LOCUS       sequence-498325-       12371 bp    DNA     circular SYN 27-JAN-2026
DEFINITION  Expresses KRAB fused to dCas9 and BFP upon cumate induction.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12371)
  AUTHORS   Fueyo R, Wang S, Crocker OJ, Swigut T, Nakauchi H, Wysocka J
  TITLE     A human-specific regulatory mechanism revealed in a pre-implantation
            model.
  JOURNAL   Nature. 2025 Oct 1. doi: 10.1038/s41586-025-09571-1.
  PUBMED    41034587
REFERENCE   2  (bases 1 to 12371)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 27, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nature. 
            2025 Oct 1. doi: 10.1038/s41586-025-09571-1."
COMMENT     Sequence Label: pB-EF1a-CuO-KRAB-dCas9-BFP-CymR-Puro
FEATURES             Location/Qualifiers
     source          1..12371
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   137..171
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(662..678)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(662..678)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(675..697)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    686..702
                     /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(710..716,717..734,735..740))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    755..776
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(893..910)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1064..1652)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1144..1163)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1823..2614,2615..2683))
                     /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     2446..2465
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2684..2788)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2814..3269)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2951..2972)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3163..3182
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3396..3418
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3410..3427
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3411..3427
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   3557..3619
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        4064..4275
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     protein_bind    4287..4314
                     /label=CuO
                     /bound_moiety="CymR"
                     /note="CymR-binding P2 operator sequence from the p-cmt 
                     operon of Pseudomonas putida (Mullick et al., 2006)"
     regulatory      4332..4341
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4368..4553
                     /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"
     CDS             4575..8678
                     /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
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAGGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             8682..8708
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             8688..8714
                     /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             8727..8747
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8754..8774
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8814..8870
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             8871..9572
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     promoter        9597..9808
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             9821..10089
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     regulatory      10117..10126
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             10129..10155
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             10135..10161
                     /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"
     primer_bind     10192..10211
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             10273..10881
                     /codon_start=1
