LOCUS       sequence-252312-       13239 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses dCas9-hEzh2-IRES-Hyg in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13239)
  AUTHORS   Ochiai H, Hayashi T, Umeda M, Yoshimura M, Harada A, Shimizu Y, 
            Nakano K, Saitoh N, Liu Z, Yamamoto T, Okamura T, Ohkawa Y, Kimura 
            H, Nikaido I
  TITLE     Genome-wide kinetic properties of transcriptional bursting in mouse 
            embryonic stem cells.
  JOURNAL   Sci Adv. 2020 Jun 17;6(25):eaaz6699. doi: 10.1126/sciadv.aaz6699. 
            eCollection 2020 Jun.
  PUBMED    32596448
REFERENCE   2  (bases 1 to 13239)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Sci Adv."; 
            date: "2020-06-17"; volume: "6(25):eaaz6699. doi"; pages: " 
            10.1126/sciadv.aaz6699. eCollection 2020 Jun"
COMMENT     Sequence Label: pLR5-CBh-dCas9-hEzh2-IRES-Hyg
FEATURES             Location/Qualifiers
     source          1..13239
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          110..337
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             358..384
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             388..408
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(418..441,442..444,445..3000,3001..3003,3004..4518)
                     /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 N863A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             join(418..441,442..444,445..2931,2932..2934,2935..4518)
                     /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="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             4522..4542
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     intron          7043..7272
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     7255..7274
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    join(7345..7919,7920..7922,7923..7931)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(7512..7529)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     7739..7758
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             7954..8979
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             7954..8979
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     polyA_signal    9107..9228
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(9144..9163)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   9486..9548
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(9599..9619)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(9599..9617)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(9638..9654)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9638..9654)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(9651..9673)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    9662..9678
                     /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(9686..9692,9693..9710,9711..9716))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9731..9752
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(9869..9886)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10040..10628)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10120..10139)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(10799..11590,11591..11659))
                     /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     11422..11441
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(11660..11764)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(11790..12245)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11927..11948)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     12139..12158
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     12372..12394
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     12386..12403
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     12387..12403
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     12413..12432
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        12413..12431
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   12461..12495
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     enhancer        12784..13069
