LOCUS       sequence-237238-       13258 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  piggyBac transposon Destination vector for dox-inducible expression 
            of Gateway cloned elements (inducible mCherry). SpCas9 is 
            constitutively expressed..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13258)
  AUTHORS   Kagawa H, Shimamoto R, Kim SI, Oceguera-Yanez F, Yamamoto T, 
            Schroeder T, Woltjen K
  TITLE     OVOL1 Influences the Determination and Expansion of iPSC 
            Reprogramming Intermediates.
  JOURNAL   Stem Cell Reports. 2018 Dec 28. pii: S2213-6711(18)30527-7. doi: 
            10.1016/j.stemcr.2018.12.008.
  PUBMED    30639212
REFERENCE   2  (bases 1 to 13258)
  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: "Stem Cell 
            Reports. 2018 Dec 28. pii: S2213-6711(18)30527-7. doi: 
            10.1016/j.stemcr.2018.12.008."
COMMENT     Sequence Label: PB-TAC-Cas9
FEATURES             Location/Qualifiers
     source          1..13258
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(28..456)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(138..159)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     350..369
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     583..605
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     597..614
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     598..614
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     621..640
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        621..639
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   706..768
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    1140..1158
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1177..1195
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1214..1232
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1251..1269
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1288..1306
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1325..1343
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1362..1380
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1399..1417
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1439..1463
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    1543..1667
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(1692..1697,1698..1715,1716..1722)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             1776..2435
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1842..1861)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             2777..3082
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     2961..2980
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(3123..3247)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     misc_feature    join(3289..3863,3864..3866,3867..3875)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3456..3473)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3683..3702
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3876..4586
                     /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(4021..4039)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4289..4308
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(4598..4617)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4663..4718
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4717..4736)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     enhancer        5155..5440
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        5442..5719
                     /label=chicken beta-actin promoter
     intron          5720..5947
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      5959..5968
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5968..6033
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             6040..6060
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6085..10185
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             6085..10185
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             10186..10233
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(10261..10278)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    10267..10474
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   10885..10919
