LOCUS       sequence-197556-       10723 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Catalytic domain [SET] of mouse Ezh2 fused to N-terminus of dCas9; 
            pCDNA3 vector backbone, mammalian expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10723)
  AUTHORS   O'Geen H, Ren C, Nicolet CM, Perez AA, Halmai J, Le VM, Mackay JP, 
            Farnham PJ, Segal DJ
  TITLE     dCas9-based epigenome editing suggests acquisition of histone 
            methylation is not sufficient for target gene repression.
  JOURNAL   Nucleic Acids Res. 2017 Sep 29;45(17):9901-9916. doi: 
            10.1093/nar/gkx578.
  PUBMED    28973434
REFERENCE   2  (bases 1 to 10723)
  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"; doi: "10"; journalName: 
            "Nucleic Acids Res"; date: "2017-09-29- 29"; volume: "45"; issue: 
            "17"; pages: "9901-9916"
COMMENT     Sequence Label: Ezh2[SET]-dCas9
FEATURES             Location/Qualifiers
     source          1..10723
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     581..605
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             702..722
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             741..806
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             join(1695..1721,1722..1724,1725..4208,4209..4214,4215..4280,
                     4281..4283,4284..5798)
                     /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(1695..1721,1722..1724,1725..4211,4212..4214,
                     4215..5798)
                     /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             1695..5798
                     /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             5874..5921
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5940..5996
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     primer_bind     complement(6060..6079)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6104..6152
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6354..6782
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6441..6460)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6651..6672
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6791..6811)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6796..7125
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6976..7111
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     7038..7057
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             7192..7986
                     /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="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(7246..7265)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     7856..7875
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    8162..8283
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8199..8218)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8253..8272
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(8332..8348)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8332..8348)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8345..8367)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8356..8372
                     /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(8380..8386,8387..8404,8405..8410))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8425..8446
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(8563..8580)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8734..9322)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8814..8833)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9493..10284,10285..10353))
                     /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     10116..10135
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10354..10458)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(10533..10552)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc
      601 agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagacac cgggaccgat
      661 ccagcctccg gactctagag gatcgaaccc ttgccaccat gcccaagaag aagaggaagg
      721 tgggaggctc cggaggaagc gactataagg accacgacgg agactacaag gatcatgata
      781 ttgattacaa agacgatgac gataagggtg gcgggtccgg cggtggatcc ggtaccatga
      841 ctgaggatgt agacactcct ccaagaaaga agaaaaggaa acatcggttg tgggctgcac
      901 actgcagaaa gatacaactg aaaaaggacg gctcctctaa ccatgtttac aactatcaac
      961 cctgtgacca tccacggcag ccttgtgaca gttcgtgccc ttgtgtgata gcacaaaatt
     1021 tttgtgaaaa gttttgtcaa tgtagttcag agtgtcaaaa ccgctttcct ggatgtcggt
     1081 gcaaagcaca atgcaacacc aaacagtgtc catgctacct ggctgtccga gagtgtgacc
     1141 ctgacctctg tctcacgtgt ggagctgctg accattggga cagtaaaaat gtatcctgta
     1201 agaactgtag cattcagcgg ggctctaaaa agcacttact gctggcaccg tctgatgtgg
     1261 caggctgggg catctttatc aaagatcctg tacagaaaaa tgaattcatc tcagaatact
     1321 gtggggagat tatttctcag gatgaagcag acagaagagg aaaagtgtat gacaaataca
     1381 tgtgcagctt tctgttcaac ttgaacaatg attttgtggt ggatgcaacc cgaaagggca
     1441 acaaaattcg ttttgctaat cattcagtaa atccaaactg ctatgcaaaa gttatgatgg
     1501 ttaatggtga ccacaggata ggcatctttg ctaagagggc tatccagact ggtgaagagt
     1561 tgttttttga ttacagatac agccaggctg atgccctgaa gtatgtgggc atcgaacgag
     1621 aaatggaaat ccctagtacc ggagggagtg gtggaagcgg cggttctggt ggctcaggcg
     1681 gcagcggccg gccaatggac aagaagtact ccattgggct cgctatcggc acaaacagcg
