LOCUS       sequence-430622-       11345 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 3xFLAG-dCas9 in mammalian cells for enChIP analysis to 
            purify specific genomic regions of interest..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11345)
  AUTHORS   Yuno M, Nagata S, Fujita T, Fujii H
  TITLE     MSCV-based retroviral plasmids expressing 3xFLAG-Sp-dCas9 for enChIP
            analysis.
  JOURNAL   Biol Methods Protoc. 2021 Jul 9;6(1):bpab013. doi: 
            10.1093/biomethods/bpab013. eCollection 2021.
  PUBMED    34409168
REFERENCE   2  (bases 1 to 11345)
  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: "Biol 
            Methods Protoc."; date: "2021-07-9"; volume: "6(1):bpab013. doi"; 
            pages: " 10.1093/biomethods/bpab013. eCollection 2021"
COMMENT     Sequence Label: 3xFLAG-dCas9/pMSCVhyg
FEATURES             Location/Qualifiers
     source          1..11345
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    579..920
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             987..1403
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1333..1355
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1371..1387
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     CDS             1452..1517
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             join(1521..1547,1548..1550,1551..4034,4035..4040,4041..4106,
                     4107..4109,4110..5624)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=Cas9m4
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the mutations D10A, D839A, 
                     H840A, and N863A (Mali et al., 2013)"
                     /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             join(1521..1547,1548..1550,1551..4037,4038..4040,
                     4041..5624)
                     /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             1521..5624
                     /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             5637..5657
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        5838..6337
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(5873..5896)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     6270..6289
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             6355..7380
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             6355..7380
                     /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"
     LTR             7738..8252
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    8414..8430
                     /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(8438..8444,8445..8462,8463..8468))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8483..8504
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(8621..8638)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8792..9380)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8872..8891)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9551..10342,10343..10411))
                     /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     10174..10193
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10412..10516)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     10584..10602
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(10640..10662)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     10762..10781
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     10975..10997
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     11125..11144
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             join(11344..11345,1..515)
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
ORIGIN
        1 tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat
       61 ggaaaataca taactgagaa tagagaagtt cagatcaagg ttaggaacag agagacagca
      121 gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga
      181 acagatggtc cccagatgcg gtcccgccct cagcagtttc tagagaacca tcagatgttt
      241 ccagggtgcc ccaaggacct gaaatgaccc tgtgccttat ttgaactaac caatcagttc
      301 gcttctcgct tctgttcgcg cgcttctgct ccccgagctc aataaaagag cccacaaccc
      361 ctcactcggc gcgccagtcc tccgatagac tgcgtcgccc gggtacccgt attcccaata
      421 aagcctcttg ctgtttgcat ccgaatcgtg gactcgctga tccttgggag ggtctcctca
      481 gattgattga ctgcccacct cgggggtctt tcatttggag gttccaccga gatttggaga
      541 cccctgccca gggaccaccg acccccccgc cgggaggtaa gctggccagc ggtcgtttcg
