LOCUS       sequence-173217-       14427 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tet-regulatable dCas9 lentiviral expression vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14427)
  AUTHORS   Kearns NA, Genga RM, Enuameh MS, Garber M, Wolfe SA, Maehr R
  TITLE     Cas9 effector-mediated regulation of transcription and 
            differentiation in human pluripotent stem cells.
  JOURNAL   Development. 2014 Jan;141(1):219-23. doi: 10.1242/dev.103341.
  PUBMED    24346702
REFERENCE   2  (bases 1 to 14427)
  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"; doi: "10.1242/dev"; 
            journalName: "Development"; date: "2014-01"; volume: "141"; issue: 
            "1"; pages: "219-23"
COMMENT     Sequence Label: pHAGE TRE dCas9
FEATURES             Location/Qualifiers
     source          1..14427
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             67..93
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     promoter        179..1390
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     1227..1246
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             join(1449..2066,2067..2072,2073..2108,2109..2111,2112..2147,
                     2148..2150,2151..2186,2187..2195)
                     /codon_start=1
                     /product="improved tetracycline-controlled transactivator"
                     /label=rtTA-Advanced
                     /note="In the Tet-On(R) system, rtTA-Advanced binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the presence of tetracycline or doxycycline."
                     /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     misc_feature    2233..2807
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2400..2417)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     CDS             2823..3617
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             2823..3617
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2877..2896)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3487..3506
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     misc_feature    3796..4384
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3849..3869)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4267..4278)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4459..4692
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(4811..4829)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4897..5001
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5002..5070,5071..5862)
                     /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(5220..5239)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6033..6621
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6522..6541
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6775..6792
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7032..7053
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7068..7073,7074..7091,7092..7098)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7106..7122
                     /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     7111..7133
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7130..7146
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7130..7146
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7196..7215
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             7407..8040
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    8087..8212
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    8709..8942
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             9127..9171
                     /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    9464..9581
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    join(9636..9654,9655..9677,9678..9696,9697..9719,9720..9738,
                     9739..9761,9762..9780,9781..9803,9804..9822,9823..9845,
                     9846..9864,9865..9887,9888..9906)
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     promoter        9939..9977
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     9974..9998
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    10107..10131
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      10135..10144
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(10141..10167,10168..10170,10171..12654,12655..12660,
                     12661..12726,12727..12729,12730..14244)
                     /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(10141..10167,10168..10170,10171..12657,12658..12660,
