgctctg tccgccgtgg gccactttac actgggctgc gtattggagg
     9601 aacaggagca tcaagtagca aaagaggaaa gagagacacc taccaccgat tctatgcccc
     9661 cacttctgag acaagcaatt gagctgttcg accggcaggg agccgaacct gccttccttt
     9721 tcggcctgga actaatcata tgtggcctgg agaaacagct aaagtgcgaa agcggcgggc
     9781 cggccgacgc ccttgacgat tttgacttag acatgctccc agccgatgcc cttgacgact
     9841 ttgaccttga tatgctgcct gctgacgctc ttgacgattt tgaccttgac atgctccccg
     9901 ggtaactaag taaggatccc gtacgaaggg tgggcgcgcc gacccagctt tcttgtacaa
     9961 agtggtgatc tagctaaaac gcggcctcga atcgaattcc gccccccccc ctctccctcc
    10021 ccccccccta acgttactgg ccgaagccgc ttggaataag gccggtgtgc gtttgtctat
    10081 atgttatttt ccaccatatt gccgtctttt ggcaatgtga gggcccggaa acctggccct
    10141 gtcttcttga cgagcattcc taggggtctt tcccctctcg ccaaaggaat gcaaggtctg
    10201 ttgaatgtcg tgaaggaagc agttcctctg gaagcttctt gaagacaaac aacgtctgta
    10261 gcgacccttt gcaggcagcg gaacccccca cctggcgaca ggtgcctctg cggccaaaag
    10321 ccacgtgtat aagatacacc tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg
    10381 atagttgtgg aaagagtcaa atggctctcc tcaagcgtat tcaacaaggg gctgaaggat
    10441 gcccagaagg taccccattg tatgggatct gatctggggc ctcggtgcac atgctttaca
    10501 tgtgtttagt cgaggttaaa aaaacgtcta ggccccccga accacgggga cgtggttttc
    10561 ctttgaaaaa cacgatgata atatggccac aaccatgacc gagtacaagc ccacggtgcg
    10621 cctcgccacc cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg cgttcgccga
    10681 ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct
    10741 gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga
    10801 cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc
    10861 cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat
    10921 ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg
    10981 cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga
    11041 ggcggccgag cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc gcaacctccc
    11101 cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg
    11161 cacctggtgc atgacccgca agcccggtgc ctgacgcccg ccccacgacc cgcagcgccc
    11221 gaccgaaagg agcgcacgac cccatgcatc gattcgaatg tacagatctg gatcctaggc
    11281 ttaagtatac ctcgagttaa attcactcct caggtgcagg ctgcctatca gaaggtggtg
    11341 gctggtgtgg ccaatgccct ggctcacaaa taccactgag atctttttcc ctctgccaaa
    11401 aattatgggg acatcatgaa gccccttgag catctgactt ctggctaata aaggaaattt
    11461 attttcattg caatagtgtg ttggaatttt ttgtgtctct cactcggaag gacatatggg
    11521 agggcaaatc atttaaaaca tcagaatgag tatttggttt agagtttggc aacatatgcc
    11581 catatgctgg ctgccatgaa caaaggttgg ctataaagag gtcatcagta tatgaaacag
    11641 ccccctgctg tccattcctt attccataga aaagccttga cttgaggtta gatttttttt
    11701 atattttgtt ttgtgttatt tttttcttta acatccctaa aattttcctt acatgtttta
    11761 ctagccagat ttttcctcct ctcctgacta ctcccagtca tagctgtccc tcttctctta
    11821 tggagatccc tcgacttaat taaccaacaa gctcgtcatc gctttgcaga agagcagaga
    11881 ggatatgctc atcgtctaaa gaactaccca ttttattata tattagtcac gatatctata
    11941 acaagaaaat atatatataa taagttatca cgtaagtaga acatgaaata acaatataat
    12001 tatcgtatga gttaaatctt aaaagtcacg taaaagataa tcatgcgtca ttttgactca
    12061 cgcggtcgtt atagttcaaa atcagtgaca cttaccgcat tgacaagcac gcctcacggg
    12121 agctccaagc ggcgactgag atgtcctaaa tgcacagcga cggattcgcg ctatttagaa
    12181 agagagagca atatttcaag aatgcatgcg tcaattttac gcagactatc tttctagggt
    12241 taatacgtat aatacatatg attcagctgc attaatgaat cggccaacgc gcggggagag
    12301 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
    12361 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
    12421 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
    12481 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
    12541 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
    12601 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
    12661 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
    12721 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
    12781 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
    12841 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
    12901 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
    12961 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
    13021 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
    13081 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
    13141 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
    13201 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
    13261 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
    13321 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
    13381 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
    13441 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
    13501 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
    13561 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
    13621 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
    13681 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
    13741 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
    13801 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
    13861 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
    13921 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
    13981 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
    14041 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
    14101 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
    14161 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
    14221 ttccgcgcac atttcccc
//