                     /gene="cymR from Pseudomonas putida"
                     /product="cumate repressor (Mullick et al., 2006)"
                     /label=CymR
                     /translation="MSPKRRTQAERAMETQGKLIAAALGVLREKGYAGFRIADVPGAAG
                     VSRGAQSHHFPTKLELLLATFEWLYEQITERSRARLAKLKPEDDVIQQMLDDAAEFFLD
                     DDFSISLDLIVAADRDPALREGIQRTVERNRFVVEDMWLGVLVSRGLSRDDAEDILWLI
                     FNSVRGLAVRSLWQKDKERFERVRNSTLEIARERYAKFKR"
     CDS             10918..10971
                     /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             10978..11577
                     /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(10978..10997)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     11474..11494
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    11624..11745
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(11661..11680)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     11715..11734
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 ttatagttca aaatcagtga cacttaccgc attgacaagc acgcctcacg ggagctccaa
       61 gcggcgactg agatgtccta aatgcacagc gacggattcg cgctatttag aaagagagag
      121 caatatttca agaatgcatg cgtcaatttt acgcagacta tctttctagg gttaatctag
      181 ctgcatcagg atcatatcgt cgggtctttt ttccggctca gtcatcgccc aagctggcgc
      241 tatctgggca tcggggagga agaagcccgt gccttttccc gcgaggttga agcggcatgg
      301 aaagagtttg ccgaggatga ctgctgctgc attgacgttg agcgaaaacg cacgtttacc
      361 atgatgattc gggaaggtgt ggccatgcac gcctttaacg gtgaactgtt cgttcaggcc
      421 acctgggata ccagttcgtc gcggcttttc cggacacagt tccggatggt cagcccgaag
      481 cgcatcagca acccgaacaa taccggcgac agccggaact gccgtgccgg tgtgcagatt
      541 aatgacagcg gtgcggcgct gggatattac gtcagcgagg acgggtatcc tggctggatg
      601 ccgcagaaat ggacatggat accccgtgag ttacccggcg ggcgcgcttg gcgtaatcat
      661 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
      721 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
      781 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
      841 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
      901 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
      961 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     1021 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     1081 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     1141 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     1201 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     1261 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     1321 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     1381 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     1441 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     1501 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     1561 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     1621 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     1681 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     1741 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     1801 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     1861 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     1921 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     1981 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     2041 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     2101 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     2161 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     2221 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     2281 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     2341 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     2401 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     2461 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     2521 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     2581 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     2641 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     2701 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     2761 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt
