LOCUS       sequence-249160-       12190 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  constitutive expression of KRAB-dCas9-BFP-KRAB from the AAVS1 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12190)
  AUTHORS   Tian R, Gachechiladze MA, Ludwig CH, Laurie MT, Hong JY, Nathaniel 
            D, Prabhu AV, Fernandopulle MS, Patel R, Abshari M, Ward ME, 
            Kampmann M
  TITLE     CRISPR Interference-Based Platform for Multimodal Genetic Screens in
            Human iPSC-Derived Neurons.
  JOURNAL   Neuron. 2019 Aug 5. pii: S0896-6273(19)30640-3. doi: 
            10.1016/j.neuron.2019.07.014.
  PUBMED    31422865
REFERENCE   2  (bases 1 to 12190)
  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: "Neuron. 
            2019 Aug 5. pii: S0896-6273(19)30640-3. doi: 
            10.1016/j.neuron.2019.07.014."
COMMENT     Sequence Label: pMTL3
FEATURES             Location/Qualifiers
     source          1..12190
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          163..1179
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1103..1125
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1187..1206
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1238..1247
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1274..1459
                     /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             join(1481..1507,1508..1510,1511..3997,3998..4000,
                     4001..5584)
                     /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             1481..5584
                     /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             5588..5614
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             5594..5620
                     /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             5633..5653
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5660..5680
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5720..6415
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     CDS             6461..6646
                     /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"
     primer_bind     complement(6741..6760)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    6806..6861
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(6860..6879)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     protein_bind    complement(7210..7243)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    7284..8120
                     /label=HA-R
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     rep_origin      complement(8168..8756)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8248..8267)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8927..9718,9719..9787))
                     /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     9550..9569
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     misc_feature    9841..10644
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     protein_bind    complement(10646..10679)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    10686..10711
                     /label=SA
                     /note="splice acceptor site"
     CDS             10735..10788
                     /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             10798..11397
                     /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(10798..10817)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     11294..11314
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(11429..11446)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    11435..11659
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(11663..11696)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     enhancer        11699..12078
