LOCUS       sequence-329033-       15412 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of dual inducible Cas9 with EFS promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 15412)
  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 15412)
  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-EFS
FEATURES             Location/Qualifiers
     source          1..15412
                     /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"
     promoter        2582..2793
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2812..2821
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2818..2820,2821..3141)
                     /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             3151..7254
                     /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             3151..7254
                     /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             7255..7302
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7303..7326
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             7342..7398
                     /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             7399..7914
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     polyA_signal    7948..8069
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7985..8004)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             8076..8102
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     promoter        8188..9399
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     9236..9255
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             join(9458..10075,10076..10081,10082..10117,10118..10120,
                     10121..10156,10157..10159,10160..10195,10196..10204)
                     /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    10242..10815
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(10409..10426)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     10636..10655
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             10831..11625
                     /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             10831..11625
                     /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(10885..10904)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     11495..11514
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     misc_feature    11802..12390
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(11855..11875)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(12273..12284)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             12465..12698
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(12817..12835)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        12903..13007
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(13008..13076,13077..13868)
                     /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(13226..13245)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      14039..14627
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     14528..14547
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     14781..14798
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    15038..15059
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(15074..15079,15080..15097,15098..15104)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    15112..15128
                     /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     15117..15139
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     15136..15152
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     15136..15152
                     /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 gagctagcta ggtcttgaaa ggagtgggaa ttggctccgg tgcccgtcag
     2581 tgggcagagc gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga
     2641 tccggtgcct agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc
     2701 cgcctttttc ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt
     2761 ctttttcgca acgggtttgc cgccagaaca caggaccggt tctagagcgc tgccaccatg
     2821 ggagtgcagg tggaaaccat ctccccaggt gacgggcgca ccttccccaa gcgcggccag
     2881 acctgtgtgg tgcactacac cgggatgctt gaagatggaa agaaagtcga ttcctcccgg
     2941 gacagaaaca agccctttaa gtttatgcta ggcaagcagg aggtgatccg aggctgggaa
     3001 gaaggggttg cccagatgag tgtgggtcag agagccaaac tgactatatc tccagattat
     3061 gcctatggtg ccactgggca cccaggcatc atcccaccac atgccactct cgtcttcgat
     3121 gtggagcttc taaaaccgga accggctagc atggacaaga agtacagcat cggcctggac
     3181 atcggcacca actctgtggg ctgggccgtg atcaccgacg agtacaaggt gcccagcaag
     3241 aaattcaagg tgctgggcaa caccgaccgg cacagcatca agaagaacct gatcggagcc
     3301 ctgctgttcg acagcggcga aacagccgag gccacccggc tgaagagaac cgccagaaga
     3361 agatacacca gacggaagaa ccggatctgc tatctgcaag agatcttcag caacgagatg
     3421 gccaaggtgg acgacagctt cttccacaga ctggaagagt ccttcctggt ggaagaggat