                     12661..14244)
                     /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             10141..14244
                     /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             14257..14277
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             14278..14367
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     protein_bind    complement(join(14417..14427,1..14))
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
ORIGIN
        1 ttgtacaaag tggttgatat ccagcacagt ggcggccgct cgagtctaga gggcccgcgg
       61 ttcgaatacc catacgacgt cccagactac gcttagtaat gattaattaa actagaaatt
      121 ctaccgggta ggggaggcgc ttttcccaag gcagtctgga gtaacccacc caagatctgg
      181 cctccgcgcc gggttttggc gcctcccgcg ggcgcccccc tcctcacggc gagcgctgcc
      241 acgtcagacg aagggcgcag cgagcgtcct gatccttccg cccggacgct caggacagcg
      301 gcccgctgct cataagactc ggccttagaa ccccagtatc agcagaagga cattttagga
      361 cgggacttgg gtgactctag ggcactggtt ttctttccag agagcggaac aggcgaggaa
      421 aagtagtccc ttctcggcga ttctgcggag ggatctccgt ggggcggtga acgccgatga
      481 ttatataagg acgcgccggg tgtggcacag ctagttccgt cgcagccggg atttgggtcg
      541 cggttcttgt ttgtggatcg ctgtgatcgt cacttggtga gtagcgggct gctgggctgg
      601 ccggggcttt cgtggccgcc gggccgctcg gtgggacgga agcgtgtgga gagaccgcca
      661 agggctgtag tctgggtccg cgagcaaggt tgccctgaac tgggggttgg ggggagcgca
      721 gcaaaatggc ggctgttccc gagtcttgaa tggaagacgc ttgtgaggcg ggctgtgagg
      781 tcgttgaaac aaggtggggg gcatggtggg cggcaagaac ccaaggtctt gaggccttcg
      841 ctaatgcggg aaagctctta ttcgggtgag atgggctggg gcaccatctg gggaccctga
      901 cgtgaagttt gtcactgact ggagaactcg gtttgtcgtc tgttgcgggg gcggcagtta
      961 tggcggtgcc gttgggcagt gcacccgtac ctttgggagc gcgcgccctc gtcgtgtcgt
     1021 gacgtcaccc gttctgttgg cttataatgc agggtggggc cacctgccgg taggtgtgcg
     1081 gtaggctttt ctccgtcgca ggacgcaggg ttcgggccta gggtaggctc tcctgaatcg
     1141 acaggcgccg gacctctggt gaggggaggg ataagtgagg cgtcagtttc tttggtcggt
     1201 tttatgtacc tatcttctta agtagctgaa gctccggttt tgaactatgc gctcggggtt
     1261 ggcgagtgtg ttttgtgaag ttttttaggc accttttgaa atgtaatcat ttgggtcaat
     1321 atgtaatttt cagtgttaga ctagtaaatt gtccgctaaa ttctggccgt ttttggcttt
     1381 tttgttagac gaagcttggt accgagctcg gatctccacc ccgtaccggt cctgcagtcg
     1441 aattcaccat gtctagactg gacaagagca aagtcataaa cggagctctg gaattactca
     1501 atggtgtcgg tatcgaaggc ctgacgacaa ggaaactcgc tcaaaagctg ggagttgagc
     1561 agcctaccct gtactggcac gtgaagaaca agcgggccct gctcgatgcc ctgccaatcg
     1621 agatgctgga caggcatcat acccacttct gccccctgga aggcgagtca tggcaagact
     1681 ttctgcggaa caacgccaag tcataccgct gtgctctcct ctcacatcgc gacggggcta
     1741 aagtgcatct cggcacccgc ccaacagaga aacagtacga aaccctggaa aatcagctcg
     1801 cgttcctgtg tcagcaaggc ttctccctgg agaacgcact gtacgctctg tccgccgtgg
     1861 gccactttac actgggctgc gtattggagg aacaggagca tcaagtagca aaagaggaaa
     1921 gagagacacc taccaccgat tctatgcccc cacttctgag acaagcaatt gagctgttcg
     1981 accggcaggg agccgaacct gccttccttt tcggcctgga actaatcata tgtggcctgg
     2041 agaaacagct aaagtgcgaa agcggcgggc cgaccgacgc ccttgacgat tttgacttag
     2101 acatgctccc agccgatgcc cttgacgact ttgaccttga tatgctgcct gctgacgctc
     2161 ttgacgattt tgaccttgac atgctccccg ggtaactaag taaggatccg cggccgcact
     2221 agaggaattc cgcccctctc cctccccccc ccctaacgtt actggccgaa gccgcttgga
     2281 ataaggccgg tgtgtgtttg tctatatgtt attttccacc atattgccgt cttttggcaa
     2341 tgtgagggcc cggaaacctg gccctgtctt cttgacgagc attcctaggg gtctttcccc
     2401 tctcgccaaa ggaatgcaag gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc
     2461 ttcttgaaga caaacaacgt ctgtagcgac cctttgcagg cagcggaacc ccccacctgg
     2521 cgacaggtgc ctctgcggcc aaaagccacg tgtataagat acacctgcaa aggcggcaca
     2581 accccagtgc cacgttgtga gttggatagt tgtggaaaga gtcaaatggc tctcctcaag
     2641 cgtagtcaac aaggggctga aggatgccca gaaggtaccc cattgtatgg gaatctgatc
     2701 tggggcctcg gtgcacatgc tttacatgtg tttagtcgag gttaaaaaaa cgtctaggcc
     2761 ccccgaacca cggggacgtg gttttccttt gaaaaacacg atgataagct taccggtcca
     2821 ccatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat
     2881 tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt
     2941 cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac
     3001 tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg
     3061 tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc
     3121 aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa
     3181 tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc
     3241 gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg
     3301 aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg agcatgcccg
     3361 acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa
     3421 atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg
     3481 acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct
     3541 tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc
     3601 ttgacgagtt cttctgatgt acaagtaaag cggccgcgac tctagatcat aatcagccat
     3661 accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg
     3721 aaacataaaa tgaatgcaat tgttgttgtt tagtccctcc caattcgata tcaagcttat
     3781 cgatcgatag atcctaatca acctctggat tacaaaattt gtgaaagatt gactggtatt
     3841 cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat
     3901 gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct
     3961 ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct
     4021 gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc
     4081 gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg
     4141 acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc
     4201 tttccttggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac
     4261 gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg
     4321 cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc
     4381 ccgcctgaga tcctttaaga ccaatgactt acaaggcagc tgtagatctt agccactttt
     4441 taaaagaaaa ggggggactg gaagggctaa ttcactccca acgaagacaa gatctgcttt
     4501 ttgcttgtac tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac
     4561 tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg
     4621 cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga
     4681 aaatctctag cagtagtagt tcatgtcatc ttattattca gtatttataa cttgcaaaga
     4741 aatgaatatc agagagtgag aggcccgggt taattaagga aagggctaga tcattcttga
     4801 agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt
     4861 tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     4921 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     4981 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     5041 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     5101 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     5161 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     5221 ctatgtggcg cggtattatc ccgtgttgac gccgggcaag agcaactcgg tcgccgcata
     5281 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     5341 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     5401 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     5461 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     5521 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     5581 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     5641 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     5701 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     5761 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     5821 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     5881 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     5941 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     6001 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     6061 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     6121 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     6181 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     6241 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     6301 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     6361 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     6421 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     6481 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     6541 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     6601 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     6661 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     6721 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     6781 tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg
     6841 ccgattcatt aatgcagcaa gctcatggct gactaatttt ttttatttat gcagaggccg
     6901 aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     6961 gcttttgcaa aaagctcccc gtggcacgac aggtttcccg actggaaagc gggcagtgag
     7021 cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg
     7081 cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc
     7141 tatgacatga ttacgaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     7201 ttgtccaaac tcatcaatgt atcttatcat gtctggatca actggataac tcaagctaac
