LOCUS       sequence-214842-       13381 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Piggyac transposon containing tamoxifen inducible Cas9 and a gRNA 
            cassette..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13381)
  TITLE     Chen lab CRISPR plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 13381)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pPB-US-ECasE
FEATURES             Location/Qualifiers
     source          1..13381
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1..105)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(131..586)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(268..289)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     480..499
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     713..735
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     727..744
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     728..744
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   874..936
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        1424..1672
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1424..1444
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1595..1614
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1691..1766
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        1858..3036
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2089..3027
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2984..3004
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     promoter        3061..3108
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     regulatory      3133..3142
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3142..4077
                     /codon_start=1
                     /product="mutated ligand-binding domain of the human 
                     estrogen receptor (Feil et al., 1997)"
                     /label=ERT2
                     /translation="AGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPIL
                     YSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEI
                     LMIGLVWRSMEHPVKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEF
                     VCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRL
                     AQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEAADAHRLHAPTSRGGASVEET
                     DQSHLATAGSTSSHSLQKYYITGEAEGFPAT"
     CDS             4090..8190
                     /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             4090..8190
                     /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             8197..9132
                     /codon_start=1
                     /product="mutated ligand-binding domain of the human 
                     estrogen receptor (Feil et al., 1997)"
                     /label=ERT2
                     /translation="AGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPIL
                     YSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEI
                     LMIGLVWRSMEHPVKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEF
                     VCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRL
                     AQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEAADAHRLHAPTSRGGASVEET
                     DQSHLATAGSTSSHSLQKYYITGEAEGFPAT"
     CDS             9145..9198
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             9199..9795
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APHNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(9199..9218)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     complement(9886..9906)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    9952..10073
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(9989..10008)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10043..10062
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   10835..10869
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(11360..11376)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11360..11376)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(11373..11395)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    11384..11400
