LOCUS       sequence-487656-       15390 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  constitutive expression of dCas9-BFP-KRAB from the CLYBL locus (Ward
            lab).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 15390)
  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 15390)
  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: pC13N-dCas9-BFP-KRAB
FEATURES             Location/Qualifiers
     source          1..15390
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(9..42)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     enhancer        113..416
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        418..695
                     /label=chicken beta-actin promoter
     intron          696..1712
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1636..1658
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1720..1739
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1779..1788
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1785..1811,1812..1814,1815..4301,4302..4304,
                     4305..5888)
                     /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             1785..5888
                     /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             5892..5918
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             5898..5924
                     /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             5937..5957
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5964..5984
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6024..6719
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     CDS             6765..6950
                     /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(7043..7062)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    7108..7163
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(7162..7181)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     protein_bind    7496..7529
                     /label=loxP511
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATAC) (Shaw et al., 2021). loxP511 sites are 
                     compatible with each other, but incompatible with wild-type
                     loxP sites."
     primer_bind     complement(9585..9602)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(9585..9601)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9594..9616)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(9810..9829)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     9929..9951
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(10082..10101)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             10093..10902
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(10160..10179)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      11080..11668
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11569..11588
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11822..11839
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11956..11977
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(11992..11997,11998..12015,12016..12022)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12030..12046
                     /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)."
     primer_bind     12035..12057
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     12054..12070
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     12054..12070
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    complement(12931..12964)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    12965..12990
                     /label=SA
                     /note="splice acceptor site"
     CDS             13014..13067
                     /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             13062..13862
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="GPRIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQG
