LOCUS       sequence-328922-       15217 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of dual inducible Cas9 with CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 15217)
  AUTHORS   Park SB, Uchida T, Tilson S, Hu Z, Ma CD, Leek M, Eichner M, Hong 
            SG, Liang TJ
  TITLE     A dual conditional CRISPR-Cas9 system to activate gene editing and 
            reduce off-target effects in human stem cells.
  JOURNAL   Mol Ther Nucleic Acids. 2022 Apr 27;28:656-669. doi: 
            10.1016/j.omtn.2022.04.013. eCollection 2022 Jun 14.
  PUBMED    35615005
REFERENCE   2  (bases 1 to 15217)
  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: "Mol Ther 
            Nucleic Acids."; date: "2022-04-27"; volume: "28:656-669. doi"; 
            pages: " 10.1016/j.omtn.2022.04.013. eCollection 2022 Jun 14"
COMMENT     Sequence Label: Lenti-Dual-iCas9-CMV
FEATURES             Location/Qualifiers
     source          1..15217
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             85..718
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    765..890
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1387..1620
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1805..1849
                     /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    2142..2259
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    join(2314..2332,2333..2355,2356..2374,2375..2397,2398..2416,
                     2417..2439,2440..2458,2459..2481,2482..2500,2501..2523,
                     2524..2542,2543..2565,2566..2584)
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     promoter        2617..2655
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2652..2676
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2687..2696
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2693..2695,2696..3016)
                     /codon_start=1
                     /product="destabilization domain that can be stabilized by 
                     Shield1 in the ProteoTuner(TM) system"
                     /label=DD
                     /note="L106P mutant of FKBP12"
                     /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNK
                     PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL
                     LKPE"
     CDS             3026..7129
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             3026..7129
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             7130..7177
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7178..7201
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             7217..7273
                     /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"
     CDS             7274..7789
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     polyA_signal    7823..7944
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7860..7879)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             7951..7977
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     promoter        8063..9274
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     9111..9130
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             join(9333..9950,9951..9956,9957..9992,9993..9995,
                     9996..10031,10032..10034,10035..10070,10071..10079)
                     /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    10131..10704
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(10298..10315)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     10525..10544
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             10720..11514
                     /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             10720..11514
                     /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(10774..10793)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     11384..11403
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     misc_feature    11691..12279
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(11744..11764)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(12162..12173)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             12354..12587
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(12706..12724)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        12792..12896
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(12897..12965,12966..13757)
                     /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(13115..13134)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      13928..14516
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     14417..14436
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     14670..14687
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    14927..14948
