LOCUS       sequence-375536-        6599 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of a sensor for EGFP CDS in mammalian cells; mTagBFP2 
            marker, mCherry output.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6599)
  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 6599)
  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: EK0393 SFFV-mTagBFP2-SGc(EGFP.s2)cSG-mCherry
            (...
FEATURES             Location/Qualifiers
     source          1..6599
                     /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..1307
                     /codon_start=1
                     /product="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /label=mTagBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRND
                     MALKLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     CDS             1347..1412
                     /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"
     primer_bind     complement(1432..1453)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1560..1625
                     /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             1632..2339
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(1774..1792)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2042..2061
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2352..2381
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(2437..2454)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2443..2667
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2839..2858)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2990..3037
                     /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             3045..4067
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3045..4067
                     /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    4197..4318
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4234..4253)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4288..4307
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4367..4383)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4367..4383)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4380..4402)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4391..4407
                     /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(4415..4421,4422..4439,4440..4445))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4460..4481
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4598..4615)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4769..5357)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4849..4868)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5528..6319,6320..6388))
                     /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     6151..6170
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6389..6493)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6568..6587)
                     /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 tgtctaaggg cgaagagctg attaaggaga acatgcacat gaagctgtac atggagggca
      661 ccgtggacaa ccatcacttc aagtgcacat ccgagggcga aggcaagccc tacgagggca
      721 cccagaccat gagaatcaag gtggtcgagg gcggccctct ccccttcgcc ttcgacatcc
      781 tggctactag cttcctctac ggcagcaaga ccttcatcaa ccacacccag ggcatccccg
      841 acttcttcaa gcagtccttc cctgagggct tcacatggga gagagtcacc acatacgagg
      901 acgggggcgt gctgaccgct acccaggaca ccagcctcca ggacggctgc ctcatctaca
      961 acgtcaagat cagaggggtg aacttcacat ccaacggccc tgtgatgcag aagaaaacac
     1021 tcggctggga ggccttcacc gaaacgctgt accccgctga cggcggcctg gaaggcagaa
     1081 acgacatggc cctgaagctc gtgggcggga gccatctgat cgcaaacgcc aagaccacat
     1141 atagatccaa gaaacccgct aagaacctca agatgcctgg cgtctactat gtggactaca
     1201 gactggaaag aatcaaggag gccaacaacg aaacctacgt cgagcagcac gaggtggcag
     1261 tggccagata ctgcgacctc cctagcaaac tggggcacaa acttaacgcg ttggaagcca
     1321 gacacaaaca gaaaattgtg gctccggtga aacagacttt gaattttgac cttctgaagc
     1381 tggcgggaga cgtcgagtcc aaccccggtc ccggtaccgg atcccggcgg tcacgaactc
     1441 cagcaggacc atgtgatcgc gcttctcgtt agggtctttg ctcagggcgg actgggtgct
     1501 caggtagtgg ttgtaccggt actagtgagg cacggcataa gcaaaagatc gtagccccag
     1561 taaagcaaac actcaacttc gatctactca aactcgcagg tgatgtggaa tctaatccag
     1621 gaccctcgag tgtgagcaag ggcgaggagg ataacatggc catcatcaag gagttcatgc
     1681 gcttcaaggt gcacatggag ggctccgtga acggccacga gttcgagatc gagggcgagg
     1741 gcgagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggtggcc
     1801 ccctgccctt cgcctgggac atcctgtccc ctcagttcat gtacggctcc aaggcctacg
     1861 tgaagcaccc cgccgacatt cccgactact tgaagctgtc cttccccgag ggcttcaagt
     1921 gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc
     1981 tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg
     2041 gccccgtaat gcagaagaag accatgggct gggaggcctc ctccgagcgg atgtaccccg
     2101 aggacggcgc cctgaagggc gagatcaagc agaggctgaa gctgaaggac ggcggccact
     2161 acgacgctga ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg cccggcgcct
     2221 acaacgtcaa catcaagttg gacatcacct cccacaacga ggactacacc atcgtggaac
     2281 agtacgaacg cgccgagggc cgccactcca ccggcggcat ggacgagctg tacaagtagg
     2341 ccggagggcc cgaacaaaaa ctcatctcag aagaggatct gaatagcgcc gtcgaccatc
     2401 atcataatca tcattgagtt taaacccgct gatcagcctc gactgtgcct tctagttgcc
     2461 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     2521 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     2581 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
     2641 atgctgggga tgcggtgggc tctatggaga tctatcgatg ttctggggat gcggtgggct
     2701 ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct
     2761 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     2821 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     2881 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     2941 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtacctag aagttcctat
     3001 tccgaagttc ctattctcta gaaagtatag gaacttcctt ggccaaaaag cctgaactca
     3061 ccgcgacgtc tgtcgagaag tttctgatcg aaaagttcga cagcgtctcc gacctgatgc
     3121 agctctcgga gggcgaagaa tctcgtgctt tcagcttcga tgtaggaggg cgtggatatg
     3181 tcctgcgggt aaatagctgc gccgatggtt tctacaaaga tcgttatgtt tatcggcact
     3241 ttgcatcggc cgcgctcccg attccggaag tgcttgacat tggggaattc agcgagagcc
     3301 tgacctattg catctcccgc cgtgcacagg gtgtcacgtt gcaagacctg cctgaaaccg
     3361 aactgcccgc tgttctgcag ccggtcgcgg aggccatgga tgcgatcgct gcggccgatc
     3421 ttagccagac gagcgggttc ggcccattcg gaccgcaagg aatcggtcaa tacactacat
     3481 ggcgtgattt catatgcgcg attgctgatc cccatgtgta tcactggcaa actgtgatgg
     3541 acgacaccgt cagtgcgtcc gtcgcgcagg ctctcgatga gctgatgctt tgggccgagg
     3601 actgccccga agtccggcac ctcgtgcacg cggatttcgg ctccaacaat gtcctgacgg
     3661 acaatggccg cataacagcg gtcattgact ggagcgaggc gatgttcggg gattcccaat
     3721 acgaggtcgc caacatcttc ttctggaggc cgtggttggc ttgtatggag cagcagacgc
     3781 gctacttcga gcggaggcat ccggagcttg caggatcgcc gcggctccgg gcgtatatgc
     3841 tccgcattgg tcttgaccaa ctctatcaga gcttggttga cggcaatttc gatgatgcag
     3901 cttgggcgca gggtcgatgc gacgcaatcg tccgatccgg agccgggact gtcgggcgta
     3961 cacaaatcgc ccgcagaagc gcggccgtct ggaccgatgg ctgtgtagaa gtactcgccg
     4021 atagtggaaa ccgacgcccc agcactcgtc cgagggcaaa ggaatagcac gtactacgag
     4081 atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg
     4141 ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact
     4201 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     4261 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     4321 atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc
     4381 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     4441 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     4501 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     4561 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     4621 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     4681 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     4741 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     4801 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     4861 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     4921 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     4981 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     5041 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     5101 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     5161 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     5221 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     5281 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     5341 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     5401 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     5461 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     5521 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     5581 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     5641 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     5701 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     5761 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     5821 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     5881 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     5941 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     6001 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     6061 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     6121 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     6181 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     6241 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     6301 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     6361 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     6421 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     6481 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc
     6541 tcccgatccc ctatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcc
//