     7261 caaaatcatc ccaaacttcc caccccatac cctattacca ctgccaatta cctgtggttt
     7321 catttactct aaacctgtga ttcctctgaa ttattttcat tttaaagaaa ttgtatttgt
     7381 taaatatgta ctacaaactt agtagttgga agggctaatt cactcccaaa gaagacaaga
     7441 tatccttgat ctgtggatct accacacaca aggctacttc cctgattagc agaactacac
     7501 accagggcca ggggtcagat atccactgac ctttggatgg tgctacaagc tagtaccagt
     7561 tgagccagat aaggtagaag aggccaataa aggagagaac accagcttgt tacaccctgt
     7621 gagcctgcat gggatggatg acccggagag agaagtgtta gagtggaggt ttgacagccg
     7681 cctagcattt catcacgtgg cccgagagct gcatccggag tacttcaaga actgctgata
     7741 tcgagcttgc tacaagggac tttccgctgg ggactttcca gggaggcgtg gcctgggcgg
     7801 gactggggag tggcgagccc tcagatcctg catataagca gctgcttttt gcctgtactg
     7861 ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac
     7921 tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt
     7981 gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca
     8041 gtggcgcccg aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag
     8101 gactcggctt gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc
     8161 aaaaattttg actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa
     8221 gcgggggaga attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa
     8281 atataaatta aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc
     8341 tggcctgtta gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct
     8401 tcagacagga tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt
     8461 gcatcaaagg atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca
     8521 aaacaaaagt aagaccaccg cacagcaagc ggccggccgc tgatcttcag acctggagga
     8581 ggagatatga gggacaattg gagaagtgaa ttatataaat ataaagtagt aaaaattgaa
     8641 ccattaggag tagcacccac caaggcaaag agaagagtgg tgcagagaga aaaaagagca
     8701 gtgggaatag gagctttgtt ccttgggttc ttgggagcag caggaagcac tatgggcgca
     8761 gcgtcaatga cgctgacggt acaggccaga caattattgt ctggtatagt gcagcagcag
     8821 aacaatttgc tgagggctat tgaggcgcaa cagcatctgt tgcaactcac agtctggggc
     8881 atcaagcagc tccaggcaag aatcctggct gtggaaagat acctaaagga tcaacagctc
     8941 ctggggattt ggggttgctc tggaaaactc atttgcacca ctgctgtgcc ttggaatgct
     9001 agttggagta ataaatctct ggaacagatt tggaatcaca cgacctggat ggagtgggac
     9061 agagaaatta acaattacac aagcttaata cactccttaa ttgaagaatc gcaaaaccag
     9121 caagaaaaga atgaacaaga attattggaa ttagataaat gggcaagttt gtggaattgg
     9181 tttaacataa caaattggct gtggtatata aaattattca taatgatagt aggaggcttg
     9241 gtaggtttaa gaatagtttt tgctgtactt tctatagtga atagagttag gcagggatat
     9301 tcaccattat cgtttcagac ccacctccca accccgaggg gacccgacag gcccgaagga
     9361 atagaagaag aaggtggaga gagagacaga gacagatcca ttcgattagt gaacggatct
     9421 cgacggtatc gccgaattca caaatggcag tattcatcca caattttaaa agaaaagggg
     9481 ggattggggg gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa
     9541 ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca
     9601 gcagagatcc agtttggact aggatccttt accactccct atcagtgata gagaaaagtg
     9661 aaagtcgagt ttaccactcc ctatcagtga tagagaaaag tgaaagtcga gtttaccact
     9721 ccctatcagt gatagagaaa agtgaaagtc gagtttacca ctccctatca gtgatagaga
     9781 aaagtgaaag tcgagtttac cactccctat cagtgataga gaaaagtgaa agtcgagttt
     9841 accactccct atcagtgata gagaaaagtg aaagtcgagt ttaccactcc ctatcagtga
     9901 tagagaaaag tgaaagtcga gctcggtacc cgggtcgagg taggcgtgta cggtgggagg
     9961 cctatataag cagagctcgt ttagtgaacc gtcagatcgc ctggagacgc catccacgct
    10021 gttttgacct ccatagaaga caccgggacc gatccagcct ccgcggcccc gaactagtcc
    10081 agtgtggtgg aattctgcag atatcaacaa gtttgtacaa aaaagcaggc tggcgccacc
    10141 atggacaaga agtactccat tgggctcgct atcggtacca acagcgtcgg ctgggccgtc
    10201 attacggacg agtacaaggt gccgagcaaa aaattcaaag ttctgggcaa taccgatcgc
    10261 cacagcataa agaagaacct cattggagcc ctcctgttcg actccgggga gacggccgaa
    10321 gccacgcggc tcaaaagaac agcacggcgc agatataccc gcagaaagaa tcggatctgc
    10381 tacctgcagg agatctttag taatgagatg gctaaggtgg atgactcttt cttccatagg
    10441 ctggaggagt cctttttggt ggaggaggat aaaaagcacg agcgccaccc aatctttggc
    10501 aatatcgtgg acgaggtggc gtaccatgaa aagtacccaa ccatatatca tctgaggaag
    10561 aagctggtag acagtactga taaggctgac ttgcggttga tctatctcgc gctggcgcac
    10621 atgatcaaat ttcggggaca cttcctcatc gagggggacc tgaacccaga caacagcgat
    10681 gtcgacaaac tctttatcca actggttcag acttacaatc agcttttcga ggagaacccg
    10741 atcaacgcat ccggcgttga cgccaaagca atcctgagcg ctaggctgtc caaatcccgg
    10801 cggctcgaaa acctcatcgc acagctccct ggggagaaga agaacggcct gtttggtaat
