LOCUS       sequence-354047-       11874 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac compatible plasmid expressing spCas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11874)
  AUTHORS   Nghi Dang , Alissa Lance-Byrne, Angela Tung, Xiaoge Guo, Ryan J 
            Cecchi, Joanna Buchthal, Alejandro Chavez, Nan Cher Yeo
  TITLE     Generation and application of a versatile CRISPR toolkit for 
            mammalian cell engineering
  JOURNAL   Synthetic Biology, Volume 6, Issue 1, 2021
REFERENCE   2  (bases 1 to 11874)
  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: "Synthetic 
            Biology, Volume 6, Issue 1, 2021"
COMMENT     Sequence Label: NAX_CAGGS_CAS9_Puro
FEATURES             Location/Qualifiers
     source          1..11874
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          117..1133
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1057..1079
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1141..1160
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1229..1238
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1235..5338
                     /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             1235..5338
                     /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             1235..5338
                     /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             5351..5371
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(5426..5445)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5470..5518
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     promoter        5750..5961
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             5977..6576
                     /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(5977..5996)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6473..6493
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    6595..6716
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6632..6651)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6686..6705
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   7479..7513
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(8004..8020)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8004..8020)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8017..8039)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8028..8044
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(8052..8058,8059..8076,8077..8082))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8097..8118
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(8235..8252)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8406..8994)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8486..8505)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9165..9956,9957..10025))
                     /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     9788..9807
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10026..10130)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(10156..10611)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10293..10314)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     10505..10524
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     10738..10760
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     10752..10769
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     10753..10769
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   10899..10961
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     enhancer        11382..11761
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt
       61 ttatggcgag gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag
      121 tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc
      181 ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg
      241 gctgtaatta gcgcttggtt taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc
      301 ttgaggggct ccgggagggc cctttgtgcg gggggagcgg ctcggggggt gcgtgcgtgt
      361 gtgtgtgcgt ggggagcgcc gcgtgcggct ccgcgctgcc cggcggctgt gagcgctgcg
      421 ggcgcggcgc ggggcttttt gcgctccgca gtgtgcgcga ggggagcgcg gccgggggcg
      481 gtgccccgcg gtgcgggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg
      541 tgggggggtg agcagggggt gtgggcgcgt cggtcgggct gcaacccccc ctgcaccccc
      601 ctccccgagt tgctgagcac ggcccggctt cgggtgcggg gctccgtacg gggcgtggcg
      661 cggggctcgc cgtgccgggc ggggggtggc ggcaggtggg ggtgccgggc ggggcggggc
      721 cgcctcgggc cggggagggc tcgggggagg ggcgcggcgg cccccggagc gccggcggct
