LOCUS       sequence-329034-       15362 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of dual inducible Cas9 with hALB promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 15362)
  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 15362)
  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-hALB
FEATURES             Location/Qualifiers
     source          1..15362
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..634
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    681..806
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1303..1536
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1721..1765
                     /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    2058..2175
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    join(2230..2248,2249..2271,2272..2290,2291..2313,2314..2332,
                     2333..2355,2356..2374,2375..2397,2398..2416,2417..2439,
                     2440..2458,2459..2481,2482..2500)
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     regulatory      2762..2771
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2768..2770,2771..3091)
                     /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             3101..7204
                     /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             3101..7204
                     /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             7205..7252
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7253..7276
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             7292..7348
                     /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             7349..7864
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     polyA_signal    7898..8019
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7935..7954)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             8026..8052
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     promoter        8138..9349
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     9186..9205
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             join(9408..10025,10026..10031,10032..10067,10068..10070,
                     10071..10106,10107..10109,10110..10145,10146..10154)
                     /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    10192..10765
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(10359..10376)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     10586..10605
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             10781..11575
                     /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             10781..11575
                     /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(10835..10854)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     11445..11464
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     misc_feature    11752..12340
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(11805..11825)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(12223..12234)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             12415..12648
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(12767..12785)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        12853..12957
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(12958..13026,13027..13818)
                     /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(13176..13195)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      13989..14577
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     14478..14497
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     14731..14748
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    14988..15009
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(15024..15029,15030..15047,15048..15054)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    15062..15078
                     /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     15067..15089
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     15086..15102
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     15086..15102
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca
       61 cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac
      121 tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca
      181 ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg
      241 agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag
      301 agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg
      361 ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat
      421 cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga
      481 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
      541 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
      601 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
      661 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
      721 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga
      781 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg
      841 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg
      901 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct
      961 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat
     1021 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca
     1081 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc
     1141 aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag
     1201 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc
     1261 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg
     1321 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc
     1381 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc
     1441 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct
     1501 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa
     1561 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca
     1621 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt
     1681 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt
     1741 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta
     1801 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt
     1861 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct
     1921 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga
     1981 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccgaa ttcacaaatg
     2041 gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa
     2101 agaatagtag acataatagc aacagacata caaactaaag aattacaaaa acaaattaca
     2161 aaaattcaaa attttcgggt ttattacagg gacagcagag atccagtttg gactaggatc
     2221 ctttaccact ccctatcagt gatagagaaa agtgaaagtc gagtttacca ctccctatca
     2281 gtgatagaga aaagtgaaag tcgagtttac cactccctat cagtgataga gaaaagtgaa
     2341 agtcgagttt accactccct atcagtgata gagaaaagtg aaagtcgagt ttaccactcc
     2401 ctatcagtga tagagaaaag tgaaagtcga gtttaccact ccctatcagt gatagagaaa
     2461 agtgaaagtc gagtttacca ctccctatca gtgatagaga aaagtgaaag tcgagctcgg
     2521 tacccgggtc gaagttccag atggtaaata tacacaaggg atttagtcaa acaatttttt
     2581 ggcaagaata ttatgaattt tgtaatcggt tggcagccaa tgaaatacaa agatgagtct
     2641 agttaataat ctacaattat tggttaaaga agtatattag tgctaatttc cctccgtttg
     2701 tcctagcttt tctcttctgt caaccccaca cgtttagtga accgtcagat cgccttctag
     2761 agccaccatg ggagtgcagg tggaaaccat ctccccaggt gacgggcgca ccttccccaa
     2821 gcgcggccag acctgtgtgg tgcactacac cgggatgctt gaagatggaa agaaagtcga
     2881 ttcctcccgg gacagaaaca agccctttaa gtttatgcta ggcaagcagg aggtgatccg
     2941 aggctgggaa gaaggggttg cccagatgag tgtgggtcag agagccaaac tgactatatc
     3001 tccagattat gcctatggtg ccactgggca cccaggcatc atcccaccac atgccactct
     3061 cgtcttcgat gtggagcttc taaaaccgga accggctagc atggacaaga agtacagcat
     3121 cggcctggac atcggcacca actctgtggg ctgggccgtg atcaccgacg agtacaaggt
     3181 gcccagcaag aaattcaagg tgctgggcaa caccgaccgg cacagcatca agaagaacct
     3241 gatcggagcc ctgctgttcg acagcggcga aacagccgag gccacccggc tgaagagaac
     3301 cgccagaaga agatacacca gacggaagaa ccggatctgc tatctgcaag agatcttcag
     3361 caacgagatg gccaaggtgg acgacagctt cttccacaga ctggaagagt ccttcctggt
     3421 ggaagaggat aagaagcacg agcggcaccc catcttcggc aacatcgtgg acgaggtggc
     3481 ctaccacgag aagtacccca ccatctacca cctgagaaag aaactggtgg acagcaccga
     3541 caaggccgac ctgcggctga tctatctggc cctggcccac atgatcaagt tccggggcca
     3601 cttcctgatc gagggcgacc tgaaccccga caacagcgac gtggacaagc tgttcatcca
     3661 gctggtgcag acctacaacc agctgttcga ggaaaacccc atcaacgcca gcggcgtgga
     3721 cgccaaggcc atcctgtctg ccagactgag caagagcaga cggctggaaa atctgatcgc
     3781 ccagctgccc ggcgagaaga agaatggcct gttcggaaac ctgattgccc tgagcctggg
     3841 cctgaccccc aacttcaaga gcaacttcga cctggccgag gatgccaaac tgcagctgag
     3901 caaggacacc tacgacgacg acctggacaa cctgctggcc cagatcggcg accagtacgc
     3961 cgacctgttt ctggccgcca agaacctgtc cgacgccatc ctgctgagcg acatcctgag
     4021 agtgaacacc gagatcacca aggcccccct gagcgcctct atgatcaaga gatacgacga
     4081 gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta
     4141 caaagagatt ttcttcgacc agagcaagaa cggctacgcc ggctacattg acggcggagc