     2821 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
     2881 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
     2941 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
     3001 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag
     3061 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
     3121 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
     3181 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
     3241 cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca ttcaggctgc gcaactgttg
     3301 ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc
     3361 tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac
     3421 ggccagtgag cgcgcctcgt tcattcacgt ttttgaaccc gtggaggacg ggcagactcg
     3481 cggtgcaaat gtgttttaca gcgtgatgga gcagatgaag atgctcgaca cgctgcagaa
     3541 cacgcagcta gattaaccct agaaagataa tcatattgtg acgtacgtta aagataatca
     3601 tgtgtaaaat tgacgcatgt gttttatcgg tctgtatatc gaggtttatt tattaatttg
     3661 aatagatatt aagttttatt atatttacac ttacatacta ataataaatt caacaaacaa
     3721 tttatttatg tttatttatt tattaaaaaa aacaaaaact caaaatttct tctataaagt
     3781 aacaaaactt ttatgaggga cagccccccc ccaaagcccc cagggatgta attacgtccc
     3841 tcccccgcta gggggcagca gcgagccgcc cggggctccg ctccggtccg gcgctccccc
     3901 cgcatccccg agccggcagc gtgcggggac agcccgggca cggggaaggt ggcacgggat
     3961 cgctttcctc tgaacgcttc tcgctgctct ttgagcctgc agacacctgg ggggatacgg
     4021 ggaaaaggcc tccacggcca gactagttcc ggtgcccgtc agtgggcaga gcgcacatcg
     4081 cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt gaacgggtgc ctagagaagg
     4141 tggcgcgggg taaactggga aagtgatgtc gtgtactggc tccgcctttt tcccgagggt
     4201 gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttcg caacgggttt
     4261 gccgccagaa cacagccccc ggtatataca aacagaccag attgtctgtt tgtttttggt
     4321 ctagagctag cgccaccatg gatgctaagt cactaactgc ctggtcccgg acactggtga
     4381 ccttcaagga tgtatttgtg gacttcacca gggaggagtg gaagctgctg gacactgctc
     4441 agcagatcgt gtacagaaat gtgatgctgg agaactataa gaacctggtt tccttgggtt
     4501 atcagcttac taagccagat gtgatcctcc ggttggagaa gggagaagag cccggcggtt
     4561 ccggcggagg gtcgatggac aagaagtaca gcatcggcct ggccatcggc accaactctg
     4621 taggctgggc cgtgatcacc gacgagtaca aggtgcccag caagaaattc aaggtgctgg
     4681 gcaacaccga ccggcacagc atcaagaaga acctgatcgg cgccctgctg ttcgacagcg
     4741 gagaaacagc cgaggccacc cggctgaaga gaaccgccag aagaagatac accagacgga
     4801 agaaccggat ctgctatctg caagagatct tcagcaacga gatggccaag gtggacgaca
     4861 gcttcttcca cagactggaa gagtccttcc tggtggaaga ggataagaag cacgagcggc
     4921 accccatctt cggcaacatc gtggacgagg tggcctacca cgagaagtac cccaccatct
     4981 accacctgag aaagaaactg gtggacagca ccgacaaggc cgacctgcgg ctgatctatc
     5041 tggccctggc ccacatgatc aagttccggg gccacttcct gatcgagggc gacctgaacc
     5101 ccgacaacag cgacgtggac aagctgttca tccagctggt gcagacctac aaccagctgt
     5161 tcgaggaaaa ccccatcaac gccggcggcg tggacgccaa ggccatcctg tctgccagac
     5221 tgagcaagag cagacggctg gaaaatctga tcgcccagct gcccggcgag aagaagaatg
     5281 gcctgttcgg caacctgatt gccctgagcc tgggcctgac ccccaacttc aagagcaact
     5341 tcgacctggc cgaggatgcc aaactgcagc tgagcaagga cacctacgac gacgacctgg
     5401 acaacctgct ggcccagatc ggcgaccagt acgccgacct gtttctggcc gccaagaacc
     5461 tgtccgacgc catcctgctg agcgacatcc tgagagtgaa caccgagatc accaaggccc
     5521 ccctgagcgc ctctatgatc aagagatacg acgagcacca ccaggacctg accctgctga
     5581 aagctctcgt gcggcagcag ctgcctgaga agtacaaaga gattttcttc gaccagagca
     5641 agaacggcta cgccggctac atcgatggcg gagccagcca ggaagagttc tacaagttca
     5701 tcaagcccat cctggaaaag atggacggca ccgaggaact gctcgtgaag ctgaacagag
     5761 aggacctgct gcggaagcag cggaccttcg acaacggcag catcccccac cagatccacc
     5821 tgggagagct gcacgccatt ctgcggcggc aggaagattt ttacccattc ctgaaggaca
     5881 accgggaaaa gatcgagaag atcctgacct tccgcatccc ctactacgtg ggccctctgg
     5941 ccaggggaaa cagcagattc gcctggatga ccagaaagag cgaggaaacc atcaccccct