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(14963..14968,14969..14986,14987..14993)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    15001..15017
                     /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     15006..15028
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     15025..15041
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     15025..15041
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 tttactctaa acctgtgatt cctctgaatt attttcattt taaagaaatt gtatttgtta
       61 aatatgtact acaaacttag tagttggaag ggctaattca ctcccaaaga agacaagata
      121 tccttgatct gtggatctac cacacacaag gctacttccc tgattagcag aactacacac
      181 cagggccagg ggtcagatat ccactgacct ttggatggtg ctacaagcta gtaccagttg
      241 agccagataa ggtagaagag gccaataaag gagagaacac cagcttgtta caccctgtga
      301 gcctgcatgg gatggatgac ccggagagag aagtgttaga gtggaggttt gacagccgcc
      361 tagcatttca tcacgtggcc cgagagctgc atccggagta cttcaagaac tgctgatatc
      421 gagcttgcta caagggactt tccgctgggg actttccagg gaggcgtggc ctgggcggga
      481 ctggggagtg gcgagccctc agatcctgca tataagcagc tgctttttgc ctgtactggg
      541 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
      601 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
      661 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt
      721 ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga
      781 ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa
      841 aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc
      901 gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat
      961 ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg
     1021 gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc
     1081 agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc
     1141 atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa
     1201 acaaaagtaa gaccaccgca cagcaagcgg ccggccgctg atcttcagac ctggaggagg
     1261 agatatgagg gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc
     1321 attaggagta gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt
     1381 gggaatagga gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc
     1441 gtcaatgacg ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa
     1501 caatttgctg agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat
     1561 caagcagctc caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct
     1621 ggggatttgg ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag
     1681 ttggagtaat aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag
     1741 agaaattaac aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca
     1801 agaaaagaat gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt
     1861 taacataaca aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt
     1921 aggtttaaga atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc
     1981 accattatcg tttcagaccc acctcccaac cccgagggga cccgacaggc ccgaaggaat
     2041 agaagaagaa ggtggagaga gagacagaga cagatccatt cgattagtga acggatctcg
     2101 acggtatcgc cgaattcaca aatggcagta ttcatccaca attttaaaag aaaagggggg
     2161 attggggggt acagtgcagg ggaaagaata gtagacataa tagcaacaga catacaaact
     2221 aaagaattac aaaaacaaat tacaaaaatt caaaattttc gggtttatta cagggacagc
     2281 agagatccag tttggactag gatcctttac cactccctat cagtgataga gaaaagtgaa
     2341 agtcgagttt accactccct atcagtgata gagaaaagtg aaagtcgagt ttaccactcc
     2401 ctatcagtga tagagaaaag tgaaagtcga gtttaccact ccctatcagt gatagagaaa
     2461 agtgaaagtc gagtttacca ctccctatca gtgatagaga aaagtgaaag tcgagtttac
     2521 cactccctat cagtgataga gaaaagtgaa agtcgagttt accactccct atcagtgata
     2581 gagaaaagtg aaagtcgagc tcggtacccg ggtcgaggta ggcgtgtacg gtgggaggcc
     2641 tatataagca gagctcgttt agtgaaccgt cagatcgcct tctagagcca ccatgggagt
     2701 gcaggtggaa accatctccc caggtgacgg gcgcaccttc cccaagcgcg gccagacctg
     2761 tgtggtgcac tacaccggga tgcttgaaga tggaaagaaa gtcgattcct cccgggacag
     2821 aaacaagccc tttaagttta tgctaggcaa gcaggaggtg atccgaggct gggaagaagg
     2881 ggttgcccag atgagtgtgg gtcagagagc caaactgact atatctccag attatgccta
     2941 tggtgccact gggcacccag gcatcatccc accacatgcc actctcgtct tcgatgtgga
     3001 gcttctaaaa ccggaaccgg ctagcatgga caagaagtac agcatcggcc tggacatcgg
     3061 caccaactct gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt
     3121 caaggtgctg ggcaacaccg accggcacag catcaagaag aacctgatcg gagccctgct
     3181 gttcgacagc ggcgaaacag ccgaggccac ccggctgaag agaaccgcca gaagaagata
     3241 caccagacgg aagaaccgga tctgctatct gcaagagatc ttcagcaacg agatggccaa
     3301 ggtggacgac agcttcttcc acagactgga agagtccttc ctggtggaag aggataagaa
     3361 gcacgagcgg caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta
     3421 ccccaccatc taccacctga gaaagaaact ggtggacagc accgacaagg ccgacctgcg