      781 gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg
      841 gacttccttt gtcccaaatc tgtgcggagc cgaaatctgg gaggcgccgc cgcaccccct
      901 ctagcgggcg cggggcgaag cggtgcggcg ccggcaggaa ggaaatgggc ggggagggcc
      961 ttcgtgcgtc gccgcgccgc cgtccccttc tccctctcca gcctcggggc tgtccgcggg
     1021 gggacggctg ccttcggggg ggacggggca gggcggggtt cggcttctgg cgtgtgaccg
     1081 gcggctctag agcctctgct aaccatgttc atgccttctt ctttttccta cagctcctgg
     1141 gcaacgtgct ggttattgtg ctgtctcatc attttggcaa agaatctaga taccctcgta
     1201 aaggtctaga gctagcgaat tcgaatttgc caccatggac aagaagtact ccattgggct
     1261 cgatatcggc acaaacagcg tcggctgggc cgtcattacg gacgagtaca aggtgccgag
     1321 caaaaaattc aaagttctgg gcaataccga tcgccacagc ataaagaaga acctcattgg
     1381 cgccctcctg ttcgactccg gggagacggc cgaagccacg cggctcaaaa gaacagcacg
     1441 gcgcagatat acccgcagaa agaatcggat ctgctacctg caggagatct ttagtaatga
     1501 gatggctaag gtggatgact ctttcttcca taggctggag gagtcctttt tggtggagga
     1561 ggataaaaag cacgagcgcc acccaatctt tggcaatatc gtggacgagg tggcgtacca
     1621 tgaaaagtac ccaaccatat atcatctgag gaagaagctt gtagacagta ctgataaggc
     1681 tgacttgcgg ttgatctatc tcgcgctggc gcatatgatc aaatttcggg gacacttcct
     1741 catcgagggg gacctgaacc cagacaacag cgatgtcgac aaactcttta tccaactggt
     1801 tcagacttac aatcagcttt tcgaagagaa cccgatcaac gcatccggag ttgacgccaa
     1861 agcaatcctg agcgctaggc tgtccaaatc ccggcggctc gaaaacctca tcgcacagct
     1921 ccctggggag aagaagaacg gcctgtttgg taatcttatc gccctgtcac tcgggctgac
     1981 ccccaacttt aaatctaact tcgacctggc cgaagatgcc aagcttcaac tgagcaaaga
     2041 cacctacgat gatgatctcg acaatctgct ggcccagatc ggcgaccagt acgcagacct
     2101 ttttttggcg gcaaagaacc tgtcagacgc cattctgctg agtgatattc tgcgagtgaa
     2161 cacggagatc accaaagctc cgctgagcgc tagtatgatc aagcgctatg atgagcacca
     2221 ccaagacttg actttgctga aggcccttgt cagacagcaa ctgcctgaga agtacaagga
     2281 aattttcttc gatcagtcta aaaatggcta cgccggatac attgacggcg gagcaagcca
     2341 ggaggaattt tacaaattta ttaagcccat cttggaaaaa atggacggca ccgaggagct
     2401 gctggtaaag cttaacagag aagatctgtt gcgcaaacag cgcactttcg acaatggaag
     2461 catcccccac cagattcacc tgggcgaact gcacgctatc ctcaggcggc aagaggattt
     2521 ctaccccttt ttgaaagata acagggaaaa gattgagaaa atcctcacat ttcggatacc
     2581 ctactatgta ggccccctcg cccggggaaa ttccagattc gcgtggatga ctcgcaaatc
     2641 agaagagacc atcactccct ggaacttcga ggaagtcgtg gataaggggg cctctgccca
     2701 gtccttcatc gaaaggatga ctaactttga taaaaatctg cctaacgaaa aggtgcttcc
     2761 taaacactct ctgctgtacg agtacttcac agtttataac gagctcacca aggtcaaata
     2821 cgtcacagaa gggatgagaa agccagcatt cctgtctgga gagcagaaga aagctatcgt
     2881 ggacctcctc ttcaagacga accggaaagt taccgtgaaa cagctcaaag aagactattt
     2941 caaaaagatt gaatgtttcg actctgttga aatcagcgga gtggaggatc gcttcaacgc
     3001 atccctggga acgtatcacg atctcctgaa aatcattaaa gacaaggact tcctggacaa
     3061 tgaggagaac gaggacattc ttgaggacat tgtcctcacc cttacgttgt ttgaagatag
     3121 ggagatgatt gaagaacgct tgaaaactta cgctcatctc ttcgacgaca aagtcatgaa
     3181 acagctcaag aggcgccgat atacaggatg ggggcggctg tcaagaaaac tgatcaatgg
     3241 gatccgagac aagcagagtg gaaagacaat cctggatttt cttaagtccg atggatttgc
     3301 caaccggaac ttcatgcagt tgatccatga tgactctctc acctttaagg aggacatcca
     3361 gaaagcacaa gtttctggcc agggggacag tcttcacgag cacatcgcta atcttgcagg
     3421 tagcccagct atcaaaaagg gaatactgca gaccgttaag gtcgtggatg aactcgtcaa
     3481 agtaatggga aggcataagc ccgagaatat cgttatcgag atggcccgag agaaccaaac
     3541 tacccagaag ggacagaaga acagtaggga aaggatgaag aggattgaag agggtataaa
     3601 agaactgggg tcccaaatcc ttaaggaaca cccagttgaa aacacccagc ttcagaatga
     3661 gaagctctac ctgtactacc tgcagaacgg cagggacatg tacgtggatc aggaactgga
     3721 catcaatcgg ctctccgact acgacgtgga tcatatcgtg ccccagtctt ttctcaaaga
     3781 tgattctatt gataataaag tgttgacaag atccgataaa aatagaggga agagtgataa
     3841 cgtcccctca gaagaagttg tcaagaaaat gaaaaattat tggcggcagc tgctgaacgc
     3901 caaactgatc acacaacgga agttcgataa tctgactaag gctgaacgag gtggcctgtc
     3961 tgagttggat aaagccggct tcatcaaaag gcagcttgtt gagacacgcc agatcaccaa
     4021 gcacgtggcc caaattctcg attcacgcat gaacaccaag tacgatgaaa atgacaaact
     4081 gattcgagag gtgaaagtta ttactctgaa gtctaagctg gtctcagatt tcagaaagga
     4141 ctttcagttt tataaggtga gagagatcaa caattaccac catgcgcatg atgcctacct
     4201 gaatgcagtg gtaggcactg cacttatcaa aaaatatccc aagcttgaat ctgaatttgt
