LOCUS       sequence-236379-       15299 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express Cas9, 2xNES, 2 tandem ERT2 and GCN4 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 15299)
  AUTHORS   Lu J, Zhao C, Zhao Y, Zhang J, Zhang Y, Chen L, Han Q, Ying Y, Peng 
            S, Ai R, Wang Y
  TITLE     Multimode drug inducible CRISPR/Cas9 devices for transcriptional 
            activation and genome editing.
  JOURNAL   Nucleic Acids Res. 2018 Mar 16;46(5):e25. doi: 10.1093/nar/gkx1222.
  PUBMED    29237052
REFERENCE   2  (bases 1 to 15299)
  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: "Nucleic 
            Acids Res."; date: "2018-03-16"; volume: "46(5):e25. doi"; pages: " 
            10.1093/nar/gkx1222"
COMMENT     Sequence Label: EF1a-Cas9-2XNES-2ERT2-GCN4-SV40-Zeo
FEATURES             Location/Qualifiers
     source          1..15299
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        130..341
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      1205..1214
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1226..5326
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMTKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5453..6388
                     /codon_start=1
                     /product="mutated ligand-binding domain of the human 
                     estrogen receptor (Feil et al., 1997)"
                     /label=ERT2
                     /translation="AGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPIL
                     YSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEI
                     LMIGLVWRSMEHPVKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEF
                     VCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRL
                     AQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEAADAHRLHAPTSRGGASVEET
                     DQSHLATAGSTSSHSLQKYYITGEAEGFPAT"
     CDS             6440..7375
                     /codon_start=1
                     /product="mutated ligand-binding domain of the human 
                     estrogen receptor (Feil et al., 1997)"
                     /label=ERT2
                     /translation="AGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPIL
                     YSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEI
                     LMIGLVWRSMEHPVKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEF
                     VCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRL
                     AQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEAADAHRLHAPTSRGGASVEET
                     DQSHLATAGSTSSHSLQKYYITGEAEGFPAT"
     CDS             7385..7411
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             7391..7417
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YDVPDYASL"
     CDS             7430..7450
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7457..7477
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7553..7609
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             7625..7681
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             7697..7753
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             7769..7825
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             7841..7897
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             7913..7969
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             7985..8041
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             8057..8113
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             8129..8185
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             8201..8257
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     primer_bind     complement(8418..8438)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        8423..8752
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      8603..8738
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             8762..9136
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     misc_feature    9222..9810
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(9275..9295)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(9693..9704)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             9897..10130