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        join(12080..12190,1..162)
                     /label=chicken beta-actin promoter
ORIGIN
        1 ggggggcgcg cgccaggcgg ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga
       61 ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg
      121 cggcggcggc ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc tgcgcgctgc
      181 cttcgccccg tgccccgctc cgccgccgcc tcgcgccgcc cgccccggct ctgactgacc
      241 gcgttactcc cacaggtgag cgggcgggac ggcccttctc ctccgggctg taattagcgc
      301 ttggtttaat gacggcttgt ttcttttctg tggctgcgtg aaagccttga ggggctccgg
      361 gagggccctt tgtgcggggg gagcggctcg gggggtgcgt gcgtgtgtgt gtgcgtgggg
      421 agcgccgcgt gcggctccgc gctgcccggc ggctgtgagc gctgcgggcg cggcgcgggg
      481 ctttgtgcgc tccgcagtgt gcgcgagggg agcgcggccg ggggcggtgc cccgcggtgc
      541 ggggggggct gcgaggggaa caaaggctgc gtgcggggtg tgtgcgtggg ggggtgagca
      601 gggggtgtgg gcgcgtcggt cgggctgcaa ccccccctgc acccccctcc ccgagttgct
      661 gagcacggcc cggcttcggg tgcggggctc cgtacggggc gtggcgcggg gctcgccgtg
      721 ccgggcgggg ggtggcggca ggtgggggtg ccgggcgggg cggggccgcc tcgggccggg
      781 gagggctcgg gggaggggcg cggcggcccc cggagcgccg gcggctgtcg aggcgcggcg
      841 agccgcagcc attgcctttt atggtaatcg tgcgagaggg cgcagggact tcctttgtcc
      901 caaatctgtg cggagccgaa atctgggagg cgccgccgca ccccctctag cgggcgcggg
      961 gcgaagcggt gcggcgccgg caggaaggaa atgggcgggg agggccttcg tgcgtcgccg
     1021 cgccgccgtc cccttctccc tctccagcct cggggctgtc cgcgggggga cggctgcctt
     1081 cgggggggac ggggcagggc ggggttcggc ttctggcgtg tgaccggcgg ctctagagcc
     1141 tctgctaacc atgttcatgc cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt
     1201 attgtgctgt ctcatcattt tggcaaagaa tgtcgacgcc accatggatg ctaagtcact
     1261 aactgcctgg tcccggacac tggtgacctt caaggatgta tttgtggact tcaccaggga
     1321 ggagtggaag ctgctggaca ctgctcagca gatcgtgtac agaaatgtga tgctggagaa
     1381 ctataagaac ctggtttcct tgggttatca gcttactaag ccagatgtga tcctccggtt
     1441 ggagaaggga gaagagcccg gcggttccgg cggagggtcg atggacaaga agtacagcat
     1501 cggcctggcc atcggcacca actctgtggg ctgggccgtg atcaccgacg agtacaaggt
     1561 gcccagcaag aaattcaagg tgctgggcaa caccgaccgg cacagcatca agaagaacct
     1621 gatcggcgcc ctgctgttcg acagcggaga aacagccgag gccacccggc tgaagagaac
     1681 cgccagaaga agatacacca gacggaagaa ccggatctgc tatctgcaag agatcttcag
     1741 caacgagatg gccaaggtgg acgacagctt cttccacaga ctggaagagt ccttcctggt
     1801 ggaagaggat aagaagcacg agcggcaccc catcttcggc aacatcgtgg acgaggtggc
     1861 ctaccacgag aagtacccca ccatctacca cctgagaaag aaactggtgg acagcaccga
     1921 caaggccgac ctgcggctga tctatctggc cctggcccac atgatcaagt tccggggcca
     1981 cttcctgatc gagggcgacc tgaaccccga caacagcgac gtggacaagc tgttcatcca
     2041 gctggtgcag acctacaacc agctgttcga ggaaaacccc atcaacgcca gcggcgtgga
     2101 cgccaaggcc atcctgtctg ccagactgag caagagcaga cggctggaaa atctgatcgc
     2161 ccagctgccc ggcgagaaga agaatggcct gttcggcaac ctgattgccc tgagcctggg
     2221 cctgaccccc aacttcaaga gcaacttcga cctggccgag gatgccaaac tgcagctgag
     2281 caaggacacc tacgacgacg acctggacaa cctgctggcc cagatcggcg accagtacgc
     2341 cgacctgttt ctggccgcca agaacctgtc cgacgccatc ctgctgagcg acatcctgag
     2401 agtgaacacc gagatcacca aggcccccct gagcgcctct atgatcaaga gatacgacga
     2461 gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta
     2521 caaagagatt ttcttcgacc agagcaagaa cggctacgcc ggctacatcg atggcggagc
     2581 cagccaggaa gagttctaca agttcatcaa gcccatcctg gaaaagatgg acggcaccga
     2641 ggaactgctc gtgaagctga acagagagga cctgctgcgg aagcagcgga ccttcgacaa
     2701 cggcagcatc ccccaccaga tccacctggg agagctgcac gccattctgc ggcggcagga
     2761 agatttttac ccattcctga aggacaaccg ggaaaagatc gagaagatcc tgaccttccg
     2821 catcccctac tacgtgggcc ctctggccag gggaaacagc agattcgcct ggatgaccag