      601 tgtctgtctc tgtctttgtg cgtgtttgtg ccggcatcta atgtttgcgc ctgcgtctgt
      661 actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac gagttctgaa
      721 cacccggccg caaccctggg agacgtccca gggactttgg gggccgtttt tgtggcccga
      781 cctgaggaag ggagtcgatg tggaatccga ccccgtcagg atatgtggtt ctggtaggag
      841 acgagaacct aaaacagttc ccgcctccgt ctgaattttt gctttcggtt tggaaccgaa
      901 gccgcgcgtc ttgtctgctg cagcgctgca gcatcgttct gtgttgtctc tgtctgactg
      961 tgtttctgta tttgtctgaa aattagggcc agactgttac cactccctta agtttgacct
     1021 taggtcactg gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga
     1081 gacgttgggt taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag
     1141 acggcacctt taaccgagac ctcatcaccc aggttaagat caaggtcttt tcacctggcc
     1201 cgcatggaca cccagaccag gtcccctaca tcgtgacctg ggaagccttg gcttttgacc
     1261 cccctccctg ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg
     1321 ccccgtctct cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag
     1381 ccctcactcc ttctctaggc gccggaatta gatctaagga tccgtttagt gaaccgtcag
     1441 aattaaccat ggactacaaa gaccatgacg gtgattataa agatcatgac atcgattaca
     1501 aggatgacga tgacaagctc atggacaaga agtactccat tgggctcgct atcggcacaa
     1561 acagcgtcgg ctgggccgtc attacggacg agtacaaggt gccgagcaaa aaattcaaag
     1621 ttctgggcaa taccgatcgc cacagcataa agaagaacct cattggcgcc ctcctgttcg
     1681 actccgggga gacggccgaa gccacgcggc tcaaaagaac agcacggcgc agatataccc
     1741 gcagaaagaa tcggatctgc tacctgcagg agatctttag taatgagatg gctaaggtgg
     1801 atgactcttt cttccatagg ctggaggagt cctttttggt ggaggaggat aaaaagcacg
     1861 agcgccaccc aatctttggc aatatcgtgg acgaggtggc gtaccatgaa aagtacccaa
     1921 ccatatatca tctgaggaag aagcttgtag acagtactga taaggctgac ttgcggttga
     1981 tctatctcgc gctggcgcat atgatcaaat ttcggggaca cttcctcatc gagggggacc
     2041 tgaacccaga caacagcgat gtcgacaaac tctttatcca actggttcag acttacaatc
     2101 agcttttcga agagaacccg atcaacgcat ccggagttga cgccaaagca atcctgagcg
     2161 ctaggctgtc caaatcccgg cggctcgaaa acctcatcgc acagctccct ggggagaaga
     2221 agaacggcct gtttggtaat cttatcgccc tgtcactcgg gctgaccccc aactttaaat
     2281 ctaacttcga cctggccgaa gatgccaagc ttcaactgag caaagacacc tacgatgatg
     2341 atctcgacaa tctgctggcc cagatcggcg accagtacgc agaccttttt ttggcggcaa
     2401 agaacctgtc agacgccatt ctgctgagtg atattctgcg agtgaacacg gagatcacca
     2461 aagctccgct gagcgctagt atgatcaagc gctatgatga gcaccaccaa gacttgactt
     2521 tgctgaaggc ccttgtcaga cagcaactgc ctgagaagta caaggaaatt ttcttcgatc
     2581 agtctaaaaa tggctacgcc ggatacattg acggcggagc aagccaggag gaattttaca
     2641 aatttattaa gcccatcttg gaaaaaatgg acggcaccga ggagctgctg gtaaagctta
     2701 acagagaaga tctgttgcgc aaacagcgca ctttcgacaa tggaagcatc ccccaccaga
     2761 ttcacctggg cgaactgcac gctatcctca ggcggcaaga ggatttctac ccctttttga
     2821 aagataacag ggaaaagatt gagaaaatcc tcacatttcg gataccctac tatgtaggcc
     2881 ccctcgcccg gggaaattcc agattcgcgt ggatgactcg caaatcagaa gagaccatca
     2941 ctccctggaa cttcgaggaa gtcgtggata agggggcctc tgcccagtcc ttcatcgaaa
     3001 ggatgactaa ctttgataaa aatctgccta acgaaaaggt gcttcctaaa cactctctgc
     3061 tgtacgagta cttcacagtt tataacgagc tcaccaaggt caaatacgtc acagaaggga
     3121 tgagaaagcc agcattcctg tctggagagc agaagaaagc tatcgtggac ctcctcttca
     3181 agacgaaccg gaaagttacc gtgaaacagc tcaaagaaga ctatttcaaa aagattgaat
     3241 gtttcgactc tgttgaaatc agcggagtgg aggatcgctt caacgcatcc ctgggaacgt
     3301 atcacgatct cctgaaaatc attaaagaca aggacttcct ggacaatgag gagaacgagg
     3361 acattcttga ggacattgtc ctcaccctta cgttgtttga agatagggag atgattgaag
     3421 aacgcttgaa aacttacgct catctcttcg acgacaaagt catgaaacag ctcaagaggc
     3481 gccgatatac aggatggggg cggctgtcaa gaaaactgat caatgggatc cgagacaagc
     3541 agagtggaaa gacaatcctg gattttctta agtccgatgg atttgccaac aggaacttca
     3601 tgcagttgat ccatgatgac tctctcacct ttaaggagga catccagaaa gcacaagttt
     3661 ctggccaggg ggacagtctt cacgagcaca tcgctaatct tgcaggtagc ccagctatca
     3721 aaaagggaat actgcagacc gttaaggtcg tggatgaact cgtcaaagta atgggaaggc
     3781 ataagcccga gaatatcgtt atcgagatgg cccgagagaa ccaaactacc cagaagggac
     3841 agaagaacag tagggaaagg atgaagagga ttgaagaggg tataaaagaa ctggggtccc
     3901 aaatccttaa ggaacaccca gttgaaaaca cccagcttca gaatgagaag ctctacctgt
     3961 actacctgca gaacggcagg gacatgtacg tggatcagga actggacatc aatcggctct