                     /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(11408..11414,11415..11432,11433..11438))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11453..11474
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(11591..11608)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11762..12350)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11842..11861)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(12521..13312,13313..13381))
                     /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     13144..13163
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
       61 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
      121 agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
      181 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
      241 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
      301 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
      361 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
      421 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
      481 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
      541 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc
      601 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg
      661 gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca
      721 cgacgttgta aaacgacggc cagtgagcgc gcctcgttca ttcacgtttt tgaacccgtg
      781 gaggacgggc agactcgcgg tgcaaatgtg ttttacagcg tgatggagca gatgaagatg
      841 ctcgacacgc tgcagaacac gcagctagat taaccctaga aagataatca tattgtgacg
      901 tacgttaaag ataatcatgc gtaaaattga cgcatgtgtt ttatcggtct gtatatcgag
      961 gtttatttat taatttgaat agatattaag ttttattata tttacactta catactaata
     1021 ataaattcaa caaacaattt atttatgttt atttatttat taaaaaaaaa caaaaactca
     1081 aaatttcttc tataaagtaa caaaactttt atgagggaca gccccccccc aaagccccca
     1141 gggatgtaat tacgtccctc ccccgctagg gggcagcagc gagccgcccg gggctccgct
     1201 ccggtccggc gctccccccg catccccgag ccggcagcgt gcggggacag cccgggcacg
     1261 gggaaggtgg cacgggatcg ctttcctctg aacgcttctc gctgctcttt gagcctgcag
     1321 acacctgggg ggatacgggg aaaaggcctc caaggccaga ctagaggtac caacgcggcc
     1381 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgtgagggcc tatttcccat
     1441 gattccttca tatttgcata tacgatacaa ggctgttaga gagataattg gaattaattt
     1501 gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg
     1561 gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg
     1621 aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccgggtcttc
     1681 gagaagacct gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac
     1741 ttgaaaaagt ggcaccgagt cggtgctttt ttgttttaga gctagaaata gcaagttaaa
     1801 ataaggctag tccgttttta gcgctcgagc ttctaggtct tgaaaggagt gggaattggc
     1861 tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa gttgggggga
     1921 ggggtcggca attgaaccgg tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat
     1981 gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat aagtgcagta
     2041 gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacaggt aagtgccgtg
     2101 tgtggttccc gcgggcctgg cctctttacg ggttatggcc cttgcgtgcc ttgaattact
     2161 tccacctggc tgcagtacgt gattcttgat cccgagcttc gggttggaag tgggtgggag
     2221 agttcgaggc cttgcgctta aggagcccct tcgcctcgtg cttgagttga ggcctggcct
     2281 gggcgctggg gccgccgcgt gcgaatctgg tggcaccttc gcgcctgtct cgctgctttc
     2341 gataagtctc tagccattta aaatttttga tgacctgctg cgacgctttt tttctggcaa
     2401 gatagtcttg taaatgcggg ccaagatctg cacactggta tttcggtttt tggggccgcg
     2461 ggcggcgacg gggcccgtgc gtcccagcgc acatgttcgg cgaggcgggg cctgcgagcg
     2521 cggccaccga gaatcggacg ggggtagtct caagctggcc ggcctgctct ggtgcctggc
     2581 ctcgcgccgc cgtgtatcgc cccgccctgg gcggcaaggc tggcccggtc ggcaccagtt
     2641 gcgtgagcgg aaagatggcc gcttcccggc cctgctgcag ggagctcaaa atggaggacg
     2701 cggcgctcgg gagagcgggc gggtgagtca cccacacaaa ggaaaagggc ctttccgtcc
     2761 tcagccgtcg cttcatgtga ctccacggag taccgggcgc cgtccaggca cctcgattag
     2821 ttctcgagct tttggagtac gtcgtcttta ggttgggggg aggggtttta tgcgatggag
     2881 tttccccaca ctgagtgggt ggagactgaa gttaggccag cttggcactt gatgtaattc
     2941 tccttggaat ttgccctttt tgagtttgga tcttggttca ttctcaagcc tcagacagtg
     3001 gttcaaagtt tttttcttcc atttcaggtg tcgtgaggaa ttagcttggt accggaccgt