     4261 ttacggagac tataaagtgt acgatgttag gaaaatgatc gcaaagtctg agcaggaaat
     4321 aggcaaggcc accgctaagt acttctttta cagcaatatt atgaattttt tcaagaccga
     4381 gattacactg gccaatggag agattcggaa gcgaccactt atcgaaacaa acggagaaac
     4441 aggagaaatc gtgtgggaca agggtaggga tttcgcgaca gtccggaagg tcctgtccat
     4501 gccgcaggtg aacatcgtta aaaagaccga agtacagacc ggaggcttct ccaaggaaag
     4561 tatcctcccg aaaaggaaca gcgacaagct gatcgcacgc aaaaaagatt gggaccccaa
     4621 gaaatacggc ggattcgatt ctcctacagt cgcttacagt gtactggttg tggccaaagt
     4681 ggagaaaggg aagtctaaaa aactcaaaag cgtcaaggaa ctgctgggca tcacaatcat
     4741 ggagcgatca agcttcgaaa aaaaccccat cgactttctc gaggcgaaag gatataaaga
     4801 ggtcaaaaaa gacctcatca ttaagcttcc caagtactct ctctttgagc ttgaaaacgg
     4861 ccggaaacga atgctcgcta gtgcgggcga gctgcagaaa ggtaacgagc tggcactgcc
     4921 ctctaaatac gttaatttct tgtatctggc cagccactat gaaaagctca aagggtctcc
     4981 cgaagataat gagcagaagc agctgttcgt ggaacaacac aaacactacc ttgatgagat
     5041 catcgagcaa ataagcgaat tctccaaaag agtgatcctc gccgacgcta acctcgataa
     5101 ggtgctttct gcttacaata agcacaggga taagcccatc agggagcagg cagaaaacat
     5161 tatccacttg tttactctga ccaacttggg cgcgcctgca gccttcaagt acttcgacac
     5221 caccatagac agaaagcggt acacctctac aaaggaggtc ctggacgcca cactgattca
     5281 tcagtcaatt acggggctct atgaaacaag aatcgacctc tctcagctcg gtggagacag
     5341 cagggctgac cccaagaaga agaggaaggt gtgaaagggt tcgatcccta ccggttagta
     5401 atgagtttaa acgggggagg ctaactgaaa cacggaagga gacaataccg gaaggaaccc
     5461 gcgctatgac ggcaataaaa agacagaata aaacgcacgg gtgttgggtc gtttgttcat
     5521 aaacgcgggg ttcggtccca gggctggcac tctgtcgata ccccaccgag accccattgg
     5581 ggccaatacg cccgcgtttc ttccttttcc ccaccccacc ccccaagttc gggtgaaggc
     5641 ccagggctcg cagccaacgt cggggcggca ggccctgcca tagcagatct gcgaaatcgg
     5701 atccgcggcc gcactgattc tagctgcaga ggctccggtg cccgtcagtg ggcagagcgc
     5761 acatcgccca cagtccccga gaagttgggg ggaggggtcg gcaattgaac cggtgcctag
     5821 agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc
     5881 gagggtgggg gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac
     5941 gggtttgccg ccagaacaca ggatcctcga gccaccatga ccgagtacaa gcccacggtg
     6001 cgcctcgcca cccgcgacga cgtcccccgg gccgtacgca ccctcgccgc cgcgttcgcc
     6061 gactaccccg ccacgcgcca caccgtcgac ccggaccgcc acatcgagcg ggtcaccgag
     6121 ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac
     6181 gacggcgccg cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc
     6241 gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag
     6301 atggaaggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc
     6361 ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg
     6421 gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc
     6481 cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg
     6541 cgcacctggt gcatgacccg caagcccggt gcctgaatct agtgtcgaca agttaacttg
     6601 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     6661 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     6721 gtctggaatt gactcaaatg atgtcaatta gtctatcaga agctcatctg gtctcccttc
     6781 cgggggacaa gacatccctg tttaatattt aaacagcagt gttcccaaac tgggttctta
     6841 tatcccttgc tctggtcaac caggttgcag ggtttcctgt cctcacagga acgaagtccc
     6901 taaagaaaca gtggcagcca ggtttagccc cggaattgac tggattcctt ttttagggcc
     6961 cattggtatg gctttttccc cgtatccccc caggtgtctg caggctcaaa gagcagcgag
     7021 aagcgttcag aggaaagcga tcccgtgcca ccttccccgt gcccgggctg tccccgcacg
     7081 ctgccggctc ggggatgcgg ggggagcgcc ggaccggagc ggagccccgg gcggctcgct
     7141 gctgccccct agcgggggag ggacgtaatt acatccctgg gggctttggg ggggggctgt
     7201 ccctgatatc tataacaaga aaatatatat ataataagtt atcacgtaag tagaacatga
     7261 aataacaata taattatcgt atgagttaaa tcttaaaagt cacgtaaaag ataatcatgc
     7321 gtcattttga ctcacgcggt cgttatagtt caaaatcagt gacacttacc gcattgacaa
     7381 gcacgcctca cgggagctcc aagcggcgac tgagatgtcc taaatgcaca gcgacggatt
     7441 cgcgctattt agaaagagag agcaatattt caagaatgca tgcgtcaatt ttacgcagac
     7501 tatctttcta gggttaatct agctgcatca ggatcatatc gtcgggtctt ttttccggct
     7561 cagtcatcgc ccaagctggc gctatctggg catcggggag gaagaagccc gtgccttttc
     7621 ccgcgaggtt gaagcggcat ggaaagagtt tgccgaggat gactgctgct gcattgacgt
     7681 tgagcgaaaa cgcacgttta ccatgatgat tcgggaaggt gtggccatgc acgcctttaa
     7741 cggtgaactg ttcgttcagg ccacctggga taccagttcg tcgcggcttt tccggacaca
     7801 gttccggatg gtcagcccga agcgcatcag caacccgaac aataccggcg acagccggaa
