LOCUS       sequence-206484-        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"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(387..473)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     522..544
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     536..553
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    569..668
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     regulatory      669..678
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             687..707
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             714..4814
                     /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             4727..4735
                     /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             4830..4850
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4866..4922
                     /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             4941..5636
                     /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    5758..5839
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5795..5814)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    complement(5844..5943)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(5960..5979)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5961..5979)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5984..6000)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5984..6000)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6102..6121)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             6113..6922
                     /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(6180..6199)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      7069..7657
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7558..7577
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac
      601 ctgttcgttg caacacattg atgagcaatg cttttttata atgccaactt tgtacaaaaa
      661 agcaggctgc caccatggct tctccaccta agaagaagag aaaggtggga agcatggata
      721 agaagtatag catcggcctg gatattggaa ctaactccgt gggttgggca gtgattacag
      781 acgactacaa ggtccctagc aagaaattta aggtgctggg taacaccgac aggcacagca
      841 tcaagaaaaa tctgattgga gccctgctgt tcggttctgg agagactgcc gaagcaacac
      901 gcctgaaaag aacagcaaga aggcgctata ccagaaggaa gaatagaatc tgttacctgc
      961 aggagatttt ctctaacgaa atggctaagg tggacgattc attctttcat aggctggagg
     1021 aaagtttcct ggtcgaggaa gataagaaac acgagcgcca tcctatcttt ggaaacattg
     1081 tggacgaggt cgcctatcac gaaaaatacc caaccatcta tcatctgcgc aagaaactgg
     1141 ctgactctac tgataaagcc gacctgagac tgatctatct ggctctggcc cacatgatta
     1201 agttcagggg tcattttctg atcgagggcg atctgaaccc cgacaattcc gatgtggaca
     1261 agctgttcat ccagctggtc cagatttaca atcagctgtt tgaggaaaac cctattaatg
     1321 cttccagagt ggacgcaaaa gctatcctgt cagccaggct gtccaagtca cgcagactgg
     1381 agaacctgat tgcacagctg cccggagaaa agaggaacgg tctgtttgga aatctgatcg
     1441 ctctgagtct gggcctgact cctaacttca aaagcaattt tgatctggct gaggacgcca
     1501 aactgcagct gtcaaaggac acatatgacg atgacctgga taacctgctg gcacagatcg
     1561 gagatcagta cgctgacctg ttcctggctg ccaaaaatct gtccgacgca atcctgctgt
     1621 cagatattct gagagtgaac agcgagatta caaaagctcc tctgagtgcc agcatgatca
     1681 agagatatga cgagcaccat caggatctga ccctgctgaa ggctctggtc aggcagcagc
     1741 tgccagagaa gtacaaggaa attttctttg atcagtccaa gaacggctac gccggttata
     1801 tcgacggagg cgcatcacag gaggaattct acaagtttat caaacctatt ctggagaaga
     1861 tggacggaac tgaggaactg ctggtgaaac tgaatagaga ggacctgctg aggaagcagc
     1921 gcacatttga taacggttcc atcccacacc agattcatct gggagagctg cacgctatcc