     3481 aagaagcacg agcggcaccc catcttcggc aacatcgtgg acgaggtggc ctaccacgag
     3541 aagtacccca ccatctacca cctgagaaag aaactggtgg acagcaccga caaggccgac
     3601 ctgcggctga tctatctggc cctggcccac atgatcaagt tccggggcca cttcctgatc
     3661 gagggcgacc tgaaccccga caacagcgac gtggacaagc tgttcatcca gctggtgcag
     3721 acctacaacc agctgttcga ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc
     3781 atcctgtctg ccagactgag caagagcaga cggctggaaa atctgatcgc ccagctgccc
     3841 ggcgagaaga agaatggcct gttcggaaac ctgattgccc tgagcctggg cctgaccccc
     3901 aacttcaaga gcaacttcga cctggccgag gatgccaaac tgcagctgag caaggacacc
     3961 tacgacgacg acctggacaa cctgctggcc cagatcggcg accagtacgc cgacctgttt
     4021 ctggccgcca agaacctgtc cgacgccatc ctgctgagcg acatcctgag agtgaacacc
     4081 gagatcacca aggcccccct gagcgcctct atgatcaaga gatacgacga gcaccaccag
     4141 gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta caaagagatt
     4201 ttcttcgacc agagcaagaa cggctacgcc ggctacattg acggcggagc cagccaggaa
     4261 gagttctaca agttcatcaa gcccatcctg gaaaagatgg acggcaccga ggaactgctc
     4321 gtgaagctga acagagagga cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc
     4381 ccccaccaga tccacctggg agagctgcac gccattctgc ggcggcagga agatttttac
     4441 ccattcctga aggacaaccg ggaaaagatc gagaagatcc tgaccttccg catcccctac
     4501 tacgtgggcc ctctggccag gggaaacagc agattcgcct ggatgaccag aaagagcgag
     4561 gaaaccatca ccccctggaa cttcgaggaa gtggtggaca agggcgcttc cgcccagagc
     4621 ttcatcgagc ggatgaccaa cttcgataag aacctgccca acgagaaggt gctgcccaag
     4681 cacagcctgc tgtacgagta cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg
     4741 accgagggaa tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc catcgtggac
     4801 ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag
     4861 aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc
     4921 ctgggcacat accacgatct gctgaaaatt atcaaggaca aggacttcct ggacaatgag
     4981 gaaaacgagg acattctgga agatatcgtg ctgaccctga cactgtttga ggacagagag
     5041 atgatcgagg aacggctgaa aacctatgcc cacctgttcg acgacaaagt gatgaagcag
     5101 ctgaagcggc ggagatacac cggctggggc aggctgagcc ggaagctgat caacggcatc
     5161 cgggacaagc agtccggcaa gacaatcctg gatttcctga agtccgacgg cttcgccaac
     5221 agaaacttca tgcagctgat ccacgacgac agcctgacct ttaaagagga catccagaaa
     5281 gcccaggtgt ccggccaggg cgatagcctg cacgagcaca ttgccaatct ggccggcagc
     5341 cccgccatta agaagggcat cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg
     5401 atgggccggc acaagcccga gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc
     5461 cagaagggac agaagaacag ccgcgagaga atgaagcgga tcgaagaggg catcaaagag
     5521 ctgggcagcc agatcctgaa agaacacccc gtggaaaaca cccagctgca gaacgagaag
     5581 ctgtacctgt actacctgca gaatgggcgg gatatgtacg tggaccagga actggacatc
     5641 aaccggctgt ccgactacga tgtggaccat atcgtgcctc agagctttct gaaggacgac
     5701 tccatcgaca acaaggtgct gaccagaagc gacaagaacc ggggcaagag cgacaacgtg
     5761 ccctccgaag aggtcgtgaa gaagatgaag aactactggc ggcagctgct gaacgccaag
     5821 ctgattaccc agagaaagtt cgacaatctg accaaggccg agagaggcgg cctgagcgaa
     5881 ctggataagg ccggcttcat caagagacag ctggtggaaa cccggcagat cacaaagcac
     5941 gtggcacaga tcctggactc ccggatgaac actaagtacg acgagaatga caagctgatc
     6001 cgggaagtga aagtgatcac cctgaagtcc aagctggtgt ccgatttccg gaaggatttc
     6061 cagttttaca aagtgcgcga gatcaacaac taccaccacg cccacgacgc ctacctgaac
     6121 gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc tggaaagcga gttcgtgtac
     6181 ggcgactaca aggtgtacga cgtgcggaag atgatcgcca agagcgagca ggaaatcggc
     6241 aaggctaccg ccaagtactt cttctacagc aacatcatga actttttcaa gaccgagatt
     6301 accctggcca acggcgagat ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg
     6361 gagatcgtgt gggataaggg ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc
     6421 caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg gcttcagcaa agagtctatc
     6481 ctgcccaaga ggaacagcga taagctgatc gccagaaaga aggactggga ccctaagaag
     6541 tacggcggct tcgacagccc caccgtggcc tattctgtgc tggtggtggc caaagtggaa
     6601 aagggcaagt ccaagaaact gaagagtgtg aaagagctgc tggggatcac catcatggaa
     6661 agaagcagct tcgagaagaa tcccatcgac tttctggaag ccaagggcta caaagaagtg
     6721 aaaaaggacc tgatcatcaa gctgcctaag tactccctgt tcgagctgga aaacggccgg
     6781 aagagaatgc tggcctctgc cggcgaactg cagaagggaa acgaactggc cctgccctcc
     6841 aaatatgtga acttcctgta cctggccagc cactatgaga agctgaaggg ctcccccgag
     6901 gataatgagc agaaacagct gtttgtggaa cagcacaagc actacctgga cgagatcatc
     6961 gagcagatca gcgagttctc caagagagtg atcctggccg acgctaatct ggacaaagtg
     7021 ctgtccgcct acaacaagca ccgggataag cccatcagag agcaggccga gaatatcatc
     7081 cacctgttta ccctgaccaa tctgggagcc cctgccgcct tcaagtactt tgacaccacc
     7141 atcgaccgga agaggtacac cagcaccaaa gaggtgctgg acgccaccct gatccaccag
     7201 agcatcaccg gcctgtacga gacacggatc gacctgtctc agctgggagg cgacaagcga