     2881 aaagagcgag gaaaccatca ccccctggaa cttcgaggaa gtggtggaca agggcgccag
     2941 cgcccagagc ttcatcgagc ggatgaccaa cttcgataag aacctgccca acgagaaggt
     3001 gctgcccaag cacagcctgc tgtacgagta cttcaccgtg tacaacgagc tgaccaaagt
     3061 gaaatacgtg accgagggaa tgagaaagcc cgccttcctg agcggcgagc agaaaaaagc
     3121 catcgtggac ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga
     3181 ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt
     3241 caacgcctcc ctgggcacat accacgatct gctgaaaatt atcaaggaca aggacttcct
     3301 ggacaatgag gaaaacgagg acattctgga agatatcgtg ctgaccctga cactgtttga
     3361 ggacagagag atgatcgagg aacggctgaa aacctatgcc cacctgttcg acgacaaagt
     3421 gatgaagcag ctgaagcggc ggagatacac cggctggggc aggctgagcc ggaagctgat
     3481 caacggcatc cgggacaagc agtccggcaa gacaatcctg gatttcctga agtccgacgg
     3541 cttcgccaac agaaacttca tgcagctgat ccacgacgac agcctgacct ttaaagagga
     3601 catccagaaa gcccaggtgt ccggccaggg cgatagcctg cacgagcaca ttgccaatct
     3661 ggccggcagc cccgccatta agaagggcat cctgcagaca gtgaaggtgg tggacgagct
     3721 cgtgaaagtg atgggccggc acaagcccga gaacatcgtg atcgaaatgg ccagagagaa
     3781 ccagaccacc cagaagggac agaagaacag ccgcgagaga atgaagcgga tcgaagaggg
     3841 catcaaagag ctgggcagcc agatcctgaa agaacacccc gtggaaaaca cccagctgca
     3901 gaacgagaag ctgtacctgt actacctgca gaatgggcgg gatatgtacg tggaccagga
     3961 actggacatc aaccggctgt ccgactacga tgtggacgct atcgtgcctc agagctttct
     4021 gaaggacgac tccatcgata acaaagtgct gactcggagc gacaagaacc ggggcaagag
     4081 cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag aactactggc gccagctgct
     4141 gaatgccaag ctgattaccc agaggaagtt cgacaatctg accaaggccg agagaggcgg
     4201 cctgagcgaa ctggataagg ccggcttcat caagagacag ctggtggaaa cccggcagat
     4261 cacaaagcac gtggcacaga tcctggactc ccggatgaac actaagtacg acgagaacga
     4321 caaactgatc cgggaagtga aagtgatcac cctgaagtcc aagctggtgt ccgatttccg
     4381 gaaggatttc cagttttaca aagtgcgcga gatcaacaac taccaccacg cccacgacgc
     4441 ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc tggaaagcga
     4501 gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag atgatcgcca agagcgagca
     4561 ggaaatcggc aaggctaccg ccaagtactt cttctacagc aacatcatga actttttcaa
     4621 gaccgagatt accctggcca acggcgagat ccggaagcgg cctctgatcg agacaaacgg
     4681 cgaaacaggc gagatcgtgt gggataaggg ccgggacttt gccaccgtgc ggaaagtgct
     4741 gtctatgccc caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg gcttcagcaa
     4801 agagtctatc ctgcccaaga ggaacagcga caagctgatc gccagaaaga aggactggga
     4861 ccctaagaag tacggcggct tcgacagccc caccgtggcc tattctgtgc tggtggtggc
     4921 caaagtggaa aagggcaagt ccaagaaact gaagagtgtg aaagagctgc tggggatcac
     4981 catcatggaa agaagcagct tcgagaagaa tcccatcgac tttctggaag ccaagggcta
     5041 caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag tactccctgt tcgagctgga
     5101 aaacggccgg aagagaatgc tggcctctgc cggcgaactg cagaagggaa acgaactggc
     5161 cctgccctcc aaatatgtga acttcctgta cctggccagc cactatgaga agctgaaggg
     5221 ctcccccgag gataatgagc agaaacagct gtttgtggaa cagcacaaac actacctgga
     5281 cgagatcatc gagcagatca gcgagttctc caagagagtg atcctggccg acgctaatct
     5341 ggacaaggtg ctgagcgcct acaacaagca cagagacaag cctatcagag agcaggccga
     5401 gaatatcatc cacctgttta ccctgaccaa tctgggagcc cctgccgcct tcaagtactt
     5461 tgacaccacc atcgaccgga agaggtacac cagcaccaaa gaggtgctgg acgccaccct
     5521 gatccaccag agcatcaccg gcctgtacga gacacggatc gacctgtctc agctgggagg
     5581 cgacgcctat ccctatgacg tgcccgatta tgccagcctg ggcagcggct cccccaagaa
     5641 aaaacgcaag gtggaagatc ctaagaaaaa gcggaaagtg gacggcattg gtagtgggag
     5701 caacggcagc agcggatcca gcgagctgat taaggagaac atgcacatga agctgtacat
     5761 ggagggcacc gtggacaacc atcacttcaa gtgcacatcc gagggcgaag gcaagcccta