     3481 gctgatctat ctggccctgg cccacatgat caagttccgg ggccacttcc tgatcgaggg
     3541 cgacctgaac cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta
     3601 caaccagctg ttcgaggaaa accccatcaa cgccagcggc gtggacgcca aggccatcct
     3661 gtctgccaga ctgagcaaga gcagacggct ggaaaatctg atcgcccagc tgcccggcga
     3721 gaagaagaat ggcctgttcg gaaacctgat tgccctgagc ctgggcctga cccccaactt
     3781 caagagcaac ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg acacctacga
     3841 cgacgacctg gacaacctgc tggcccagat cggcgaccag tacgccgacc tgtttctggc
     3901 cgccaagaac ctgtccgacg ccatcctgct gagcgacatc ctgagagtga acaccgagat
     3961 caccaaggcc cccctgagcg cctctatgat caagagatac gacgagcacc accaggacct
     4021 gaccctgctg aaagctctcg tgcggcagca gctgcctgag aagtacaaag agattttctt
     4081 cgaccagagc aagaacggct acgccggcta cattgacggc ggagccagcc aggaagagtt
     4141 ctacaagttc atcaagccca tcctggaaaa gatggacggc accgaggaac tgctcgtgaa
     4201 gctgaacaga gaggacctgc tgcggaagca gcggaccttc gacaacggca gcatccccca
     4261 ccagatccac ctgggagagc tgcacgccat tctgcggcgg caggaagatt tttacccatt
     4321 cctgaaggac aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc cctactacgt
     4381 gggccctctg gccaggggaa acagcagatt cgcctggatg accagaaaga gcgaggaaac
     4441 catcaccccc tggaacttcg aggaagtggt ggacaagggc gcttccgccc agagcttcat
     4501 cgagcggatg accaacttcg ataagaacct gcccaacgag aaggtgctgc ccaagcacag
     4561 cctgctgtac gagtacttca ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga
     4621 gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg tggacctgct
     4681 gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat
     4741 cgagtgcttc gactccgtgg aaatctccgg cgtggaagat cggttcaacg cctccctggg
     4801 cacataccac gatctgctga aaattatcaa ggacaaggac ttcctggaca atgaggaaaa
     4861 cgaggacatt ctggaagata tcgtgctgac cctgacactg tttgaggaca gagagatgat
     4921 cgaggaacgg ctgaaaacct atgcccacct gttcgacgac aaagtgatga agcagctgaa
     4981 gcggcggaga tacaccggct ggggcaggct gagccggaag ctgatcaacg gcatccggga
     5041 caagcagtcc ggcaagacaa tcctggattt cctgaagtcc gacggcttcg ccaacagaaa
     5101 cttcatgcag ctgatccacg acgacagcct gacctttaaa gaggacatcc agaaagccca
     5161 ggtgtccggc cagggcgata gcctgcacga gcacattgcc aatctggccg gcagccccgc
     5221 cattaagaag ggcatcctgc agacagtgaa ggtggtggac gagctcgtga aagtgatggg
     5281 ccggcacaag cccgagaaca tcgtgatcga aatggccaga gagaaccaga ccacccagaa
     5341 gggacagaag aacagccgcg agagaatgaa gcggatcgaa gagggcatca aagagctggg
     5401 cagccagatc ctgaaagaac accccgtgga aaacacccag ctgcagaacg agaagctgta
     5461 cctgtactac ctgcagaatg ggcgggatat gtacgtggac caggaactgg acatcaaccg
     5521 gctgtccgac tacgatgtgg accatatcgt gcctcagagc tttctgaagg acgactccat
     5581 cgacaacaag gtgctgacca gaagcgacaa gaaccggggc aagagcgaca acgtgccctc
     5641 cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag ctgctgaacg ccaagctgat
     5701 tacccagaga aagttcgaca atctgaccaa ggccgagaga ggcggcctga gcgaactgga
     5761 taaggccggc ttcatcaaga gacagctggt ggaaacccgg cagatcacaa agcacgtggc
     5821 acagatcctg gactcccgga tgaacactaa gtacgacgag aatgacaagc tgatccggga
     5881 agtgaaagtg atcaccctga agtccaagct ggtgtccgat ttccggaagg atttccagtt
     5941 ttacaaagtg cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt
     6001 cgtgggaacc gccctgatca aaaagtaccc taagctggaa agcgagttcg tgtacggcga
     6061 ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc
     6121 taccgccaag tacttcttct acagcaacat catgaacttt ttcaagaccg agattaccct
     6181 ggccaacggc gagatccgga agcggcctct gatcgagaca aacggcgaaa ccggggagat
     6241 cgtgtgggat aagggccggg attttgccac cgtgcggaaa gtgctgagca tgccccaagt
     6301 gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc agcaaagagt ctatcctgcc
     6361 caagaggaac agcgataagc tgatcgccag aaagaaggac tgggacccta agaagtacgg
     6421 cggcttcgac agccccaccg tggcctattc tgtgctggtg gtggccaaag tggaaaaggg
     6481 caagtccaag aaactgaaga gtgtgaaaga gctgctgggg atcaccatca tggaaagaag
     6541 cagcttcgag aagaatccca tcgactttct ggaagccaag ggctacaaag aagtgaaaaa
     6601 ggacctgatc atcaagctgc ctaagtactc cctgttcgag ctggaaaacg gccggaagag
     6661 aatgctggcc tctgccggcg aactgcagaa gggaaacgaa ctggccctgc cctccaaata
     6721 tgtgaacttc ctgtacctgg ccagccacta tgagaagctg aagggctccc ccgaggataa
     6781 tgagcagaaa cagctgtttg tggaacagca caagcactac ctggacgaga tcatcgagca
     6841 gatcagcgag ttctccaaga gagtgatcct ggccgacgct aatctggaca aagtgctgtc
     6901 cgcctacaac aagcaccggg ataagcccat cagagagcag gccgagaata tcatccacct
     6961 gtttaccctg accaatctgg gagcccctgc cgccttcaag tactttgaca ccaccatcga
     7021 ccggaagagg tacaccagca ccaaagaggt gctggacgcc accctgatcc accagagcat
     7081 caccggcctg tacgagacac ggatcgacct gtctcagctg ggaggcgaca agcgacctgc
     7141 cgccacaaag aaggctggac aggctaagaa gaagaaagat tacaaagacg atgacgataa