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        join(13071..13239,1..109)
                     /label=chicken beta-actin promoter
ORIGIN
        1 gcggagaggt gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc cttttatggc
       61 gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg gagtcgctgc
      121 gcgctgcctt cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc cccggctctg
      181 actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc cgggctgtaa
      241 ttagctgagc aagaggtaag ggtttaaggg atggttggtt ggtggggtat taatgtttaa
      301 ttacctggag cacctgcctg aaatcacttt ttttcaggtt ggaccggtgc caccatgtac
      361 ccatacgatg ttccagatta cgcttcgccg aagaaaaagc gcaaggtcga agcgtccgac
      421 aagaagtaca gcatcggcct ggccatcggc accaactctg tgggctgggc cgtgatcacc
      481 gacgagtaca aggtgcccag caagaaattc aaggtgctgg gcaacaccga ccggcacagc
      541 atcaagaaga acctgatcgg agccctgctg ttcgacagcg gcgaaacagc cgaggccacc
      601 cggctgaaga gaaccgccag aagaagatac accagacgga agaaccggat ctgctatctg
      661 caagagatct tcagcaacga gatggccaag gtggacgaca gcttcttcca cagactggaa
      721 gagtccttcc tggtggaaga ggataagaag cacgagcggc accccatctt cggcaacatc
      781 gtggacgagg tggcctacca cgagaagtac cccaccatct accacctgag aaagaaactg
      841 gtggacagca ccgacaaggc cgacctgcgg ctgatctatc tggccctggc ccacatgatc
      901 aagttccggg gccacttcct gatcgagggc gacctgaacc ccgacaacag cgacgtggac
      961 aagctgttca tccagctggt gcagacctac aaccagctgt tcgaggaaaa ccccatcaac
     1021 gccagcggcg tggacgccaa ggccatcctg tctgccagac tgagcaagag cagacggctg
     1081 gaaaatctga tcgcccagct gcccggcgag aagaagaatg gcctgttcgg caacctgatt
     1141 gccctgagcc tgggcctgac ccccaacttc aagagcaact tcgacctggc cgaggatgcc
     1201 aaactgcagc tgagcaagga cacctacgac gacgacctgg acaacctgct ggcccagatc
     1261 ggcgaccagt acgccgacct gtttctggcc gccaagaacc tgtccgacgc catcctgctg
     1321 agcgacatcc tgagagtgaa caccgagatc accaaggccc ccctgagcgc ctctatgatc
     1381 aagagatacg acgagcacca ccaggacctg accctgctga aagctctcgt gcggcagcag
     1441 ctgcctgaga agtacaaaga gattttcttc gaccagagca agaacggcta cgccggctac
     1501 attgacggcg gagccagcca ggaagagttc tacaagttca tcaagcccat cctggaaaag
     1561 atggacggca ccgaggaact gctcgtgaag ctgaacagag aggacctgct gcggaagcag
     1621 cggaccttcg acaacggcag catcccccac cagatccacc tgggagagct gcacgccatt
     1681 ctgcggcggc aggaagattt ttacccattc ctgaaggaca accgggaaaa gatcgagaag
     1741 atcctgacct tccgcatccc ctactacgtg ggccctctgg ccaggggaaa cagcagattc
     1801 gcctggatga ccagaaagag cgaggaaacc atcaccccct ggaacttcga ggaagtggtg
     1861 gacaagggcg cttcagccca gagcttcatc gagcggatga ccaacttcga taagaacctg
     1921 cccaacgaga aggtgctgcc caagcacagc ctgctgtacg agtacttcac cgtgtataac
     1981 gagctgacca aagtgaaata cgtgaccgag ggaatgagaa agcccgcctt cctgagcggc
     2041 gagcagaaaa aggccatcgt ggacctgctg ttcaagacca accggaaagt gaccgtgaag
     2101 cagctgaaag aggactactt caagaaaatc gagtgcttcg actccgtgga aatctccggc
     2161 gtggaagatc ggttcaacgc ctccctgggc acataccacg atctgctgaa aattatcaag
     2221 gacaaggact tcctggacaa tgaggaaaac gaggacattc tggaagatat cgtgctgacc
     2281 ctgacactgt ttgaggacag agagatgatc gaggaacggc tgaaaaccta tgcccacctg
     2341 ttcgacgaca aagtgatgaa gcagctgaag cggcggagat acaccggctg gggcaggctg
     2401 agccggaagc tgatcaacgg catccgggac aagcagtccg gcaagacaat cctggatttc
     2461 ctgaagtccg acggcttcgc caacagaaac ttcatgcagc tgatccacga cgacagcctg
     2521 acctttaaag aggacatcca gaaagcccag gtgtccggcc agggcgatag cctgcacgag
     2581 cacattgcca atctggccgg cagccccgcc attaagaagg gcatcctgca gacagtgaag
     2641 gtggtggacg agctcgtgaa agtgatgggc cggcacaagc ccgagaacat cgtgatcgaa
     2701 atggccagag agaaccaaac cacccagaag ggacagaaga acagccgcga gagaatgaag
     2761 cggatcgaag agggcatcaa agagctgggc agccagatcc tgaaagaaca ccccgtggaa
     2821 aacacccagc tgcagaacga gaagctgtac ctgtactacc tgcagaatgg gcgggatatg
     2881 tacgtggacc aggaactgga catcaaccgg ctgtccgact acgatgtgga cgccatcgtg
     2941 cctcagagct ttctgaagga cgactccatc gacaacaagg tgctgaccag aagcgacaag
     3001 aaccggggca agagcgacaa cgtgccctcc gaagaggtcg tgaagaagat gaagaactac