                     RPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL
                     LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEE
                     HQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQD
                     IALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(13122..13141)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     13732..13751
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    13897..14121
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(14122..14155)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
ORIGIN
        1 cggtcgacat aacttcgtat aggatacttt atacgaagtt atcaattcag gctagttatt
       61 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
      121 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
      181 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
      241 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
      301 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct
      361 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtcg
      421 aggtgagccc cacgttctgc ttcactctcc ccatctcccc cccctcccca cccccaattt
      481 tgtatttatt tattttttaa ttattttgtg cagcgatggg ggcggggggg gggggggggc
      541 gcgcgccagg cggggcgggg cggggcgagg ggcggggcgg ggcgaggcgg agaggtgcgg
      601 cggcagccaa tcagagcggc gcgctccgaa agtttccttt tatggcgagg cggcggcggc
      661 ggcggcccta taaaaagcga agcgcgcggc gggcgggagt cgctgcgcgc tgccttcgcc
      721 ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg accgcgttac
      781 tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag cgcttggttt
      841 aatgacggct tgtttctttt ctgtggctgc gtgaaagcct tgaggggctc cgggagggcc
      901 ctttgtgcgg ggggagcggc tcggggggtg cgtgcgtgtg tgtgtgcgtg gggagcgccg
      961 cgtgcggctc cgcgctgccc ggcggctgtg agcgctgcgg gcgcggcgcg gggctttgtg
     1021 cgctccgcag tgtgcgcgag gggagcgcgg ccgggggcgg tgccccgcgg tgcggggggg
     1081 gctgcgaggg gaacaaaggc tgcgtgcggg gtgtgtgcgt gggggggtga gcagggggtg
     1141 tgggcgcgtc ggtcgggctg caaccccccc tgcacccccc tccccgagtt gctgagcacg
     1201 gcccggcttc gggtgcgggg ctccgtacgg ggcgtggcgc ggggctcgcc gtgccgggcg
     1261 gggggtggcg gcaggtgggg gtgccgggcg gggcggggcc gcctcgggcc ggggagggct
     1321 cgggggaggg gcgcggcggc ccccggagcg ccggcggctg tcgaggcgcg gcgagccgca
     1381 gccattgcct tttatggtaa tcgtgcgaga gggcgcaggg acttcctttg tcccaaatct
     1441 gtgcggagcc gaaatctggg aggcgccgcc gcaccccctc tagcgggcgc ggggcgaagc
     1501 ggtgcggcgc cggcaggaag gaaatgggcg gggagggcct tcgtgcgtcg ccgcgccgcc
     1561 gtccccttct ccctctccag cctcggggct gtccgcgggg ggacggctgc cttcgggggg
     1621 gacggggcag ggcggggttc ggcttctggc gtgtgaccgg cggctctaga gcctctgcta
     1681 accatgttca tgccttcttc tttttcctac agctcctggg caacgtgctg gttattgtgc
     1741 tgtctcatca ttttggcaaa gaattgtgta caactagtgc caccatggac aagaagtaca
     1801 gcatcggcct ggccatcggc accaactctg tgggctgggc cgtgatcacc gacgagtaca
     1861 aggtgcccag caagaaattc aaggtgctgg gcaacaccga ccggcacagc atcaagaaga
     1921 acctgatcgg cgccctgctg ttcgacagcg gagaaacagc cgaggccacc cggctgaaga
     1981 gaaccgccag aagaagatac accagacgga agaaccggat ctgctatctg caagagatct
     2041 tcagcaacga gatggccaag gtggacgaca gcttcttcca cagactggaa gagtccttcc
     2101 tggtggaaga ggataagaag cacgagcggc accccatctt cggcaacatc gtggacgagg
     2161 tggcctacca cgagaagtac cccaccatct accacctgag aaagaaactg gtggacagca
     2221 ccgacaaggc cgacctgcgg ctgatctatc tggccctggc ccacatgatc aagttccggg
     2281 gccacttcct gatcgagggc gacctgaacc ccgacaacag cgacgtggac aagctgttca
     2341 tccagctggt gcagacctac aaccagctgt tcgaggaaaa ccccatcaac gccagcggcg
     2401 tggacgccaa ggccatcctg tctgccagac tgagcaagag cagacggctg gaaaatctga
     2461 tcgcccagct gcccggcgag aagaagaatg gcctgttcgg caacctgatt gccctgagcc
     2521 tgggcctgac ccccaacttc aagagcaact tcgacctggc cgaggatgcc aaactgcagc
     2581 tgagcaagga cacctacgac gacgacctgg acaacctgct ggcccagatc ggcgaccagt
     2641 acgccgacct gtttctggcc gccaagaacc tgtccgacgc catcctgctg agcgacatcc
