LOCUS       sequence-160882-       14081 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses ABA-inducible KRAB-Sp dCas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14081)
  AUTHORS   Gao Y, Xiong X, Wong S, Charles EJ, Lim WA, Qi LS
  TITLE     Complex transcriptional modulation with orthogonal and inducible 
            dCas9 regulators.
  JOURNAL   Nat Methods. 2016 Oct 24. doi: 10.1038/nmeth.4042.
  PUBMED    27776111
REFERENCE   2  (bases 1 to 14081)
  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: "Nat 
            Methods. 2016 Oct 24. doi: 10.1038/nmeth.4042."
COMMENT     Sequence Label: pSLQ2818 pPB: CAG-PYL1-KRAB-IRES-Puro-WPRE-SV4...
FEATURES             Location/Qualifiers
     source          1..14081
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        7..284
                     /label=chicken beta-actin promoter
     intron          286..1303
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1227..1249
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1311..1330
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1358..1367
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1985..2170
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEP"
     misc_feature    join(2188..2761,2762..2764,2765..2773)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2355..2372)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2582..2601
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2795..3394
                     /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(2795..2814)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3291..3311
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3432..4020
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3485..3505)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3903..3914)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4059..4078)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4113..4132
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        4174..4673
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(4209..4232)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     4606..4625
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             5633..6331
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     regulatory      6347..6356
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(6353..6379,6380..6382,6383..8869,8870..8872,
                     8873..10456)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             6353..10456
                     /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="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             10460..10486
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             10466..10492
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YDVPDYASL"
     CDS             10505..10525
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             10532..10552
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     repeat_region   10769..10831
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(10882..10902)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(10882..10900)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(10921..10937)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10921..10937)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10934..10956)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10945..10961
                     /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(10969..10975,10976..10993,10994..10999))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11014..11035
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(11152..11169)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11323..11911)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11403..11422)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(12082..12873,12874..12942))
                     /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     12705..12724
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12943..13047)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(13073..13528)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(13210..13231)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     13422..13441
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     13655..13677
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     13669..13686