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    10202..10323
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      10342..10477
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(10497..10516)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(10498..10516)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(10526..10543)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(10526..10542)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10535..10557)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      10684..11139
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10771..10790)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     10981..11002
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        11165..11269
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(11270..11338,11339..12130)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(11488..11507)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      12301..12889
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     12790..12809
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     13043..13060
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    13177..13198
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(13213..13218,13219..13236,13237..13243)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    13251..13267
                     /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     13256..13278
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     13275..13291
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     13275..13291
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     13310..13330
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        13312..13330
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        13358..13584
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             13585..13765
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    13812..13937
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    14430..14663
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             14848..14892
                     /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    15159..15276
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
ORIGIN
        1 atagcccggg ctcgaccata tgataagctt tgcaaagatg gataaagttt taaacagaga
       61 ggaatctttg cagctaatgg accttctagg tcttgaaagg agtgggaatt ggctccggtg
      121 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg
      181 gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt
      241 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg
      301 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt
      361 cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct
      421 ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga
      481 ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct
      541 ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt
      601 ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc
      661 ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg
      721 acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac
      781 cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc
      841 cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag
      901 cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct
      961 cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg
     1021 tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga
     1081 gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc
     1141 acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctcctttc
     1201 tagagccacc atgggtggcg cgcctgacaa gaagtacagc atcggcctgg acatcggcac
     1261 caactctgtg ggctgggccg tgatcaccga cgagtacaag gtgcccagca agaaattcaa
     1321 ggtgctgggc aacaccgacc ggcacagcat caagaagaac ctgatcggag ccctgctgtt
     1381 cgacagcggc gaaacagccg aggccacccg gctgaagaga accgccagaa gaagatacac
     1441 cagacggaag aaccggatct gctatctgca agagatcttc agcaacgaga tgaccaaggt
     1501 ggacgacagc ttcttccaca gactggaaga gtccttcctg gtggaagagg ataagaagca