     4201 cagccaggaa gagttctaca agttcatcaa gcccatcctg gaaaagatgg acggcaccga
     4261 ggaactgctc gtgaagctga acagagagga cctgctgcgg aagcagcgga ccttcgacaa
     4321 cggcagcatc ccccaccaga tccacctggg agagctgcac gccattctgc ggcggcagga
     4381 agatttttac ccattcctga aggacaaccg ggaaaagatc gagaagatcc tgaccttccg
     4441 catcccctac tacgtgggcc ctctggccag gggaaacagc agattcgcct ggatgaccag
     4501 aaagagcgag gaaaccatca ccccctggaa cttcgaggaa gtggtggaca agggcgcttc
     4561 cgcccagagc ttcatcgagc ggatgaccaa cttcgataag aacctgccca acgagaaggt
     4621 gctgcccaag cacagcctgc tgtacgagta cttcaccgtg tataacgagc tgaccaaagt
     4681 gaaatacgtg accgagggaa tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc
     4741 catcgtggac ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga
     4801 ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt
     4861 caacgcctcc ctgggcacat accacgatct gctgaaaatt atcaaggaca aggacttcct
     4921 ggacaatgag gaaaacgagg acattctgga agatatcgtg ctgaccctga cactgtttga
     4981 ggacagagag atgatcgagg aacggctgaa aacctatgcc cacctgttcg acgacaaagt
     5041 gatgaagcag ctgaagcggc ggagatacac cggctggggc aggctgagcc ggaagctgat
     5101 caacggcatc cgggacaagc agtccggcaa gacaatcctg gatttcctga agtccgacgg
     5161 cttcgccaac agaaacttca tgcagctgat ccacgacgac agcctgacct ttaaagagga
     5221 catccagaaa gcccaggtgt ccggccaggg cgatagcctg cacgagcaca ttgccaatct
     5281 ggccggcagc cccgccatta agaagggcat cctgcagaca gtgaaggtgg tggacgagct
     5341 cgtgaaagtg atgggccggc acaagcccga gaacatcgtg atcgaaatgg ccagagagaa
     5401 ccagaccacc cagaagggac agaagaacag ccgcgagaga atgaagcgga tcgaagaggg
     5461 catcaaagag ctgggcagcc agatcctgaa agaacacccc gtggaaaaca cccagctgca
     5521 gaacgagaag ctgtacctgt actacctgca gaatgggcgg gatatgtacg tggaccagga
     5581 actggacatc aaccggctgt ccgactacga tgtggaccat atcgtgcctc agagctttct
     5641 gaaggacgac tccatcgaca acaaggtgct gaccagaagc gacaagaacc ggggcaagag
     5701 cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag aactactggc ggcagctgct
     5761 gaacgccaag ctgattaccc agagaaagtt cgacaatctg accaaggccg agagaggcgg
     5821 cctgagcgaa ctggataagg ccggcttcat caagagacag ctggtggaaa cccggcagat
     5881 cacaaagcac gtggcacaga tcctggactc ccggatgaac actaagtacg acgagaatga
     5941 caagctgatc cgggaagtga aagtgatcac cctgaagtcc aagctggtgt ccgatttccg
     6001 gaaggatttc cagttttaca aagtgcgcga gatcaacaac taccaccacg cccacgacgc
     6061 ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc tggaaagcga
     6121 gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag atgatcgcca agagcgagca
     6181 ggaaatcggc aaggctaccg ccaagtactt cttctacagc aacatcatga actttttcaa
     6241 gaccgagatt accctggcca acggcgagat ccggaagcgg cctctgatcg agacaaacgg
     6301 cgaaaccggg gagatcgtgt gggataaggg ccgggatttt gccaccgtgc ggaaagtgct
     6361 gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg gcttcagcaa
     6421 agagtctatc ctgcccaaga ggaacagcga taagctgatc gccagaaaga aggactggga
     6481 ccctaagaag tacggcggct tcgacagccc caccgtggcc tattctgtgc tggtggtggc
     6541 caaagtggaa aagggcaagt ccaagaaact gaagagtgtg aaagagctgc tggggatcac
     6601 catcatggaa agaagcagct tcgagaagaa tcccatcgac tttctggaag ccaagggcta
     6661 caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag tactccctgt tcgagctgga
     6721 aaacggccgg aagagaatgc tggcctctgc cggcgaactg cagaagggaa acgaactggc
     6781 cctgccctcc aaatatgtga acttcctgta cctggccagc cactatgaga agctgaaggg
     6841 ctcccccgag gataatgagc agaaacagct gtttgtggaa cagcacaagc actacctgga
     6901 cgagatcatc gagcagatca gcgagttctc caagagagtg atcctggccg acgctaatct
     6961 ggacaaagtg ctgtccgcct acaacaagca ccgggataag cccatcagag agcaggccga
     7021 gaatatcatc cacctgttta ccctgaccaa tctgggagcc cctgccgcct tcaagtactt
     7081 tgacaccacc atcgaccgga agaggtacac cagcaccaaa gaggtgctgg acgccaccct
     7141 gatccaccag agcatcaccg gcctgtacga gacacggatc gacctgtctc agctgggagg
     7201 cgacaagcga cctgccgcca caaagaaggc tggacaggct aagaagaaga aagattacaa
     7261 agacgatgac gataagggat cggatccgga ggctactaac ttcagcctgc tgaagcaggc
     7321 tggggacgtg gaggagaacc ctggacctat ggtcttcaca ctcgaagatt tcgttgggga
     7381 ctggcgacag acagccggct acaacctgga ccaagtcctt gaacagggag gtgtgtccag
     7441 tttgtttcag aatctcgggg tgtccgtaac tccgatccaa aggattgtcc tgagcggtga
     7501 aaatgggctg aagatcgaca tccatgtcat catcccgtat gaaggtctga gcggcgacca
     7561 aatgggccag atcgaaaaaa tttttaaggt ggtgtaccct gtggatgatc atcactttaa
     7621 ggtgatcctg cactatggca cactggtaat cgacggggtt acgccgaaca tgatcgacta
     7681 tttcggacgg ccgtatgaag gcatcgccgt gttcgacggc aaaaagatca ctgtaacagg