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     13670..13686
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     13696..13715
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        13696..13714
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   13744..13778
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
ORIGIN
        1 ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac
       61 ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg
      121 ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga
      181 gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc
      241 ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcggggagt cgctgcgacg
      301 ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact
      361 gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta
      421 gcgcttggtt taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc ttgaggggct
      481 ccgggagggc cctttgtgcg gggggagcgg ctcggggggt gcgtgcgtgt gtgtgtgcgt
      541 ggggagcgcc gcgtgcggct ccgcgctgcc cggcggctgt gagcgctgcg ggcgcggcgc
      601 ggggctttgt gcgctccgca gtgtgcgcga ggggagcgcg gccgggggcg gtgccccgcg
      661 gtgcgggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg tgggggggtg
      721 agcagggggt gtgggcgcgt cggtcgggct gcaacccccc ctgcaccccc ctccccgagt
      781 tgctgagcac ggcccggctt cgggtgcggg gctccgtacg gggcgtggcg cggggctcgc
      841 cgtgccgggc ggggggtggc ggcaggtggg ggtgccgggc ggggcggggc cgcctcgggc
      901 cggggagggc tcgggggagg ggcgcggcgg cccccggagc gccggcggct gtcgaggcgc
      961 ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg gacttccttt
     1021 gtcccaaatc tgtgcggagc cgaaatctgg gaggcgccgc cgcaccccct ctagcgggcg
     1081 cggggcgaag cggtgcggcg ccggcaggaa ggaaatgggc ggggagggcc ttcgtgcgtc
     1141 gccgcgccgc cgtccccttc tccctctcca gcctcggggc tgtccgcggg gggacggctg
     1201 ccttcggggg ggacggggca gggcggggtt cggcttctgg cgtgtgaccg gcggctctag
     1261 agcctctgct aaccatgttc atgccttctt ctttttccta cagctcctgg gcaacgtgct
     1321 ggttattgtg ctgtctcatc attttggcaa agaattcgcc accatgggtg ggggcgcgcc
     1381 aactcaagac gaattcaccc aactctccca atcaatcgcc gagttccaca cgtaccaact
     1441 cggtaacggc cgttgctcat ctctcctagc tcagcgaatc cacgcgccgc cggaaacagt
     1501 atggtccgtg gtgagacgtt tcgataggcc acagatttac aaacacttca tcaaaagctg
     1561 taacgtgagt gaagatttcg agatgcgagt gggatgcacg cgcgacgtga acgtgataag
     1621 tggattaccg gcgaatacgt ctcgagagag attagatctg ttggacgatg atcggagagt
     1681 gactgggttt agtataaccg gtggtgaaca taggctgagg aattataaat cggttacgac
     1741 ggttcataga tttgagaaag aagaagaaga agaaaggatc tggaccgttg ttttggaatc
     1801 ttatgttgtt gatgtaccgg aaggtaattc ggaggaagat acgagattgt ttgctgatac
     1861 ggttattaga ttgaatcttc agaaacttgc ttcgatcact gaagctatga acgaattctc
     1921 aagcgctgca ggtactagtg gcggtggcgg agggatggat gctaagtcac taactgcctg
     1981 gtcccggaca ctggtgacct tcaaggatgt atttgtggac ttcaccaggg aggagtggaa
     2041 gctgctggac actgctcagc agatcgtgta cagaaatgtg atgctggaga actataagaa
     2101 cctggtttcc ttgggttatc agcttactaa gccagatgtg atcctccggt tggagaaggg
     2161 agaagagccc tagatttaaa taattcgccc ctctccctcc ccccccccta acgttactgg
     2221 ccgaagccgc ttggaataag gccggtgtgc gtttgtctat atgttatttt ccaccatatt
     2281 gccgtctttt ggcaatgtga gggcccggaa acctggccct gtcttcttga cgagcattcc
     2341 taggggtctt tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc
     2401 agttcctctg gaagcttctt gaagacaaac aacgtctgta gcgacccttt gcaggcagcg
     2461 gaacccccca cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat aagatacacc
     2521 tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa
     2581 atggctctcc tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg taccccattg
     2641 tatgggatct gatctggggc ctcggtgcac atgctttacg tgtgtttagt cgaggttaaa
     2701 aaacgtctag gccccccgaa ccacggggac gtggttttcc tttgaaaaac acgatgataa
     2761 tatggccaca accggatccg gagctaccat gaccatgacc gagtacaagc ccacggtgcg
     2821 cctcgccacc cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg cgttcgccga
     2881 ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct
     2941 gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga
     3001 cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc
     3061 cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat
     3121 ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg
     3181 cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga
     3241 ggcggccgag cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc gcaacctccc
     3301 cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg
     3361 cacctggtgc atgacccgca agcccggtgc ctgagtttaa accatgcaag cttgatatca
     3421 agcttatcga taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta
     3481 actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta
     3541 ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt
     3601 atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg
     3661 caacccccac tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt
     3721 tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag
     3781 gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc
     3841 cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc
     3901 cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc
     3961 ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc
     4021 atccttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     4081 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     4141 ttatcatgtc tggatcctgc aggtaattct accgggtagg ggaggcgctt ttcccaaggc
     4201 agtctggagc atgcgcttta gcagccccgc tgggcacttg gcgctacaca agtggcctct
     4261 ggcctcgcac acattccaca tccaccggta ggcgccaacc ggctccgttc tttggtggcc
     4321 ccttcgcgcc accttctact cctcccctag tcaggaagtt cccccccgcc ccgcagctcg
     4381 cgtcgtgcag gacgtgacaa atggaagtag cacgtctcac taggctcgtg cagatggaca
     4441 gcaccgctga gcaatggaag cgggtaggcc tttggggcag cggccaatag cagctttgct
     4501 ccttcgcttt ctgggctcag aggctgggaa ggggtgggtc cgggggcggg ctcaggggcg
     4561 ggctcagggg cggggcgggc gcccgaaggt cctccggagg cccggcattc tgcacgcttc
     4621 aaaagcgcac gtctgccgcg ctgttctcct cttcctcatc tccgggcctt tcgacctgca
     4681 gcccaagctt gtcgacgcca ccatgacgcg tgtgcctttg tatggtttta cttcgatttg
     4741 tggaagaaga cctgagatgg aagctgctgt ttcgactata ccaagattcc ttcaatcttc
     4801 ctctggttcg atgttagatg gtcggtttga tcctcaatcc gccgctcatt tcttcggtgt
     4861 ttacgacggc catggcggtt ctcaggtagc gaactattgt agagagagga tgcatttggc
     4921 tttggcggag gagatagcta aggagaaacc gatgctctgc gatggtgata cgtggctgga
     4981 gaagtggaag aaagctcttt tcaactcgtt cctgagagtt gactcggaga ttgagtcagt
     5041 tgcgccggag acggttgggt caacgtcggt ggttgccgtt gttttcccgt ctcacatctt
     5101 cgtcgctaac tgcggtgact ctagagccgt tctttgccgc ggcaaaactg cacttccatt
     5161 atccgttgac cataaaccgg atagagaaga tgaagctgcg aggattgaag ccgcaggagg
     5221 gaaagtgatt cagtggaatg gagctcgtgt tttcggtgtt ctcgccatgt cgagatccat
     5281 tggcgataga tacttgaaac catccatcat tcctgatccg gaagtgacgg ctgtgaagag
     5341 agtaaaagaa gatgattgtc tgattttggc gagtgacggg gtttgggatg taatgacgga
     5401 tgaagaagcg tgtgagatgg caaggaagcg gattctcttg tggcacaaga aaaacgcggt
     5461 ggctggggat gcatcgttgc tcgcggatga gcggagaaag gaagggaaag atcctgcggc
     5521 gatgtccgcg gctgagtatt tgtcaaagct ggcgatacag agaggaagca aagacaacat
     5581 aagtgtggtg gtggttgatt tgaagcatat gtcaagcgct gcaggtgcta ccatgagcga
     5641 gctgattaag gagaacatgc acatgaagct gtacatggag ggcaccgtgg acaaccatca
     5701 cttcaagtgc acatccgagg gcgaaggcaa gccctacgag ggcacccaga ccatgagaat
     5761 caaggtggtc gagggcggcc ctctcccctt cgccttcgac atcctggcta ctagcttcct
     5821 ctacggcagc aagaccttca tcaaccacac ccagggcatc cccgacttct tcaagcagtc
     5881 cttccctgag ggcttcacat gggagagagt caccacatac gaagacgggg gcgtgctgac
     5941 cgctacccag gacaccagcc tccaggacgg ctgcctcatc tacaacgtca agatcagagg
     6001 ggtgaacttc acatccaacg gccctgtgat gcagaagaaa acactcggct gggaggcctt
     6061 caccgagacg ctgtaccccg ctgacggcgg cctggaaggc agaaacgaca tggccctgaa
     6121 gctcgtgggc gggagccatc tgatcgcaaa catcaagacc acatatagat ccaagaaacc
     6181 cgctaagaac ctcaagatgc ctggcgtcta ctatgtggac tacagactgg aaagaatcaa
     6241 ggaggccaac aacgagacct acgtcgagca gcacgaggtg gcagtggcca gatactgcga
     6301 cctccctagc aaactggggc acaagcttaa ttcaagcgct gcaggtgcca ccatggacaa
     6361 gaagtacagc atcggcctgg ccatcggcac caactctgtg ggctgggccg tgatcaccga
     6421 cgagtacaag gtgcccagca agaaattcaa ggtgctgggc aacaccgacc ggcacagcat
     6481 caagaagaac ctgatcggcg ccctgctgtt cgacagcgga gaaacagccg aggccacccg
     6541 gctgaagaga accgccagaa gaagatacac cagacggaag aaccggatct gctatctgca
     6601 agagatcttc agcaacgaga tggccaaggt ggacgacagc ttcttccaca gactggaaga
     6661 gtccttcctg gtggaagagg ataagaagca cgagcggcac cccatcttcg gcaacatcgt
     6721 ggacgaggtg gcctaccacg agaagtaccc caccatctac cacctgagaa agaaactggt