     1561 cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg gcctaccacg agaagtaccc
     1621 caccatctac cacctgagaa agaaactggt ggacagcacc gacaaggccg acctgcggct
     1681 gatctatctg gccctggccc acatgatcaa gttccggggc cacttcctga tcgagggcga
     1741 cctgaacccc gacaacagcg acgtggacaa gctgttcatc cagctggtgc agacctacaa
     1801 ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg gacgccaagg ccatcctgtc
     1861 tgccagactg agcaagagca gacggctgga aaatctgatc gcccagctgc ccggcgagaa
     1921 gaagaatggc ctgttcggaa acctgattgc cctgagcctg ggcctgaccc ccaacttcaa
     1981 gagcaacttc gacctggccg aggatgccaa actgcagctg agcaaggaca cctacgacga
     2041 cgacctggac aacctgctgg cccagatcgg cgaccagtac gccgacctgt ttctggccgc
     2101 caagaacctg tccgacgcca tcctgctgag cgacatcctg agagtgaaca ccgagatcac
     2161 caaggccccc ctgagcgcct ctatgatcaa gagatacgac gagcaccacc aggacctgac
     2221 cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag tacaaagaga ttttcttcga
     2281 ccagagcaag aacggctacg ccggctacat tgacggcgga gccagccagg aagagttcta
     2341 caagttcatc aagcccatcc tggaaaagat ggacggcacc gaggaactgc tcgtgaagct
     2401 gaacagagag gacctgctgc ggaagcagcg gaccttcgac aacggcagca tcccccacca
     2461 gatccacctg ggagagctgc acgccattct gcggcggcag gaagattttt acccattcct
     2521 gaaggacaac cgggaaaaga tcgagaagat cctgaccttc cgcatcccct actacgtggg
     2581 ccctctggcc aggggaaaca gcagattcgc ctggatgacc agaaagagcg aggaaaccat
     2641 caccccctgg aacttcgagg aagtggtgga caagggcgct tccgcccaga gcttcatcga
     2701 gcggatgacc aacttcgata agaacctgcc caacgagaag gtgctgccca agcacagcct
     2761 gctgtacgag tacttcaccg tgtataacga gctgaccaaa gtgaaatacg tgaccgaggg
     2821 aatgagaaag cccgccttcc tgagcggcga gcagaaaaag gccatcgtgg acctgctgtt
     2881 caagaccaac cggaaagtga ccgtgaagca gctgaaagag gactacttca agaaaatcga
     2941 gtgcttcgac tccgtggaaa tctccggcgt ggaagatcgg ttcaacgcct ccctgggcac
     3001 ataccacgat ctgctgaaaa ttatcaagga caaggacttc ctggacaatg aggaaaacga
     3061 ggacattctg gaagatatcg tgctgaccct gacactgttt gaggacagag agatgatcga
     3121 ggaacggctg aaaacctatg cccacctgtt cgacgacaaa gtgatgaagc agctgaagcg
     3181 gcggagatac accggctggg gcaggctgag ccggaagctg atcaacggca tccgggacaa
     3241 gcagtccggc aagacaatcc tggatttcct gaagtccgac ggcttcgcca acagaaactt
     3301 catgcagctg atccacgacg acagcctgac ctttaaagag gacatccaga aagcccaggt
     3361 gtccggccag ggcgatagcc tgcacgagca cattgccaat ctggccggca gccccgccat
     3421 taagaagggc atcctgcaga cagtgaaggt ggtggacgag ctcgtgaaag tgatgggccg
     3481 gcacaagccc gagaacatcg tgatcgaaat ggccagagag aaccagacca cccagaaggg
     3541 acagaagaac agccgcgaga gaatgaagcg gatcgaagag ggcatcaaag agctgggcag
     3601 ccagatcctg aaagaacacc ccgtggaaaa cacccagctg cagaacgaga agctgtacct
     3661 gtactacctg cagaatgggc gggatatgta cgtggaccag gaactggaca tcaaccggct
     3721 gtccgactac gatgtggacc atatcgtgcc tcagagcttt ctgaaggacg actccatcga
     3781 caacaaggtg ctgaccagaa gcgacaagaa ccggggcaag agcgacaacg tgccctccga
     3841 agaggtcgtg aagaagatga agaactactg gcggcagctg ctgaacgcca agctgattac
     3901 ccagagaaag ttcgacaatc tgaccaaggc cgagagaggc ggcctgagcg aactggataa
     3961 ggccggcttc atcaagagac agctggtgga aacccggcag atcacaaagc acgtggcaca
     4021 gatcctggac tcccggatga acactaagta cgacgagaat gacaagctga tccgggaagt
     4081 gaaagtgatc accctgaagt ccaagctggt gtccgatttc cggaaggatt tccagtttta
     4141 caaagtgcgc gagatcaaca actaccacca cgcccacgac gcctacctga acgccgtcgt
     4201 gggaaccgcc ctgatcaaaa agtaccctaa gctggaaagc gagttcgtgt acggcgacta
     4261 caaggtgtac gacgtgcgga agatgatcgc caagagcgag caggaaatcg gcaaggctac
     4321 cgccaagtac ttcttctaca gcaacatcat gaactttttc aagaccgaga ttaccctggc
     4381 caacggcgag atccggaagc ggcctctgat cgagacaaac ggcgaaaccg gggagatcgt
     4441 gtgggataag ggccgggatt ttgccaccgt gcggaaagtg ctgagcatgc cccaagtgaa
     4501 tatcgtgaaa aagaccgagg tgcagacagg cggcttcagc aaagagtcta tcctgcccaa
     4561 gaggaacagc gataagctga tcgccagaaa gaaggactgg gaccctaaga agtacggcgg
     4621 cttcgacagc cccaccgtgg cctattctgt gctggtggtg gccaaagtgg aaaagggcaa
     4681 gtccaagaaa ctgaagagtg tgaaagagct gctggggatc accatcatgg aaagaagcag
     4741 cttcgagaag aatcccatcg actttctgga agccaagggc tacaaagaag tgaaaaagga
     4801 cctgatcatc aagctgccta agtactccct gttcgagctg gaaaacggcc ggaagagaat