     7201 gggatcggat ccggaggcta ctaacttcag cctgctgaag caggctgggg acgtggagga
     7261 gaaccctgga cctatggtct tcacactcga agatttcgtt ggggactggc gacagacagc
     7321 cggctacaac ctggaccaag tccttgaaca gggaggtgtg tccagtttgt ttcagaatct
     7381 cggggtgtcc gtaactccga tccaaaggat tgtcctgagc ggtgaaaatg ggctgaagat
     7441 cgacatccat gtcatcatcc cgtatgaagg tctgagcggc gaccaaatgg gccagatcga
     7501 aaaaattttt aaggtggtgt accctgtgga tgatcatcac tttaaggtga tcctgcacta
     7561 tggcacactg gtaatcgacg gggttacgcc gaacatgatc gactatttcg gacggccgta
     7621 tgaaggcatc gccgtgttcg acggcaaaaa gatcactgta acagggaccc tgtggaacgg
     7681 caacaaaatt atcgacgagc gcctgatcaa ccccgacggc tccctgctgt tccgagtaac
     7741 catcaacgga gtgaccggct ggcggctgtg cgaacgcatt ctggcgtaac tcgagtcgag
     7801 tctagagggc ccgcggttcg aaaacttgtt tattgcagct tataatggtt acaaataaag
     7861 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     7921 gtccaaactc atcaatgtat cttattcgaa tacccatacg acgtcccaga ctacgcttag
     7981 taatgattaa ttaaactaga aattctaccg ggtaggggag gcgcttttcc caaggcagtc
     8041 tggagtaacc cacccaagat ctggcctccg cgccgggttt tggcgcctcc cgcgggcgcc
     8101 cccctcctca cggcgagcgc tgccacgtca gacgaagggc gcagcgagcg tcctgatcct
     8161 tccgcccgga cgctcaggac agcggcccgc tgctcataag actcggcctt agaaccccag
     8221 tatcagcaga aggacatttt aggacgggac ttgggtgact ctagggcact ggttttcttt
     8281 ccagagagcg gaacaggcga ggaaaagtag tcccttctcg gcgattctgc ggagggatct
     8341 ccgtggggcg gtgaacgccg atgattatat aaggacgcgc cgggtgtggc acagctagtt
     8401 ccgtcgcagc cgggatttgg gtcgcggttc ttgtttgtgg atcgctgtga tcgtcacttg
     8461 gtgagtagcg ggctgctggg ctggccgggg ctttcgtggc cgccgggccg ctcggtggga
     8521 cggaagcgtg tggagagacc gccaagggct gtagtctggg tccgcgagca aggttgccct
     8581 gaactggggg ttggggggag cgcagcaaaa tggcggctgt tcccgagtct tgaatggaag
     8641 acgcttgtga ggcgggctgt gaggtcgttg aaacaaggtg gggggcatgg tgggcggcaa
     8701 gaacccaagg tcttgaggcc ttcgctaatg cgggaaagct cttattcggg tgagatgggc
     8761 tggggcacca tctggggacc ctgacgtgaa gtttgtcact gactggagaa ctcggtttgt
     8821 cgtctgttgc gggggcggca gttatggcgg tgccgttggg cagtgcaccc gtacctttgg
     8881 gagcgcgcgc cctcgtcgtg tcgtgacgtc acccgttctg ttggcttata atgcagggtg
     8941 gggccacctg ccggtaggtg tgcggtaggc ttttctccgt cgcaggacgc agggttcggg
     9001 cctagggtag gctctcctga atcgacaggc gccggacctc tggtgagggg agggataagt
     9061 gaggcgtcag tttctttggt cggttttatg tacctatctt cttaagtagc tgaagctccg
     9121 gttttgaact atgcgctcgg ggttggcgag tgtgttttgt gaagtttttt aggcaccttt
     9181 tgaaatgtaa tcatttgggt caatatgtaa ttttcagtgt tagactagta aattgtccgc
     9241 taaattctgg ccgtttttgg cttttttgtt agacgaagct tggtaccgag ctcggatctc
     9301 caccccgtac cggtcctgca gtcgaattca ccatgtctag actggacaag agcaaagtca
     9361 taaacggagc tctggaatta ctcaatggtg tcggtatcga aggcctgacg acaaggaaac
     9421 tcgctcaaaa gctgggagtt gagcagccta ccctgtactg gcacgtgaag aacaagcggg
     9481 ccctgctcga tgccctgcca atcgagatgc tggacaggca tcatacccac ttctgccccc
     9541 tggaaggcga gtcatggcaa gactttctgc ggaacaacgc caagtcatac cgctgtgctc
     9601 tcctctcaca tcgcgacggg gctaaagtgc atctcggcac ccgcccaaca gagaaacagt
     9661 acgaaaccct ggaaaatcag ctcgcgttcc tgtgtcagca aggcttctcc ctggagaacg
     9721 cactgtacgc tctgtccgcc gtgggccact ttacactggg ctgcgtattg gaggaacagg
     9781 agcatcaagt agcaaaagag gaaagagaga cacctaccac cgattctatg cccccacttc
     9841 tgagacaagc aattgagctg ttcgaccggc agggagccga acctgccttc cttttcggcc
     9901 tggaactaat catatgtggc ctggagaaac agctaaagtg cgaaagcggc gggccgaccg
     9961 acgcccttga cgattttgac ttagacatgc tcccagccga tgcccttgac gactttgacc
    10021 ttgatatgct gcctgctgac gctcttgacg attttgacct tgacatgctc cccgggtaac
    10081 taagtaagga tccgcggccg cactagagga attccgcccc tctccccccc cccctctccc
    10141 tccccccccc ctaacgttac tggccgaagc cgcttggaat aaggccggtg tgcgtttgtc
    10201 tatatgttat tttccaccat attgccgtct tttggcaatg tgagggcccg gaaacctggc
    10261 cctgtcttct tgacgagcat tcctaggggt ctttcccctc tcgccaaagg aatgcaaggt
    10321 ctgttgaatg tcgtgaagga agcagttcct ctggaagctt cttgaagaca aacaacgtct
    10381 gtagcgaccc tttgcaggca gcggaacccc ccacctggcg acaggtgcct ctgcggccaa
    10441 aagccacgtg tataagatac acctgcaaag gcggcacaac cccagtgcca cgttgtgagt
    10501 tggatagttg tggaaagagt caaatggctc tcctcaagcg tattcaacaa ggggctgaag
    10561 gatgcccaga aggtacccca ttgtatggga tctgatctgg ggcctcggtg cacatgcttt
    10621 acatgtgttt agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg ggacgtggtt
    10681 ttcctttgaa aaacacgatg ataagcttac cggtccacca tgattgaaca agatggattg
    10741 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
    10801 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
    10861 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aagacgaggc agcgcggcta
    10921 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