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        complement(11068..11086)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(11107..11123)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11107..11123)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(11120..11142)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    11131..11147
                     /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(11155..11161,11162..11179,11180..11185))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11200..11221
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(11338..11355)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11509..12097)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11589..11608)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(12268..13059,13060..13128))
                     /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     12891..12910
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(13129..13233)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat
       61 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga
      121 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
      181 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct
      241 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
      301 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
      361 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
      421 cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc aggctgcgca
      481 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg
      541 gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta
      601 aaacgacggc cagtgaattg taatacgact cactataggg cgaattggag ctcggtattc
      661 acgacagcag gctgaataat aaaaaaatta gaaactatta tttaacccta gaaagataat
      721 catattgtga cgtacgttaa agataatcat gcgtaaaatt gacgcatgtg ttttatcggt
      781 ctgtatatcg aggtttattt attaatttga atagatatta agttttatta tatttacact
      841 tacatactaa taataaattc aacaaacaat ttatttatgt ttatttattt attaaaaaaa
      901 aacaaaaact caaaatttct tctataaagt aacaaaactt ttaaacattc tctcttttac
      961 aaaaataaac ttattttgta ctttaaaaac agtcatgttg tattataaaa taagtaatta
     1021 gcttaaccta tacataatag aaacaaatta tacttattag tcagtcagaa acaactttgg
     1081 cacatatcaa tattatgctc tcgttaatta actcgaggga tccaagctgg ccgcacgtct
     1141 ccctatcagt gatagagaag tcgacacgtc tcgagctccc tatcagtgat agagaaggta
     1201 cgtctagaac gtctccctat cagtgataga gaagtcgaca cgtctcgagc tccctatcag
     1261 tgatagagaa ggtacgtcta gaacgtctcc ctatcagtga tagagaagtc gacacgtctc
     1321 gagctcccta tcagtgatag agaaggtacg tctagaacgt ctccctatca gtgatagaga
     1381 agtcgacacg tctcgagctc cctatcagtg atagagaagg taccccctat ataagcagag
     1441 ctcgtttagt gaaccgtcag atcgcctgga gacgccatcc acgctgtttt gacctccata
     1501 gaagacaccg ggaccgatcc agcctccgcg gtggcggcca tcacaagttt gtacaaaaaa
     1561 gctgaacgag aaacgtaaaa tgatataaat atcaatatat taaattagat tttgcataaa
     1621 aaacagacta cataatactg taaaacacaa catatccagt cactatggcg gccgcattag
     1681 gcaccccagg ctttacactt tatgcttccg gctcgtataa tgtgtggatt ttgagttagg
     1741 atccgtcgag attttcagga gctaaggaag ctaaaatgga gaaaaaaatc actggatata
     1801 ccaccgttga tatatcccaa tggcatcgta aagaacattt tgaggcattt cagtcagttg
     1861 ctcaatgtac ctataaccag accgttcagc tggatattac ggccttttta aagaccgtaa
     1921 agaaaaataa gcacaagttt tatccggcct ttattcacat tcttgcccgc ctgatgaatg
     1981 ctcatccgga attccgtatg gcaatgaaag acggtgagct ggtgatatgg gatagtgttc
     2041 acccttgtta caccgttttc catgagcaaa ctgaaacgtt ttcatcgctc tggagtgaat
     2101 accacgacga tttccggcag tttctacaca tatattcgca agatgtggcg tgttacggtg
     2161 aaaacctggc ctatttccct aaagggttta ttgagaatat gtttttcgtc tcagccaatc
     2221 cctgggtgag tttcaccagt tttgatttaa acgtggccaa tatggacaac ttcttcgccc
     2281 ccgttttcac catgggcaaa tattatacgc aaggcgacaa ggtgctgatg ccgctggcga
     2341 ttcaggttca tcatgccgtt tgtgatggct tccatgtcgg cagaatgctt aatgaattac
     2401 aacagtactg cgatgagtgg cagggcgggg cgtaaacgcg tggatccggc ttactaaaag
     2461 ccagataaca gtatgcgtat ttgcgcgctg atttttgcgg tataagaata tatactgata
     2521 tgtatacccg aagtatgtca aaaagaggta tgctatgaag cagcgtatta cagtgacagt
     2581 tgacagcgac agctatcagt tgctcaaggc atatatgatg tcaatatctc cggtctggta
     2641 agcacaacca tgcagaatga agcccgtcgt ctgcgtgccg aacgctggaa agcggaaaat