     4861 gctggcctct gccggcgaac tgcagaaggg aaacgaactg gccctgccct ccaaatatgt
     4921 gaacttcctg tacctggcca gccactatga gaagctgaag ggctcccccg aggataatga
     4981 gcagaaacag ctgtttgtgg aacagcacaa gcactacctg gacgagatca tcgagcagat
     5041 cagcgagttc tccaagagag tgatcctggc cgacgctaat ctggacaaag tgctgtccgc
     5101 ctacaacaag caccgggata agcccatcag agagcaggcc gagaatatca tccacctgtt
     5161 taccctgacc aatctgggag cccctgccgc cttcaagtac tttgacacca ccatcgaccg
     5221 gaagaggtac accagcacca aagaggtgct ggacgccacc ctgatccacc agagcatcac
     5281 cggcctgtac gagacacgga tcgacctgtc tcagctggga ggcgacggag gtagtggagg
     5341 tactagttta gccttgaaat tagcaggtct tgatatcgct agcttagcct tgaaattagc
     5401 aggtcttgat atcgctagcc gcctgctgga agatggcgat ctcgagccat ctgctggaga
     5461 catgagagct gccaaccttt ggccaagccc gctcatgatc aaacgctcta agaagaacag
     5521 cctggccttg tccctgacgg ccgaccagat ggtcagtgcc ttgttggatg ctgagccccc
     5581 catactctat tccgagtatg atcctaccag acccttcagt gaagcttcga tgatgggctt
     5641 actgaccaac ctggcagaca gggagctggt tcacatgatc aactgggcga agagggtgcc
     5701 aggctttgtg gatttgaccc tccatgatca ggtccacctt ctagaatgtg cctggctaga
     5761 gatcctgatg attggtctcg tctggcgctc catggagcac ccagtgaagc tactgtttgc
     5821 tcctaacttg ctcttggaca ggaaccaggg aaaatgtgta gagggcatgg tggagatctt
     5881 cgacatgctg ctggctacat catctcggtt ccgcatgatg aatctgcagg gagaggagtt
     5941 tgtgtgcctc aaatctatta ttttgcttaa ttctggagtg tacacatttc tgtccagcac
     6001 cctgaagtct ctggaagaga aggaccatat ccaccgagtc ctggacaaga tcacagacac
     6061 tttgatccac ctgatggcca aggcaggcct gaccctgcag cagcagcacc agcggctggc
     6121 ccagctcctc ctcatcctct cccacatcag gcacatgagt aacaaaggca tggagcatct
     6181 gtacagcatg aagtgcaaga acgtggtgcc cctctatgac ctgctgctgg aggcggcgga
     6241 cgcccaccgc ctacatgcgc ccactagccg tggaggggca tccgtggagg agacggacca
     6301 aagccacttg gccactgcgg gctctacttc atcgcattcc ttgcaaaagt attacatcac
     6361 gggggaggca gagggtttcc ctgccacagc tggatcccgc actagtcgcc tgctggaaga
     6421 tggcgatctc gagccatctg ctggagacat gagagctgcc aacctttggc caagcccgct
     6481 catgatcaaa cgctctaaga agaacagcct ggccttgtcc ctgacggccg accagatggt
     6541 cagtgccttg ttggatgctg agccccccat actctattcc gagtatgatc ctaccagacc
     6601 cttcagtgaa gcttcgatga tgggcttact gaccaacctg gcagacaggg agctggttca
     6661 catgatcaac tgggcgaaga gggtgccagg ctttgtggat ttgaccctcc atgatcaggt
     6721 ccaccttcta gaatgtgcct ggctagagat cctgatgatt ggtctcgtct ggcgctccat
     6781 ggagcaccca gtgaagctac tgtttgctcc taacttgctc ttggacagga accagggaaa
     6841 atgtgtagag ggcatggtgg agatcttcga catgctgctg gctacatcat ctcggttccg
     6901 catgatgaat ctgcagggag aggagtttgt gtgcctcaaa tctattattt tgcttaattc
     6961 tggagtgtac acatttctgt ccagcaccct gaagtctctg gaagagaagg accatatcca
     7021 ccgagtcctg gacaagatca cagacacttt gatccacctg atggccaagg caggcctgac
     7081 cctgcagcag cagcaccagc ggctggccca gctcctcctc atcctctccc acatcaggca
     7141 catgagtaac aaaggcatgg agcatctgta cagcatgaag tgcaagaacg tggtgcccct
     7201 ctatgacctg ctgctggagg cggcggacgc ccaccgccta catgcgccca ctagccgtgg
     7261 aggggcatcc gtggaggaga cggaccaaag ccacttggcc actgcgggct ctacttcatc
     7321 gcattccttg caaaagtatt acatcacggg ggaggcagag ggtttccctg ccacagctgg
     7381 tacctatccc tatgacgtgc ccgattatgc cagcctgggc agcggctccc ccaagaaaaa
     7441 acgcaaggtg gaagatccta agaaaaagcg gaaagtggac ggcattggta gtgggagcaa
     7501 cggcagcagc ggttccggcg ccatcataca cgcggccgca acaagcggtc cggaagaact
     7561 tttgagcaag aattatcatc ttgagaacga agtggctcgt cttaagaaag gttctggcag
     7621 tggagaagaa ctgctttcaa agaattacca cctggaaaat gaggtagcta gactgaaaaa
     7681 ggggagcgga agtggggagg agttgctgag caaaaattat catttggaga acgaagtagc
     7741 acgactaaag aaagggtccg gatcgggtga ggagttactc tcgaaaaatt atcatctcga
     7801 aaacgaagtg gctcggctaa aaaagggcag tggttctgga gaagagctat tatctaaaaa
     7861 ctaccacctc gaaaatgagg tggcacgctt aaaaaaggga agtggcagtg gtgaagagct
     7921 actatccaag aattatcatc ttgagaacga ggtagcgcgt ttgaagaagg gttccggctc
     7981 aggagaggaa ctgctctcga agaactatca tcttgaaaat gaggtcgctc gattaaaaaa
     8041 gggatcgggc agtggtgagg aactactttc aaagaattac cacctcgaaa acgaagtagc
     8101 tcgattaaag aaaggttcag ggtcgggtga agaattactg agtaaaaatt atcatctgga
