LOCUS       sequence-206189-       12560 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral vector for expressing effector domains fused to dCas9..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12560)
  AUTHORS   Goubert D, Koncz M, Kiss A, Rots MG
  TITLE     Establishment of Cell Lines Stably Expressing dCas9-Fusions to 
            Address Kinetics of Epigenetic Editing.
  JOURNAL   Methods Mol Biol. 2018;1767:395-415. doi: 
            10.1007/978-1-4939-7774-1_22.
  PUBMED    29524148
REFERENCE   2  (bases 1 to 12560)
  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: "Methods Mol
            Biol."; date: "2018"; volume: "1767:395-415. doi"; pages: " 
            10.1007/978-1-4939-7774-1_22"
COMMENT     Sequence Label: pHAGE EF1-alpha dCas9-NED
FEATURES             Location/Qualifiers
     source          1..12560
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             34..60
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     promoter        93..592
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(128..151)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     525..544
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             613..1212
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(613..632)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1109..1129
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    1229..1817
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1282..1302)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1700..1711)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             1892..2125
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(2244..2262)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2330..2434
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2435..2503,2504..3295)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2653..2672)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3466..4054
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3955..3974
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4208..4225
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4465..4486
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4501..4506,4507..4524,4525..4531)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4539..4555
                     /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     4544..4566
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4563..4579
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4563..4579
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     4629..4648
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             4840..5473
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    5520..5645
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6142..6375
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6560..6604
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    6897..7014
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        7060..8238
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          7291..8229
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     8186..8206
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    8273..8297
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      8301..8310
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(8307..8333,8334..8336,8337..10820,10821..10826,
                     10827..10892,10893..10895,10896..12410)
                     /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(8307..8333,8334..8336,8337..10823,10824..10826,
                     10827..12410)
                     /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             8307..12410
                     /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             12423..12443
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             12444..12533
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
ORIGIN
        1 ggccgctcga gtctagaggg cccgcggttc gaatacccat acgacgtccc agactacgct
       61 tagtaatgat taattaaact agaaattcta ccgggtaggg gaggcgcttt tcccaaggca
      121 gtctggagca tgcgctttag cagccccgct gggcacttgg cgctacacaa gtggcctctg
      181 gcctcgcaca cattccacat ccaccggtag gcgccaaccg gctccgttct ttggtggccc
      241 cttcgcgcca ccttctactc ctcccctagt caggaagttc ccccccgccc cgcagctcgc
      301 gtcgtgcagg acgtgacaaa tggaagtagc acgtctcact agtctcgtgc agatggacag
      361 caccgctgag caatggaagc gggtaggcct ttggggcagc ggccaatagc agctttgctc
      421 cttcgctttc tgggctcaga ggctgggaag gggtgggtcc gggggcgggc tcaggggcgg
      481 gctcaggggc ggggcgggcg cccgaaggtc ctccggaggc ccggcattct gcacgcttca
      541 aaagcgcacg tctgccgcgc tgttctcctc ttcctcatct ccgggccttt cgacctgcag
      601 cccaagctta ccatgaccga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc
      661 cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc
      721 gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt cctcacgcgc
      781 gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg
      841 accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc
      901 gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct ggcgccgcac
      961 cggcccaagg agcccgcgtg gttcctggcc accgtcggcg tctcgcccga ccaccagggc
     1021 aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg cgccggggtg
     1081 cccgccttcc tggagacctc cgcgccccgc aacctcccct tctacgagcg gctcggcttc
     1141 accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag
     1201 cccggtgcct gaggtaccga tagatcctaa tcaacctctg gattacaaaa tttgtgaaag
     1261 attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat
     1321 gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc
     1381 ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg
     1441 cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct
     1501 ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct
     1561 tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg
     1621 gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac
     1681 gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct
     1741 gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct
     1801 ttgggccgcc tccccgcctg agatccttta agaccaatga cttacaaggc agctgtagat
     1861 cttagccact ttttaaaaga aaagggggga ctggaagggc taattcactc ccaacgaaga
     1921 caagatctgc tttttgcttg tactgggtct ctctggttag accagatctg agcctgggag
     1981 ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt
     2041 caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt
     2101 tagtcagtgt ggaaaatctc tagcagtagt agttcatgtc atcttattat tcagtattta
     2161 taacttgcaa agaaatgaat atcagagagt gagaggcccg ggttaattaa ggaaagggct
     2221 agatcattct tgaagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat
     2281 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     2341 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     2401 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     2461 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     2521 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     2581 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     2641 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc aagagcaact
     2701 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     2761 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     2821 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     2881 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     2941 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     3001 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     3061 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     3121 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     3181 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     3241 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     3301 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     3361 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     3421 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     3481 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     3541 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     3601 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     3661 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     3721 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     3781 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     3841 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     3901 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     3961 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     4021 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     4081 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     4141 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     4201 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct
     4261 ccccgcgcgt tggccgattc attaatgcag caagctcatg gctgactaat tttttttatt
     4321 tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt
     4381 tttggaggcc taggcttttg caaaaagctc cccgtggcac gacaggtttc ccgactggaa
     4441 agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc
     4501 tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca
     4561 cacaggaaac agctatgaca tgattacgaa tttcacaaat aaagcatttt tttcactgca
     4621 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgga tcaactggat
     4681 aactcaagct aaccaaaatc atcccaaact tcccacccca taccctatta ccactgccaa
     4741 ttacctgtgg tttcatttac tctaaacctg tgattcctct gaattatttt cattttaaag
     4801 aaattgtatt tgttaaatat gtactacaaa cttagtagtt ggaagggcta attcactccc
     4861 aaagaagaca agatatcctt gatctgtgga tctaccacac acaaggctac ttccctgatt
     4921 agcagaacta cacaccaggg ccaggggtca gatatccact gacctttgga tggtgctaca