     1741 tcggctgggc cgtcattacg gacgagtaca aggtgccgag caaaaaattc aaagttctgg
     1801 gcaataccga tcgccacagc ataaagaaga acctcattgg cgccctcctg ttcgactccg
     1861 gggagacggc cgaagccacg cggctcaaaa gaacagcacg gcgcagatat acccgcagaa
     1921 agaatcggat ctgctacctg caggagatct ttagtaatga gatggctaag gtggatgact
     1981 ctttcttcca taggctggag gagtcctttt tggtggagga ggataaaaag cacgagcgcc
     2041 acccaatctt tggcaatatc gtggacgagg tggcgtacca tgaaaagtac ccaaccatat
     2101 atcatctgag gaagaagctt gtagacagta ctgataaggc tgacttgcgg ttgatctatc
     2161 tcgcgctggc gcatatgatc aaatttcggg gacacttcct catcgagggg gacctgaacc
     2221 cagacaacag cgatgtcgac aaactcttta tccaactggt tcagacttac aatcagcttt
     2281 tcgaagagaa cccgatcaac gcatccggag ttgacgccaa agcaatcctg agcgctaggc
     2341 tgtccaaatc ccggcggctc gaaaacctca tcgcacagct ccctggggag aagaagaacg
     2401 gcctgtttgg taatcttatc gccctgtcac tcgggctgac ccccaacttt aaatctaact
     2461 tcgacctggc cgaagatgcc aagcttcaac tgagcaaaga cacctacgat gatgatctcg
     2521 acaatctgct ggcccagatc ggcgaccagt acgcagacct ttttttggcg gcaaagaacc
     2581 tgtcagacgc cattctgctg agtgatattc tgcgagtgaa cacggagatc accaaagctc
     2641 cgctgagcgc tagtatgatc aagcgctatg atgagcacca ccaagacttg actttgctga
     2701 aggcccttgt cagacagcaa ctgcctgaga agtacaagga aattttcttc gatcagtcta
     2761 aaaatggcta cgccggatac attgacggcg gagcaagcca ggaggaattt tacaaattta
     2821 ttaagcccat cttggaaaaa atggacggca ccgaggagct gctggtaaag cttaacagag
     2881 aagatctgtt gcgcaaacag cgcactttcg acaatggaag catcccccac cagattcacc
     2941 tgggcgaact gcacgctatc ctcaggcggc aagaggattt ctaccccttt ttgaaagata
     3001 acagggaaaa gattgagaaa atcctcacat ttcggatacc ctactatgta ggccccctcg
     3061 cccggggaaa ttccagattc gcgtggatga ctcgcaaatc agaagagacc atcactccct
     3121 ggaacttcga ggaagtcgtg gataaggggg cctctgccca gtccttcatc gaaaggatga
     3181 ctaactttga taaaaatctg cctaacgaaa aggtgcttcc taaacactct ctgctgtacg
     3241 agtacttcac agtttataac gagctcacca aggtcaaata cgtcacagaa gggatgagaa
     3301 agccagcatt cctgtctgga gagcagaaga aagctatcgt ggacctcctc ttcaagacga
     3361 accggaaagt taccgtgaaa cagctcaaag aagactattt caaaaagatt gaatgtttcg
     3421 actctgttga aatcagcgga gtggaggatc gcttcaacgc atccctggga acgtatcacg
     3481 atctcctgaa aatcattaaa gacaaggact tcctggacaa tgaggagaac gaggacattc
     3541 ttgaggacat tgtcctcacc cttacgttgt ttgaagatag ggagatgatt gaagaacgct
     3601 tgaaaactta cgctcatctc ttcgacgaca aagtcatgaa acagctcaag aggcgccgat
     3661 atacaggatg ggggcggctg tcaagaaaac tgatcaatgg gatccgagac aagcagagtg
     3721 gaaagacaat cctggatttt cttaagtccg atggatttgc caacaggaac ttcatgcagt
     3781 tgatccatga tgactctctc acctttaagg aggacatcca gaaagcacaa gtttctggcc
     3841 agggggacag tcttcacgag cacatcgcta atcttgcagg tagcccagct atcaaaaagg
     3901 gaatactgca gaccgttaag gtcgtggatg aactcgtcaa agtaatggga aggcataagc
     3961 ccgagaatat cgttatcgag atggcccgag agaaccaaac tacccagaag ggacagaaga
     4021 acagtaggga aaggatgaag aggattgaag agggtataaa agaactgggg tcccaaatcc
     4081 ttaaggaaca cccagttgaa aacacccagc ttcagaatga gaagctctac ctgtactacc
     4141 tgcagaacgg cagggacatg tacgtggatc aggaactgga catcaatcgg ctctccgact
     4201 acgacgtgga tgctatcgtg ccccagtctt ttctcaaaga tgattctatt gataataaag
     4261 tgttgacaag atccgataaa aatagaggga agagtgataa cgtcccctca gaagaagttg
     4321 tcaagaaaat gaaaaattat tggcggcagc tgctgaacgc caaactgatc acacaacgga
     4381 agttcgataa tctgactaag gctgaacgag gtggcctgtc tgagttggat aaagccggct
     4441 tcatcaaaag gcagcttgtt gagacacgcc agatcaccaa gcacgtggcc caaattctcg
     4501 attcacgcat gaacaccaag tacgatgaaa atgacaaact gattcgagag gtgaaagtta
     4561 ttactctgaa gtctaagctg gtctcagatt tcagaaagga ctttcagttt tataaggtga
     4621 gagagatcaa caattaccac catgcgcatg atgcctacct gaatgcagtg gtaggcactg
     4681 cacttatcaa aaaatatccc aagcttgaat ctgaatttgt ttacggagac tataaagtgt
     4741 acgatgttag gaaaatgatc gcaaagtctg agcaggaaat aggcaaggcc accgctaagt
     4801 acttctttta cagcaatatt atgaattttt tcaagaccga gattacactg gccaatggag
     4861 agattcggaa gcgaccactt atcgaaacaa acggagaaac aggagaaatc gtgtgggaca
     4921 agggtaggga tttcgcgaca gtccggaagg tcctgtccat gccgcaggtg aacatcgtta
     4981 aaaagaccga agtacagacc ggaggcttct ccaaggaaag tatcctcccg aaaaggaaca
     5041 gcgacaagct gatcgcacgc aaaaaagatt gggaccccaa gaaatacggc ggattcgatt
     5101 ctcctacagt cgcttacagt gtactggttg tggccaaagt ggagaaaggg aagtctaaaa
     5161 aactcaaaag cgtcaaggaa ctgctgggca tcacaatcat ggagcgatca agcttcgaaa
     5221 aaaaccccat cgactttctc gaggcgaaag gatataaaga ggtcaaaaaa gacctcatca
     5281 ttaagcttcc caagtactct ctctttgagc ttgaaaacgg ccggaaacga atgctcgcta
     5341 gtgcgggcga gctgcagaaa ggtaacgagc tggcactgcc ctctaaatac gttaatttct
     5401 tgtatctggc cagccactat gaaaagctca aagggtctcc cgaagataat gagcagaagc
     5461 agctgttcgt ggaacaacac aaacactacc ttgatgagat catcgagcaa ataagcgaat
     5521 tctccaaaag agtgatcctc gccgacgcta acctcgataa ggtgctttct gcttacaata
     5581 agcacaggga taagcccatc agggagcagg cagaaaacat tatccacttg tttactctga
     5641 ccaacttggg cgcgcctgca gccttcaagt acttcgacac caccatagac agaaagcggt
     5701 acacctctac aaaggaggtc ctggacgcca cactgattca tcagtcaatt acggggctct
     5761 atgaaacaag aatcgacctc tctcagctcg gtggagacgg tggtagtgga ggttcaggag
     5821 gatccggggg gagcggaggg agcgctagcg gtggaggctc cggaggtgga tctaaaaggc