     6781 ggacagcacc gacaaggccg acctgcggct gatctatctg gccctggccc acatgatcaa
     6841 gttccggggc cacttcctga tcgagggcga cctgaacccc gacaacagcg acgtggacaa
     6901 gctgttcatc cagctggtgc agacctacaa ccagctgttc gaggaaaacc ccatcaacgc
     6961 cagcggcgtg gacgccaagg ccatcctgtc tgccagactg agcaagagca gacggctgga
     7021 aaatctgatc gcccagctgc ccggcgagaa gaagaatggc ctgttcggca acctgattgc
     7081 cctgagcctg ggcctgaccc ccaacttcaa gagcaacttc gacctggccg aggatgccaa
     7141 actgcagctg agcaaggaca cctacgacga cgacctggac aacctgctgg cccagatcgg
     7201 cgaccagtac gccgacctgt ttctggccgc caagaacctg tccgacgcca tcctgctgag
     7261 cgacatcctg agagtgaaca ccgagatcac caaggccccc ctgagcgcct ctatgatcaa
     7321 gagatacgac gagcaccacc aggacctgac cctgctgaaa gctctcgtgc ggcagcagct
     7381 gcctgagaag tacaaagaga ttttcttcga ccagagcaag aacggctacg ccggctacat
     7441 cgatggcgga gccagccagg aagagttcta caagttcatc aagcccatcc tggaaaagat
     7501 ggacggcacc gaggaactgc tcgtgaagct gaacagagag gacctgctgc ggaagcagcg
     7561 gaccttcgac aacggcagca tcccccacca gatccacctg ggagagctgc acgccattct
     7621 gcggcggcag gaagattttt acccattcct gaaggacaac cgggaaaaga tcgagaagat
     7681 cctgaccttc cgcatcccct actacgtggg ccctctggcc aggggaaaca gcagattcgc
     7741 ctggatgacc agaaagagcg aggaaaccat caccccctgg aacttcgagg aagtggtgga
     7801 caagggcgcc agcgcccaga gcttcatcga gcggatgacc aacttcgata agaacctgcc
     7861 caacgagaag gtgctgccca agcacagcct gctgtacgag tacttcaccg tgtacaacga
     7921 gctgaccaaa gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc tgagcggcga
     7981 gcagaaaaaa gccatcgtgg acctgctgtt caagaccaac cggaaagtga ccgtgaagca
     8041 gctgaaagag gactacttca agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt
     8101 ggaagatcgg ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa ttatcaagga
     8161 caaggacttc ctggacaatg aggaaaacga ggacattctg gaagatatcg tgctgaccct
     8221 gacactgttt gaggacagag agatgatcga ggaacggctg aaaacctatg cccacctgtt
     8281 cgacgacaaa gtgatgaagc agctgaagcg gcggagatac accggctggg gcaggctgag
     8341 ccggaagctg atcaacggca tccgggacaa gcagtccggc aagacaatcc tggatttcct
     8401 gaagtccgac ggcttcgcca acagaaactt catgcagctg atccacgacg acagcctgac
     8461 ctttaaagag gacatccaga aagcccaggt gtccggccag ggcgatagcc tgcacgagca
     8521 cattgccaat ctggccggca gccccgccat taagaagggc atcctgcaga cagtgaaggt
     8581 ggtggacgag ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg tgatcgaaat
     8641 ggccagagag aaccagacca cccagaaggg acagaagaac agccgcgaga gaatgaagcg
     8701 gatcgaagag ggcatcaaag agctgggcag ccagatcctg aaagaacacc ccgtggaaaa
     8761 cacccagctg cagaacgaga agctgtacct gtactacctg cagaatgggc gggatatgta
     8821 cgtggaccag gaactggaca tcaaccggct gtccgactac gatgtggacg ctatcgtgcc
     8881 tcagagcttt ctgaaggacg actccatcga taacaaagtg ctgactcgga gcgacaagaa
     8941 ccggggcaag agcgacaacg tgccctccga agaggtcgtg aagaagatga agaactactg
     9001 gcgccagctg ctgaatgcca agctgattac ccagaggaag ttcgacaatc tgaccaaggc
     9061 cgagagaggc ggcctgagcg aactggataa ggccggcttc atcaagagac agctggtgga
     9121 aacccggcag atcacaaagc acgtggcaca gatcctggac tcccggatga acactaagta
     9181 cgacgagaac gacaaactga tccgggaagt gaaagtgatc accctgaagt ccaagctggt
     9241 gtccgatttc cggaaggatt tccagtttta caaagtgcgc gagatcaaca actaccacca
     9301 cgcccacgac gcctacctga acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa
     9361 gctggaaagc gagttcgtgt acggcgacta caaggtgtac gacgtgcgga agatgatcgc
     9421 caagagcgag caggaaatcg gcaaggctac cgccaagtac ttcttctaca gcaacatcat
     9481 gaactttttc aagaccgaga ttaccctggc caacggcgag atccggaagc ggcctctgat
     9541 cgagacaaac ggcgaaacag gcgagatcgt gtgggataag ggccgggact ttgccaccgt
     9601 gcggaaagtg ctgtctatgc cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg
     9661 cggcttcagc aaagagtcta tcctgcccaa gaggaacagc gacaagctga tcgccagaaa
     9721 gaaggactgg gaccctaaga agtacggcgg cttcgacagc cccaccgtgg cctattctgt
     9781 gctggtggtg gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct
     9841 gctggggatc accatcatgg aaagaagcag cttcgagaag aatcccatcg actttctgga
     9901 agccaagggc tacaaagaag tgaaaaagga cctgatcatc aagctgccta agtactccct
     9961 gttcgagctg gaaaacggcc ggaagagaat gctggcctct gccggcgaac tgcagaaggg
    10021 aaacgaactg gccctgccct ccaaatatgt gaacttcctg tacctggcca gccactatga
    10081 gaagctgaag ggctcccccg aggataatga gcagaaacag ctgtttgtgg aacagcacaa
    10141 acactacctg gacgagatca tcgagcagat cagcgagttc tccaagagag tgatcctggc