     4021 ccgactacga cgtggatgcc atcgtgcccc agtcttttct caaagatgat tctattgata
     4081 ataaagtgtt gacaagatcc gataaaaata gagggaagag tgataacgtc ccctcagaag
     4141 aagttgtcaa gaaaatgaaa aattattggc ggcagctgct gaacgccaaa ctgatcacac
     4201 aacggaagtt cgataatctg actaaggctg aacgaggtgg cctgtctgag ttggataaag
     4261 ccggcttcat caaaaggcag cttgttgaga cacgccagat caccaagcac gtggcccaaa
     4321 ttctcgattc acgcatgaac accaagtacg atgaaaatga caaactgatt cgagaggtga
     4381 aagttattac tctgaagtct aagctggtct cagatttcag aaaggacttt cagttttata
     4441 aggtgagaga gatcaacaat taccaccatg cgcatgatgc ctacctgaat gcagtggtag
     4501 gcactgcact tatcaaaaaa tatcccaagc ttgaatctga atttgtttac ggagactata
     4561 aagtgtacga tgttaggaaa atgatcgcaa agtctgagca ggaaataggc aaggccaccg
     4621 ctaagtactt cttttacagc aatattatga attttttcaa gaccgagatt acactggcca
     4681 atggagagat tcggaagcga ccacttatcg aaacaaacgg agaaacagga gaaatcgtgt
     4741 gggacaaggg tagggatttc gcgacagtcc ggaaggtcct gtccatgccg caggtgaaca
     4801 tcgttaaaaa gaccgaagta cagaccggag gcttctccaa ggaaagtatc ctcccgaaaa
     4861 ggaacagcga caagctgatc gcacgcaaaa aagattggga ccccaagaaa tacggcggat
     4921 tcgattctcc tacagtcgct tacagtgtac tggttgtggc caaagtggag aaagggaagt
     4981 ctaaaaaact caaaagcgtc aaggaactgc tgggcatcac aatcatggag cgatcaagct
     5041 tcgaaaaaaa ccccatcgac tttctcgagg cgaaaggata taaagaggtc aaaaaagacc
     5101 tcatcattaa gcttcccaag tactctctct ttgagcttga aaacggccgg aaacgaatgc
     5161 tcgctagtgc gggcgagctg cagaaaggta acgagctggc actgccctct aaatacgtta
     5221 atttcttgta tctggccagc cactatgaaa agctcaaagg gtctcccgaa gataatgagc
     5281 agaagcagct gttcgtggaa caacacaaac actaccttga tgagatcatc gagcaaataa
     5341 gcgaattctc caaaagagtg atcctcgccg acgctaacct cgataaggtg ctttctgctt
     5401 acaataagca cagggataag cccatcaggg agcaggcaga aaacattatc cacttgttta
     5461 ctctgaccaa cttgggcgcg cctgcagcct tcaagtactt cgacaccacc atagacagaa
     5521 agcggtacac ctctacaaag gaggtcctgg acgccacact gattcatcag tcaattacgg
     5581 ggctctatga aacaagaatc gacctctctc agctcggtgg agacagcagg gctgacccca
     5641 agaagaagag gaaggtgtga aagggttcga tccctaccgg atcgaattcc agctgagcgc
     5701 cggtcgctac cattaccagt tggtctggtg tcaaaaataa taataaccgg gcaggccatg
     5761 tctgcccgta tttcgcgtaa ggaaatccat tatgtactat ttaaactcga gcggcctcga
     5821 ggttaacgaa ttctaccggg taggggaggc gcttttccca aggcagtctg gagcatgcgc
     5881 tttagcagcc ccgctgggca cttggcgcta cacaagtggc ctctggcctc gcacacattc
     5941 cacatccacc ggtaggcgcc aaccggctcc gttctttggt ggccccttcg cgccaccttc
     6001 tactcctccc ctagtcagga agttcccccc cgccccgcag ctcgcgtcgt gcaggacgtg
     6061 acaaatggaa gtagcacgtc tcactagtct cgtgcagatg gacagcaccg ctgagcaatg
     6121 gaagcgggta ggcctttggg gcagcggcca atagcagctt tgctccttcg ctttctgggc
     6181 tcagaggctg ggaaggggtg ggtccggggg cgggctcagg ggcgggctca ggggcggggc
     6241 gggcgcccga aggtcctccg gaggcccggc attctgcacg cttcaaaagc gcacgtctgc
     6301 cgcgctgttc tcctcttcct catctccggg cctttcgacc tgcatcccgc caccatgaaa
     6361 aagcctgaac tcaccgcgac gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtc
     6421 tccgacctga tgcagctctc ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga
     6481 gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg gtttctacaa agatcgttat
     6541 gtttatcggc actttgcatc ggccgcgctc ccgattccgg aagtgcttga cattggggaa
     6601 ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac
     6661 ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg cggaggccat ggatgcgatc
     6721 gctgcggccg atcttagcca gacgagcggg ttcggcccat tcggaccgca aggaatcggt
     6781 caatacacta catggcgtga tttcatatgc gcgattgctg atccccatgt gtatcactgg
     6841 caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg
     6901 ctttgggccg aggactgccc cgaagtccgg cacctcgtgc acgcggattt cggctccaac
     6961 aatgtcctga cggacaatgg ccgcataaca gcggtcattg actggagcga ggcgatgttc
     7021 ggggattccc aatacgaggt cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg
     7081 gagcagcaga cgcgctactt cgagcggagg catccggagc ttgcaggatc gccgcggctc
     7141 cgggcgtata tgctccgcat tggtcttgac caactctatc agagcttggt tgacggcaat
     7201 ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg
     7261 actgtcgggc gtacacaaat cgcccgcaga agcgcggccg tctggaccga tggctgtgta
     7321 gaagtactcg ccgatagtgg aaaccgacgc cccagcactc gtccgagggc aaaggaatag
     7381 agtagatgcc gaccgaacaa gagctgattt cgagaacgcc tcagccagca actcgcgcga
     7441 gcctagcaag gcaaatgcga gagaacggcc ttacgcttgg tggcacagtt ctcgtccaca