     7741 gaccctgtgg aacggcaaca aaattatcga cgagcgcctg atcaaccccg acggctccct
     7801 gctgttccga gtaaccatca acggagtgac cggctggcgg ctgtgcgaac gcattctggc
     7861 gtaactcgag tcgagtctag agggcccgcg gttcgaaaac ttgtttattg cagcttataa
     7921 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     7981 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat tcgaataccc atacgacgtc
     8041 ccagactacg cttagtaatg attaattaaa ctagaaattc taccgggtag gggaggcgct
     8101 tttcccaagg cagtctggag taacccaccc aagatctggc ctccgcgccg ggttttggcg
     8161 cctcccgcgg gcgcccccct cctcacggcg agcgctgcca cgtcagacga agggcgcagc
     8221 gagcgtcctg atccttccgc ccggacgctc aggacagcgg cccgctgctc ataagactcg
     8281 gccttagaac cccagtatca gcagaaggac attttaggac gggacttggg tgactctagg
     8341 gcactggttt tctttccaga gagcggaaca ggcgaggaaa agtagtccct tctcggcgat
     8401 tctgcggagg gatctccgtg gggcggtgaa cgccgatgat tatataagga cgcgccgggt
     8461 gtggcacagc tagttccgtc gcagccggga tttgggtcgc ggttcttgtt tgtggatcgc
     8521 tgtgatcgtc acttggtgag tagcgggctg ctgggctggc cggggctttc gtggccgccg
     8581 ggccgctcgg tgggacggaa gcgtgtggag agaccgccaa gggctgtagt ctgggtccgc
     8641 gagcaaggtt gccctgaact gggggttggg gggagcgcag caaaatggcg gctgttcccg
     8701 agtcttgaat ggaagacgct tgtgaggcgg gctgtgaggt cgttgaaaca aggtgggggg
     8761 catggtgggc ggcaagaacc caaggtcttg aggccttcgc taatgcggga aagctcttat
     8821 tcgggtgaga tgggctgggg caccatctgg ggaccctgac gtgaagtttg tcactgactg
     8881 gagaactcgg tttgtcgtct gttgcggggg cggcagttat ggcggtgccg ttgggcagtg
     8941 cacccgtacc tttgggagcg cgcgccctcg tcgtgtcgtg acgtcacccg ttctgttggc
     9001 ttataatgca gggtggggcc acctgccggt aggtgtgcgg taggcttttc tccgtcgcag
     9061 gacgcagggt tcgggcctag ggtaggctct cctgaatcga caggcgccgg acctctggtg
     9121 aggggaggga taagtgaggc gtcagtttct ttggtcggtt ttatgtacct atcttcttaa
     9181 gtagctgaag ctccggtttt gaactatgcg ctcggggttg gcgagtgtgt tttgtgaagt
     9241 tttttaggca ccttttgaaa tgtaatcatt tgggtcaata tgtaattttc agtgttagac
     9301 tagtaaattg tccgctaaat tctggccgtt tttggctttt ttgttagacg aagcttggta
     9361 ccgagctcgg atctccaccc cgtaccggtc ctgcagtcga attcaccatg tctagactgg
     9421 acaagagcaa agtcataaac ggagctctgg aattactcaa tggtgtcggt atcgaaggcc
     9481 tgacgacaag gaaactcgct caaaagctgg gagttgagca gcctaccctg tactggcacg
     9541 tgaagaacaa gcgggccctg ctcgatgccc tgccaatcga gatgctggac aggcatcata
     9601 cccacttctg ccccctggaa ggcgagtcat ggcaagactt tctgcggaac aacgccaagt
     9661 cataccgctg tgctctcctc tcacatcgcg acggggctaa agtgcatctc ggcacccgcc
     9721 caacagagaa acagtacgaa accctggaaa atcagctcgc gttcctgtgt cagcaaggct
     9781 tctccctgga gaacgcactg tacgctctgt ccgccgtggg ccactttaca ctgggctgcg
     9841 tattggagga acaggagcat caagtagcaa aagaggaaag agagacacct accaccgatt
     9901 ctatgccccc acttctgaga caagcaattg agctgttcga ccggcaggga gccgaacctg
     9961 ccttcctttt cggcctggaa ctaatcatat gtggcctgga gaaacagcta aagtgcgaaa
    10021 gcggcgggcc gaccgacgcc cttgacgatt ttgacttaga catgctccca gccgatgccc
    10081 ttgacgactt tgaccttgat atgctgcctg ctgacgctct tgacgatttt gaccttgaca
    10141 tgctccccgg gtaactaagt aaggatccgc ggccgcacta gaggaattcc gcccctctcc
    10201 cycccccccc cctaacgtta ctggccgaag ccgcttggaa taaggccggt gtgcgtttgt
    10261 ctatatgtta ttttccacca tattgccgtc ttttggcaat gtgagggccc ggaaacctgg
    10321 ccctgtcttc ttgacgagca ttcctagggg tctttcccct ctcgccaaag gaatgcaagg
    10381 tctgttgaat gtcgtgaagg aagcagttcc tctggaagct tcttgaagac aaacaacgtc
    10441 tgtagcgacc ctttgcaggc agcggaaccc cccacctggc gacaggtgcc tctgcggcca
    10501 aaagccacgt gtataagata cacctgcaaa ggcggcacaa ccccagtgcc acgttgtgag
    10561 ttggatagtt gtggaaagag tcaaatggct ctcctcaagc gtattcaaca aggggctgaa
    10621 ggatgcccag aaggtacccc attgtatggg atctgatctg gggcctcggt gcacatgctt
    10681 tacatgtgtt tagtcgaggt taaaaaaacg tctaggcccc ccgaaccacg gggacgtggt
    10741 tttcctttga aaaacacgat gataagctta ccggtccacc atgattgaac aagatggatt
    10801 gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca
    10861 gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct
    10921 ttttgtcaag accgacctgt ccggtgccct gaatgaactg caagacgagg cagcgcggct
    10981 atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc
    11041 gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct
    11101 tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga
    11161 tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg
    11221 gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc
    11281 agccgaactg ttcgccaggc tcaaggcgag catgcccgac ggcgaggatc tcgtcgtgac