     6001 ggaacttcga ggaagtggtg gacaagggcg ccagcgccca gagcttcatc gagcggatga
     6061 ccaacttcga taagaacctg cccaacgaga aggtgctgcc caagcacagc ctgctgtacg
     6121 agtacttcac cgtgtacaac gagctgacca aagtgaaata cgtgaccgag ggaatgagaa
     6181 agcccgcctt cctgagcggc gagcagaaaa aagccatcgt ggacctgctg ttcaagacca
     6241 accggaaagt gaccgtgaag cagctgaaag aggactactt caagaaaatc gagtgcttcg
     6301 actccgtgga aatctccggc gtggaagatc ggttcaacgc ctccctgggc acataccacg
     6361 atctgctgaa aattatcaag gacaaggact tcctggacaa tgaggaaaac gaggacattc
     6421 tggaagatat cgtgctgacc ctgacactgt ttgaggacag agagatgatc gaggaacggc
     6481 tgaaaaccta tgcccacctg ttcgacgaca aagtgatgaa gcagctgaag cggcggagat
     6541 acaccggctg gggcaggctg agccggaagc tgatcaacgg catccgggac aagcagtccg
     6601 gcaagacaat cctggatttc ctgaagtccg acggcttcgc caacagaaac ttcatgcagc
     6661 tgatccacga cgacagcctg acctttaaag aggacatcca gaaagcccag gtgtccggcc
     6721 agggcgatag cctgcacgag cacattgcca atctggccgg cagccccgcc attaagaagg
     6781 gcatcctgca gacagtgaag gtggtggacg agctcgtgaa agtgatgggc cggcacaagc
     6841 ccgagaacat cgtgatcgaa atggccagag agaaccagac cacccagaag ggacagaaga
     6901 acagccgcga gagaatgaag cggatcgaag agggcatcaa agagctgggc agccagatcc
     6961 tgaaagaaca ccccgtggaa aacacccagc tgcagaacga gaagctgtac ctgtactacc
     7021 tgcagaatgg gcgggatatg tacgtggacc aggaactgga catcaaccgg ctgtccgact
     7081 acgatgtgga cgctatcgtg cctcagagct ttctgaagga cgactccatc gataacaaag
     7141 tgctgactcg gagcgacaag aaccggggca agagcgacaa cgtgccctcc gaagaggtcg
     7201 tgaagaagat gaagaactac tggcgccagc tgctgaatgc caagctgatt acccagagga
     7261 agttcgacaa tctgaccaag gccgagagag gcggcctgag cgaactggat aaggccggct
     7321 tcatcaagag acagctggtg gaaacccggc agatcacaaa gcacgtggca cagatcctgg
     7381 actcccggat gaacactaag tacgacgaga acgacaaact gatccgggaa gtgaaagtga
     7441 tcaccctgaa gtccaagctg gtgtccgatt tccggaagga tttccagttt tacaaagtgc
     7501 gcgagatcaa caactaccac cacgcccacg acgcctacct gaacgccgtc gtgggaaccg
     7561 ccctgatcaa aaagtaccct aagctggaaa gcgagttcgt gtacggcgac tacaaggtgt
     7621 acgacgtgcg gaagatgatc gccaagagcg agcaggaaat cggcaaggct accgccaagt
     7681 acttcttcta cagcaacatc atgaactttt tcaagaccga gattaccctg gccaacggcg
     7741 agatccggaa gcggcctctg atcgagacaa acggcgaaac aggcgagatc gtgtgggata
     7801 agggccggga ctttgccacc gtgcggaaag tgctgtctat gccccaagtg aatatcgtga
     7861 aaaagaccga ggtgcagaca ggcggcttca gcaaagagtc tatcctgccc aagaggaaca
     7921 gcgacaagct gatcgccaga aagaaggact gggaccctaa gaagtacggc ggcttcgaca
     7981 gccccaccgt ggcctattct gtgctggtgg tggccaaagt ggaaaagggc aagtccaaga
     8041 aactgaagag tgtgaaagag ctgctgggga tcaccatcat ggaaagaagc agcttcgaga
     8101 agaatcccat cgactttctg gaagccaagg gctacaaaga agtgaaaaag gacctgatca
     8161 tcaagctgcc taagtactcc ctgttcgagc tggaaaacgg ccggaagaga atgctggcct
     8221 ctgccggcga actgcagaag ggaaacgaac tggccctgcc ctccaaatat gtgaacttcc
     8281 tgtacctggc cagccactat gagaagctga agggctcccc cgaggataat gagcagaaac
     8341 agctgtttgt ggaacagcac aaacactacc tggacgagat catcgagcag atcagcgagt
     8401 tctccaagag agtgatcctg gccgacgcta atctggacaa ggtgctgagc gcctacaaca
     8461 agcacagaga caagcctatc agagagcagg ccgagaatat catccacctg tttaccctga
     8521 ccaatctggg agcccctgcc gccttcaagt actttgacac caccatcgac cggaagaggt
     8581 acaccagcac caaagaggtg ctggacgcca ccctgatcca ccagagcatc accggcctgt
     8641 acgagacacg gatcgacctg tctcagctgg gaggcgacgc ctatccctat gacgtgcccg
     8701 attatgccag cctgggcagc ggctccccca agaaaaaacg caaggtggaa gatcctaaga
     8761 aaaagcggaa agtggacggc attggtagtg ggagcaacgg cagcagcgga tccgctacta
     8821 acttcagcct gctgaagcag gctggagacg tggaggagaa ccctggacct atgagcgagc
     8881 tgattaagga gaacatgcac atgaagctgt acatggaggg caccgtggac aaccatcact
     8941 tcaagtgcac atccgagggc gaaggcaagc cctacgaggg cacccagacc atgagaatca
     9001 aggtggtcga gggcggccct ctccccttcg ccttcgacat cctggctact agcttcctct
     9061 acggcagcaa gaccttcatc aaccacaccc agggcatccc cgacttcttc aagcagtcct