     7501 gttcgctaag ctcgctcggc tgggtcgcgg gagggccggt cgcagtgatt caggcccttc
     7561 tggattgtgt tggtccccag ggcacgattg tcatgcccac gcactcgggt gatctgactg
     7621 atcccgcaga ttggagatcg ccgcccgtgc ctgccgattg ggtgcagatc cgtcgacctg
     7681 cagccaagct tatcgataaa ataaaagatt ttatttagtc tccagaaaaa ggggggaatg
     7741 aaagacccca cctgtaggtt tggcaagcta gcttaagtaa cgccattttg caaggcatgg
     7801 aaaatacata actgagaata gagaagttca gatcaaggtt aggaacagag agacagcaga
     7861 atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag ggccaagaac
     7921 agatggtccc cagatgcggt cccgccctca gcagtttcta gagaaccatc agatgtttcc
     7981 agggtgcccc aaggacctga aatgaccctg tgccttattt gaactaacca atcagttcgc
     8041 ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc cacaacccct
     8101 cactcggcgc gccagtcctc cgatagactg cgtcgcccgg gtacccgtgt atccaataaa
     8161 ccctcttgca gttgcatccg acttgtggtc tcgctgttcc ttgggagggt ctcctctgag
     8221 tgattgacta cccgtcagcg ggggtctttc atgggtaaca gtttcttgaa gttggagaac
     8281 aacattctga gggtaggagt cgaatattaa gtaatcctga ctcaattagc cactgttttg
     8341 aatccacata ctccaatact cctgaaatag ttcattatgg acagcgcaga aagagctggg
     8401 gagaattgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     8461 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     8521 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     8581 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     8641 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     8701 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     8761 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     8821 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     8881 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     8941 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     9001 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     9061 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     9121 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     9181 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     9241 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     9301 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     9361 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     9421 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     9481 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     9541 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     9601 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     9661 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     9721 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     9781 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     9841 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     9901 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     9961 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
    10021 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
    10081 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
    10141 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
    10201 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
    10261 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
    10321 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
    10381 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
    10441 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
    10501 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta
    10561 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt
    10621 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc
    10681 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt
    10741 gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc
    10801 ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc cattcgccat
    10861 tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc
    10921 tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt
    10981 cacgacgttg taaaacgacg gcgcaaggaa tggtgcatgc aaggagatgg cgcccaacag
    11041 tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa
    11101 gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc
    11161 tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggcgattag tccaatttgt
    11221 taaagacagg atatcagtgg tccaggctct agttttgact caacaatatc accagctgaa
    11281 gcctatagag tacgagccat agataaaata aaagatttta tttagtctcc agaaaaaggg
    11341 gggaa
//