     2701 caggaaggga tggctgaggt cgcccggttt attgaaatga acggctcttt tgctgacgag
     2761 aacaggggct ggtgaaatgc agtttaaggt ttacacctat aaaagagaga gccgttatcg
     2821 tctgtttgtg gatgtacaga gtgatattat tgacacgccc gggcgacgga tggtgatccc
     2881 cctggccagt gcacgtctgc tgtcagataa agtctcccgt gaactttacc cggtggtgca
     2941 tatcggggat gaaagctggc gcatgatgac caccgatatg gccagtgtgc cggtctccgt
     3001 tatcggggaa gaagtggctg atctcagcca ccgcgaaaat gacatcaaaa acgccattaa
     3061 cctgatgttc tggggaatat aaatgtcagg ctcccttata cacagccagt ctgcaggtcg
     3121 accatagtga ctggatatgt tgtgttttac agtattatgt agtctgtttt ttatgcaaaa
     3181 tctaatttaa tatattgata tttatatcat tttacgtttc tcgttcagct ttcttgtaca
     3241 aagtggtgat ctagctaaaa cgcggcctcg aatcgaattc cgcccccccc cctctccctc
     3301 ccccccccct aacgttactg gccgaagccg cttggaataa ggccggtgtg cgtttgtcta
     3361 tatgttattt tccaccatat tgccgtcttt tggcaatgtg agggcccgga aacctggccc
     3421 tgtcttcttg acgagcattc ctaggggtct ttcccctctc gccaaaggaa tgcaaggtct
     3481 gttgaatgtc gtgaaggaag cagttcctct ggaagcttct tgaagacaaa caacgtctgt
     3541 agcgaccctt tgcaggcagc ggaacccccc acctggcgac aggtgcctct gcggccaaaa
     3601 gccacgtgta taagatacac ctgcaaaggc ggcacaaccc cagtgccacg ttgtgagttg
     3661 gatagttgtg gaaagagtca aatggctctc ctcaagcgta ttcaacaagg ggctgaagga
     3721 tgcccagaag gtaccccatt gtatgggatc tgatctgggg cctcggtgca catgctttac
     3781 atgtgtttag tcgaggttaa aaaaacgtct aggccccccg aaccacgggg acgtggtttt
     3841 cctttgaaaa acacgatgat aatatggcca caaccatggt gagcaagggc gaggaggata
     3901 acatggccat catcaaggag ttcatgcgct tcaaggtgca catggagggc tccgtgaacg
     3961 gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc acccagaccg
     4021 ccaagctgaa ggtgaccaag ggtggccccc tgcccttcgc ctgggacatc ctgtcccctc
     4081 agttcatgta cggctccaag gcctacgtga agcaccccgc cgacatcccc gactacttga
     4141 agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg
     4201 tggtgaccgt gacccaggac tcctccctgc aggacggcga gttcatctac aaggtgaagc
     4261 tgcgcggcac caacttcccc tccgacggcc ccgtaatgca gaagaagacc atgggctggg
     4321 aggcctcctc cgagcggatg taccccgagg acggcgccct gaagggcgag atcaagcaga
     4381 ggctgaagct gaaggacggc ggccactacg acgctgaggt caagaccacc tacaaggcca
     4441 agaagcccgt gcagctgccc ggcgcctaca acgtcaacat caagttggac atcacctccc
     4501 acaacgagga ctacaccatc gtggaacagt acgaacgcgc cgagggccgc cactccaccg
     4561 gcggcatgga cgagctgtac aagtaaagcg gccgcgatct ttttccctct gccaaaaatt
     4621 atggggacat catgaagccc cttgagcatc tgacttctgg ctaataaagg aaatttattt
     4681 tcattgcaat agtgtgttgg aattttttgt gtctctcact cggaaggaca tatgggaggg
     4741 caaatcattt aaaacatcag aatgagtatt tggtttagag tttggcaaca tatgcccata
     4801 tgctggctgc catgaacaaa ggttggctat aaagaggtca tcagtatatg aaacagcccc
     4861 ctgctgtcca ttccttattc catagaaaag ccttgacttg aggttagatt ttttttatat
     4921 tttgttttgt gttatttttt tctttaacat ccctaaaatt ttccttacat gttttactag
     4981 ccagattttt cctcctctcc tgactactcc cagtcatagc tgtccctctt ctcttatgga
     5041 gatccctcga cctgcaggaa ttcgattact taagcctagg atccagatct gtacattcga
     5101 atcgatgaat tcgttaacca tggaagcttc tgcagcggcc gcactagagg tacccgttac
     5161 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc
     5221 aatagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac
     5281 tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa
     5341 tgacggtaaa tggcccgcct ggcattgtgc ccagtacatg accttatggg actttcctac
     5401 ttggcagtac atctacgtat tagtcatcgc tattaccatg gtcgaggtga gccccacgtt
     5461 ctgcttcact ctccccatct cccccccctc cccaccccca attttgtatt tatttatttt
     5521 ttaattattt tgtgcagcga tgggggcggg gggggggggg gggcgcgcgc crggsggggs
     5581 ggggsggggs grggggsggg gsggggcgag gcggagaggt gcggcggcag ccaatcagag
     5641 cggcgcgctc cgaaagtttc cttttatggc gaggcggcgg cggcggcggc cctataaaaa
     5701 gcgaagcgcg cggcgggcgg gagtcgctgc gcgctgcctt cgccccgtgc cccgctccgc
     5761 cgccgcctcg cgccgcccgc cccggctctg actgaccgcg ttactcccac aggtgagcgg
     5821 gcgggacggc ccttctcctc cgggctgtaa ttagctgagc aagaggtaag ggtttaaggg
     5881 atggttggtt ggtggggtat taatgtttaa ttacctggag cacctgcctg aaatcacttt
     5941 ttttcaggtt ggaccggtgc caccatggac tataaggacc acgacggaga ctacaaggat
     6001 catgatattg attacaaaga cgatgacgat aagatggccc caaagaagaa gcggaaggtc
     6061 ggtatccacg gagtcccagc agccgacaag aagtacagca tcggcctgga catcggcacc
