LOCUS       sequence-197195-        7078 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Encodes dCas9(N) fused to transmembrane tether.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 7078)
  AUTHORS   Baeumler TA, Ahmed AA, Fulga TA
  TITLE     Engineering Synthetic Signaling Pathways with Programmable 
            dCas9-Based Chimeric Receptors.
  JOURNAL   Cell Rep. 2017 Sep 12;20(11):2639-2653. doi: 
            10.1016/j.celrep.2017.08.044.
  PUBMED    28903044
REFERENCE   2  (bases 1 to 7078)
  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"; doi: 
            "10.1016/j.celrep.2017.08"; journalName: "Cell Rep"; date: 
            "2017-09-12- 12"; volume: "20"; issue: "11"; pages: "2639-2653"
COMMENT     Sequence Label: pTMt_NES_TCS(Q'G)_HA-dCas9(N)_P2A-Puro-WPRE
FEATURES             Location/Qualifiers
     source          1..7078
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     intron          112..339
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     sig_peptide     361..420
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="METDTLLLWVLLLWVPGSTG"
     CDS             463..492
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             496..642
                     /codon_start=1
                     /product="transmembrane domain from platelet derived growth
                     factor receptor beta"
                     /label=PDGFR-beta TM domain
                     /translation="AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQ
                     KKPR"
     CDS             739..759
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             778..804
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(820..843,844..846,847..2421)
                     /codon_start=1
                     /product="N-terminal portion of catalytically inactive 
                     Streptococcus pyogenes dCas9 carrying the D10A mutation 
                     (Zetsche et al., 2015)"
                     /label=dCas9(N)
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMR"
     CDS             2452..2508
                     /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"
     primer_bind     complement(2515..2534)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3011..3031
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(3148..3168)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3566..3577)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3708..3725)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3714..3921
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3930..4070
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3930..4059
                     /label=AAV2 ITR
     rep_origin      4145..4600
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4232..4251)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4442..4463
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4617..4636)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4736..4758
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4796..4814)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4882..4986
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4987..5055,5056..5847)
                     /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(5205..5224)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6018..6606
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6507..6526
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        6677..6962
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
ORIGIN
        1 aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt tccttttatg
       61 gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc gggagtcgct
      121 gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc gccccggctc
      181 tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc tccgggctgt
      241 aattagctga gcaagaggta agggtttaag ggatggttgg ttggtggggt attaatgttt
      301 aattacctgg agcacctgcc tgaaatcact ttttttcagg ttggaccggt gatatccacc
      361 atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt
      421 gaccattctg gtggcggaag tgggggaggc agcggtcgac aagaacaaaa actcatctca
      481 gaagaggatc tgaatgctgt gggccaggac acgcaggagg tcatcgtggt gccacactcc
      541 ttgcccttta aggtggtggt gatctcagcc atcctggccc tggtggtgct caccatcatc
      601 tcccttatca tcctcatcat gctttggcag aagaagccac gtctgcagag cggcagcgag
      661 actcccggga cctcagagtc cgccacaccc gaaagtgcta gcctagattt agctagctta
      721 atcctcggca agcttggcga aaacctgtat tttcaggggg ggggaggcag cactagttac
      781 ccatacgacg taccagatta cgctggaggt tctggatccg acaagaagta cagcatcggc
      841 ctggccatcg gcaccaactc tgtgggctgg gccgtgatca ccgacgagta caaggtgccc
      901 agcaagaaat tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa gaacctgatc
      961 ggagccctgc tgttcgacag cggcgaaaca gccgaggcca cccggctgaa gagaaccgcc
     1021 agaagaagat acaccagacg gaagaaccgg atctgctatc tgcaagagat cttcagcaac
     1081 gagatggcca aggtggacga cagcttcttc cacagactgg aagagtcctt cctggtggaa
     1141 gaggataaga agcacgagcg gcaccccatc ttcggcaaca tcgtggacga ggtggcctac
     1201 cacgagaagt accccaccat ctaccacctg agaaagaaac tggtggacag caccgacaag
     1261 gccgacctgc ggctgatcta tctggccctg gcccacatga tcaagttccg gggccacttc
     1321 ctgatcgagg gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt catccagctg
     1381 gtgcagacct acaaccagct gttcgaggaa aaccccatca acgccagcgg cgtggacgcc
     1441 aaggccatcc tgtctgccag actgagcaag agcagacggc tggaaaatct gatcgcccag
     1501 ctgcccggcg agaagaagaa tggcctgttc ggcaacctga ttgccctgag cctgggcctg
     1561 acccccaact tcaagagcaa cttcgacctg gccgaggatg ccaaactgca gctgagcaag
     1621 gacacctacg acgacgacct ggacaacctg ctggcccaga tcggcgacca gtacgccgac
     1681 ctgtttctgg ccgccaagaa cctgtccgac gccatcctgc tgagcgacat cctgagagtg
     1741 aacaccgaga tcaccaaggc ccccctgagc gcctctatga tcaagagata cgacgagcac
     1801 caccaggacc tgaccctgct gaaagctctc gtgcggcagc agctgcctga gaagtacaaa