     5821 cgagggcacc cagaccatga gaatcaaggt ggtcgagggc ggccctctcc ccttcgcctt
     5881 cgacatcctg gctactagct tcctctacgg cagcaagacc ttcatcaacc acacccaggg
     5941 catccccgac ttcttcaagc agtccttccc tgagggcttc acatgggaga gagtcaccac
     6001 atacgaagac gggggcgtgc tgaccgctac ccaggacacc agcctccagg acggctgcct
     6061 catctacaac gtcaagatca gaggggtgaa cttcacatcc aacggccctg tgatgcagaa
     6121 gaaaacactc ggctgggagg ccttcaccga gacgctgtac cccgctgacg gcggcctgga
     6181 aggcagaaac gacatggccc tgaagctcgt gggcgggagc catctgatcg caaacatcaa
     6241 gaccacatat agatccaaga aacccgctaa gaacctcaag atgcctggcg tctactatgt
     6301 ggactacaga ctggaaagaa tcaaggaggc caacaacgag acctacgtcg agcagcacga
     6361 ggtggcagtg gccagatact gcgacctccc tagcaaactg gggcacaagc ttaatggcgg
     6421 tggcggaggg atggatgcta agtcactaac tgcctggtcc cggacactgg tgaccttcaa
     6481 ggatgtattt gtggacttca ccagggagga gtggaagctg ctggacactg ctcagcagat
     6541 cgtgtacaga aatgtgatgc tggagaacta taagaacctg gtttccttgg gttatcagct
     6601 tactaagcca gatgtgatcc tccggttgga gaagggagaa gagccctagt aacgcgtgaa
     6661 ttcactcctc aggtgcaggc tgcctatcag aaggtggtgg ctggtgtggc caatgccctg
     6721 gctcacaaat accactgaga tctttttccc tctgccaaaa attatgggga catcatgaag
     6781 ccccttgagc atctgacttc tggctaataa aggaaattta ttttcattgc aatagtgtgt
     6841 tggaattttt tgtgtctctc actcggaagg acatatggga gggcaaatca tttaaaacat
     6901 cagaatgagt atttggttta gagtttggca acatatgccc atatgctggc tgccatgaac
     6961 aaaggttggc tataaagagg tcatcagtat atgaaacagc cccctgctgt ccattcctta
     7021 ttccatagaa aagccttgac ttgaggttag atttttttta tattttgttt tgtgttattt
     7081 ttttctttaa catccctaaa attttcctta catgttttac tagccagatt tttcctcctc
     7141 tcctgactac tcccagtcat agctgtccct cttctcttat ggagatccct cgacctgcag
     7201 cccaagctta taacttcgta tagcatacat tatacgaagt tatgcggccg caccggtatg
     7261 catttaatta agatatcagc tttactaggg acaggattgg tgacagaaaa gccccatcct
     7321 taggcctcct ccttcctagt ctcctgatat tgggtctaac ccccacctcc tgttaggcag
     7381 attccttatc tggtgacaca cccccatttc ctggagccat ctctctcctt gccagaacct
     7441 ctaaggtttg cttacgatgg agccagagag gatcctggga gggagagctt ggcagggggt
     7501 gggagggaag ggggggatgc gtgacctgcc cggttctcag tggccaccct gcgctaccct
     7561 ctcccagaac ctgagctgct ctgacgcggc tgtctggtgc gtttcactga tcctggtgct
     7621 gcagcttcct tacacttccc aagaggagaa gcagtttgga aaaacaaaat cagaataagt
     7681 tggtcctgag ttctaacttt ggctcttcac ctttctagtc cccaatttat attgttcctc
     7741 cgtgcgtcag ttttacctgt gagataaggc cagtagccag ccccgtcctg gcagggctgt
     7801 ggtgaggagg ggggtgtccg tgtggaaaac tccctttgtg agaatggtgc gtcctaggtg
     7861 ttcaccaggt cgtggccgcc tctactccct ttctctttct ccatccttct ttccttaaag
     7921 agtccccagt gctatctggg acatattcct ccgcccagag cagggtcccg cttccctaag
     7981 gccctgctct gggcttctgg gtttgagtcc ttggcaagcc caggagaggc gctcaggctt
     8041 ccctgtcccc cttcctcgtc caccatctca tgcccctggc tctcctgccc cttccctaca
     8101 ggggttcctg gctctgctct aagggcgaat tcgccaggaa ccgtaaaaag gccgcgttgc
     8161 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     8221 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     8281 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     8341 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     8401 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     8461 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     8521 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     8581 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     8641 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     8701 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     8761 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     8821 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     8881 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     8941 