     3061 gttgacaatt aatcatcggc atagtatatc ggcatagtat aatacgacaa ggtgaggaac
     3121 taaaccggat ccgccaccat ggctggagac atgagagctg ccaacctttg gccaagcccg
     3181 ctcatgatca aacgctctaa gaagaacagc ctggccttgt ccctgacggc cgaccagatg
     3241 gtcagtgcct tgttggatgc tgagcccccc atactctatt ccgagtatga tcctaccaga
     3301 cccttcagtg aagcttcgat gatgggctta ctgaccaacc tggcagacag ggagctggtt
     3361 cacatgatca actgggcgaa gagggtgcca ggctttgtgg atttgaccct ccatgatcag
     3421 gtccaccttc tagaatgtgc ctggctagag atcctgatga ttggtctcgt ctggcgctcc
     3481 atggagcacc cagtgaagct actgtttgct cctaacttgc tcttggacag gaaccaggga
     3541 aaatgtgtag agggcatggt ggagatcttc gacatgctgc tggctacatc atctcggttc
     3601 cgcatgatga atctgcaggg agaggagttt gtgtgcctca aatctattat tttgcttaat
     3661 tctggagtgt acacatttct gtccagcacc ctgaagtctc tggaagagaa ggaccatatc
     3721 caccgagtcc tggacaagat cacagacact ttgatccacc tgatggccaa ggcaggcctg
     3781 accctgcagc agcagcacca gcggctggcc cagctcctcc tcatcctctc ccacatcagg
     3841 cacatgagta acaaaggcat ggagcatctg tacagcatga agtgcaagaa cgtggtgccc
     3901 ctctatgacc tgctgctgga ggcggcggac gcccaccgcc tacatgcgcc cactagccgt
     3961 ggaggggcat ccgtggagga gacggaccaa agccacttgg ccactgcggg ctctacttca
     4021 tcgcattcct tgcaaaagta ttacatcacg ggggaggcag agggtttccc tgccacagct
     4081 taccggtttg acaagaagta cagcatcggc ctggacatcg gcaccaactc tgtgggctgg
     4141 gccgtgatca ccgacgagta caaggtgccc agcaagaaat tcaaggtgct gggcaacacc
     4201 gaccggcaca gcatcaagaa gaacctgatc ggagccctgc tgttcgacag cggcgaaaca
     4261 gccgaggcca cccggctgaa gagaaccgcc agaagaagat acaccagacg gaagaaccgg
     4321 atctgctatc tgcaagagat cttcagcaac gagatggcca aggtggacga cagcttcttc
     4381 cacagactgg aagagtcctt cctggtggaa gaggataaga agcacgagcg gcaccccatc
     4441 ttcggcaaca tcgtggacga ggtggcctac cacgagaagt accccaccat ctaccacctg
     4501 agaaagaaac tggtggacag caccgacaag gccgacctgc ggctgatcta tctggccctg
     4561 gcccacatga tcaagttccg gggccacttc ctgatcgagg gcgacctgaa ccccgacaac
     4621 agcgacgtgg acaagctgtt catccagctg gtgcagacct acaaccagct gttcgaggaa
     4681 aaccccatca acgccagcgg cgtggacgcc aaggccatcc tgtctgccag actgagcaag
     4741 agcagacggc tggaaaatct gatcgcccag ctgcccggcg agaagaagaa tggcctgttc
     4801 ggaaacctga ttgccctgag cctgggcctg acccccaact tcaagagcaa cttcgacctg
     4861 gccgaggatg ccaaactgca gctgagcaag gacacctacg acgacgacct ggacaacctg
     4921 ctggcccaga tcggcgacca gtacgccgac ctgtttctgg ccgccaagaa cctgtccgac
     4981 gccatcctgc tgagcgacat cctgagagtg aacaccgaga tcaccaaggc ccccctgagc
     5041 gcctctatga tcaagagata cgacgagcac caccaggacc tgaccctgct gaaagctctc
     5101 gtgcggcagc agctgcctga gaagtacaaa gagattttct tcgaccagag caagaacggc
     5161 tacgccggct acattgacgg cggagccagc caggaagagt tctacaagtt catcaagccc
     5221 atcctggaaa agatggacgg caccgaggaa ctgctcgtga agctgaacag agaggacctg
     5281 ctgcggaagc agcggacctt cgacaacggc agcatccccc accagatcca cctgggagag
     5341 ctgcacgcca ttctgcggcg gcaggaagat ttttacccat tcctgaagga caaccgggaa
     5401 aagatcgaga agatcctgac cttccgcatc ccctactacg tgggccctct ggccagggga
     5461 aacagcagat tcgcctggat gaccagaaag agcgaggaaa ccatcacccc ctggaacttc
     5521 gaggaagtgg tggacaaggg cgcttccgcc cagagcttca tcgagcggat gaccaacttc
     5581 gataagaacc tgcccaacga gaaggtgctg cccaagcaca gcctgctgta cgagtacttc
     5641 accgtgtata acgagctgac caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc
     5701 ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc tgttcaagac caaccggaaa
     5761 gtgaccgtga agcagctgaa agaggactac ttcaagaaaa tcgagtgctt cgactccgtg
     5821 gaaatctccg gcgtggaaga tcggttcaac gcctccctgg gcacatacca cgatctgctg
     5881 aaaattatca aggacaagga cttcctggac aatgaggaaa acgaggacat tctggaagat
     5941 atcgtgctga ccctgacact gtttgaggac agagagatga tcgaggaacg gctgaaaacc
     6001 tatgcccacc tgttcgacga caaagtgatg aagcagctga agcggcggag atacaccggc
     6061 tggggcaggc tgagccggaa gctgatcaac ggcatccggg acaagcagtc cggcaagaca
     6121 atcctggatt tcctgaagtc cgacggcttc gccaacagaa acttcatgca gctgatccac
     6181 gacgacagcc tgacctttaa agaggacatc cagaaagccc aggtgtccgg ccagggcgat
     6241 agcctgcacg agcacattgc caatctggcc ggcagccccg ccattaagaa gggcatcctg
     6301 cagacagtga aggtggtgga cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac
     6361 atcgtgatcg aaatggccag agagaaccag accacccaga agggacagaa gaacagccgc
     6421 gagagaatga agcggatcga agagggcatc aaagagctgg gcagccagat cctgaaagaa
     6481 caccccgtgg aaaacaccca gctgcagaac gagaagctgt acctgtacta cctgcagaat
     6541 gggcgggata tgtacgtgga ccaggaactg gacatcaacc ggctgtccga ctacgatgtg
     6601 gaccatatcg tgcctcagag ctttctgaag gacgactcca tcgacaacaa ggtgctgacc
     6661 agaagcgaca agaaccgggg caagagcgac aacgtgccct ccgaagaggt cgtgaagaag
     6721 atgaagaact actggcggca gctgctgaac gccaagctga ttacccagag aaagttcgac
     6781 aatctgacca aggccgagag aggcggcctg agcgaactgg ataaggccgg cttcatcaag
     6841 agacagctgg tggaaacccg gcagatcaca aagcacgtgg cacagatcct ggactcccgg
     6901 atgaacacta agtacgacga gaatgacaag ctgatccggg aagtgaaagt gatcaccctg
     6961 aagtccaagc tggtgtccga tttccggaag gatttccagt tttacaaagt gcgcgagatc
     7021 aacaactacc accacgccca cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc
     7081 aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg actacaaggt gtacgacgtg
     7141 cggaagatga tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc
     7201 tacagcaaca tcatgaactt tttcaagacc gagattaccc tggccaacgg cgagatccgg
     7261 aagcggcctc tgatcgagac aaacggcgaa accggggaga tcgtgtggga taagggccgg
     7321 gattttgcca ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt gaaaaagacc
     7381 gaggtgcaga caggcggctt cagcaaagag tctatcctgc ccaagaggaa cagcgataag
     7441 ctgatcgcca gaaagaagga ctgggaccct aagaagtacg gcggcttcga cagccccacc
     7501 gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag
     7561 agtgtgaaag agctgctggg gatcaccatc atggaaagaa gcagcttcga gaagaatccc
     7621 atcgactttc tggaagccaa gggctacaaa gaagtgaaaa aggacctgat catcaagctg
     7681 cctaagtact ccctgttcga gctggaaaac ggccggaaga gaatgctggc ctctgccggc
     7741 gaactgcaga agggaaacga actggccctg ccctccaaat atgtgaactt cctgtacctg
     7801 gccagccact atgagaagct gaagggctcc cccgaggata atgagcagaa acagctgttt
     7861 gtggaacagc acaagcacta cctggacgag atcatcgagc agatcagcga gttctccaag
     7921 agagtgatcc tggccgacgc taatctggac aaagtgctgt ccgcctacaa caagcaccgg
     7981 gataagccca tcagagagca ggccgagaat atcatccacc tgtttaccct gaccaatctg
     8041 ggagcccctg ccgccttcaa gtactttgac accaccatcg accggaagag gtacaccagc
     8101 accaaagagg tgctggacgc caccctgatc caccagagca tcaccggcct gtacgagaca
     8161 cggatcgacc tgtctcagct gggaggcgac accggtgctg gagacatgag agctgccaac
     8221 ctttggccaa gcccgctcat gatcaaacgc tctaagaaga acagcctggc cttgtccctg
     8281 acggccgacc agatggtcag tgccttgttg gatgctgagc cccccatact ctattccgag
     8341 tatgatccta ccagaccctt cagtgaagct tcgatgatgg gcttactgac caacctggca
     8401 gacagggagc tggttcacat gatcaactgg gcgaagaggg tgccaggctt tgtggatttg
     8461 accctccatg atcaggtcca ccttctagaa tgtgcctggc tagagatcct gatgattggt
     8521 ctcgtctggc gctccatgga gcacccagtg aagctactgt ttgctcctaa cttgctcttg
     8581 gacaggaacc agggaaaatg tgtagagggc atggtggaga tcttcgacat gctgctggct
     8641 acatcatctc ggttccgcat gatgaatctg cagggagagg agtttgtgtg cctcaaatct
     8701 attattttgc ttaattctgg agtgtacaca tttctgtcca gcaccctgaa gtctctggaa
     8761 gagaaggacc atatccaccg agtcctggac aagatcacag acactttgat ccacctgatg
     8821 gccaaggcag gcctgaccct gcagcagcag caccagcggc tggcccagct cctcctcatc
     8881 ctctcccaca tcaggcacat gagtaacaaa ggcatggagc atctgtacag catgaagtgc
     8941 aagaacgtgg tgcccctcta tgacctgctg ctggaggcgg cggacgccca ccgcctacat
     9001 gcgcccacta gccgtggagg ggcatccgtg gaggagacgg accaaagcca cttggccact
     9061 gcgggctcta cttcatcgca ttccttgcaa aagtattaca tcacggggga ggcagagggt
     9121 ttccctgcca cagcttatgt cgacgagggc agaggaagtc tgctaacatg cggtgacgtc
     9181 gaggagaatc ctggcccaat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac
     9241 gacgtcccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc
     9301 cacaccgtcg atccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc
     9361 acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg
     9421 gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc
     9481 atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg
     9541 ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac
     9601 cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc
     9661 ggggtgcccg ccttcctgga gacctccgcg ccccacaacc tccccttcta cgagcggctc