    11341 ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat
    11401 cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga
    11461 tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc
    11521 cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgatgtac
    11581 aagtaaagcg gccgcgactc tagatcataa tcagccatac cacatttgta gaggttttac
    11641 ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg
    11701 ttgttgttta gtccctccca attcgatatc aagcttatcg cgatagatcc taatcaacct
    11761 ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg
    11821 ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc
    11881 attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt
    11941 gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc
    12001 attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg
    12061 gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact
    12121 gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt
    12181 gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg
    12241 gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc
    12301 cctcagacga gtcggatctc cctttgggcc gcctccccgc ctgagatcct ttaagaccaa
    12361 tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag
    12421 ggctaattca ctcccaacga agacaagatc tgctttttgc ttgtactggg tctctctggt
    12481 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
    12541 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
    12601 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt agtagttcat
    12661 gtcatcttat tattcagtat ttataacttg caaagaaatg aatatcagag agtgagaggc
    12721 ccgggttaat taaggaaagg gctagatcat tcttgaagac gaaagggcct cgtgatacgc
    12781 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
    12841 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
    12901 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
    12961 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
    13021 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
    13081 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
    13141 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
    13201 gttgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
    13261 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
    13321 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
    13381 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
    13441 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
    13501 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
    13561 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
    13621 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
    13681 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
    13741 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
    13801 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
    13861 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
    13921 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
    13981 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
    14041 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
    14101 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
    14161 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
    14221 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
    14281 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
    14341 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
    14401 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
    14461 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
    14521 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
    14581 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
    14641 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
    14701 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
    14761 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagcaagctc
    14821 atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat
    14881 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctccccgtgg
    14941 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag
    15001 ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga
    15061 attgtgagcg gataacaatt tcacacagga aacagctatg acatgattac gaatttcaca
    15121 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
    15181 tatcatgtct ggatcaactg gataactcaa gctaaccaaa atcatcccaa acttcccacc
    15241 ccatacccta ttaccactgc caattacctg tggtttcatt tactctaaac ctgtgattcc
    15301 tctgaattat tttcatttta aagaaattgt atttgttaaa tatgtactac aaacttagta
    15361 gt
//
