LOCUS       sequence-197196-        7130 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes dCas9(N) fused to transmembrane tether.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7130)
  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 7130)
  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..7130
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        12..297
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        299..576
                     /label=chicken beta-actin promoter
     intron          577..804
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     sig_peptide     826..885
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="METDTLLLWVLLLWVPGSTG"
     CDS             928..957
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             961..1107
                     /codon_start=1
                     /product="transmembrane domain from platelet derived growth
                     factor receptor beta"
                     /label=PDGFR-beta TM domain
                     /translation="AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQ
                     KKPR"
     CDS             1204..1224
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1243..1269
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(1285..1308,1309..1311,1312..2886)
                     /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             2917..2973
                     /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(2980..2999)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3476..3496
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(3613..3633)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4031..4042)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4173..4190)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4179..4386
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4395..4535
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4395..4524
                     /label=AAV2 ITR
     rep_origin      4610..5065
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4697..4716)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4907..4928
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5082..5101)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5201..5223
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5261..5279)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5347..5451
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5452..5520,5521..6312)
                     /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(5670..5689)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6483..7071
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6972..6991
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctagaggtac ccgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac
       61 ccccgcccat tgacgtcaat agtaacgcca atagggactt tccattgacg tcaatgggtg
      121 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
      181 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attgtgccca gtacatgacc
      241 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtc
      301 gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc acccccaatt
      361 ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg gggggggggg
      421 cgcgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg
      481 gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg
      541 cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg ctgccttcgc
      601 cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta
      661 ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta gctgagcaag
      721 aggtaagggt ttaagggatg gttggttggt ggggtattaa tgtttaatta cctggagcac
      781 ctgcctgaaa tcactttttt tcaggttgga ccggtgatat ccaccatgga gacagacaca
      841 ctcctgctat gggtactgct gctctgggtt ccaggttcca ctggtgacca ttctggtggc
      901 ggaagtgggg gaggcagcgg tcgacaagaa caaaaactca tctcagaaga ggatctgaat
      961 gctgtgggcc aggacacgca ggaggtcatc gtggtgccac actccttgcc ctttaaggtg
     1021 gtggtgatct cagccatcct ggccctggtg gtgctcacca tcatctccct tatcatcctc
     1081 atcatgcttt ggcagaagaa gccacgtctg cagagcggca gcgagactcc cgggacctca
     1141 gagtccgcca cacccgaaag tgctagccta gatttagcta gcttaatcct cggcaagctt
     1201 ggcgaaaacc tgtattttca ggggggggga ggcagcacta gttacccata cgacgtacca
     1261 gattacgctg gaggttctgg atccgacaag aagtacagca tcggcctggc catcggcacc
     1321 aactctgtgg gctgggccgt gatcaccgac gagtacaagg tgcccagcaa gaaattcaag
     1381 gtgctgggca acaccgaccg gcacagcatc aagaagaacc tgatcggagc cctgctgttc
     1441 gacagcggcg aaacagccga ggccacccgg ctgaagagaa ccgccagaag aagatacacc
     1501 agacggaaga accggatctg ctatctgcaa gagatcttca gcaacgagat ggccaaggtg
     1561 gacgacagct tcttccacag actggaagag tccttcctgg tggaagagga taagaagcac
     1621 gagcggcacc ccatcttcgg caacatcgtg gacgaggtgg cctaccacga gaagtacccc
     1681 accatctacc acctgagaaa gaaactggtg gacagcaccg acaaggccga cctgcggctg
     1741 atctatctgg ccctggccca catgatcaag ttccggggcc acttcctgat cgagggcgac
     1801 ctgaaccccg acaacagcga cgtggacaag ctgttcatcc agctggtgca gacctacaac
     1861 cagctgttcg aggaaaaccc catcaacgcc agcggcgtgg acgccaaggc catcctgtct
     1921 gccagactga gcaagagcag acggctggaa aatctgatcg cccagctgcc cggcgagaag
     1981 aagaatggcc tgttcggcaa cctgattgcc ctgagcctgg gcctgacccc caacttcaag
     2041 agcaacttcg acctggccga ggatgccaaa ctgcagctga gcaaggacac ctacgacgac
     2101 gacctggaca acctgctggc ccagatcggc gaccagtacg ccgacctgtt tctggccgcc
     2161 aagaacctgt ccgacgccat cctgctgagc gacatcctga gagtgaacac cgagatcacc
     2221 aaggcccccc tgagcgcctc tatgatcaag agatacgacg agcaccacca ggacctgacc
     2281 ctgctgaaag ctctcgtgcg gcagcagctg cctgagaagt acaaagagat tttcttcgac
     2341 cagagcaaga acggctacgc cggctacatt gacggcggag ccagccagga agagttctac
     2401 aagttcatca agcccatcct ggaaaagatg gacggcaccg aggaactgct cgtgaagctg
     2461 aacagagagg acctgctgcg gaagcagcgg accttcgaca acggcagcat cccccaccag
     2521 atccacctgg gagagctgca cgccattctg cggcggcagg aagattttta cccattcctg
     2581 aaggacaacc gggaaaagat cgagaagatc ctgaccttcc gcatccccta ctacgtgggc
     2641 cctctggcca ggggaaacag cagattcgcc tggatgacca gaaagagcga ggaaaccatc
     2701 accccctgga acttcgagga agtggtggac aagggcgctt ccgcccagag cttcatcgag
     2761 cggatgacca acttcgataa gaacctgccc aacgagaagg tgctgcccaa gcacagcctg
     2821 ctgtacgagt acttcaccgt gtataacgag ctgaccaaag tgaaatacgt gaccgaggga
     2881 atgagaggtg gcggtggctc gggtaccgga tctggagcca ccaatttttc tctgctgaag
     2941 caggctggtg atgttgaaga aaaccctgga cctgaattca tgaccgagta caagcccacg
     3001 gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc
     3061 gccgactacc ccgccacgcg ccacaccgtc gatccggacc gccacatcga gcgggtcacc
     3121 gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg
     3181 gacgacggcg ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg
     3241 ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa
     3301 cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc
     3361 gtcggcgtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga
     3421 gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacctccgc gccccgcaac
     3481 ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga
     3541 ccgcgcacct ggtgcatgac ccgcaagggg taactaagta aggatcgatc caagatatcg
     3601 tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta
     3661 tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct
     3721 gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt
     3781 tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac
     3841 tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg
     3901 ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaagctgac
     3961 gtcctttcca tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg
     4021 ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct
     4081 gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc
     4141 ctccccgcca attcctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc
     4201 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     4261 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     4321 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagagaata gcaggcatgc
     4381 tggggagcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg
     4441 ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc
     4501 ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt attttctcct
     4561 tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt
     4621 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     4681 agcgccttag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     4741 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     4801 cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga
     4861 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     4921 caaactggaa caacactcaa ctctatctcg ggctattctt ttgatttata agggattttg
     4981 ccgatttcgg tctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     5041 aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc
     5101 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     5161 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     5221 aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt
     5281 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     5341 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     5401 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     5461 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     5521 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     5581 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     5641 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     5701 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     5761 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     5821 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     5881 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     5941 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     6001 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     6061 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     6121 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtggaag ccgcggtatc
     6181 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     6241 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     6301 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     6361 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     6421 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     6481 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     6541 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     6601 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     6661 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     6721 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     6781 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     6841 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     6901 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     6961 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     7021 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     7081 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
//
