LOCUS       sequence-375479-        6674 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of a sensor for p53 mutant R248Q in mammalian cells; 
            mCherry marker, EGFP output.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6674)
  AUTHORS   Kaseniit KE, Katz N, Kolber NS, Call CC, Wengier DL, Cody WB, 
            Sattely ES, Gao XJ
  TITLE     Modular, programmable RNA sensing using ADAR editing in living 
            cells.
  JOURNAL   Nat Biotechnol. 2022 Oct 5. pii: 10.1038/s41587-022-01493-x. doi: 
            10.1038/s41587-022-01493-x.
  PUBMED    36198772
REFERENCE   2  (bases 1 to 6674)
  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 
            Biotechnol. 2022 Oct 5. pii: 10.1038/s41587-022-01493-x. doi: 
            10.1038/s41587-022-01493-x."
COMMENT     Sequence Label: NSK024 SFFV-mCherry-SG(p53R248Q.s2)SG-EGFP (FL...
FEATURES             Location/Qualifiers
     source          1..6674
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        176..583
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     regulatory      591..600
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             597..1304
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(742..760)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1010..1029
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1341..1406
                     /codon_start=1
                     /product="2A peptide from foot-and-mouth disease virus 
                     polyprotein"
                     /label=F2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="VKQTLNFDLLKLAGDVESNPGP"
     CDS             1626..1691
                     /codon_start=1
                     /product="2A peptide from foot-and-mouth disease virus 
                     polyprotein"
                     /label=F2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="VKQTLNFDLLKLAGDVESNPGP"
     CDS             join(1692..1694,1695..2405)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             2427..2456
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(2512..2529)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2518..2742
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2914..2933)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3065..3112
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             3120..4142
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3120..4142
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    4272..4393
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4309..4328)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4363..4382
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4442..4458)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4442..4458)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4455..4477)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4466..4482
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4490..4496,4497..4514,4515..4520))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4535..4556
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4673..4690)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4844..5432)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4924..4943)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5603..6394,6395..6463))
                     /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     6226..6245
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6464..6568)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6643..6662)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag
       61 ctacaacaag gcaaggcttg accgacaatt gccgataaaa taaaagattt tatttagtct
      121 ccagaaaaag gggggaatga aagaccccac ctgtaggttt ggcaagctag ctgcagtaac
      181 gccattttgc aaggcatgga aaaataccaa accaagaata gagaagttca gatcaagggc
      241 gggtacatga aaatagctaa cgttgggcca aacaggatat ctgcggtgag cagtttcggc
      301 cccggcccgg ggccaagaac agatggtcac cgcagtttcg gccccggccc gaggccaaga
      361 acagatggtc cccagatatg gcccaaccct cagcagtttc ttaagaccca tcagatgttt
      421 ccaggctccc ccaaggacct gaaatgaccc tgcgccttat ttgaattaac caatcagcct
      481 gcttctcgct tctgttcgcg cgcttctgct tcccgagctc tataaaagag ctcacaaccc
      541 ctcactcggc gcgccagtcc tccgacagac tgagtcgccc gggaagcttc gccaccatgg
      601 tgagcaaggg cgaggaggat aacatggcca tcatcaagga gttcatgcgc ttcaaggtgc
      661 acatggaggg ctccgtgaac ggccacgagt tcgagatcga gggcgagggc gagggccgcc
      721 cctacgaggg cacccagacc gccaagctga aggtgaccaa gggtggcccc ctgcccttcg
      781 cctgggacat cctgtcccct cagttcatgt acggctccaa ggcctacgtg aagcaccccg
      841 ccgacattcc cgactacttg aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga
      901 tgaacttcga ggacggcggc gtggtgaccg tgacccagga ctcctccctg caggacggcg
      961 agttcatcta caaggtgaag ctgcgcggca ccaacttccc ctccgacggc cccgtaatgc
     1021 agaagaagac catgggctgg gaggcctcct ccgagcggat gtaccccgag gacggcgccc
     1081 tgaagggcga gatcaagcag aggctgaagc tgaaggacgg cggccactac gacgctgagg
     1141 tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc cggcgcctac aacgtcaaca
     1201 tcaagttgga catcacctcc cacaacgagg actacaccat cgtggaacag tacgaacgcg
     1261 ccgagggccg ccactccacc ggcggcatgg acgagctgta caagagcgaa gccagacaca
     1321 aacagaaaat tgtggctccg gtgaaacaga ctttgaattt tgaccttctg aagctggcgg
     1381 gagacgtcga gtccaacccc ggtcccggat ccggctcccc tttcttgcgg agattctctt
     1441 cctctgtgcg ccggtctctc ccaggacagg cacaaacacg cacctcaaag ctgttccgtc
     1501 ccagtagatt accactggag tcttccagtg tgatgatggt gaggatgggc ctctagttca
     1561 tgccgcccat gcaggaactg ttacacaccg gtgaggcacg gcataagcaa aagatcgtag
     1621 ccccagtaaa gcaaacactc aacttcgatc tactcaaact cgcaggtgat gtggaatcta
     1681 atccaggacc tgtctcaaag ggcgaggaac tttttacggg cgttgttccg atcctcgtcg