     2701 tgagagtgaa caccgagatc accaaggccc ccctgagcgc ctctatgatc aagagatacg
     2761 acgagcacca ccaggacctg accctgctga aagctctcgt gcggcagcag ctgcctgaga
     2821 agtacaaaga gattttcttc gaccagagca agaacggcta cgccggctac atcgatggcg
     2881 gagccagcca ggaagagttc tacaagttca tcaagcccat cctggaaaag atggacggca
     2941 ccgaggaact gctcgtgaag ctgaacagag aggacctgct gcggaagcag cggaccttcg
     3001 acaacggcag catcccccac cagatccacc tgggagagct gcacgccatt ctgcggcggc
     3061 aggaagattt ttacccattc ctgaaggaca accgggaaaa gatcgagaag atcctgacct
     3121 tccgcatccc ctactacgtg ggccctctgg ccaggggaaa cagcagattc gcctggatga
     3181 ccagaaagag cgaggaaacc atcaccccct ggaacttcga ggaagtggtg gacaagggcg
     3241 ccagcgccca gagcttcatc gagcggatga ccaacttcga taagaacctg cccaacgaga
     3301 aggtgctgcc caagcacagc ctgctgtacg agtacttcac cgtgtacaac gagctgacca
     3361 aagtgaaata cgtgaccgag ggaatgagaa agcccgcctt cctgagcggc gagcagaaaa
     3421 aagccatcgt ggacctgctg ttcaagacca accggaaagt gaccgtgaag cagctgaaag
     3481 aggactactt caagaaaatc gagtgcttcg actccgtgga aatctccggc gtggaagatc
     3541 ggttcaacgc ctccctgggc acataccacg atctgctgaa aattatcaag gacaaggact
     3601 tcctggacaa tgaggaaaac gaggacattc tggaagatat cgtgctgacc ctgacactgt
     3661 ttgaggacag agagatgatc gaggaacggc tgaaaaccta tgcccacctg ttcgacgaca
     3721 aagtgatgaa gcagctgaag cggcggagat acaccggctg gggcaggctg agccggaagc
     3781 tgatcaacgg catccgggac aagcagtccg gcaagacaat cctggatttc ctgaagtccg
     3841 acggcttcgc caacagaaac ttcatgcagc tgatccacga cgacagcctg acctttaaag
     3901 aggacatcca gaaagcccag gtgtccggcc agggcgatag cctgcacgag cacattgcca
     3961 atctggccgg cagccccgcc attaagaagg gcatcctgca gacagtgaag gtggtggacg
     4021 agctcgtgaa agtgatgggc cggcacaagc ccgagaacat cgtgatcgaa atggccagag
     4081 agaaccagac cacccagaag ggacagaaga acagccgcga gagaatgaag cggatcgaag
     4141 agggcatcaa agagctgggc agccagatcc tgaaagaaca ccccgtggaa aacacccagc
     4201 tgcagaacga gaagctgtac ctgtactacc tgcagaatgg gcgggatatg tacgtggacc
     4261 aggaactgga catcaaccgg ctgtccgact acgatgtgga cgctatcgtg cctcagagct
     4321 ttctgaagga cgactccatc gataacaaag tgctgactcg gagcgacaag aaccggggca
     4381 agagcgacaa cgtgccctcc gaagaggtcg tgaagaagat gaagaactac tggcgccagc
     4441 tgctgaatgc caagctgatt acccagagga agttcgacaa tctgaccaag gccgagagag
     4501 gcggcctgag cgaactggat aaggccggct tcatcaagag acagctggtg gaaacccggc
     4561 agatcacaaa gcacgtggca cagatcctgg actcccggat gaacactaag tacgacgaga
     4621 acgacaaact gatccgggaa gtgaaagtga tcaccctgaa gtccaagctg gtgtccgatt
     4681 tccggaagga tttccagttt tacaaagtgc gcgagatcaa caactaccac cacgcccacg
     4741 acgcctacct gaacgccgtc gtgggaaccg ccctgatcaa aaagtaccct aagctggaaa
     4801 gcgagttcgt gtacggcgac tacaaggtgt acgacgtgcg gaagatgatc gccaagagcg
     4861 agcaggaaat cggcaaggct accgccaagt acttcttcta cagcaacatc atgaactttt
     4921 tcaagaccga gattaccctg gccaacggcg agatccggaa gcggcctctg atcgagacaa
     4981 acggcgaaac aggcgagatc gtgtgggata agggccggga ctttgccacc gtgcggaaag
     5041 tgctgtctat gccccaagtg aatatcgtga aaaagaccga ggtgcagaca ggcggcttca
     5101 gcaaagagtc tatcctgccc aagaggaaca gcgacaagct gatcgccaga aagaaggact
     5161 gggaccctaa gaagtacggc ggcttcgaca gccccaccgt ggcctattct gtgctggtgg
     5221 tggccaaagt ggaaaagggc aagtccaaga aactgaagag tgtgaaagag ctgctgggga
     5281 tcaccatcat ggaaagaagc agcttcgaga agaatcccat cgactttctg gaagccaagg
     5341 gctacaaaga agtgaaaaag gacctgatca tcaagctgcc taagtactcc ctgttcgagc
     5401 tggaaaacgg ccggaagaga atgctggcct ctgccggcga actgcagaag ggaaacgaac
     5461 tggccctgcc ctccaaatat gtgaacttcc tgtacctggc cagccactat gagaagctga
     5521 agggctcccc cgaggataat gagcagaaac agctgtttgt ggaacagcac aaacactacc
     5581 tggacgagat catcgagcag atcagcgagt tctccaagag agtgatcctg gccgacgcta
     5641 atctggacaa ggtgctgagc gcctacaaca agcacagaga caagcctatc agagagcagg
     5701 ccgagaatat catccacctg tttaccctga ccaatctggg agcccctgcc gccttcaagt