     8161 aaatgaggta gcgagactaa aaaaggggag tggttctggc gaggaattgc tatcgaaaaa
     8221 ttatcatctt gagaacgaag ttgctaggct caaaaagggc tcaggctcag gctgagttta
     8281 aactaattgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     8341 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     8401 tcttatgtcg accctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc
     8461 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
     8521 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
     8581 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     8641 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga
     8701 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagcttac
     8761 catggccaag ttgaccagtg ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt
     8821 cgagttctgg accgaccggc tcgggttctc ccgggacttc gtggaggacg acttcgccgg
     8881 tgtggtccgg gacgacgtga ccctgttcat cagcgcggtc caggaccagg tggtgccgga
     8941 caacaccctg gcctgggtgt gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga
     9001 ggtcgtgtcc acgaacttcc gggacgcctc cgggccggcc atgaccgaga tcggcgagca
     9061 gccgtggggg cgggagttcg ccctgcgcga cccggccggc aactgcgtgc acttcgtggc
     9121 cgaggagcag gactgaccga tcgataaaat aaaagatttt atttagtctc cagaaaaagg
     9181 ggggaatgaa agaccccacc tgtaggtttg gcaagctcga caatcaacct ctggattaca
     9241 aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat
     9301 acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct
     9361 ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac
     9421 gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca
     9481 cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca
     9541 tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg
     9601 tggtgttgtc ggggaagctg acgtcctttc catggctgct cgcctgtgtt gccacctgga
     9661 ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt
     9721 cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga
     9781 gtcggatctc cctttgggcc gcctccccgc ctggaattcg agctcagtac ctttaagacc
     9841 aatgacttac aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga
     9901 agggctaatt cactcccaac gaagacaaga tctgcttttt gcttgtactg ggtctctctg
     9961 gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc
    10021 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg
    10081 taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gtagtagttc
    10141 atgtcatctt attattcagt atttataact tgcaaagaaa tgaatatcag agagtgagag
    10201 gaacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
    10261 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
    10321 ttatcatgtc tggctctagc tatcccgccc ctaactccgc ccagttccgc ccattctccg
    10381 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag
    10441 ctattccaga agtagtgagg aggctttttt ggaggcctag ggacgtaccc aattcgccct
    10501 atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa
    10561 accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta
    10621 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat
    10681 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
    10741 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
    10801 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
    10861 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
    10921 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
    10981 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
    11041 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
    11101 ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa
    11161 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
    11221 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
    11281 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
    11341 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
    11401 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
    11461 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
    11521 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
    11581 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
    11641 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
    11701 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
