LOCUS       sequence-375483-        6659 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of a sensor for synthetic sequence 1 (EK0208) with three 
            MS2 sequences in the coding sequence in mammalian cells. Similar to 
            EK0438, but with MS2 sequences directly after the sensor sequence, 
            rather than 3' UTR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6659)
  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 6659)
  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: NK10 SFFV-mCherry-SG(s70587-3xMS2)SG-EGFP (FLP...
FEATURES             Location/Qualifiers
     source          1..6659
                     /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             1611..1676
                     /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(1677..1679,1680..2390)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             2412..2441
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(2497..2514)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2503..2727
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2899..2918)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3050..3097
                     /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             3105..4127
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3105..4127
                     /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    4257..4378
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4294..4313)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4348..4367
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4427..4443)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4427..4443)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4440..4462)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4451..4467
                     /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(4475..4481,4482..4499,4500..4505))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4520..4541
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4658..4675)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4829..5417)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4909..4928)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5588..6379,6380..6448))
                     /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     6211..6230
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6449..6553)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6628..6647)
                     /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 ccaccaagtc gacacggatg agtgtttcct
     1441 tctatccgcg tcattgttag gccttacgta ccgatagacc actggtgcaa atagagagct
     1501 ataccggtat agttcagcat cagcgaacta tttgtgagga gtacccacaa atacatgagg
     1561 attacccatg tactagtgag gcacggcata agcaaaagat cgtagcccca gtaaagcaaa
     1621 cactcaactt cgatctactc aaactcgcag gtgatgtgga atctaatcca ggacctgtct
     1681 caaagggcga ggaacttttt acgggcgttg ttccgatcct cgtcgaattg gacggggacg
     1741 ttaatgggca taaattctca gttagtggtg agggtgaggg ggacgcgaca tatgggaaac
     1801 ttacgctgaa gttcatatgt accaccggaa aattgccagt gccgtggcct actctcgtca
     1861 ccaccctgac atatggtgtt cagtgtttta gccggtatcc agaccatatg aaacagcacg
     1921 atttttttaa atccgctatg cctgaaggtt acgtacagga gcgaacgata ttttttaaag
     1981 acgacggaaa ctacaaaact agagccgagg taaaattcga gggtgacact ctcgtgaacc
     2041 ggatcgaact gaagggcata gattttaagg aggatggcaa tatactgggc cacaagctcg
     2101 aatacaacta taattctcat aatgtctata tcatggcgga caaacagaaa aacggcatca
     2161 aggtaaactt taaaatccga cacaatattg aggacggctc tgtccagctt gcggaccatt
     2221 accaacagaa cacgccgata ggggatggtc cagtacttct ccctgataat cactatttgt
     2281 ctactcagtc agctcttagc aaggacccaa acgaaaaacg ggatcacatg gtccttcttg
     2341 agttcgtcac tgcggctggg ataacactcg ggatggacga actgtataag taggccgctc
     2401 gaggagggcc cgaacaaaaa ctcatctcag aagaggatct gaatagcgcc gtcgaccatc
     2461 atcataatca tcattgagtt taaacccgct gatcagcctc gactgtgcct tctagttgcc
     2521 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     2581 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     2641 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
     2701 atgctgggga tgcggtgggc tctatggaga tctatcgatg ttctggggat gcggtgggct
     2761 ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct
     2821 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     2881 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     2941 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     3001 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtacctag aagttcctat
     3061 tccgaagttc ctattctcta gaaagtatag gaacttcctt ggccaaaaag cctgaactca
     3121 ccgcgacgtc tgtcgagaag tttctgatcg aaaagttcga cagcgtctcc gacctgatgc
     3181 agctctcgga gggcgaagaa tctcgtgctt tcagcttcga tgtaggaggg cgtggatatg
     3241 tcctgcgggt aaatagctgc gccgatggtt tctacaaaga tcgttatgtt tatcggcact
     3301 ttgcatcggc cgcgctcccg attccggaag tgcttgacat tggggaattc agcgagagcc
     3361 tgacctattg catctcccgc cgtgcacagg gtgtcacgtt gcaagacctg cctgaaaccg
     3421 aactgcccgc tgttctgcag ccggtcgcgg aggccatgga tgcgatcgct gcggccgatc
     3481 ttagccagac gagcgggttc ggcccattcg gaccgcaagg aatcggtcaa tacactacat
     3541 ggcgtgattt catatgcgcg attgctgatc cccatgtgta tcactggcaa actgtgatgg
     3601 acgacaccgt cagtgcgtcc gtcgcgcagg ctctcgatga gctgatgctt tgggccgagg
     3661 actgccccga agtccggcac ctcgtgcacg cggatttcgg ctccaacaat gtcctgacgg
     3721 acaatggccg cataacagcg gtcattgact ggagcgaggc gatgttcggg gattcccaat
     3781 acgaggtcgc caacatcttc ttctggaggc cgtggttggc ttgtatggag cagcagacgc
     3841 gctacttcga gcggaggcat ccggagcttg caggatcgcc gcggctccgg gcgtatatgc
     3901 tccgcattgg tcttgaccaa ctctatcaga gcttggttga cggcaatttc gatgatgcag
     3961 cttgggcgca gggtcgatgc gacgcaatcg tccgatccgg agccgggact gtcgggcgta
     4021 cacaaatcgc ccgcagaagc gcggccgtct ggaccgatgg ctgtgtagaa gtactcgccg
     4081 atagtggaaa ccgacgcccc agcactcgtc cgagggcaaa ggaatagcac gtactacgag
     4141 atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg
     4201 ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact
     4261 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     4321 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     4381 atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc
     4441 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     4501 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     4561 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     4621 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     4681 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     4741 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     4801 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     4861 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     4921 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     4981 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     5041 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     5101 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     5161 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     5221 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     5281 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     5341 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     5401 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     5461 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     5521 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     5581 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     5641 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     5701 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     5761 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     5821 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     5881 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     5941 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     6001 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     6061 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     6121 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     6181 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     6241 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     6301 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     6361 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     6421 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     6481 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     6541 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc
     6601 tcccgatccc ctatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcc
//
