LOCUS       sequence-346512-        7725 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Multisite gateway vector for expression of nCas9n with blue 
            reporter. Parton lab clone ENT.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7725)
  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 7725)
  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-TagBFP2-pA
FEATURES             Location/Qualifiers
     source          1..7725
                     /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"
     polyA_signal    5764..5845
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5801..5820)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    complement(5850..5949)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(5966..5985)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5967..5985)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5990..6006)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5990..6006)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6108..6127)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             6119..6928
                     /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(6186..6205)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      7075..7663
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7564..7583
                     /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 caacaaattg 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 gtcagtgaat tgataaagga gaatatgcat atgaaactgt
     4981 acatggaggg tacagtagat aaccaccact tcaagtgtac aagtgaaggc gagggaaaac
     5041 cgtacgaggg aactcagacg atgaggatca aagtagtgga gggtggacct cttcctttcg
     5101 ctttcgacat cctggctacc tctttcctgt atggaagtaa aacttttata aatcacacac
     5161 aaggtatccc cgattttttc aagcagtcat tccccgaagg cttcacctgg gaaagagtca
     5221 caacctacga ggatggcggc gtcctgacag ccactcagga tacatcattg caagacggat
     5281 gtctgatata taacgttaaa atcagaggcg taaactttac ctctaatgga cccgtaatgc
     5341 agaaaaaaac cctgggctgg gaggctttta ccgagacgct gtatcctgca gacggaggtc
     5401 tcgaggggag aaatgacatg gccctgaagc ttgtgggggg gagtcacctg attgccaacg
     5461 ccaaaactac gtataggtcc aaaaaacctg caaagaacct caaaatgccc ggcgtgtact
     5521 acgtggatta ccgtctggag cgcattaagg aagccaacaa cgagacttac gtagagcagc
     5581 atgaagttgc tgtggctaga tattgcgact tgcccagtaa attggggcac aagttgaatt
     5641 gagcgaccgc gactctagat cataatcagc cataccacat ttgtagaggt tttacttgct
     5701 ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt
     5761 gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     5821 acaaataaag catttttttc actgcgtcga cccagctttc ttgtacaaag ttggcattat
     5881 aagaaagcat tgcttatcaa tttgttgcaa cgaacaggtc actatcagtc aaaataaaat
     5941 cattatttgc catccagctg atatccccta tagtgagtcg tattacatgg tcatagctgt
     6001 ttcctggcag ctctggcccg tgtctcaaaa tctctgatgt tacattgcac aagataaaaa
     6061 tatatcatca tgaacaataa aactgtctgc ttacataaac agtaatacaa ggggtgttat
     6121 gagccatatt caacgggaaa cgtcgaggcc gcgattaaat tccaacatgg atgctgattt
     6181 atatgggtat aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa tctatcgctt
     6241 gtatgggaag cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa
     6301 tgatgttaca gatgagatgg tcagactaaa ctggctgacg gaatttatgc ctcttccgac
     6361 catcaagcat tttatccgta ctcctgatga tgcatggtta ctcaccactg cgatccccgg
     6421 aaaaacagca ttccaggtat tagaagaata tcctgattca ggtgaaaata ttgttgatgc
     6481 gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc cttttaacag
     6541 cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg aataacggtt tggttgatgc
     6601 gagtgatttt gatgacgagc gtaatggctg gcctgttgaa caagtctgga aagaaatgca
     6661 taaacttttg ccattctcac cggattcagt cgtcactcat ggtgatttct cacttgataa
     6721 ccttattttt gacgagggga aattaatagg ttgtattgat gttggacgag tcggaatcgc
     6781 agaccgatac caggatcttg ccatcctatg gaactgcctc ggtgagtttt ctccttcatt
     6841 acagaaacgg ctttttcaaa aatatggtat tgataatcct gatatgaata aattgcagtt
     6901 tcatttgatg ctcgatgagt ttttctaatc agaattggtt aattggttgt aacactggca
     6961 gagcattacg ctgacttgac gggacggcgc aagctcatga ccaaaatccc ttaacgtgag
     7021 ttacgcgtcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     7081 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     7141 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     7201 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     7261 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     7321 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     7381 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     7441 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     7501 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     7561 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     7621 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     7681 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgtt
//