     1981 tgaggcgcca ggaagacttc tacccctttc tgaaagataa ccgcgagaag atcgaaaaaa
     2041 ttctgacctt cagaatccct tactatgtgg gtccactggc tcgcggaaac agcagatttg
     2101 cctggatgac tcgcaaatcc gaggaaacca ttactccttg gaacttcgag gaagtggtcg
     2161 ataagggcgc ctctgcacag tccttcatcg agagaatgac taattttgac aaaaacctgc
     2221 ccaatgagaa agtgctgcct aagcactccc tgctgtacga gtatttcact gtctataacg
     2281 aactgacaaa ggtgaaatac gtcaccgagg gcatgagaaa gccagccttc ctgtcaggag
     2341 agcagaagaa agcaatcgtg gatctgctgt ttaaaaccaa caggaaagtg actgtcaagc
     2401 agctgaagga ggactacttc aagaaaattg aatgcttcga ttccgtggag atcagcggag
     2461 tcgaagacag atttaacgca agcctgggcg cttaccacga tctgctgaag atcattaagg
     2521 ataaagactt cctggacaac gaggaaaatg aggatatcct ggaagacatt gtgctgacac
     2581 tgaccctgtt tgaggacaga ggaatgatcg aggaaagact gaaaacctat gctcatctgt
     2641 tcgatgacaa ggtgatgaaa cagctgaaga gaaggcgcta cactggctgg ggtagactga
     2701 gcaggaagct gatcaacggc attagggata aacagtcagg aaagacaatc ctggactttc
     2761 tgaaaagtga tggcttcgcc aaccgcaatt ttatgcagct gattcacgat gacagtctga
     2821 ccttcaaaga ggacatccag aaggctcagg tgtctggaca gggccactcc ctgcatgagc
     2881 agattgcaaa cctggctgga agcccagcca tcaagaaagg cattctgcag acagtgaaaa
     2941 tcgtcgatga gctggtgaaa gtcatgggcc ataagcccga aaacatcgtg attgagatgg
     3001 ctcgcgaaaa tcagacaacc cagaagggtc agaagaacag tagagagagg atgaaaagaa
     3061 tcgaggaagg cattaaggag ctgggtagcc agatcctgaa agagcaccca gtggaaaaca
     3121 cacagctgca gaatgagaag ctgtatctgt actatctgca gaatggaaga gatatgtacg
     3181 tggaccagga gctggatatt aacaggctgt ctgattacga cgtggatcat atcgtccccc
     3241 agagtttcat caaagatgac agcattgaca acaaggtgct gaccaggtcc gacaaaaaca
     3301 gaggaaaatc agataatgtc cctagtgagg aagtggtcaa gaaaatgaag aactactgga
     3361 gacagctgct gaatgccaaa ctgatcactc agaggaagtt tgataacctg acaaaagcag
     3421 agcgcggtgg actgtcagaa ctggacaaag ctggattcat caagaggcag ctggtggaaa
     3481 cacgccagat cactaaacac gtcgcacaga ttctggatag tcgcatgaac acaaagtacg
     3541 atgagaatga caaactgatc agagaagtga aggtcattac cctgaagagt aaactggtca
     3601 gcgactttag gaaagatttc cagttttata aggtccgcga gattaacaat tatcaccatg
     3661 cccatgacgc atacctgaac gccgtggtcg gtaccgcact gatcaagaaa tacccaaaac
     3721 tggagagcga attcgtgtac ggagactata aggtgtacga tgtcagaaaa atgatcgcca
     3781 agtccgagca ggaaattgga aaagctactg ccaagtattt cttttactca aacatcatga
     3841 atttctttaa gacagagatc accctggcca atggagaaat ccgcaaaagg cccctgattg
     3901 agacaaacgg agagacaggc gaaatcgtgt gggacaaagg cagagatttt gcaaccgtga
     3961 ggaaggtcct gagcatgcct caagtgaata tcgtcaagaa aactgaggtg cagacaggcg
     4021 gtttctcaaa agaaagtatt ctgccaaaac gcaactctga taagctgatc gctagaaaga
     4081 aagactggga ccctaagaag tatggaggct ttgactctcc cactgtggca tactccgtcc
     4141 tggtggtcgc taaggtggag aagggcaaaa gcaagaaact gaaatctgtc aaggagctgc
     4201 tgggtatcac aattatggag agaagctctt tcgagaagaa cccaatcgat tttctggagg
     4261 ccaaaggtta taaggaagtg aagaaagacc tgatcattaa actgcccaag tacagtctgt
     4321 ttgagctgga aaacggcagg aaacgcatgc tggcaagcgc tggagagctg cagaaaggca
     4381 atgaactggc cctgccttct aagtacgtga acttcctgta tctggcaagc cactacgaga
     4441 agctgaaagg atctccagag gataacgaac agaaacagct gtttgtggag cagcacaagc
     4501 attatctgga cgagatcatt gaacagatta gcgagttctc taaaagagtg atcctggccg
     4561 acgcaaatct ggataaggtc ctgtctgctt acaacaaaca cagagataag cccatcaggg
     4621 agcaggccga aaatatcatt catctgttca ctctgacaaa cctgggcgca cctgcagctt
     4681 tcaagtactt cgacactaca atcgatagaa agaggtacac ctccactaag gaggtgctgg
     4741 acgctacact gatccatcag agtattaccg gcctgtacga aacaaggatt gacctgtctc