     1861 gagattttct tcgaccagag caagaacggc tacgccggct acattgacgg cggagccagc
     1921 caggaagagt tctacaagtt catcaagccc atcctggaaa agatggacgg caccgaggaa
     1981 ctgctcgtga agctgaacag agaggacctg ctgcggaagc agcggacctt cgacaacggc
     2041 agcatccccc accagatcca cctgggagag ctgcacgcca ttctgcggcg gcaggaagat
     2101 ttttacccat tcctgaagga caaccgggaa aagatcgaga agatcctgac cttccgcatc
     2161 ccctactacg tgggccctct ggccagggga aacagcagat tcgcctggat gaccagaaag
     2221 agcgaggaaa ccatcacccc ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc
     2281 cagagcttca tcgagcggat gaccaacttc gataagaacc tgcccaacga gaaggtgctg
     2341 cccaagcaca gcctgctgta cgagtacttc accgtgtata acgagctgac caaagtgaaa
     2401 tacgtgaccg agggaatgag aggtggcggt ggctcgggta ccggatctgg agccaccaat
     2461 ttttctctgc tgaagcaggc tggtgatgtt gaagaaaacc ctggacctga attcatgacc
     2521 gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc
     2581 ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac
     2641 atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc
     2701 aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc
     2761 gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg
     2821 ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg
     2881 tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc
     2941 gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc
     3001 tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc
     3061 gaggtgcccg aaggaccgcg cacctggtgc atgacccgca aggggtaact aagtaaggat
     3121 cgatccaaga tatcgtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc
     3181 tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta
     3241 taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt
     3301 ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca
     3361 gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc
     3421 ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt
     3481 gtcggggaag ctgacgtcct ttccatggct gctcgcctgt gttgccacct ggattctgcg
     3541 cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg
     3601 cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat
     3661 ctccctttgg gccgcctccc cgccaattcc tagagctcgc tgatcagcct cgactgtgcc
     3721 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     3781 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     3841 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     3901 gaatagcagg catgctgggg agcggccgca ggaaccccta gtgatggagt tggccactcc
     3961 ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg
     4021 ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat
     4081 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca
     4141 tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     4201 accgctacac ttgccagcgc cttagcgccc gctcctttcg ctttcttccc ttcctttctc
     4261 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     4321 tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt
     4381 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     4441 agtggactct tgttccaaac tggaacaaca ctcaactcta tctcgggcta ttcttttgat
     4501 ttataaggga ttttgccgat ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa
     4561 tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca
     4621 atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg
     4681 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     4741 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc
     4801 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt
     4861 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     4921 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     4981 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     5041 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     5101 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     5161 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     5221 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     5281 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     5341 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     5401 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     5461 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     5521 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     5581 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     5641 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     5701 ggaagccgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     5761 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     5821 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     5881 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     5941 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     6001 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     6061 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     6121 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     6181 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     6241 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     6301 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     6361 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     6421 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     6481 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     6541 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     6601 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     6661 cacatctaga ggtacccgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
     6721 acgacccccg cccattgacg tcaatagtaa cgccaatagg gactttccat tgacgtcaat
     6781 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
     6841 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattgt gcccagtaca
     6901 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
     6961 tggtcgaggt gagccccacg ttctgcttca ctctccccat ctcccccccc tccccacccc
     7021 caattttgta tttatttatt ttttaattat tttgtgcagc gatgggggcg gggggggg
//