     4981 agctagtacc agttgagcca gataaggtag aagaggccaa taaaggagag aacaccagct
     5041 tgttacaccc tgtgagcctg catgggatgg atgacccgga gagagaagtg ttagagtgga
     5101 ggtttgacag ccgcctagca tttcatcacg tggcccgaga gctgcatccg gagtacttca
     5161 agaactgctg atatcgagct tgctacaagg gactttccgc tggggacttt ccagggaggc
     5221 gtggcctggg cgggactggg gagtggcgag ccctcagatc ctgcatataa gcagctgctt
     5281 tttgcctgta ctgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa
     5341 ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt
     5401 gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg
     5461 aaaatctcta gcagtggcgc ccgaacaggg acttgaaagc gaaagggaaa ccagaggagc
     5521 tctctcgacg caggactcgg cttgctgaag cgcgcacggc aagaggcgag gggcggcgac
     5581 tggtgagtac gccaaaaatt ttgactagcg gaggctagaa ggagagagat gggtgcgaga
     5641 gcgtcagtat taagcggggg agaattagat cgcgatggga aaaaattcgg ttaaggccag
     5701 ggggaaagaa aaaatataaa ttaaaacata tagtatgggc aagcagggag ctagaacgat
     5761 tcgcagttaa tcctggcctg ttagaaacat cagaaggctg tagacaaata ctgggacagc
     5821 tacaaccatc ccttcagaca ggatcagaag aacttagatc attatataat acagtagcaa
     5881 ccctctattg tgtgcatcaa aggatagaga taaaagacac caaggaagct ttagacaaga
     5941 tagaggaaga gcaaaacaaa agtaagacca ccgcacagca agcggccggc cgctgatctt
     6001 cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata aatataaagt
     6061 agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag tggtgcagag
     6121 agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag cagcaggaag
     6181 cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat tgtctggtat
     6241 agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc tgttgcaact
     6301 cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa gatacctaaa
     6361 ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca ccactgctgt
     6421 gccttggaat gctagttgga gtaataaatc tctggaacag atttggaatc acacgacctg
     6481 gatggagtgg gacagagaaa ttaacaatta cacaagctta atacactcct taattgaaga
     6541 atcgcaaaac cagcaagaaa agaatgaaca agaattattg gaattagata aatgggcaag
     6601 tttgtggaat tggtttaaca taacaaattg gctgtggtat ataaaattat tcataatgat
     6661 agtaggaggc ttggtaggtt taagaatagt ttttgctgta ctttctatag tgaatagagt
     6721 taggcaggga tattcaccat tatcgtttca gacccacctc ccaaccccga ggggacccga
     6781 caggcccgaa ggaatagaag aagaaggtgg agagagagac agagacagat ccattcgatt
     6841 agtgaacgga tctcgacggt atcgccgaat tcacaaatgg cagtattcat ccacaatttt
     6901 aaaagaaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca
     6961 acagacatac aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt
     7021 tattacaggg acagcagaga tccagtttgg actagcgatg gctccggtgc ccgtcagtgg
     7081 gcagagcgca catcgcccac agtccccgag aagttgtggg gaggggtcgg caattgaacc
     7141 ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc
     7201 ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt gaacgttctt
     7261 tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg tgtgtggttc ccgcgggcct
     7321 ggcctcttta cgggttatgg cccttgcgtg ccttgaatta cttccacctg gctgcagtac
     7381 gtgattcttg atcccgagct tcgggttgga agtgggtggg agagttcgag gccttgcgct
     7441 taaggagccc cttcgcctcg tgcttgagtt gaggcctggc ctgggcgctg gggccgccgc
     7501 gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt tcgataagtc tctagccatt
     7561 taaaattttt gatgacctgc tgcgacgctt tttttctggc aagatagtct tgtaaatgcg
     7621 ggccaagatc tgcacactgg tatttcggtt tttggggccg cgggcggcga cggggcccgt
     7681 gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag cgcggccacc gagaatcgga
     7741 cgggggtagt ctcaagctgg ccggcctgct ctggtgcctg gcctcgcgcc gccgtgtatc
     7801 gccccgccct gggcggcaag gctggcccgg tcggcaccag ttgcgtgagc ggaaagatgg
     7861 ccgcttcccg gccctgctgc agggagctca aaatggagga cgcggcgctc gggagagcgg
     7921 gcgggtgagt cacccacaca aaggaaaagg gcctttccgt cctcagccgt cgcttcatgt
     7981 gactccacgg agtaccgggc gccgtccagg cacctcgatt agttctcgag cttttggagt
     8041 acgtcgtctt taggttgggg ggaggggttt tatgcgatgg agtttcccca cactgagtgg
     8101 gtggagactg aagttaggcc agcttggcac ttgatgtaat tctccttgga atttgccctt
     8161 tttgagtttg gatcttggtt cattctcaag cctcagacag tggttcaaag tttttttctt
     8221 ccatttcagg tgtcgtgaac tagtccagtg tggtggaatt ctgcagatat caacaagttt
     8281 gtacaaaaaa gcaggctggc gccaccatgg acaagaagta ctccattggg ctcgctatcg
     8341 gtaccaacag cgtcggctgg gccgtcatta cggacgagta caaggtgccg agcaaaaaat
     8401 tcaaagttct gggcaatacc gatcgccaca gcataaagaa gaacctcatt ggagccctcc
     8461 tgttcgactc cggggagacg gccgaagcca cgcggctcaa aagaacagca cggcgcagat
     8521 atacccgcag aaagaatcgg atctgctacc tgcaggagat ctttagtaat gagatggcta
     8581 aggtggatga ctctttcttc cataggctgg aggagtcctt tttggtggag gaggataaaa
     8641 agcacgagcg ccacccaatc tttggcaata tcgtggacga ggtggcgtac catgaaaagt
     8701 acccaaccat atatcatctg aggaagaagc tggtagacag tactgataag gctgacttgc