     5761 actttgacac caccatcgac cggaagaggt acaccagcac caaagaggtg ctggacgcca
     5821 ccctgatcca ccagagcatc accggcctgt acgagacacg gatcgacctg tctcagctgg
     5881 gaggcgacgc ctatccctat gacgtgcccg attatgccag cctgggcagc ggctccccca
     5941 agaaaaaacg caaggtggaa gatcctaaga aaaagcggaa agtggacggc attggtagtg
     6001 ggagcaacgg cagcagcgga tccagcgagc tgattaagga gaacatgcac atgaagctgt
     6061 acatggaggg caccgtggac aaccatcact tcaagtgcac atccgagggc gaaggcaagc
     6121 cctacgaggg cacccagacc atgagaatca aggtggtcga gggcggccct ctccccttcg
     6181 ccttcgacat cctggctact agcttcctct acggcagcaa gaccttcatc aaccacaccc
     6241 agggcatccc cgacttcttc aagcagtcct tccctgaggg cttcacatgg gagagagtca
     6301 ccacatacga agacgggggc gtgctgaccg ctacccagga caccagcctc caggacggct
     6361 gcctcatcta caacgtcaag atcagagggg tgaacttcac atccaacggc cctgtgatgc
     6421 agaagaaaac actcggctgg gaggccttca ccgagacgct gtaccccgct gacggcggcc
     6481 tggaaggcag aaacgacatg gccctgaagc tcgtgggcgg gagccatctg atcgcaaaca
     6541 tcaagaccac atatagatcc aagaaacccg ctaagaacct caagatgcct ggcgtctact
     6601 atgtggacta cagactggaa agaatcaagg aggccaacaa cgagacctac gtcgagcagc
     6661 acgaggtggc agtggccaga tactgcgacc tccctagcaa actggggcac aagcttaatg
     6721 gcggtggcgg agggatggat gctaagtcac taactgcctg gtcccggaca ctggtgacct
     6781 tcaaggatgt atttgtggac ttcaccaggg aggagtggaa gctgctggac actgctcagc
     6841 agatcgtgta cagaaatgtg atgctggaga actataagaa cctggtttcc ttgggttatc
     6901 agcttactaa gccagatgtg atcctccggt tggagaaggg agaagagccc tagacgcgtg
     6961 aattcactcc tcaggtgcag gctgcctatc agaaggtggt ggctggtgtg gccaatgccc
     7021 tggctcacaa ataccactga gatctttttc cctctgccaa aaattatggg gacatcatga
     7081 agccccttga gcatctgact tctggctaat aaaggaaatt tattttcatt gcaatagtgt
     7141 gttggaattt tttgtgtctc tcactcggaa ggacatatgg gagggcaaat catttaaaac
     7201 atcagaatga gtatttggtt tagagtttgg caacatatgc ccatatgctg gctgccatga
     7261 acaaaggttg gctataaaga ggtcatcagt atatgaaaca gccccctgct gtccattcct
     7321 tattccatag aaaagccttg acttgaggtt agattttttt tatattttgt tttgtgttat
     7381 ttttttcttt aacatcccta aaattttcct tacatgtttt actagccaga tttttcctcc
     7441 tctcctgact actcccagtc atagctgtcc ctcttctctt atggagatca ccggtataac
     7501 ttcgtataat gtatactata cgaagttatg gtaccttaat taagttaaca tgcatagtac
     7561 tgagctcacg gggacagccc ccccccaaag cccccaggga tgtaattacg tccctccccc
     7621 gctagggggc agcagcgagc cgcccggggc tccgctccgg tccggcgctc cccccgcatc
     7681 cccgagccgg cagcgtgcgg ggacagcccg ggcacgggga aggtggcacg ggatcgcttt
     7741 cctctgaacg cttctcgctg ctctttgagc ctgcagacac ctggggggat acggggaaaa
     7801 agctttaggc tgaaagagag atttagaatg acagaatcat agaacggcct gggttgcaaa
     7861 ggagcacagt gctcatccag atccaacccc ctgctatgtg cagggtcatc aaccagcagc
     7921 ccaggctgcc cagagccaca tccagcctgg ccttgaatgc ctgcagggat ggggcatcca
     7981 cagcctcctt gggcaacctg ttcagtgcgt caccaccctc tgggggaaaa actgcctcct
     8041 catatccaac ccaaacctcc cctgtctcag tgtaaagcca ttcccccttg tcctatcaag
     8101 ggggagtttg ctgtgacatt gttggtctgg ggtgacacat gtttgccaat tcagtgcatc
     8161 acggagaggc agatcttggg gataaggaag tgcaggacag catggacgtg ggacatgcag
     8221 gtgttgaggg ctctgggaca ctctccaagt cacagcgttc agaacagcct taaggataag
     8281 aagataggat agaaggacaa agagcaagtt aaaacccagc atggagagga gcacaaaaag
     8341 gccacagaca ctgctggtcc ctgtgtctga gcctgcatgt ttgatggtgt ctggatgcaa
     8401 gcagaagggg tggaagagct tgcctggaga gatacagctg ggtcagtagg actgggacag
     8461 gcagctggag aattgccatg tagatgttca tacaatcgtc aaatcatgaa ggctggaaaa
     8521 gccctccaag atccccaaga ccaaccccaa cccacccacc gtgcccactg gccatgtccc
     8581 tcagtgccac atccccacag ttcttcatca cctccaggga cggtgacccc cccacctccg
     8641 tgggcagctg tgccactgca gcaccgctct ttggagaagg taaatcttgc taaatccagc
     8701 ccgaccctcc cctggcacaa cgtaaggcca ttatctctca tccaactcca ggacggagtc
     8761 agtgagaata ttgtttaaac tcctcatcct tccaacataa atatattttg ggattatatc
     8821 aacattcaat gttacttaaa gtgaccttgt aaatattttc acaactgagc catgtttgat