     7261 cctgccgcca caaagaaggc tggacaggct aagaagaaga aagattacaa agacgatgac
     7321 gataagggat cggatccgga ggctactaac ttcagcctgc tgaagcaggc tggggacgtg
     7381 gaggagaacc ctggacctat ggtcttcaca ctcgaagatt tcgttgggga ctggcgacag
     7441 acagccggct acaacctgga ccaagtcctt gaacagggag gtgtgtccag tttgtttcag
     7501 aatctcgggg tgtccgtaac tccgatccaa aggattgtcc tgagcggtga aaatgggctg
     7561 aagatcgaca tccatgtcat catcccgtat gaaggtctga gcggcgacca aatgggccag
     7621 atcgaaaaaa tttttaaggt ggtgtaccct gtggatgatc atcactttaa ggtgatcctg
     7681 cactatggca cactggtaat cgacggggtt acgccgaaca tgatcgacta tttcggacgg
     7741 ccgtatgaag gcatcgccgt gttcgacggc aaaaagatca ctgtaacagg gaccctgtgg
     7801 aacggcaaca aaattatcga cgagcgcctg atcaaccccg acggctccct gctgttccga
     7861 gtaaccatca acggagtgac cggctggcgg ctgtgcgaac gcattctggc gtaactcgag
     7921 tcgagtctag agggcccgcg gttcgaaaac ttgtttattg cagcttataa tggttacaaa
     7981 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     8041 ggtttgtcca aactcatcaa tgtatcttat tcgaataccc atacgacgtc ccagactacg
     8101 cttagtaatg attaattaaa ctagaaattc taccgggtag gggaggcgct tttcccaagg
     8161 cagtctggag taacccaccc aagatctggc ctccgcgccg ggttttggcg cctcccgcgg
     8221 gcgcccccct cctcacggcg agcgctgcca cgtcagacga agggcgcagc gagcgtcctg
     8281 atccttccgc ccggacgctc aggacagcgg cccgctgctc ataagactcg gccttagaac
     8341 cccagtatca gcagaaggac attttaggac gggacttggg tgactctagg gcactggttt
     8401 tctttccaga gagcggaaca ggcgaggaaa agtagtccct tctcggcgat tctgcggagg
     8461 gatctccgtg gggcggtgaa cgccgatgat tatataagga cgcgccgggt gtggcacagc
     8521 tagttccgtc gcagccggga tttgggtcgc ggttcttgtt tgtggatcgc tgtgatcgtc
     8581 acttggtgag tagcgggctg ctgggctggc cggggctttc gtggccgccg ggccgctcgg
     8641 tgggacggaa gcgtgtggag agaccgccaa gggctgtagt ctgggtccgc gagcaaggtt
     8701 gccctgaact gggggttggg gggagcgcag caaaatggcg gctgttcccg agtcttgaat
     8761 ggaagacgct tgtgaggcgg gctgtgaggt cgttgaaaca aggtgggggg catggtgggc
     8821 ggcaagaacc caaggtcttg aggccttcgc taatgcggga aagctcttat tcgggtgaga
     8881 tgggctgggg caccatctgg ggaccctgac gtgaagtttg tcactgactg gagaactcgg
     8941 tttgtcgtct gttgcggggg cggcagttat ggcggtgccg ttgggcagtg cacccgtacc
     9001 tttgggagcg cgcgccctcg tcgtgtcgtg acgtcacccg ttctgttggc ttataatgca
     9061 gggtggggcc acctgccggt aggtgtgcgg taggcttttc tccgtcgcag gacgcagggt
     9121 tcgggcctag ggtaggctct cctgaatcga caggcgccgg acctctggtg aggggaggga
     9181 taagtgaggc gtcagtttct ttggtcggtt ttatgtacct atcttcttaa gtagctgaag
     9241 ctccggtttt gaactatgcg ctcggggttg gcgagtgtgt tttgtgaagt tttttaggca
     9301 ccttttgaaa tgtaatcatt tgggtcaata tgtaattttc agtgttagac tagtaaattg
     9361 tccgctaaat tctggccgtt tttggctttt ttgttagacg aagcttggta ccgagctcgg
     9421 atctccaccc cgtaccggtc ctgcagtcga attcaccatg tctagactgg acaagagcaa
     9481 agtcataaac ggagctctgg aattactcaa tggtgtcggt atcgaaggcc tgacgacaag
     9541 gaaactcgct caaaagctgg gagttgagca gcctaccctg tactggcacg tgaagaacaa
     9601 gcgggccctg ctcgatgccc tgccaatcga gatgctggac aggcatcata cccacttctg
     9661 ccccctggaa ggcgagtcat ggcaagactt tctgcggaac aacgccaagt cataccgctg
     9721 tgctctcctc tcacatcgcg acggggctaa agtgcatctc ggcacccgcc caacagagaa
     9781 acagtacgaa accctggaaa atcagctcgc gttcctgtgt cagcaaggct tctccctgga
     9841 gaacgcactg tacgctctgt ccgccgtggg ccactttaca ctgggctgcg tattggagga
     9901 acaggagcat caagtagcaa aagaggaaag agagacacct accaccgatt ctatgccccc
     9961 acttctgaga caagcaattg agctgttcga ccggcaggga gccgaacctg ccttcctttt
    10021 cggcctggaa ctaatcatat gtggcctgga gaaacagcta aagtgcgaaa gcggcgggcc
    10081 gaccgacgcc cttgacgatt ttgacttaga catgctccca gccgatgccc ttgacgactt
    10141 tgaccttgat atgctgcctg ctgacgctct tgacgatttt gaccttgaca tgctccccgg
    10201 gtaactaagt aaggatccgc ggccgcacta gaggaattcc gcccctctcc cycccccccc
    10261 cctaacgtta ctggccgaag ccgcttggaa taaggccggt gtgcgtttgt ctatatgtta
    10321 ttttccacca tattgccgtc ttttggcaat gtgagggccc ggaaacctgg ccctgtcttc
    10381 ttgacgagca ttcctagggg tctttcccct ctcgccaaag gaatgcaagg tctgttgaat
    10441 gtcgtgaagg aagcagttcc tctggaagct tcttgaagac aaacaacgtc tgtagcgacc
    10501 ctttgcaggc agcggaaccc cccacctggc gacaggtgcc tctgcggcca aaagccacgt
    10561 gtataagata cacctgcaaa ggcggcacaa ccccagtgcc acgttgtgag ttggatagtt
    10621 gtggaaagag tcaaatggct ctcctcaagc gtattcaaca aggggctgaa ggatgcccag
    10681 aaggtacccc attgtatggg atctgatctg gggcctcggt gcacatgctt tacatgtgtt
    10741 tagtcgaggt taaaaaaacg tctaggcccc ccgaaccacg gggacgtggt tttcctttga
    10801 aaaacacgat gataagctta ccggtccacc atgattgaac aagatggatt gcacgcaggt
    10861 tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc
    10921 tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag
    10981 accgacctgt ccggtgccct gaatgaactg caagacgagg cagcgcggct atcgtggctg
    11041 gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac
    11101 tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc
    11161 gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc
    11221 tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc
    11281 ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg
    11341 ttcgccaggc tcaaggcgag catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat
    11401 gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc
    11461 cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa
    11521 gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat
    11581 tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgatgtac aagtaaagcg
    11641 gccgcgactc tagatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa
    11701 aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttta
    11761 gtccctccca attcgatatc aagcttatcg cgatagatcc taatcaacct ctggattaca
    11821 aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat
    11881 acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct
    11941 ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac
    12001 gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca
    12061 cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca
    12121 tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg
    12181 tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga
    12241 ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt
    12301 cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga
    12361 gtcggatctc cctttgggcc gcctccccgc ctgagatcct ttaagaccaa tgacttacaa
    12421 ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca
    12481 ctcccaacga agacaagatc tgctttttgc ttgtactggg tctctctggt tagaccagat
    12541 ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt
    12601 gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc
    12661 cctcagaccc ttttagtcag tgtggaaaat ctctagcagt agtagttcat gtcatcttat
    12721 tattcagtat ttataacttg caaagaaatg aatatcagag agtgagaggc ccgggttaat
    12781 taaggaaagg gctagatcat tcttgaagac gaaagggcct cgtgatacgc ctatttttat
    12841 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
    12901 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
    12961 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
    13021 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
    13081 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
    13141 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
    13201 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt gttgacgccg
    13261 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
    13321 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
    13381 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
    13441 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
    13501 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
    13561 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
    13621 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
    13681 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
    13741 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
    13801 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
    13861 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
    13921 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
    13981 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
    14041 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
    14101 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
    14161 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
    14221 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
    14281 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
    14341 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
    14401 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
    14461 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
    14521 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
    14581 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
    14641 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
    14701 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
    14761 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac
    14821 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagcaagctc atggctgact
    14881 aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta
    14941 gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctccccgtgg cacgacaggt
    15001 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt
    15061 aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg
    15121 gataacaatt tcacacagga aacagctatg acatgattac gaatttcaca aataaagcat
    15181 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
    15241 ggatcaactg gataactcaa gctaaccaaa atcatcccaa acttcccacc ccatacccta
    15301 ttaccactgc caattacctg tggtttcatt tactctaaac ctgtgattcc tctgaattat
    15361 tttcatttta aagaaattgt atttgttaaa tatgtactac aaacttagta gt
//