     8761 ggttgatcta tctcgcgctg gcgcacatga tcaaatttcg gggacacttc ctcatcgagg
     8821 gggacctgaa cccagacaac agcgatgtcg acaaactctt tatccaactg gttcagactt
     8881 acaatcagct tttcgaggag aacccgatca acgcatccgg cgttgacgcc aaagcaatcc
     8941 tgagcgctag gctgtccaaa tcccggcggc tcgaaaacct catcgcacag ctccctgggg
     9001 agaagaagaa cggcctgttt ggtaatctta tcgccctgtc actcgggctg acccccaact
     9061 ttaaatctaa cttcgacctg gccgaagatg ccaagctgca actgagcaaa gacacctacg
     9121 atgatgatct cgacaatctg ctggcccaga tcggcgacca gtacgcagac ctttttttgg
     9181 cggcaaagaa cctgtcagac gccattctgc tgagtgatat tctgcgagtg aacacggaga
     9241 tcaccaaagc tccgctgagc gctagtatga tcaagcgcta tgatgagcac caccaagact
     9301 tgactttgct gaaggccctt gtcagacagc aactgcctga gaagtacaag gaaattttct
     9361 tcgatcagtc taaaaatggc tacgccggat acattgacgg cggagcaagc caggaggaat
     9421 tttacaaatt tattaagccc atcttggaaa aaatggacgg caccgaggag ctgctggtaa
     9481 agctgaacag agaagatctg ttgcgcaaac agcgcacttt cgacaatgga agcatccccc
     9541 accagattca cctgggcgaa ctgcacgcta tcctcaggcg gcaagaggat ttctacccct
     9601 ttttgaaaga taacagggaa aagattgaga aaatcctcac atttcggata ccctactatg
     9661 taggccccct cgctcgggga aattccagat tcgcgtggat gactcgcaaa tcagaagaga
     9721 ccatcactcc ctggaacttc gaggaagtcg tggataaggg ggcctctgcc cagtccttca
     9781 tcgaaaggat gactaacttt gataaaaatc tgcctaacga aaaggtgctt cctaaacact
     9841 ctctgctgta cgagtacttc acagtttata acgagctcac caaggtcaaa tacgtcacag
     9901 aagggatgag aaagccagca ttcctgtctg gagagcagaa gaaagctatc gtggacctcc
     9961 tcttcaagac gaaccggaaa gttaccgtga aacagctcaa agaagactat ttcaaaaaga
    10021 ttgaatgttt cgactctgtt gaaatcagcg gagtggagga tcgcttcaac gcatccctgg
    10081 gaacgtatca cgatctcctg aaaatcatta aagacaagga cttcctggac aatgaggaga
    10141 acgaggacat tcttgaggac attgtcctca cccttacgtt gtttgaagat agggagatga
    10201 ttgaagaacg cttgaaaact tacgctcatc tcttcgacga caaagtcatg aaacagctca
    10261 agagacgccg atatacagga tgggggcggc tgtcaagaaa actgatcaat ggcatccgag
    10321 acaagcagag tggaaagaca atcctggatt ttcttaagtc cgatggattt gccaaccgga
    10381 acttcatgca gttgatccat gatgactctc tcacctttaa ggaggacatc cagaaagcac
    10441 aagtttctgg ccagggggac agtcttcacg agcacatcgc taatcttgca ggtagcccag
    10501 ctatcaaaaa gggaatactg cagaccgtta aggtcgtgga tgaactcgtc aaagtaatgg
    10561 gaaggcataa gcccgagaat atcgttatcg agatggcccg agagaaccaa actacccaga
    10621 agggacagaa gaacagtagg gaaaggatga agaggattga agagggtata aaagaactgg
    10681 ggtcccaaat ccttaaggaa cacccagttg aaaacaccca gcttcagaat gagaagctct
    10741 acctgtacta cctgcagaac ggcagggaca tgtacgtgga tcaggaactg gacatcaacc
    10801 ggttgtccga ctacgacgtg gatgctatcg tgccccaaag ctttctcaaa gatgattcta
    10861 ttgataataa agtgttgaca agatccgata aaaatagagg gaagagtgat aacgtcccct
    10921 cagaagaagt tgtcaagaaa atgaaaaatt attggcggca gctgctgaac gccaaactga
    10981 tcacacaacg gaagttcgat aatctgacta aggctgaacg aggtggcctg tctgagttgg
    11041 ataaagccgg cttcatcaaa aggcagcttg ttgagacacg ccagatcacc aagcacgtgg
    11101 cccaaattct cgattcacgc atgaacacca agtacgatga aaatgacaaa ctgattcgag
    11161 aggtgaaagt tattactctg aagtctaagc tggtctcaga tttcagaaag gactttcagt
    11221 tttataaggt gagagagatc aacaattacc accatgcgca tgatgcctac ctgaatgcag
    11281 tggtaggcac tgcacttatc aaaaaatatc ccaagctgga atctgaattt gtttacggag
    11341 actataaagt gtacgatgtt aggaaaatga tcgcaaagtc tgagcaggaa ataggcaagg
    11401 ccaccgctaa gtacttcttt tacagcaata ttatgaattt tttcaagacc gagattacac
    11461 tggccaatgg agagattcgg aagcgaccac ttatcgaaac aaacggagaa acaggagaaa
    11521 tcgtgtggga caagggtagg gatttcgcga cagtccgcaa ggtcctgtcc atgccgcagg
    11581 tgaacatcgt taaaaagacc gaagtacaga ccggaggctt ctccaaggaa agtatcctcc
    11641 cgaaaaggaa cagcgacaag ctgatcgcac gcaaaaaaga ttgggacccc aagaaatacg
    11701 gcggattcga ttctcctaca gtcgcttaca gtgtactggt tgtggccaaa gtggagaaag
    11761 ggaagtctaa aaaactcaaa agcgtcaagg aactgctggg catcacaatc atggagcgat
    11821 ccagcttcga gaaaaacccc atcgactttc tcgaagcgaa aggatataaa gaggtcaaaa
    11881 aagacctcat cattaagctg cccaagtact ctctctttga gcttgaaaac ggccggaaac
    11941 gaatgctcgc tagtgcgggc gagctgcaga aaggtaacga gctggcactg ccctctaaat
    12001 acgttaattt cttgtatctg gccagccact atgaaaagct caaagggtct cccgaagata
    12061 atgagcagaa gcagctgttc gtggaacaac acaaacacta ccttgatgag atcatcgagc
    12121 aaataagcga gttctccaaa agagtgatcc tcgccgacgc taacctcgat aaggtgcttt
    12181 ctgcttacaa taagcacagg gataagccca tcagggagca ggcagaaaac attatccact
    12241 tgtttactct gaccaacttg ggcgcgcctg cagccttcaa gtacttcgac accaccatag
    12301 acagaaagcg gtacacctct acaaaggagg tcctggacgc cacactgatt catcagtcaa
    12361 ttacggggct ctatgaaaca agaatcgacc tctctcagct cggtggagac ggcaccggcg
    12421 ggcccaagaa gaagaggaag gtatacccat acgatgttcc tgactatgcg ggctatccct
    12481 atgacgtccc ggactatgca ggatcgtatc cttatgacgt tccagattac gctggatcca
    12541 cacgcgttaa gcgatcgcgc
//