     3061 tggcggcagc tgctgaacgc caagctgatt acccagagaa agttcgacaa tctgaccaag
     3121 gccgagagag gcggcctgag cgaactggat aaggccggct tcatcaagag acagctggtg
     3181 gaaacccggc agatcacaaa gcacgtggca cagatcctgg actcccggat gaacactaag
     3241 tacgacgaga atgacaagct gatccgggaa gtgaaagtga tcaccctgaa gtccaagctg
     3301 gtgtccgatt tccggaagga tttccagttt tacaaagtgc gcgagatcaa caactaccac
     3361 cacgcccacg acgcctacct gaacgccgtc gtgggaaccg ccctgatcaa aaagtaccct
     3421 aagctggaaa gcgagttcgt gtacggcgac tacaaggtgt acgacgtgcg gaagatgatc
     3481 gccaagagcg agcaggaaat cggcaaggct accgccaagt acttcttcta cagcaacatc
     3541 atgaactttt tcaagaccga gattaccctg gccaacggcg agatccggaa gcggcctctg
     3601 atcgagacaa acggcgaaac cggggagatc gtgtgggata agggccggga ttttgccacc
     3661 gtgcggaaag tgctgagcat gccccaagtg aatatcgtga aaaagaccga ggtgcagaca
     3721 ggcggcttca gcaaagagtc tatcctgccc aagaggaaca gcgataagct gatcgccaga
     3781 aagaaggact gggaccctaa gaagtacggc ggcttcgaca gccccaccgt ggcctattct
     3841 gtgctggtgg tggccaaagt ggaaaagggc aagtccaaga aactgaagag tgtgaaagag
     3901 ctgctgggga tcaccatcat ggaaagaagc agcttcgaga agaatcccat cgactttctg
     3961 gaagccaagg gctacaaaga agtgaaaaag gacctgatca tcaagctgcc taagtactcc
     4021 ctgttcgagc tggaaaacgg ccggaagaga atgctggcct ctgccggcga actgcagaag
     4081 ggaaacgaac tggccctgcc ctccaaatat gtgaacttcc tgtacctggc cagccactat
     4141 gagaagctga agggctcccc cgaggataat gagcagaaac agctgtttgt ggaacagcac
     4201 aagcactacc tggacgagat catcgagcag atcagcgagt tctccaagag agtgatcctg
     4261 gccgacgcta atctggacaa agtgctgtcc gcctacaaca agcaccggga taagcccatc
     4321 agagagcagg ccgagaatat catccacctg tttaccctga ccaatctggg agcccctgcc
     4381 gccttcaagt actttgacac caccatcgac cggaagaggt acaccagcac caaagaggtg
     4441 ctggacgcca ccctgatcca ccagagcatc accggcctgt acgagacacg gatcgacctg
     4501 tctcagctgg gaggcgacag ccccaagaag aagagaaagg tggaggccag cctctcgagg
     4561 ccggaccccg cgctggccgc actcggaggc ggcggaagcg gtggcggagg gtcaggcgga
     4621 ggtggatcca tgaaatcctg cggagtgatg ggccagactg ggaagaaatc tgagaaggga
     4681 ccagtttgtt ggcggaagcg tgtaaaatca gagtacatgc gactgagaca gctcaagagg
     4741 ttcagacgag ctgatgaagt aaagagtatg tttagttcca atcgtcagaa aattttggaa
     4801 agaacggaaa tcttaaacca agaatggaaa cagcgaagga tacagcctgt gcacatcctg
     4861 acttctgtga gctcattgcg cgggactagg gagtgttcgg tgaccagtga cttggatttt
     4921 ccaacacaag tcatcccatt aaagactctg aatgcagttg cttcagtacc cataatgtat
     4981 tcttggtctc ccctacagca gaattttatg gtggaagatg aaactgtttt acataacatt
     5041 ccttatatgg gagatgaagt tttagatcag gatggtactt tcattgaaga actaataaaa
     5101 aattatgatg ggaaagtaca cggggataga gaatgtgggt ttataaatga tgaaattttt
     5161 gtggagttgg tgaatgccct tggtcaatat aatgatgatg acgatgatga tgatggagac
     5221 gatcctgaag aaagagaaga aaagcagaaa gatctggagg atcaccgaga tgataaagaa
     5281 agccgcccac ctcggaaatt tccttctgat aaaatttttg aagccatttc ctcaatgttt
     5341 ccagataagg gcacagcaga agaactaaag gaaaaatata aagaactcac cgaacagcag
     5401 ctcccaggcg cacttcctcc tgaatgtacc cccaacatag atggaccaaa tgctaaatct
     5461 gttcagagag agcaaagctt acactccttt catacgcttt tctgtaggcg atgttttaaa
     5521 tatgactgct tcctacatcg taagtgcaat tattcttttc atgcaacacc caacacttat
     5581 aagcggaaga acacagaaac agctctagac aacaaacctt gtggaccaca gtgttaccag
     5641 catttggagg gagcaaagga gtttgctgct gctctcaccg ctgagcggat aaagacccca
     5701 ccaaaacgtc caggaggccg cagaagagga cggcttccca ataacagtag caggcccagc
     5761 acccccacca ttaatgtgct ggaatcaaag gatacagaca gtgataggga agcagggact
     5821 gaaacggggg gagagaacaa tgataaagaa gaagaagaga agaaagatga aacttcgagc
     5881 tcctctgaag caaattctcg gtgtcaaaca ccaataaaga tgaagccaaa tattgaacct
     5941 cctgagaatg tggagtggag tggtgctgaa gcctcaatgt ttagagtcct cattggcact
     6001 tactatgaca atttctgtgc cattgctagg ttaattggga ccaaaacatg tagacaggtg
     6061 tatgagttta gagtcaaaga atctagcatc atagctccag ctcccgctga ggatgtggat
     6121 actcctccaa ggaaaaagaa gaggaaacac cggttgtggg ctgcacactg cagaaagata
     6181 cagctgaaaa aggacggctc ctctaaccat gtttacaact atcaaccctg tgatcatcca
     6241 cggcagcctt gtgacagttc gtgcccttgt gtgatagcac aaaatttttg tgaaaagttt
     6301 tgtcaatgta gttcagagtg tcaaaaccgc tttccgggat gccgctgcaa agcacagtgc