    10981 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
    11041 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
    11101 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
    11161 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
    11221 gccgaactgt tcgccaggct caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc
    11281 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
    11341 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
    11401 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
    11461 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgatgtaca
    11521 agtaaagcgg ccgcgactct agatcataat cagccatacc acatttgtag aggttttact
    11581 tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt
    11641 tgttgtttag tccctcccaa ttcgatatca agcttatcgc gatagatcct aatcaacctc
    11701 tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc
    11761 tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca
    11821 ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg
    11881 tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca
    11941 ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg
    12001 cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg
    12061 acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg
    12121 ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg
    12181 accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc
    12241 ctcagacgag tcggatctcc ctttgggccg cctccccgcc tgagatcctt taagaccaat
    12301 gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg gactggaagg
    12361 gctaattcac tcccaacgaa gacaagatct gctttttgct tgtactgggt ctctctggtt
    12421 agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca
    12481 ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa
    12541 ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagta gtagttcatg
    12601 tcatcttatt attcagtatt tataacttgc aaagaaatga atatcagaga gtgagaggcc
    12661 cgggttaatt aaggaaaggg ctagatcatt cttgaagacg aaagggcctc gtgatacgcc
    12721 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc
    12781 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
    12841 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
    12901 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
    12961 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
    13021 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
    13081 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgtg
    13141 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
    13201 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
    13261 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
    13321 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
    13381 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
    13441 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
    13501 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
    13561 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
    13621 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
    13681 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
    13741 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
    13801 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
    13861 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
    13921 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
    13981 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
    14041 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
    14101 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
    14161 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
    14221 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
    14281 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
    14341 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
    14401 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
    14461 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
    14521 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
    14581 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
    14641 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
    14701 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agcaagctca
    14761 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt
    14821 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tccccgtggc
    14881 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
    14941 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
    15001 ttgtgagcgg ataacaattt cacacaggaa acagctatga catgattacg aatttcacaa
    15061 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
    15121 atcatgtctg gatcaactgg ataactcaag ctaaccaaaa tcatcccaaa cttcccaccc
    15181 cataccctat taccactgcc aattacctgt ggtttca
//
