LOCUS       sequence-223869-        7719 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Multisite gateway vector for expression of nCas9n with bicistronic 
            expression of mKate2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7719)
  AUTHORS   Hall TE, Martel N, Ariotti N, Xiong Z, Lo HP, Ferguson C, Rae J, Lim
            YW, Parton RG
  TITLE     In vivo cell biological screening identifies an endocytic capture 
            mechanism for T-tubule formation.
  JOURNAL   Nat Commun. 2020 Jul 24;11(1):3711. doi: 10.1038/s41467-020-17486-w.
  PUBMED    32709891
REFERENCE   2  (bases 1 to 7719)
  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: "Nat 
            Commun."; date: "2020-07-24"; volume: "11(1):3711. doi"; pages: " 
            10.1038/s41467-020-17486-w"
COMMENT     Sequence Label: pME-nCas9n-P2A-mKate2-pA
FEATURES             Location/Qualifiers
     source          1..7719
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..100
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     regulatory      101..110
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             119..139
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             146..4246
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes MGAS15252 Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFGSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLADSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQIYNQLFEENPINASRVDAKAILSARLSKSRRL
                     ENLIAQLPGEKRNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNSEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGAYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRGMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGHSLHEQIANLAGSPAIKKGILQTVKIVDELVKVMGH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFIKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     RBS             4159..4167
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             4262..4282
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4298..4354
                     /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             4373..5068
                     /codon_start=1
                     /product="monomeric far-red fluorescent protein (Shcherbo 
                     et al., 2009)"
                     /label=mKate2
                     /note="mammalian codon-optimized"
                     /translation="VSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKA
                     VEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMALK
                     LVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARYC
                     DLPSKLGHR"
     polyA_signal    5190..5271
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5227..5246)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    complement(5276..5375)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(5392..5411)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5393..5411)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5416..5432)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5416..5432)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5534..5553)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5545..6354
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(5612..5631)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      6501..7089
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6990..7009
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7243..7260
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(7419..7446)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(7538..7624)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     7673..7695
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7687..7704
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7688..7704
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 caaataatga ttttattttg actgatagtg acctgttcgt tgcaacaaat tgatgagcaa
       61 tgctttttta taatgccaac tttgtacaaa aaagcaggct gccaccatgg cttctccacc
      121 taagaagaag agaaaggtgg gaagcatgga taagaagtat agcatcggcc tggatattgg
      181 aactaactcc gtgggttggg cagtgattac agacgactac aaggtcccta gcaagaaatt
      241 taaggtgctg ggtaacaccg acaggcacag catcaagaaa aatctgattg gagccctgct
      301 gttcggttct ggagagactg ccgaagcaac acgcctgaaa agaacagcaa gaaggcgcta
      361 taccagaagg aagaatagaa tctgttacct gcaggagatt ttctctaacg aaatggctaa
      421 ggtggacgat tcattctttc ataggctgga ggaaagtttc ctggtcgagg aagataagaa