     6361 aacaccaagc agtgcccgtg ctacctggct gtccgagagt gtgaccctga cctctgtctt
     6421 acttgtggag ccgctgacca ttgggacagt aaaaatgtgt cctgcaagaa ctgcagtatt
     6481 cagcggggct ccaaaaagca tctattgctg gcaccatctg acgtggcagg ctgggggatt
     6541 tttatcaaag atcctgtgca gaaaaatgaa ttcatctcag aatactgtgg agagattatt
     6601 tctcaagatg aagctgacag aagagggaaa gtgtatgata aatacatgtg cagctttctg
     6661 ttcaacttga acaatgattt tgtggtggat gcaacccgca agggtaacaa aattcgtttt
     6721 gcaaatcatt cggtaaatcc aaactgctat gcaaaagtta tgatggttaa cggtgatcac
     6781 aggataggta tttttgccaa gagagccatc cagactggcg aagagctgtt ttttgattac
     6841 agatacagcc aggctgatgc cctgaagtat gtcggcatcg aaagagaaat ggaaatccct
     6901 tgatctagaa tgctgatggg ctagctaaga attcctagag ctgatccgcg tttaaactcg
     6961 aggttaatta atgagcggcc gcatagataa ctgatccagt gtgctggaat taattcgctg
     7021 tctgcgaggg ccagctgttg gggtgagtac tccctctcaa aagcgggcat gacttctgcg
     7081 ctaagattgt cagtttccaa aaacgaggag gatttgatat tcacctggcc cgcggtgatg
     7141 cctttgaggg tggccgcgtc catctggtca gaaaagacaa tctttttgtt gtcaagcttg
     7201 aggtgtggca ggcttgagat ctggccatac acttgagtga caatgacatc cactttgcct
     7261 ttctctccac aggtgtccac tcccaggtcc aactgcaggt cgagcatgca tctagggcgg
     7321 ccaattccgc ccctctcccc cccccccctc tccctccccc ccccctaacg ttactggccg
     7381 aagccgcttg gaataaggcc ggtgtgcgtt tgtctatatg ttattttcca ccatattgcc
     7441 gtcttttggc aatgtgaggg cccggaaacc tggccctgtc ttcttgacga gcattcctag
     7501 gggtctttcc cctctcgcca aaggaatgca aggtctgttg aatgtcgtga aggaagcagt
     7561 tcctctggaa gcttcttgaa gacaaacaac gtctgtagcg accctttgca ggcagcggaa
     7621 ccccccacct ggcgacaggt gcctctgcgg ccaaaagcca cgtgtataag atacacctgc
     7681 aaaggcggca caaccccagt gccacgttgt gagttggata gttgtggaaa gagtcaaatg
     7741 gctctcctca agcgtattca acaaggggct gaaggatgcc cagaaggtac cccattgtat
     7801 gggatctgat ctggggcctc ggtgcacatg ctttacatgt gtttagtcga ggttaaaaaa
     7861 acgtctaggc cccccgaacc acggggacgt ggttttcctt tgaaaaacac gatgataagc
     7921 ttgccacaac ccgggataat tcctgcagcc aatatgaaaa agcctgaact caccgcgacg
     7981 tctgtcgaga agtttctgat cgaaaagttc gacagcgtct ccgacctgat gcagctctcg
     8041 gagggcgaag aatctcgtgc tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg
     8101 gtaaatagct gcgccgatgg tttctacaaa gatcgttatg tttatcggca ctttgcatcg
     8161 gccgcgctcc cgattccgga agtgcttgac attggggaat tcagcgagag cctgacctat
     8221 tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc
     8281 gctgttctgc agccggtcgc ggaggccatg gatgcgatcg ctgcggccga tcttagccag
     8341 acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc aatacactac atggcgtgat
     8401 ttcatatgcg cgattgctga tccccatgtg tatcactggc aaactgtgat ggacgacacc
     8461 gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc tttgggccga ggactgcccc
     8521 gaagtccggc acctcgtgca cgcggatttc ggctccaaca atgtcctgac ggacaatggc
     8581 cgcataacag cggtcattga ctggagcgag gcgatgttcg gggattccca atacgaggtc
     8641 gccaacatct tcttctggag gccgtggttg gcttgtatgg agcagcagac gcgctacttc
     8701 gagcggaggc atccggagct tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt
     8761 ggtcttgacc aactctatca gagcttggtt gacggcaatt tcgatgatgc agcttgggcg
     8821 cagggtcgat gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc
     8881 gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc cgatagtgga
     8941 aaccgacgcc ccagcactcg tccgagggca aaggaatagg gatcaattct ctagataact
     9001 gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca
     9061 cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc
     9121 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     9181 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttaac gcgtcgagtg
     9241 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg
     9301 acatactagt taaaagtttt gttactttat agaagaaatt ttgagttttt gttttttttt
     9361 aataaataaa taaacataaa taaattgttt gttgaattta ttattagtat gtaagtgtaa
     9421 atataataaa acttaatatc tattcaaatt aataaataaa cctcgatata cagaccgata
     9481 aaacacatgc gtcaatttta cgcatgatta tctttaacgt acgtcacaat atgattatct
     9541 ttctagggtt aatctagtat acgcgtatgc ggccgcttaa ttaatccagc ttttgttccc