     6121 aactctgtgg gctgggccgt gatcaccgac gagtacaagg tgcccagcaa gaaattcaag
     6181 gtgctgggca acaccgaccg gcacagcatc aagaagaacc tgatcggagc cctgctgttc
     6241 gacagcggcg aaacagccga ggccacccgg ctgaagagaa ccgccagaag aagatacacc
     6301 agacggaaga accggatctg ctatctgcaa gagatcttca gcaacgagat ggccaaggtg
     6361 gacgacagct tcttccacag actggaagag tccttcctgg tggaagagga taagaagcac
     6421 gagcggcacc ccatcttcgg caacatcgtg gacgaggtgg cctaccacga gaagtacccc
     6481 accatctacc acctgagaaa gaaactggtg gacagcaccg acaaggccga cctgcggctg
     6541 atctatctgg ccctggccca catgatcaag ttccggggcc acttcctgat cgagggcgac
     6601 ctgaaccccg acaacagcga cgtggacaag ctgttcatcc agctggtgca gacctacaac
     6661 cagctgttcg aggaaaaccc catcaacgcc agcggcgtgg acgccaaggc catcctgtct
     6721 gccagactga gcaagagcag acggctggaa aatctgatcg cccagctgcc cggcgagaag
     6781 aagaatggcc tgttcggaaa cctgattgcc ctgagcctgg gcctgacccc caacttcaag
     6841 agcaacttcg acctggccga ggatgccaaa ctgcagctga gcaaggacac ctacgacgac
     6901 gacctggaca acctgctggc ccagatcggc gaccagtacg ccgacctgtt tctggccgcc
     6961 aagaacctgt ccgacgccat cctgctgagc gacatcctga gagtgaacac cgagatcacc
     7021 aaggcccccc tgagcgcctc tatgatcaag agatacgacg agcaccacca ggacctgacc
     7081 ctgctgaaag ctctcgtgcg gcagcagctg cctgagaagt acaaagagat tttcttcgac
     7141 cagagcaaga acggctacgc cggctacatt gacggcggag ccagccagga agagttctac
     7201 aagttcatca agcccatcct ggaaaagatg gacggcaccg aggaactgct cgtgaagctg
     7261 aacagagagg acctgctgcg gaagcagcgg accttcgaca acggcagcat cccccaccag
     7321 atccacctgg gagagctgca cgccattctg cggcggcagg aagattttta cccattcctg
     7381 aaggacaacc gggaaaagat cgagaagatc ctgaccttcc gcatccccta ctacgtgggc
     7441 cctctggcca ggggaaacag cagattcgcc tggatgacca gaaagagcga ggaaaccatc
     7501 accccctgga acttcgagga agtggtggac aagggcgctt ccgcccagag cttcatcgag
     7561 cggatgacca acttcgataa gaacctgccc aacgagaagg tgctgcccaa gcacagcctg
     7621 ctgtacgagt acttcaccgt gtataacgag ctgaccaaag tgaaatacgt gaccgaggga
     7681 atgagaaagc ccgccttcct gagcggcgag cagaaaaagg ccatcgtgga cctgctgttc
     7741 aagaccaacc ggaaagtgac cgtgaagcag ctgaaagagg actacttcaa gaaaatcgag
     7801 tgcttcgact ccgtggaaat ctccggcgtg gaagatcggt tcaacgcctc cctgggcaca
     7861 taccacgatc tgctgaaaat tatcaaggac aaggacttcc tggacaatga ggaaaacgag
     7921 gacattctgg aagatatcgt gctgaccctg acactgtttg aggacagaga gatgatcgag
     7981 gaacggctga aaacctatgc ccacctgttc gacgacaaag tgatgaagca gctgaagcgg
     8041 cggagataca ccggctgggg caggctgagc cggaagctga tcaacggcat ccgggacaag
     8101 cagtccggca agacaatcct ggatttcctg aagtccgacg gcttcgccaa cagaaacttc
     8161 atgcagctga tccacgacga cagcctgacc tttaaagagg acatccagaa agcccaggtg
     8221 tccggccagg gcgatagcct gcacgagcac attgccaatc tggccggcag ccccgccatt
     8281 aagaagggca tcctgcagac agtgaaggtg gtggacgagc tcgtgaaagt gatgggccgg
     8341 cacaagcccg agaacatcgt gatcgaaatg gccagagaga accagaccac ccagaaggga
     8401 cagaagaaca gccgcgagag aatgaagcgg atcgaagagg gcatcaaaga gctgggcagc
     8461 cagatcctga aagaacaccc cgtggaaaac acccagctgc agaacgagaa gctgtacctg
     8521 tactacctgc agaatgggcg ggatatgtac gtggaccagg aactggacat caaccggctg
     8581 tccgactacg atgtggacca tatcgtgcct cagagctttc tgaaggacga ctccatcgac
     8641 aacaaggtgc tgaccagaag cgacaagaac cggggcaaga gcgacaacgt gccctccgaa
     8701 gaggtcgtga agaagatgaa gaactactgg cggcagctgc tgaacgccaa gctgattacc
     8761 cagagaaagt tcgacaatct gaccaaggcc gagagaggcg gcctgagcga actggataag
     8821 gccggcttca tcaagagaca gctggtggaa acccggcaga tcacaaagca cgtggcacag
     8881 atcctggact cccggatgaa cactaagtac gacgagaatg acaagctgat ccgggaagtg
     8941 aaagtgatca ccctgaagtc caagctggtg tccgatttcc ggaaggattt ccagttttac
     9001 aaagtgcgcg agatcaacaa ctaccaccac gcccacgacg cctacctgaa cgccgtcgtg
     9061 ggaaccgccc tgatcaaaaa gtaccctaag ctggaaagcg agttcgtgta cggcgactac
     9121 aaggtgtacg acgtgcggaa gatgatcgcc aagagcgagc aggaaatcgg caaggctacc
     9181 gccaagtact tcttctacag caacatcatg aactttttca agaccgagat taccctggcc
     9241 aacggcgaga tccggaagcg gcctctgatc gagacaaacg gcgaaaccgg ggagatcgtg
     9301 tgggataagg gccgggattt tgccaccgtg cggaaagtgc tgagcatgcc ccaagtgaat
     9361 atcgtgaaaa agaccgaggt gcagacaggc ggcttcagca aagagtctat cctgcccaag
     9421 aggaacagcg ataagctgat cgccagaaag aaggactggg accctaagaa gtacggcggc
     9481 ttcgacagcc ccaccgtggc ctattctgtg ctggtggtgg ccaaagtgga aaagggcaag