      481 acacgagcgc catcctatct ttggaaacat tgtggacgag gtcgcctatc acgaaaaata
      541 cccaaccatc tatcatctgc gcaagaaact ggctgactct actgataaag ccgacctgag
      601 actgatctat ctggctctgg cccacatgat taagttcagg ggtcattttc tgatcgaggg
      661 cgatctgaac cccgacaatt ccgatgtgga caagctgttc atccagctgg tccagattta
      721 caatcagctg tttgaggaaa accctattaa tgcttccaga gtggacgcaa aagctatcct
      781 gtcagccagg ctgtccaagt cacgcagact ggagaacctg attgcacagc tgcccggaga
      841 aaagaggaac ggtctgtttg gaaatctgat cgctctgagt ctgggcctga ctcctaactt
      901 caaaagcaat tttgatctgg ctgaggacgc caaactgcag ctgtcaaagg acacatatga
      961 cgatgacctg gataacctgc tggcacagat cggagatcag tacgctgacc tgttcctggc
     1021 tgccaaaaat ctgtccgacg caatcctgct gtcagatatt ctgagagtga acagcgagat
     1081 tacaaaagct cctctgagtg ccagcatgat caagagatat gacgagcacc atcaggatct
     1141 gaccctgctg aaggctctgg tcaggcagca gctgccagag aagtacaagg aaattttctt
     1201 tgatcagtcc aagaacggct acgccggtta tatcgacgga ggcgcatcac aggaggaatt
     1261 ctacaagttt atcaaaccta ttctggagaa gatggacgga actgaggaac tgctggtgaa
     1321 actgaataga gaggacctgc tgaggaagca gcgcacattt gataacggtt ccatcccaca
     1381 ccagattcat ctgggagagc tgcacgctat cctgaggcgc caggaagact tctacccctt
     1441 tctgaaagat aaccgcgaga agatcgaaaa aattctgacc ttcagaatcc cttactatgt
     1501 gggtccactg gctcgcggaa acagcagatt tgcctggatg actcgcaaat ccgaggaaac
     1561 cattactcct tggaacttcg aggaagtggt cgataagggc gcctctgcac agtccttcat
     1621 cgagagaatg actaattttg acaaaaacct gcccaatgag aaagtgctgc ctaagcactc
     1681 cctgctgtac gagtatttca ctgtctataa cgaactgaca aaggtgaaat acgtcaccga
     1741 gggcatgaga aagccagcct tcctgtcagg agagcagaag aaagcaatcg tggatctgct
     1801 gtttaaaacc aacaggaaag tgactgtcaa gcagctgaag gaggactact tcaagaaaat
     1861 tgaatgcttc gattccgtgg agatcagcgg agtcgaagac agatttaacg caagcctggg
     1921 cgcttaccac gatctgctga agatcattaa ggataaagac ttcctggaca acgaggaaaa
     1981 tgaggatatc ctggaagaca ttgtgctgac actgaccctg tttgaggaca gaggaatgat
     2041 cgaggaaaga ctgaaaacct atgctcatct gttcgatgac aaggtgatga aacagctgaa
     2101 gagaaggcgc tacactggct ggggtagact gagcaggaag ctgatcaacg gcattaggga
     2161 taaacagtca ggaaagacaa tcctggactt tctgaaaagt gatggcttcg ccaaccgcaa
     2221 ttttatgcag ctgattcacg atgacagtct gaccttcaaa gaggacatcc agaaggctca
     2281 ggtgtctgga cagggccact ccctgcatga gcagattgca aacctggctg gaagcccagc
     2341 catcaagaaa ggcattctgc agacagtgaa aatcgtcgat gagctggtga aagtcatggg
     2401 ccataagccc gaaaacatcg tgattgagat ggctcgcgaa aatcagacaa cccagaaggg
     2461 tcagaagaac agtagagaga ggatgaaaag aatcgaggaa ggcattaagg agctgggtag
     2521 ccagatcctg aaagagcacc cagtggaaaa cacacagctg cagaatgaga agctgtatct
     2581 gtactatctg cagaatggaa gagatatgta cgtggaccag gagctggata ttaacaggct
     2641 gtctgattac gacgtggatc atatcgtccc ccagagtttc atcaaagatg acagcattga
     2701 caacaaggtg ctgaccaggt ccgacaaaaa cagaggaaaa tcagataatg tccctagtga
     2761 ggaagtggtc aagaaaatga agaactactg gagacagctg ctgaatgcca aactgatcac
     2821 tcagaggaag tttgataacc tgacaaaagc agagcgcggt ggactgtcag aactggacaa
     2881 agctggattc atcaagaggc agctggtgga aacacgccag atcactaaac acgtcgcaca
     2941 gattctggat agtcgcatga acacaaagta cgatgagaat gacaaactga tcagagaagt
     3001 gaaggtcatt accctgaaga gtaaactggt cagcgacttt aggaaagatt tccagtttta
     3061 taaggtccgc gagattaaca attatcacca tgcccatgac gcatacctga acgccgtggt
     3121 cggtaccgca ctgatcaaga aatacccaaa actggagagc gaattcgtgt acggagacta
     3181 taaggtgtac gatgtcagaa aaatgatcgc caagtccgag caggaaattg gaaaagctac
     3241 tgccaagtat ttcttttact caaacatcat gaatttcttt aagacagaga tcaccctggc
     3301 caatggagaa atccgcaaaa ggcccctgat tgagacaaac ggagagacag gcgaaatcgt
     3361 gtgggacaaa ggcagagatt ttgcaaccgt gaggaaggtc ctgagcatgc ctcaagtgaa
     3421 tatcgtcaag aaaactgagg tgcagacagg cggtttctca aaagaaagta ttctgccaaa
     3481 acgcaactct gataagctga tcgctagaaa gaaagactgg gaccctaaga agtatggagg
     3541 ctttgactct cccactgtgg catactccgt cctggtggtc gctaaggtgg agaagggcaa
     3601 aagcaagaaa ctgaaatctg tcaaggagct gctgggtatc acaattatgg agagaagctc
     3661 tttcgagaag aacccaatcg attttctgga ggccaaaggt tataaggaag tgaagaaaga
     3721 cctgatcatt aaactgccca agtacagtct gtttgagctg gaaaacggca ggaaacgcat
     3781 gctggcaagc gctggagagc tgcagaaagg caatgaactg gccctgcctt ctaagtacgt
     3841 gaacttcctg tatctggcaa gccactacga gaagctgaaa ggatctccag aggataacga
     3901 acagaaacag ctgtttgtgg agcagcacaa gcattatctg gacgagatca ttgaacagat
     3961 tagcgagttc tctaaaagag tgatcctggc cgacgcaaat ctggataagg tcctgtctgc