     4801 agctgggtgg cgactcaccg gtgagatctc ctaagaagaa gagaaaggtg cctaggggca
     4861 gcggcgccac caacttcagc ctgctgaagc aggccggcga cgtggaggag aaccccggcc
     4921 ccgcccccgg cagcctcgag gtgagcgagc tgattaagga gaacatgcac atgaagctgt
     4981 acatggaggg caccgtgaac aaccaccact tcaagtgcac atccgagggc gaaggcaagc
     5041 cctacgaggg cacccagacc atgagaatca aggcggtcga gggcggccct ctccccttcg
     5101 ccttcgacat cctggctacc agcttcatgt acggcagcaa aaccttcatc aaccacaccc
     5161 agggcatccc cgacttcttt aagcagtcct tccccgaggg cttcacatgg gagagagtca
     5221 ccacatacga agacgggggc gtgctgaccg ctacccagga caccagcctc caggacggct
     5281 gcctcatcta caacgtcaag atcagagggg tgaacttccc atccaacggc cctgtgatgc
     5341 agaagaaaac actcggctgg gaggcctcca ccgagaccct gtaccccgct gacggcggcc
     5401 tggaaggcag agccgacatg gccctgaagc tcgtgggcgg gggccacctg atctgcaact
     5461 tgaagaccac atacagatcc aagaaacccg ctaagaacct caagatgccc ggcgtctact
     5521 atgtggacag aagactggaa agaatcaagg aggccgacaa agagacctac gtcgagcagc
     5581 acgaggtggc tgtggccaga tactgcgacc tccctagcaa actggggcac agatgagcgg
     5641 ccgcgactct agatcataat cagccatacc acatttgtag aggttttact tgctttaaaa
     5701 aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac
     5761 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     5821 aaagcatttt tttcactgcg tcgacccagc tttcttgtac aaagttggca ttataagaaa
     5881 gcattgctta tcaatttgtt gcaacgaaca ggtcactatc agtcaaaata aaatcattat
     5941 ttgccatcca gctgatatcc cctatagtga gtcgtattac atggtcatag ctgtttcctg
     6001 gcagctctgg cccgtgtctc aaaatctctg atgttacatt gcacaagata aaataatatc
     6061 atcatgaaca ataaaactgt ctgcttacat aaacagtaat acaaggggtg ttatgagcca
     6121 tattcaacgg gaaacgtcga ggccgcgatt aaattccaac atggatgctg atttatatgg
     6181 gtataaatgg gctcgcgata atgtcgggca atcaggtgcg acaatctatc gcttgtatgg
     6241 gaagcccgat gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt
     6301 tacagatgag atggtcagac taaactggct gacggaattt atgcctcttc cgaccatcaa
     6361 gcattttatc cgtactcctg atgatgcatg gttactcacc actgcgatcc ccggaaaaac
     6421 agcattccag gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc
     6481 agtgttcctg cgccggttgc attcgattcc tgtttgtaat tgtcctttta acagcgatcg
     6541 cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac ggtttggttg atgcgagtga
     6601 ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcataaact
     6661 tttgccattc tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat
     6721 ttttgacgag gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg
     6781 ataccaggat cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa
     6841 acggcttttt caaaaatatg gtattgataa tcctgatatg aataaattgc agtttcattt
     6901 gatgctcgat gagtttttct aatcagaatt ggttaattgg ttgtaacact ggcagagcat
     6961 tacgctgact tgacgggacg gcgcaagctc atgaccaaaa tcccttaacg tgagttacgc
     7021 gtcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     7081 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     7141 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     7201 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     7261 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     7321 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     7381 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     7441 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     7501 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     7561 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     7621 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     7681 acggttcctg gccttttgct ggccttttgc tcacatgtt
//