    10201 cgacgctaat ctggacaagg tgctgagcgc ctacaacaag cacagagaca agcctatcag
    10261 agagcaggcc gagaatatca tccacctgtt taccctgacc aatctgggag cccctgccgc
    10321 cttcaagtac tttgacacca ccatcgaccg gaagaggtac accagcacca aagaggtgct
    10381 ggacgccacc ctgatccacc agagcatcac cggcctgtac gagacacgga tcgacctgtc
    10441 tcagctggga ggcgacgcct atccctatga cgtgcccgat tatgccagcc tgggcagcgg
    10501 ctcccccaag aaaaaacgca aggtggaaga tcctaagaaa aagcggaaag tggacggcat
    10561 tggtagtggg agcaacggat gactcgagct agttaaaagt tttgttactt tatagaagaa
    10621 attttgagtt tttgtttttt tttaataaat aaataaacat aaataaattg tttgttgaat
    10681 ttattattag tatgtaagtg taaatataat aaaacttaat atctattcaa attaataaat
    10741 aaacctcgat atacagaccg ataaaacaca tgcgtcaatt ttacgcatga ttatctttaa
    10801 cgtacgtcac aatatgatta tctttctagg gttaatctag tatacgcgta tgcggccgct
    10861 taattaatcc agcttttgtt ccctttagtg agggttaatt gcgcgcttgg cgtaatcatg
    10921 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
    10981 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
    11041 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
    11101 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
    11161 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
    11221 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
    11281 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
    11341 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
    11401 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
    11461 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
    11521 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
    11581 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
    11641 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
    11701 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
    11761 aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
    11821 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
    11881 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
    11941 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
    12001 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
    12061 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
    12121 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
    12181 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
    12241 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
    12301 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
    12361 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
    12421 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
    12481 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
    12541 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
    12601 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
    12661 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
    12721 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
    12781 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
    12841 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
    12901 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
    12961 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
    13021 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctaaattgta
    13081 agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac
    13141 caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg
    13201 agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa
    13261 gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt
    13321 tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt
    13381 agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga
    13441 gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc
    13501 gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg caactgttgg
    13561 gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct
    13621 gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg
    13681 gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggggcgcgc cattctagat
    13741 taaccctaga aagatagtct gcgtaaaatt gacgcatgca ttcttgaaat attgctctct
    13801 ctttctaaat agcgcgaatc cgtcgctgtg catttaggac atctcagtcg ccgcttggag
    13861 ctcccgtgag gcgtgcttgt caatgcggta agtgtcactg attttgaact ataacgaccg
    13921 cgtgagtcaa aatgacgcat gattatcttt tacgtgactt ttaagattta actcatacga
    13981 taattatatt gttatttcat gttctactta cgtgataact tattatatat atattttctt
    14041 gttatagata tcaactagaa tgctagcatg ggcccagatc t
//