     4021 ttacaacaaa cacagagata agcccatcag ggagcaggcc gaaaatatca ttcatctgtt
     4081 cactctgaca aacctgggcg cacctgcagc tttcaagtac ttcgacacta caatcgatag
     4141 aaagaggtac acctccacta aggaggtgct ggacgctaca ctgatccatc agagtattac
     4201 cggcctgtac gaaacaagga ttgacctgtc tcagctgggt ggcgactcac cggtgagatc
     4261 tcctaagaag aagagaaagg tgcctagggg cagcggcgcc accaacttca gcctgctgaa
     4321 gcaggccggc gacgtggagg agaaccccgg ccccgccccc ggcagcctcg aggtgagcga
     4381 gctgattaag gagaacatgc acatgaagct gtacatggag ggcaccgtga acaaccacca
     4441 cttcaagtgc acatccgagg gcgaaggcaa gccctacgag ggcacccaga ccatgagaat
     4501 caaggcggtc gagggcggcc ctctcccctt cgccttcgac atcctggcta ccagcttcat
     4561 gtacggcagc aaaaccttca tcaaccacac ccagggcatc cccgacttct ttaagcagtc
     4621 cttccccgag ggcttcacat gggagagagt caccacatac gaagacgggg gcgtgctgac
     4681 cgctacccag gacaccagcc tccaggacgg ctgcctcatc tacaacgtca agatcagagg
     4741 ggtgaacttc ccatccaacg gccctgtgat gcagaagaaa acactcggct gggaggcctc
     4801 caccgagacc ctgtaccccg ctgacggcgg cctggaaggc agagccgaca tggccctgaa
     4861 gctcgtgggc gggggccacc tgatctgcaa cttgaagacc acatacagat ccaagaaacc
     4921 cgctaagaac ctcaagatgc ccggcgtcta ctatgtggac agaagactgg aaagaatcaa
     4981 ggaggccgac aaagagacct acgtcgagca gcacgaggtg gctgtggcca gatactgcga
     5041 cctccctagc aaactggggc acagatgagc ggccgcgact ctagatcata atcagccata
     5101 ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga
     5161 aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca
     5221 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cgtcgaccca
     5281 gctttcttgt acaaagttgg cattataaga aagcattgct tatcaatttg ttgcaacgaa
     5341 caggtcacta tcagtcaaaa taaaatcatt atttgccatc cagctgatat cccctatagt
     5401 gagtcgtatt acatggtcat agctgtttcc tggcagctct ggcccgtgtc tcaaaatctc
     5461 tgatgttaca ttgcacaaga taaaaatata tcatcatgaa caataaaact gtctgcttac
     5521 ataaacagta atacaagggg tgttatgagc catattcaac gggaaacgtc gaggccgcga
     5581 ttaaattcca acatggatgc tgatttatat gggtataaat gggctcgcga taatgtcggg
     5641 caatcaggtg cgacaatcta tcgcttgtat gggaagcccg atgcgccaga gttgtttctg
     5701 aaacatggca aaggtagcgt tgccaatgat gttacagatg agatggtcag actaaactgg
     5761 ctgacggaat ttatgcctct tccgaccatc aagcatttta tccgtactcc tgatgatgca
     5821 tggttactca ccactgcgat ccccggaaaa acagcattcc aggtattaga agaatatcct
     5881 gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt
     5941 cctgtttgta attgtccttt taacagcgat cgcgtatttc gtctcgctca ggcgcaatca
     6001 cgaatgaata acggtttggt tgatgcgagt gattttgatg acgagcgtaa tggctggcct
     6061 gttgaacaag tctggaaaga aatgcataaa cttttgccat tctcaccgga ttcagtcgtc
     6121 actcatggtg atttctcact tgataacctt atttttgacg aggggaaatt aataggttgt
     6181 attgatgttg gacgagtcgg aatcgcagac cgataccagg atcttgccat cctatggaac
     6241 tgcctcggtg agttttctcc ttcattacag aaacggcttt ttcaaaaata tggtattgat
     6301 aatcctgata tgaataaatt gcagtttcat ttgatgctcg atgagttttt ctaatcagaa
     6361 ttggttaatt ggttgtaaca ctggcagagc attacgctga cttgacggga cggcgcaagc
     6421 tcatgaccaa aatcccttaa cgtgagttac gcgtcgttcc actgagcgtc agaccccgta
     6481 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     6541 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     6601 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     6661 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     6721 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     6781 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     6841 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     6901 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     6961 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     7021 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     7081 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     7141 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     7201 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     7261 gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
     7321 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
     7381 acgcgtaccg ctagccagga agagtttgta gaaacgcaaa aaggccatcc gtcaggatgg
     7441 ccttctgctt agtttgatgc ctggcagttt atggcgggcg tcctgcccgc caccctccgg
     7501 gccgttgctt cacaacgttc aaatccgctc ccggcggatt tgtcctactc aggagagcgt
     7561 tcaccgacaa acaacagata aaacgaaagg cccagtcttc cgactgagcc tttcgtttta
     7621 tttgatgcct ggcagttccc tactctcgcg ttaacgctag catggatgtt ttcccagtca
     7681 cgacgttgta aaacgacggc cagtcttaag ctcgggccc
//