     7861 ctgccgtgcc ggtgtgcaga ttaatgacag cggtgcggcg ctgggatatt acgtcagcga
     7921 ggacgggtat cctggctgga tgccgcagaa atggacatgg ataccccgtg agttacccgg
     7981 cgggcgcgct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     8041 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     8101 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     8161 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     8221 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     8281 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     8341 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     8401 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     8461 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     8521 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     8581 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     8641 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     8701 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     8761 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     8821 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     8881 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     8941 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     9001 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     9061 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     9121 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     9181 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     9241 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     9301 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     9361 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     9421 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     9481 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     9541 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     9601 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     9661 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     9721 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     9781 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     9841 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     9901 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     9961 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
    10021 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
    10081 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
    10141 cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt
    10201 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa
    10261 aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa
    10321 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac
    10381 gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga
    10441 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
    10501 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc
    10561 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc
    10621 cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc
    10681 agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc
    10741 agtcacgacg ttgtaaaacg acggccagtg agcgcgcctc gttcattcac gtttttgaac
    10801 ccgtggagga cgggcagact cgcggtgcaa atgtgtttta cagcgtgatg gagcagatga
    10861 agatgctcga cacgctgcag aacacgcagc tagattaacc ctagaaagat aatcatattg
    10921 tgacgtacgt taaagataat catgtgtaaa attgacgcat gtgttttatc ggtctgtata
    10981 tcgaggttta tttattaatt tgaatagata ttaagtttta ttatatttac acttacatac
    11041 taataataaa ttcaacaaac aatttattta tgtttattta tttattaaaa aaaacaaaaa
    11101 ctcaaaattt cttctataaa gtaacaaaac ttttatgagg gacagccccc ccccaaagcc
    11161 cccagggatg taattacgtc cctcccccgc tagggggcag cagcgagccg cccggggctc
    11221 cgctccggtc cggcgctccc cccgcatccc cgagccggca gcgtgcgggg acagcccggg
    11281 cacggggaag gtggcacggg atcgctttcc tctgaacgct tctcgctgct ctttgagcct
    11341 gcagacacct ggggggatac ggggaaaagg cctccacggc cgacattgat tattgactag
    11401 ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt
    11461 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
    11521 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg
    11581 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag
    11641 tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat
    11701 gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat
    11761 ggtcgaggtg agccccacgt tctgcttcac tctccccatc tcccccccct ccccaccccc
    11821 aattttgtat ttatttattt tttaattatt ttgtgcagcg atgggggcgg gggg
//