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     9001 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     9061 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     9121 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     9181 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     9241 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     9301 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     9361 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     9421 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     9481 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     9541 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     9601 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     9661 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     9721 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     9781 tactcatact cttccttttt caatattatt gaagcattta tcagggttga attcgccctt
     9841 tgctttctct gaccagcatt ctctcccctg ggcctgtgcc gctttctgtc tgcagcttgt
     9901 ggcctgggtc acctctacgg ctggcccaga tccttccctg ccgcctcctt caggttccgt
     9961 cttcctccac tccctcttcc ccttgctctc tgctgtgttg ctgcccaagg atgctctttc
    10021 cggagcactt ccttctcggc gctgcaccac gtgatgtcct ctgagcggat cctccccgtg
    10081 tctgggtcct ctccgggcat ctctcctccc tcacccaacc ccatgccgtc ttcactcgct
    10141 gggttccctt ttccttctcc ttctggggcc tgtgccatct ctcgtttctt aggatggcct
    10201 tctccgacgg atgtctccct tgcgtcccgc ctccccttct tgtaggcctg catcatcacc
    10261 gtttttctgg acaaccccaa agtaccccgt ctccctggct ttagccacct ctccatcctc
    10321 ttgctttctt tgcctggaca ccccgttctc ctgtggattc gggtcacctc tcactccttt
    10381 catttgggca gctcccctac cccccttacc tctctagtct gtgctagctc ttccagcccc
    10441 ctgtcatggc atcttccagg ggtccgagag ctcagctagt cttcttcctc caacccgggc
    10501 ccctatgtcc acttcaggac agcatgtttg ctgcctccag ggatcctgtg tccccgagct
    10561 gggaccacct tatattccca gggccggtta atgtggctct ggttctgggt acttttatct
    10621 gtcccctcca ccccacagtg gggcaataac ttcgtatagc atacattata cgaagttata
    10681 agcttctgac ctcttctctt cctcccacag ggcctcgaga gatctggcag cggagagggc
    10741 agaggaagtc ttctaacatg cggtgacgtg gaggagaatc ccggccctag gctcgagatg
    10801 accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc
    10861 accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga tccggaccgc
    10921 cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc
    10981 ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc
    11041 gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc
    11101 cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc
    11161 gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc agggcaaggg tctgggcagc
    11221 gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag
    11281 acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac
    11341 gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcctgatct
    11401 agagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct
    11461 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
    11521 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
    11581 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg
    11641 gatgcggtgg gctctatggg tcataacttc gtatagcata cattatacga agttattcga
    11701 cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca
    11761 tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac
    11821 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact
    11881 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
    11941 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
    12001 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
    12061 gtcatcgcta ttaccatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc
    12121 cccccctccc cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg
    12181 ggggcggggg
//