     9121 tccctgaggg cttcacatgg gagagagtca ccacatacga agacgggggc gtgctgaccg
     9181 ctacccagga caccagcctc caggacggct gcctcatcta caacgtcaag atcagagggg
     9241 tgaacttcac atccaacggc cctgtgatgc agaagaaaac actcggctgg gaggccttca
     9301 ccgagacgct gtaccccgct gacggcggcc tggaaggcag aaacgacatg gccctgaagc
     9361 tcgtgggcgg gagccatctg atcgcaaaca tcaagaccac atatagatcc aagaaacccg
     9421 ctaagaacct caagatgcct ggcgtctact atgtggacta cagactggaa agaatcaagg
     9481 aggccaacaa cgagacctac gtcgagcagc acgaggtggc agtggccaga tactgcgacc
     9541 tccctagcaa actggggcac aagcttaatt aagcgatcgc tccggtgccc gtcagtgggc
     9601 agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca attgaacggg
     9661 tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct
     9721 ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt
     9781 tcgcaacggg tttgccgcca gaacacagct gaagcttcga ggggctcgca tctctccttc
     9841 acgcgcccgc cgccctacct gaggccgcca tccacgccgg ttgagtcgcg ttctgccgcc
     9901 tcccgcctgt ggtgcctcct gaactgcgtc cgccgtctag gtaagtttaa agctcaggtc
     9961 gagaccgggc ctttgtccgg cgctcccttg gagcctacct agactcagcc ggctctccac
    10021 gctttgcctg accctgcttg ctcaactcta cgtctttgtt tcgttttctg ttctgcgccg
    10081 ttacagatcc aagctgtgac cggcgcctac tctagagccg ccatggctta cccatacgat
    10141 gttccagatt acgctagttt ggtgaggcag cagagaccga tgacatccac tttgcctttc
    10201 tctccacagg tgtccactcc caggtccaag tttggaagat ctaccatgcc aaagagaccc
    10261 agacccgtga tcatgagtcc aaagagaaga acacaggcag agcgcgcaat ggagacccag
    10321 ggcaagttga ttgcagcggc cctgggggtt ttacgggaaa aaggttacgc gggattccgg
    10381 atcgcagatg tgcccggtgc tgcaggtgtc tcgagaggag cgcagagcca tcatttcccg
    10441 acaaagcttg agcttctgct tgccactttt gaatggcttt acgaacagat caccgaacgc
    10501 agtcgggctc gattagcgaa attgaagcca gaggatgacg tcatccagca aatgctggac
    10561 gacgccgccg aatttttcct cgacgatgac ttctctatca gccttgattt gattgtggct
    10621 gccgaccggg atccagcgtt acgcgagggt attcagcgca cggtagagag gaatcggttt
    10681 gtcgtcgagg atatgtggct tggtgttctg gtgagccgtg gtctttcgcg tgatgatgca
    10741 gaagatatcc tttggttgat attcaattcg gtgcgtgggc ttgctgttcg tagcctatgg
    10801 cagaaggaca aagaacgctt tgagcgtgtc aggaactcga cactcgaaat tgcgcgagag
    10861 cggtacgcga aattcaagcg cgcggccaaa ccctctggcc ggtctctctc tgttcgagag
    10921 ggcagaggaa gtcttctaac atgcggtgac gtggaggaga atcccggccc tgaattcatg
    10981 accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc
    11041 accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga tccggaccgc
    11101 cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc
    11161 ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc
    11221 gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc
    11281 cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc
    11341 gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc agggcaaggg tctgggcagc
    11401 gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag
    11461 acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac
    11521 gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcctgagtc
    11581 gacaatcaac ctctggatta caaaatttgt ggttaactaa ctaaacttgt ttattgcagc
    11641 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
    11701 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggaattg
    11761 actcaaatga tgtcaattag tctatcagaa gctcatctgg tctcccttcc gggggacaag
    11821 acatccctgt ttaatattta aacagcagtg ttcccaaact gggttcttat atcccttgct
    11881 ctggtcaacc aggttgcagg gtttcctgtc ctcacaggaa cgaagtccct aaagaaacag
    11941 tggcagccag gtttagcccc ggaattgact ggattccttt tttagggccc attggtatgg
    12001 ctttttcccc gtatcccccc aggtgtctgc aggctcaaag agcagcgaga agcgttcaga
    12061 ggaaagcgat cccgtgccac cttccccgtg cccgggctgt ccccgcacgc tgccggctcg
    12121 gggatgcggg gggagcgccg gaccggagcg gagccccggg cggctcgctg ctgcccccta
    12181 gcgggggagg gacgtaatta catccctggg ggctttgggg gggggctgtc cctgatatct
    12241 ataacaagaa aatatatata taataagtta tcacgtaagt agaacatgaa ataacaatat
    12301 aattatcgta tgagttaaat cttaaaagtc acgtaaaaga taatcatgcg tcattttgac
    12361 tcacgcggtc g
//