     9541 tccaagaaac tgaagagtgt gaaagagctg ctggggatca ccatcatgga aagaagcagc
     9601 ttcgagaaga atcccatcga ctttctggaa gccaagggct acaaagaagt gaaaaaggac
     9661 ctgatcatca agctgcctaa gtactccctg ttcgagctgg aaaacggccg gaagagaatg
     9721 ctggcctctg ccggcgaact gcagaaggga aacgaactgg ccctgccctc caaatatgtg
     9781 aacttcctgt acctggccag ccactatgag aagctgaagg gctcccccga ggataatgag
     9841 cagaaacagc tgtttgtgga acagcacaag cactacctgg acgagatcat cgagcagatc
     9901 agcgagttct ccaagagagt gatcctggcc gacgctaatc tggacaaagt gctgtccgcc
     9961 tacaacaagc accgggataa gcccatcaga gagcaggccg agaatatcat ccacctgttt
    10021 accctgacca atctgggagc ccctgccgcc ttcaagtact ttgacaccac catcgaccgg
    10081 aagaggtaca ccagcaccaa agaggtgctg gacgccaccc tgatccacca gagcatcacc
    10141 ggcctgtacg agacacggat cgacctgtct cagctgggag gcgacaaaag gccggcggcc
    10201 acgaaaaagg ccggccaggc aaaaaagaaa aagtaagaat tcctagagct cgctgatcag
    10261 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
    10321 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
    10381 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
    10441 aggattggga agagaatagc aggcatgctg gggagcggcc gcactagtct agtctagagc
    10501 gatcgcccgg gtttaaacgc tagccaacaa gctcgtcatc gctttgcaga agagcagaga
    10561 ggatatgctc atcgtctaaa gaactaccca ttttattata tattagtcac gatatctata
    10621 acaagaaaat atatatataa taagttatca cgtaagtaga acatgaaata acaatataat
    10681 tatcgtatga gttaaatctt aaaagtcacg taaaagataa tcatgcgtca ttttgactca
    10741 cgcggtcgtt atagttcaaa atcagtgaca cttaccgcat tgacaagcac gcctcacggg
    10801 agctccaagc ggcgactgag atgtcctaaa tgcacagcga cggattcgcg ctatttagaa
    10861 agagagagca atatttcaag aatgcatgcg tcaattttac gcagactatc tttctagggt
    10921 taaaaaagat ttgcgcttta ctcgacctaa actttaaaca cgtcatagaa tcttcgtttg
    10981 acaaaaacca cattgtggcc aagctgtgtg acgcgacgcg cgctaaagaa tggcaaacca
    11041 agtcgcgcga ggtaccagct tttgttccct ttagtgaggg ttaatttcga gcttggcgta
    11101 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
    11161 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
    11221 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
    11281 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
    11341 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
    11401 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
    11461 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
    11521 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
    11581 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
    11641 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
    11701 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
    11761 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
    11821 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
    11881 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
    11941 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
    12001 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
    12061 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
    12121 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
    12181 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
    12241 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
    12301 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
    12361 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
    12421 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
    12481 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
    12541 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
    12601 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
    12661 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
    12721 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
    12781 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
    12841 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
    12901 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
    12961 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
    13021 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
    13081 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
    13141 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
    13201 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacct
//