     5881 cggcggccac gaaaaaggcc ggtcaggcaa aaaagaaaaa gggtggtagt ggaagcggag
     5941 ctactaactt cagcctgctg aagcaggctg gagacgtgga ggagaaccct ggacctgcgg
     6001 ccgcatgaaa gggttcgatc cctaccggtt agtaatgagt ttaaacgggg gaggctaact
     6061 gaaacacgga aggagacaat accggaagga acccgcgcta tgacggcaat aaaaagacag
     6121 aataaaacgc acgggtgttg ggtcgtttgt tcataaacgc ggggttcggt cccagggctg
     6181 gcactctgtc gataccccac cgagacccca ttggggccaa tacgcccgcg tttcttcctt
     6241 ttccccaccc caccccccaa gttcgggtga aggcccaggg ctcgcagcca acgtcggggc
     6301 ggcaggccct gccatagcag atctgcgcag ctggggctct agggggtatc cccacgcgcc
     6361 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     6421 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     6481 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     6541 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     6601 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     6661 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     6721 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     6781 ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc
     6841 agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc
     6901 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg
     6961 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     7021 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc
     7081 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct
     7141 tgtatatcca ttttcggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa
     7201 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     7261 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     7321 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag
     7381 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     7441 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     7501 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     7561 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     7621 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     7681 gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat
     7741 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     7801 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     7861 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     7921 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     7981 ttctgagcgg gactctgggg ttcgcgaaat gaccgaccaa gcgacgccca acctgccatc
     8041 acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg
     8101 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc
     8161 caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     8221 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     8281 ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct
     8341 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     8401 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     8461 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     8521 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     8581 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     8641 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     8701 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     8761 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     8821 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     8881 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     8941 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     9001 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     9061 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     9121 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     9181 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     9241 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     9301 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     9361 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     9421 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     9481 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     9541 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     9601 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     9661 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     9721 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     9781 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     9841 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     9901 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     9961 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
    10021 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
    10081 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
    10141 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
    10201 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
    10261 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
    10321 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
    10381 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
    10441 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg acggatcggg
    10501 agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
    10561 agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt
    10621 taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct tagggttagg
    10681 cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gtt
//