     9721 ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc
     9781 cgcaagcccg gtgccacgcg ttaactaagt aagcggccgc cccggtatcg acaatcaacc
     9841 tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac
     9901 gctatgtgga tacgctgctt taatgccttt gtatcatgcg ttaactaact aaacttgttt
     9961 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
    10021 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
    10081 tggaattgac tcaaatgatg tcaattagtc tatcagaagc tatctggtct cccttccggg
    10141 ggacaagaca tccctgttta atatttaaac agcagtgttc ccaaactggg ttcttatatc
    10201 ccttgctctg gtcaaccagg ttgcagggtt tcctgtcctc acaggaacga agtccctaaa
    10261 gaaacagtgg cagccaggtt tagccccgga attgactgga ttcctttttt agggcccatt
    10321 ggtatggctt tttccccgta tccccccagg tgtctgcagg ctcaaagagc agcgagaagc
    10381 gttcagagga aagcgatccc gtgccacctt ccccgtgccc gggctgtccc cgcacgctgc
    10441 cggctcgggg atgcgggggg agcgccggac cggagcggag ccccgggcgg ctcgctgctg
    10501 ccccctagcg ggggagggac gtaattacat ccctgggggc tttggggggg ggctgtccct
    10561 gatatctata acaagaaaat atatatataa taagttatca cgtaagtaga acatgaaata
    10621 acaatataat tatcgtatga gttaaatctt aaaagtcacg taaaagataa tcatgcgtca
    10681 ttttgactca cgcggtcgtt atagttcaaa atcagtgaca cttaccgcat tgacaagcac
    10741 gcctcacggg agctccaagc ggcgactgag atgtcctaaa tgcacagcga cggattcgcg
    10801 ctatttagaa agagagagca atatttcaag aatgcatgcg tcaattttac gcagactatc
    10861 tttctagggt taatctagct gcatcaggat catatcgtcg ggtctttttt ccggctcagt
    10921 catcgcccaa gctggcgcta tctgggcatc ggggaggaag aagcccgtgc cttttcccgc
    10981 gaggttgaag cggcatggaa agagtttgcc gaggatgact gctgctgcat tgacgttgag
    11041 cgaaaacgca cgtttaccat gatgattcgg gaaggtgtgg ccatgcacgc ctttaacggt
    11101 gaactgttcg ttcaggccac ctgggatacc agttcgtcgc ggcttttccg gacacagttc
    11161 cggatggtca gcccgaagcg catcagcaac ccgaacaata ccggcgacag ccggaactgc
    11221 cgtgccggtg tgcagattaa tgacagcggt gcggcgctgg gatattacgt cagcgaggac
    11281 gggtatcctg gctggatgcc gcagaaatgg acatggatac cccgtgagtt acccggcggg
    11341 cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
    11401 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
    11461 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
    11521 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
    11581 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
    11641 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
    11701 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
    11761 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
    11821 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
    11881 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
    11941 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
    12001 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
    12061 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
    12121 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
    12181 ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta
    12241 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
    12301 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
    12361 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
    12421 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
    12481 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
    12541 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
    12601 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
    12661 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
    12721 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
    12781 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
    12841 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
    12901 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
    12961 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
    13021 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
    13081 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
    13141 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
    13201 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
    13261 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
    13321 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
    13381 t
//