     8881 ttgtatactt atgtttactt tactgttttt cctgaagtta ataattgcct tgaatttatt
     8941 tatttcttta aaaatgtttc attactcagg actgtagttt acattacgat tctttgtgtt
     9001 atacagttga tgggtttctt ttctttctta atttctttaa aaaatagaga tggggtctta
     9061 ctatattacc caggctggtc ttgaagtcct gggctcaagt gatcttcctg tctcagccta
     9121 ccaagtagct gagactatag gtgcaaaaaa gccactatac ctggctagtt tacaggtttt
     9181 aacaaatgca ttatgccacg tatccattat tacaggatca cacaagatat tttcattacc
     9241 ctgaaagcat ccctgtgttc caccaattca tcctgcctcc atgagccgct ggcaaccact
     9301 gatctctata gttttgcctt ttctaaaatg tcatataatt ggaatcatac agtctgtagc
     9361 attttcagac tagcttttaa aatttggcaa tatgcattta aggttcctcc ttaaatgtga
     9421 agggcatagc caatgtggct tgatagctca tttcttttta ttggtgaata tttcattgtc
     9481 tggatgttcc acagtttgtt tatccattca cctattcaat ttgctttttt tctgtgtatc
     9541 tatcactaat tcaatactgg actctccaac agagccgttt aaacactggc cgtcgtttta
     9601 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc
     9661 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg
     9721 cgcagcctga atggcgaatg gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     9781 atttcacacc gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     9841 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     9901 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     9961 tcatcaccga aacgcgcgat gcagctctgg cccgtgtctc aaaatctctg atgttacatt
    10021 gcacaagata aaaatatatc atcatgaaca ataaaactgt ctgcttacat aaacagtaat
    10081 acaaggggtg ttatgagcca tattcaacgg gaaacgtcga ggccgcgatt aaattccaac
    10141 atggatgctg atttatatgg gtataaatgg gctcgcgata atgtcgggca atcaggtgcg
    10201 acaatctatc gcttgtatgg gaagcccgat gcgccagagt tgtttctgaa acatggcaaa
    10261 ggtagcgttg ccaatgatgt tacagatgag atggtcagac taaactggct gacggaattt
    10321 atgcctcttc cgaccatcaa gcattttatc cgtactcctg atgatgcatg gttactcacc
    10381 actgcgatcc ccggaaaaac agcattccag gtattagaag aatatcctga ttcaggtgaa
    10441 aatattgttg atgcgctggc agtgttcctg cgccggttgc attcgattcc tgtttgtaat
    10501 tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac
    10561 ggtttggttg atgcgagtga ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc
    10621 tggaaagaaa tgcataaact tttgccattc tcaccggatt cagtcgtcac tcatggtgat
    10681 ttctcacttg ataaccttat ttttgacgag gggaaattaa taggttgtat tgatgttgga
    10741 cgagtcggaa tcgcagaccg ataccaggat cttgccatcc tatggaactg cctcggtgag
    10801 ttttctcctt cattacagaa acggcttttt caaaaatatg gtattgataa tcctgatatg
    10861 aataaattgc agtttcattt gatgctcgat gagtttttct aatcagaatt ggttaattgg
    10921 ttgtaacatt attcagattg ggcttgattt aaaacttcat ttttaattta aaaggatcta
    10981 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
    11041 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
    11101 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
    11161 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
    11221 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
    11281 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
    11341 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
    11401 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
    11461 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
    11521 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
    11581 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
    11641 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
    11701 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
    11761 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
    11821 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc
    11881 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
    11941 tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt
    12001 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
    12061 cagctatgac catgattacg ccgcggccgc agcaggagaa tcatgccaat gggccaatat
    12121 acattctgac ccacagtttc ataataaaat aaaatggttg tggttgtaag ccactatgtt