    11761 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
    11821 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
    11881 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
    11941 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
    12001 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
    12061 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
    12121 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
    12181 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
    12241 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
    12301 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
    12361 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
    12421 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
    12481 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
    12541 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
    12601 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
    12661 ctacaccaaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
    12721 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
    12781 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
    12841 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
    12901 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
    12961 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
    13021 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
    13081 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
    13141 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
    13201 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
    13261 ataacaattt cacacaggaa acagctatga ccatgattac gccaagcgcg caattaaccc
    13321 tcactaaagg gaacaaaagc tggagctgca agcttaatgt agtcttatgc aatactcttg
    13381 tagtcttgca acatggtaac gatgagttag caacatgcct tacaaggaga gaaaaagcac
    13441 cgtgcatgcc gattggtgga agtaaggtgg tacgatcgtg ccttattagg aaggcaacag
    13501 acgggtctga catggattgg acgaaccact gaattgccgc attgcagaga tattgtattt
    13561 aagtgcctag ctcgatacat aaacgggtct ctctggttag accagatctg agcctgggag
    13621 ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt
    13681 caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt
    13741 tagtcagtgt ggaaaatctc tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga
    13801 aaccagagga gctctctcga cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg
    13861 aggggcggcg actggtgagt acgccaaaaa ttttgactag cggaggctag aaggagagag
    13921 atgggtgcga gagcgtcagt attaagcggg ggagaattag atcgcgatgg gaaaaaattc
    13981 ggttaaggcc agggggaaag aaaaaatata aattaaaaca tatagtatgg gcaagcaggg
    14041 agctagaacg attcgcagtt aatcctggcc tgttagaaac atcagaaggc tgtagacaaa
    14101 tactgggaca gctacaacca tcccttcaga caggatcaga agaacttaga tcattatata
    14161 atacagtagc aaccctctat tgtgtgcatc aaaggataga gataaaagac accaaggaag
    14221 ctttagacaa gatagaggaa gagcaaaaca aaagtaagac caccgcacag caagcggccg
    14281 ctgatcttca gacctggagg aggagatatg agggacaatt ggagaagtga attatataaa
    14341 tataaagtag taaaaattga accattagga gtagcaccca ccaaggcaaa gagaagagtg
    14401 gtgcagagag aaaaaagagc agtgggaata ggagctttgt tccttgggtt cttgggagca
    14461 gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg tacaggccag acaattattg
    14521 tctggtatag tgcagcagca gaacaatttg ctgagggcta ttgaggcgca acagcatctg
    14581 ttgcaactca cagtctgggg catcaagcag ctccaggcaa gaatcctggc tgtggaaaga
    14641 tacctaaagg atcaacagct cctggggatt tggggttgct ctggaaaact catttgcacc
    14701 actgctgtgc cttggaatgc tagttggagt aataaatctc tggaacagat ttggaatcac
    14761 acgacctgga tggagtggga cagagaaatt aacaattaca caagcttaat acactcctta
    14821 attgaagaat cgcaaaacca gcaagaaaag aatgaacaag aattattgga attagataaa
    14881 tgggcaagtt tgtggaattg gtttaacata acaaattggc tgtggtatat aaaattattc
    14941 ataatgatag taggaggctt ggtaggttta agaatagttt ttgctgtact ttctatagtg
    15001 aatagagtta ggcagggata ttcaccatta tcgtttcaga cccacctccc aaccccgagg
    15061 ggacccgaca ggcccgaagg aatagaagaa gaaggtggag agagagacag agacagatcc
    15121 attcgattag tgaacggatc tcgacggtat cggttaactt ttaaaagaaa aggggggatt
    15181 ggggggtaca gtgcagggga aagaatagta gacataatag caacagacat acaaactaaa
    15241 gaattacaaa aacaaattac aaaaattcaa aattttatcg atcacgagac tagcctcga
//