     9601 tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     9661 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     9721 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     9781 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     9841 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     9901 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     9961 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
    10021 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
    10081 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
    10141 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
    10201 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
    10261 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
    10321 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
    10381 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
    10441 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
    10501 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
    10561 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
    10621 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
    10681 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
    10741 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
    10801 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
    10861 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
    10921 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
    10981 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
    11041 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
    11101 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
    11161 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
    11221 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
    11281 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
    11341 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
    11401 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
    11461 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
    11521 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
    11581 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
    11641 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
    11701 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
    11761 cgcgcacatt tccccgaaaa gtgccaccta aattgtaagc gttaatattt tgttaaaatt
    11821 cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat
    11881 cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa
    11941 gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg
    12001 cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa
    12061 agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc
    12121 gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag
    12181 tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg
    12241 cgcgtcccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc
    12301 ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac
    12361 gccagggttt tcccagtcac gacgttgtaa aacgacggcc agtgagcgcg cgtaatacga
    12421 ctcactatag ggcgaattgg ggcgcgccat tctagattaa ccctagaaag atagtctgcg
    12481 taaaattgac gcatgcattc ttgaaatatt gctctctctt tctaaatagc gcgaatccgt
    12541 cgctgtgcat ttaggacatc tcagtcgccg cttggagctc ccgtgaggcg tgcttgtcaa
    12601 tgcggtaagt gtcactgatt ttgaactata acgaccgcgt gagtcaaaat gacgcatgat
    12661 tatcttttac gtgactttta agatttaact catacgataa ttatattgtt atttcatgtt
    12721 ctacttacgt gataacttat tatatatata ttttcttgtt atagatatca actagaatgc
    12781 tagcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc
    12841 attgacgtca atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
    12901 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
    12961 tgacgtcaat gacggtaaat ggcccgcctg gcattgtgcc cagtacatga ccttatggga
    13021 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tcgaggtgag
    13081 ccccacgttc tgcttcactc tccccatctc ccccccctcc ccacccccaa ttttgtattt
    13141 atttattttt taattatttt gtgcagcgat gggggcgggg gggggggggg ggcgcgcgcc
    13201 aggcggggcg gggcggggcg aggggcgggg cggggcgag
//
