LOCUS       sequence-375604-        6590 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Sensor 2 (for EK0211) in mammalian cells; mCherry 
            marker, EGFP output..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6590)
  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 6590)
  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: EK0289 SFFV-mCherry-SG(s17d9b)SG-EGFP (FLP-IN)
FEATURES             Location/Qualifiers
     source          1..6590
                     /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             1542..1607
                     /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(1608..1610,1611..2321)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             2343..2372
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(2428..2445)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2434..2658
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2830..2849)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2981..3028
                     /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             3036..4058
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3036..4058
                     /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    4188..4309
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4225..4244)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4279..4298
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4358..4374)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4358..4374)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4371..4393)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4382..4398
                     /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(4406..4412,4413..4430,4431..4436))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4451..4472
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4589..4606)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4760..5348)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4840..4859)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5519..6310,6311..6379))
                     /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     6142..6161
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6380..6484)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6559..6578)
                     /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 cccgtccgga ctcgaaaggg atggtagcac
     1441 cctgtcgaat ccggccttag aatgtcagac ggtcgaccta cataatgaat ctcctaagca
     1501 ggaccggtga ggcacggcat aagcaaaaga tcgtagcccc agtaaagcaa acactcaact
     1561 tcgatctact caaactcgca ggtgatgtgg aatctaatcc aggacctgtc tcaaagggcg
     1621 aggaactttt tacgggcgtt gttccgatcc tcgtcgaatt ggacggggac gttaatgggc
     1681 ataaattctc agttagtggt gagggtgagg gggacgcgac atatgggaaa cttacgctga
     1741 agttcatatg taccaccgga aaattgccag tgccgtggcc tactctcgtc accaccctga
     1801 catatggtgt tcagtgtttt agccggtatc cagaccatat gaaacagcac gattttttta
     1861 aatccgctat gcctgaaggt tacgtacagg agcgaacgat attttttaaa gacgacggaa
     1921 actacaaaac tagagccgag gtaaaattcg agggtgacac tctcgtgaac cggatcgaac
     1981 tgaagggcat agattttaag gaggatggca atatactggg ccacaagctc gaatacaact
     2041 ataattctca taatgtctat atcatggcgg acaaacagaa aaacggcatc aaggtaaact
     2101 ttaaaatccg acacaatatt gaggacggct ctgtccagct tgcggaccat taccaacaga
     2161 acacgccgat aggggatggt ccagtacttc tccctgataa tcactatttg tctactcagt
     2221 cagctcttag caaggaccca aacgaaaaac gggatcacat ggtccttctt gagttcgtca
     2281 ctgcggctgg gataacactc gggatggacg aactgtataa gtaggccgct cgaggagggc
     2341 ccgaacaaaa actcatctca gaagaggatc tgaatagcgc cgtcgaccat catcataatc
     2401 atcattgagt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
     2461 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     2521 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     2581 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg
     2641 atgcggtggg ctctatggag atctatcgat gttctgggga tgcggtgggc tctatggctt
     2701 ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg
     2761 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     2821 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     2881 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     2941 accccaaaaa acttgattag ggtgatggtt cacgtaccta gaagttccta ttccgaagtt
     3001 cctattctct agaaagtata ggaacttcct tggccaaaaa gcctgaactc accgcgacgt
     3061 ctgtcgagaa gtttctgatc gaaaagttcg acagcgtctc cgacctgatg cagctctcgg
     3121 agggcgaaga atctcgtgct ttcagcttcg atgtaggagg gcgtggatat gtcctgcggg
     3181 taaatagctg cgccgatggt ttctacaaag atcgttatgt ttatcggcac tttgcatcgg
     3241 ccgcgctccc gattccggaa gtgcttgaca ttggggaatt cagcgagagc ctgacctatt
     3301 gcatctcccg ccgtgcacag ggtgtcacgt tgcaagacct gcctgaaacc gaactgcccg
     3361 ctgttctgca gccggtcgcg gaggccatgg atgcgatcgc tgcggccgat cttagccaga
     3421 cgagcgggtt cggcccattc ggaccgcaag gaatcggtca atacactaca tggcgtgatt
     3481 tcatatgcgc gattgctgat ccccatgtgt atcactggca aactgtgatg gacgacaccg
     3541 tcagtgcgtc cgtcgcgcag gctctcgatg agctgatgct ttgggccgag gactgccccg
     3601 aagtccggca cctcgtgcac gcggatttcg gctccaacaa tgtcctgacg gacaatggcc
     3661 gcataacagc ggtcattgac tggagcgagg cgatgttcgg ggattcccaa tacgaggtcg
     3721 ccaacatctt cttctggagg ccgtggttgg cttgtatgga gcagcagacg cgctacttcg
     3781 agcggaggca tccggagctt gcaggatcgc cgcggctccg ggcgtatatg ctccgcattg
     3841 gtcttgacca actctatcag agcttggttg acggcaattt cgatgatgca gcttgggcgc
     3901 agggtcgatg cgacgcaatc gtccgatccg gagccgggac tgtcgggcgt acacaaatcg
     3961 cccgcagaag cgcggccgtc tggaccgatg gctgtgtaga agtactcgcc gatagtggaa
     4021 accgacgccc cagcactcgt ccgagggcaa aggaatagca cgtactacga gatttcgatt
     4081 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     4141 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg
     4201 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     4261 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
     4321 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     4381 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     4441 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     4501 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     4561 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     4621 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     4681 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     4741 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     4801 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     4861 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     4921 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     4981 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     5041 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     5101 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     5161 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     5221 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     5281 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     5341 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     5401 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     5461 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     5521 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     5581 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     5641 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     5701 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     5761 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     5821 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     5881 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     5941 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     6001 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     6061 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     6121 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     6181 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     6241 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     6301 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     6361 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     6421 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     6481 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc
     6541 cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
//