    10861 cttatcgccc tgtcactcgg gctgaccccc aactttaaat ctaacttcga cctggccgaa
    10921 gatgccaagc tgcaactgag caaagacacc tacgatgatg atctcgacaa tctgctggcc
    10981 cagatcggcg accagtacgc agaccttttt ttggcggcaa agaacctgtc agacgccatt
    11041 ctgctgagtg atattctgcg agtgaacacg gagatcacca aagctccgct gagcgctagt
    11101 atgatcaagc gctatgatga gcaccaccaa gacttgactt tgctgaaggc ccttgtcaga
    11161 cagcaactgc ctgagaagta caaggaaatt ttcttcgatc agtctaaaaa tggctacgcc
    11221 ggatacattg acggcggagc aagccaggag gaattttaca aatttattaa gcccatcttg
    11281 gaaaaaatgg acggcaccga ggagctgctg gtaaagctga acagagaaga tctgttgcgc
    11341 aaacagcgca ctttcgacaa tggaagcatc ccccaccaga ttcacctggg cgaactgcac
    11401 gctatcctca ggcggcaaga ggatttctac ccctttttga aagataacag ggaaaagatt
    11461 gagaaaatcc tcacatttcg gataccctac tatgtaggcc ccctcgctcg gggaaattcc
    11521 agattcgcgt ggatgactcg caaatcagaa gagaccatca ctccctggaa cttcgaggaa
    11581 gtcgtggata agggggcctc tgcccagtcc ttcatcgaaa ggatgactaa ctttgataaa
    11641 aatctgccta acgaaaaggt gcttcctaaa cactctctgc tgtacgagta cttcacagtt
    11701 tataacgagc tcaccaaggt caaatacgtc acagaaggga tgagaaagcc agcattcctg
    11761 tctggagagc agaagaaagc tatcgtggac ctcctcttca agacgaaccg gaaagttacc
    11821 gtgaaacagc tcaaagaaga ctatttcaaa aagattgaat gtttcgactc tgttgaaatc
    11881 agcggagtgg aggatcgctt caacgcatcc ctgggaacgt atcacgatct cctgaaaatc
    11941 attaaagaca aggacttcct ggacaatgag gagaacgagg acattcttga ggacattgtc
    12001 ctcaccctta cgttgtttga agatagggag atgattgaag aacgcttgaa aacttacgct
    12061 catctcttcg acgacaaagt catgaaacag ctcaagagac gccgatatac aggatggggg
    12121 cggctgtcaa gaaaactgat caatggcatc cgagacaagc agagtggaaa gacaatcctg
    12181 gattttctta agtccgatgg atttgccaac cggaacttca tgcagttgat ccatgatgac
    12241 tctctcacct ttaaggagga catccagaaa gcacaagttt ctggccaggg ggacagtctt
    12301 cacgagcaca tcgctaatct tgcaggtagc ccagctatca aaaagggaat actgcagacc
    12361 gttaaggtcg tggatgaact cgtcaaagta atgggaaggc ataagcccga gaatatcgtt
    12421 atcgagatgg cccgagagaa ccaaactacc cagaagggac agaagaacag tagggaaagg
    12481 atgaagagga ttgaagaggg tataaaagaa ctggggtccc aaatccttaa ggaacaccca
    12541 gttgaaaaca cccagcttca gaatgagaag ctctacctgt actacctgca gaacggcagg
    12601 gacatgtacg tggatcagga actggacatc aaccggttgt ccgactacga cgtggatgct
    12661 atcgtgcccc aaagctttct caaagatgat tctattgata ataaagtgtt gacaagatcc
    12721 gataaaaata gagggaagag tgataacgtc ccctcagaag aagttgtcaa gaaaatgaaa
    12781 aattattggc ggcagctgct gaacgccaaa ctgatcacac aacggaagtt cgataatctg
    12841 actaaggctg aacgaggtgg cctgtctgag ttggataaag ccggcttcat caaaaggcag
    12901 cttgttgaga cacgccagat caccaagcac gtggcccaaa ttctcgattc acgcatgaac
    12961 accaagtacg atgaaaatga caaactgatt cgagaggtga aagttattac tctgaagtct
    13021 aagctggtct cagatttcag aaaggacttt cagttttata aggtgagaga gatcaacaat
    13081 taccaccatg cgcatgatgc ctacctgaat gcagtggtag gcactgcact tatcaaaaaa
    13141 tatcccaagc tggaatctga atttgtttac ggagactata aagtgtacga tgttaggaaa
    13201 atgatcgcaa agtctgagca ggaaataggc aaggccaccg ctaagtactt cttttacagc
    13261 aatattatga attttttcaa gaccgagatt acactggcca atggagagat tcggaagcga
    13321 ccacttatcg aaacaaacgg agaaacagga gaaatcgtgt gggacaaggg tagggatttc
    13381 gcgacagtcc gcaaggtcct gtccatgccg caggtgaaca tcgttaaaaa gaccgaagta
    13441 cagaccggag gcttctccaa ggaaagtatc ctcccgaaaa ggaacagcga caagctgatc
    13501 gcacgcaaaa aagattggga ccccaagaaa tacggcggat tcgattctcc tacagtcgct
    13561 tacagtgtac tggttgtggc caaagtggag aaagggaagt ctaaaaaact caaaagcgtc
    13621 aaggaactgc tgggcatcac aatcatggag cgatccagct tcgagaaaaa ccccatcgac
    13681 tttctcgaag cgaaaggata taaagaggtc aaaaaagacc tcatcattaa gctgcccaag
    13741 tactctctct ttgagcttga aaacggccgg aaacgaatgc tcgctagtgc gggcgagctg
    13801 cagaaaggta acgagctggc actgccctct aaatacgtta atttcttgta tctggccagc
    13861 cactatgaaa agctcaaagg gtctcccgaa gataatgagc agaagcagct gttcgtggaa
    13921 caacacaaac actaccttga tgagatcatc gagcaaataa gcgagttctc caaaagagtg
    13981 atcctcgccg acgctaacct cgataaggtg ctttctgctt acaataagca cagggataag
    14041 cccatcaggg agcaggcaga aaacattatc cacttgttta ctctgaccaa cttgggcgcg
    14101 cctgcagcct tcaagtactt cgacaccacc atagacagaa agcggtacac ctctacaaag
    14161 gaggtcctgg acgccacact gattcatcag tcaattacgg ggctctatga aacaagaatc
    14221 gacctctctc agctcggtgg agacggcacc ggcgggccca agaagaagag gaaggtatac
    14281 ccatacgatg ttcctgacta tgcgggctat ccctatgacg tcccggacta tgcaggatcg
    14341 tatccttatg acgttccaga ttacgctgga tcctaatgat tcgaatccgg ataagcggcc
    14401 gcctcgagat atctagaccc agctttc
//