    12181 tcagagtggt ttgttacaca gcaataaata actaatatag taggcatacc atcaagtcca
    12241 aagtaggtag agaagaatgt aaatagcaga gcaaaacagc atgactggtg gctgggaggc
    12301 ttaaaactgg gacaggatca gagtcatgaa agaagtcaaa gaaatggttc agaagtaagg
    12361 ctgagactga cttacaaaag ctgaaagtcc ctttaagttg gtgtttggtg cattggcagg
    12421 ggcaggtatg gtgacttaaa agagccatgc tcaacaagat caagcacaac acaatcacgg
    12481 gtcaccccag cagaccttag cgagtctagc catttctttg gtggtggtca cagtcatgct
    12541 tcagcccagt ttccacttgg acaaatggta catattttca atgagatgaa aattaagata
    12601 caatccatgt gctcagagag tgatcacagc tctgactaaa cactgtgccc caaagtgttg
    12661 aggaattggg aaaacctagc tgagttagtg gtctcttttc tgttacaata aagctcataa
    12721 tgaaaattag ccttctttgt tcttccccaa gtctttcttt ctagacgaaa ctactttcaa
    12781 ctgttttaac ttccttactg ttaacttcca tatttctaga tagtatggtc agactgttat
    12841 ttcttgactt ttaaatgtaa gatattatct actgacttcc ttctatgtaa gatgaggatt
    12901 gagctctctt acccttctcc cagcggccgc ataacttcgt atagcataca ttatacgaag
    12961 ttatctgacc tcttctcttc ctcccacagg gcctcgagag atctggcagc ggagagggca
    13021 gaggaagtct tctaacatgc ggtgacgtgg aggagaatcc cggccctagg attgaacaag
    13081 atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg
    13141 cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc
    13201 cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag
    13261 cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca
    13321 ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat
    13381 ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata
    13441 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac
    13501 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc
    13561 tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg
    13621 tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg
    13681 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta
    13741 cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg
    13801 gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct
    13861 aacacctggt gcatgacccg caagcccggt gcctgactgt gccttctagt tgccagccat
    13921 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
    13981 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
    14041 ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg
    14101 gggatgcggt gggctctatg gataacttcg tatagcatac attatacgaa gttatgagct
    14161 cacggggaca gccccccccc aaagccccca gggatgtaat tacgtccctc ccccgctagg
    14221 gggcagcagc gagccgcccg gggctccgct ccggtccggc gctccccccg catccccgag
    14281 ccggcagcgt gcggggacag cccgggcacg gggaaggtgg cacgggatcg ctttcctctg
    14341 aacgcttctc gctgctcttt gagcctgcag acacctgggg ggatacgggg aaaaagcttt
    14401 aggctgaaag agagatttag aatgacagaa tcatagaacg gcctgggttg caaaggagca
    14461 cagtgctcat ccagatccaa ccccctgcta tgtgcagggt catcaaccag cagcccaggc
    14521 tgcccagagc cacatccagc ctggccttga atgcctgcag ggatggggca tccacagcct
    14581 ccttgggcaa cctgttcagt gcgtcaccac cctctggggg aaaaactgcc tcctcatatc
    14641 caacccaaac ctcccctgtc tcagtgtaaa gccattcccc cttgtcctat caagggggag
    14701 tttgctgtga cattgttggt ctggggtgac acatgtttgc caattcagtg catcacggag
    14761 aggcagatct tggggataag gaagtgcagg acagcatgga cgtgggacat gcaggtgttg
    14821 agggctctgg gacactctcc aagtcacagc gttcagaaca gccttaagga taagaagata
    14881 ggatagaagg acaaagagca agttaaaacc cagcatggag aggagcacaa aaaggccaca
    14941 gacactgctg gtccctgtgt ctgagcctgc atgtttgatg gtgtctggat gcaagcagaa
    15001 ggggtggaag agcttgcctg gagagataca gctgggtcag taggactggg acaggcagct
    15061 ggagaattgc catgtagatg ttcatacaat cgtcaaatca tgaaggctgg aaaagccctc
    15121 caagatcccc aagaccaacc ccaacccacc caccgtgccc actggccatg tccctcagtg
    15181 ccacatcccc acagttcttc atcacctcca gggacggtga cccccccacc tccgtgggca
    15241 gctgtgccac tgcagcaccg ctctttggag aaggtaaatc ttgctaaatc cagcccgacc
    15301 ctcccctggc acaacgtaag gccattatct ctcatccaac tccaggacgg agtcagtgag
    15361 aatattggcg cgccgaattc tcgcgacgat
//