     1741 aattggacgg ggacgttaat gggcataaat tctcagttag tggtgagggt gagggggacg
     1801 cgacatatgg gaaacttacg ctgaagttca tatgtaccac cggaaaattg ccagtgccgt
     1861 ggcctactct cgtcaccacc ctgacatatg gtgttcagtg ttttagccgg tatccagacc
     1921 atatgaaaca gcacgatttt tttaaatccg ctatgcctga aggttacgta caggagcgaa
     1981 cgatattttt taaagacgac ggaaactaca aaactagagc cgaggtaaaa ttcgagggtg
     2041 acactctcgt gaaccggatc gaactgaagg gcatagattt taaggaggat ggcaatatac
     2101 tgggccacaa gctcgaatac aactataatt ctcataatgt ctatatcatg gcggacaaac
     2161 agaaaaacgg catcaaggta aactttaaaa tccgacacaa tattgaggac ggctctgtcc
     2221 agcttgcgga ccattaccaa cagaacacgc cgatagggga tggtccagta cttctccctg
     2281 ataatcacta tttgtctact cagtcagctc ttagcaagga cccaaacgaa aaacgggatc
     2341 acatggtcct tcttgagttc gtcactgcgg ctgggataac actcgggatg gacgaactgt
     2401 ataagtaggc cgctcgagga gggcccgaac aaaaactcat ctcagaagag gatctgaata
     2461 gcgccgtcga ccatcatcat aatcatcatt gagtttaaac ccgctgatca gcctcgactg
     2521 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     2581 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     2641 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     2701 aagacaatag caggcatgct ggggatgcgg tgggctctat ggagatctat cgatgttctg
     2761 gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt
     2821 atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     2881 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     2941 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     3001 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     3061 cctagaagtt cctattccga agttcctatt ctctagaaag tataggaact tccttggcca
     3121 aaaagcctga actcaccgcg acgtctgtcg agaagtttct gatcgaaaag ttcgacagcg
     3181 tctccgacct gatgcagctc tcggagggcg aagaatctcg tgctttcagc ttcgatgtag
     3241 gagggcgtgg atatgtcctg cgggtaaata gctgcgccga tggtttctac aaagatcgtt
     3301 atgtttatcg gcactttgca tcggccgcgc tcccgattcc ggaagtgctt gacattgggg
     3361 aattcagcga gagcctgacc tattgcatct cccgccgtgc acagggtgtc acgttgcaag
     3421 acctgcctga aaccgaactg cccgctgttc tgcagccggt cgcggaggcc atggatgcga
     3481 tcgctgcggc cgatcttagc cagacgagcg ggttcggccc attcggaccg caaggaatcg
     3541 gtcaatacac tacatggcgt gatttcatat gcgcgattgc tgatccccat gtgtatcact
     3601 ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc gcaggctctc gatgagctga
     3661 tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat ttcggctcca
     3721 acaatgtcct gacggacaat ggccgcataa cagcggtcat tgactggagc gaggcgatgt
     3781 tcggggattc ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg ttggcttgta
     3841 tggagcagca gacgcgctac ttcgagcgga ggcatccgga gcttgcagga tcgccgcggc
     3901 tccgggcgta tatgctccgc attggtcttg accaactcta tcagagcttg gttgacggca
     3961 atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga tccggagccg
     4021 ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc gatggctgtg
     4081 tagaagtact cgccgatagt ggaaaccgac gccccagcac tcgtccgagg gcaaaggaat
     4141 agcacgtact acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa
     4201 tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct
     4261 tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     4321 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     4381 tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat
     4441 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     4501 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     4561 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     4621 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     4681 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     4741 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     4801 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     4861 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     4921 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     4981 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     5041 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     5101 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     5161 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     5221 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     5281 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     5341 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     5401 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     5461 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     5521 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     5581 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     5641 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     5701 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     5761 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     5821 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     5881 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     5941 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     6001 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     6061 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     6121 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     6181 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     6241 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     6301 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     6361 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     6421 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     6481 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     6541 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg
     6601 acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc
     6661 cgcatagtta agcc
//
