LOCUS       sequence-375495-        6905 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of EK0209 with frameshift mutation (EGFP is 
            frameshifted)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6905)
  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 6905)
  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: EK0261 CMV-TO-mCherry-SG(s70587)SG-fs(EGFP) (F...
FEATURES             Location/Qualifiers
     source          1..6905
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        384..587
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     538..558
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    589..607
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    610..628
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     638..662
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      754..763
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             760..1467
                     /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(905..923)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1173..1192
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1504..1569
                     /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             1706..1771
                     /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(1772..1774,1775..2485)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             2507..2536
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(2592..2609)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2598..2822
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2994..3013)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3145..3192
                     /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             3200..4222
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3200..4222
                     /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    4352..4473
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4389..4408)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4443..4462
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4522..4538)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4522..4538)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4535..4557)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4546..4562
                     /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(4570..4576,4577..4594,4595..4600))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4615..4636
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4753..4770)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4924..5512)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5004..5023)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5683..6474,6475..6543))
                     /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     6306..6325
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6544..6648)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6723..6742)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
      421 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
      481 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc
      541 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc cctatcagtg
      601 atagagatct ccctatcagt gatagagatc gtcgacgagc tcgtttagtg aaccgtcaga
      661 tcgcctggag acgccatcca cgctgttttg acctccatag aagacaccgg gaccgatcca
      721 gcctccggac tctagcgttt aaacttaagc ttcgccacca tggtgagcaa gggcgaggag
      781 gataacatgg ccatcatcaa ggagttcatg cgcttcaagg tgcacatgga gggctccgtg
      841 aacggccacg agttcgagat cgagggcgag ggcgagggcc gcccctacga gggcacccag
      901 accgccaagc tgaaggtgac caagggtggc cccctgccct tcgcctggga catcctgtcc
      961 cctcagttca tgtacggctc caaggcctac gtgaagcacc ccgccgacat tcccgactac
     1021 ttgaagctgt ccttccccga gggcttcaag tgggagcgcg tgatgaactt cgaggacggc
     1081 ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat ctacaaggtg
     1141 aagctgcgcg gcaccaactt cccctccgac ggccccgtaa tgcagaagaa gaccatgggc
     1201 tgggaggcct cctccgagcg gatgtacccc gaggacggcg ccctgaaggg cgagatcaag
     1261 cagaggctga agctgaagga cggcggccac tacgacgctg aggtcaagac cacctacaag
     1321 gccaagaagc ccgtgcagct gcccggcgcc tacaacgtca acatcaagtt ggacatcacc
     1381 tcccacaacg aggactacac catcgtggaa cagtacgaac gcgccgaggg ccgccactcc
     1441 accggcggca tggacgagct gtacaagagc gaagccagac acaaacagaa aattgtggct
     1501 ccggtgaaac agactttgaa ttttgacctt ctgaagctgg cgggagacgt cgagtccaac
     1561 cccggtcccg gatcctacca agtcgacacg gatgagtgtt tccttctatc cgcgtcattg
     1621 ttaggcctta cgtaccgata gaccactggt gcaaatagag agctataccg gtgaggcacg
     1681 gcataagcaa aagatcgtag ccccagtaaa gcaaacactc aacttcgatc tactcaaact
     1741 cgcaggtgat gtggaatcta atccaggacc tgtctcaaag ggcgaggaac tttttacggg
     1801 cgttgttccg atcctcgtcg aattggacgg ggacgttaat gggcataaat tctcagttag
     1861 tggtgagggt gagggggacg cgacatatgg gaaacttacg ctgaagttca tatgtaccac
     1921 cggaaaattg ccagtgccgt ggcctactct cgtcaccacc ctgacatatg gtgttcagtg
     1981 ttttagccgg tatccagacc atatgaaaca gcacgatttt tttaaatccg ctatgcctga
     2041 aggttacgta caggagcgaa cgatattttt taaagacgac ggaaactaca aaactagagc
     2101 cgaggtaaaa ttcgagggtg acactctcgt gaaccggatc gaactgaagg gcatagattt
     2161 taaggaggat ggcaatatac tgggccacaa gctcgaatac aactataatt ctcataatgt
     2221 ctatatcatg gcggacaaac agaaaaacgg catcaaggta aactttaaaa tccgacacaa
     2281 tattgaggac ggctctgtcc agcttgcgga ccattaccaa cagaacacgc cgatagggga
     2341 tggtccagta cttctccctg ataatcacta tttgtctact cagtcagctc ttagcaagga
     2401 cccaaacgaa aaacgggatc acatggtcct tcttgagttc gtcactgcgg ctgggataac
     2461 actcgggatg gacgaactgt ataagtaggc cgctcgagga gggcccgaac aaaaactcat
     2521 ctcagaagag gatctgaata gcgccgtcga ccatcatcat aatcatcatt gagtttaaac
     2581 ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     2641 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     2701 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     2761 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat
     2821 ggagatctat cgatgttctg gggatgcggt gggctctatg gcttctgagg cggaaagaac
     2881 cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg
     2941 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     3001 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     3061 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     3121 ttagggtgat ggttcacgta cctagaagtt cctattccga agttcctatt ctctagaaag
     3181 tataggaact tccttggcca aaaagcctga actcaccgcg acgtctgtcg agaagtttct
     3241 gatcgaaaag ttcgacagcg tctccgacct gatgcagctc tcggagggcg aagaatctcg
     3301 tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata gctgcgccga
     3361 tggtttctac aaagatcgtt atgtttatcg gcactttgca tcggccgcgc tcccgattcc
     3421 ggaagtgctt gacattgggg aattcagcga gagcctgacc tattgcatct cccgccgtgc
     3481 acagggtgtc acgttgcaag acctgcctga aaccgaactg cccgctgttc tgcagccggt
     3541 cgcggaggcc atggatgcga tcgctgcggc cgatcttagc cagacgagcg ggttcggccc
     3601 attcggaccg caaggaatcg gtcaatacac tacatggcgt gatttcatat gcgcgattgc
     3661 tgatccccat gtgtatcact ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc
     3721 gcaggctctc gatgagctga tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt
     3781 gcacgcggat ttcggctcca acaatgtcct gacggacaat ggccgcataa cagcggtcat
     3841 tgactggagc gaggcgatgt tcggggattc ccaatacgag gtcgccaaca tcttcttctg
     3901 gaggccgtgg ttggcttgta tggagcagca gacgcgctac ttcgagcgga ggcatccgga
     3961 gcttgcagga tcgccgcggc tccgggcgta tatgctccgc attggtcttg accaactcta
     4021 tcagagcttg gttgacggca atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc
     4081 aatcgtccga tccggagccg ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc
     4141 cgtctggacc gatggctgtg tagaagtact cgccgatagt ggaaaccgac gccccagcac
     4201 tcgtccgagg gcaaaggaat agcacgtact acgagatttc gattccaccg ccgccttcta
     4261 tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg
     4321 ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta
     4381 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     4441 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag
     4501 ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     4561 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     4621 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     4681 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     4741 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     4801 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     4861 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     4921 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     4981 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     5041 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     5101 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     5161 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     5221 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     5281 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     5341 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     5401 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     5461 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     5521 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     5581 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     5641 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     5701 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     5761 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     5821 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     5881 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     5941 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     6001 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     6061 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     6121 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     6181 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     6241 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     6301 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     6361 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     6421 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     6481 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     6541 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     6601 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     6661 aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg gtgcactctc
     6721 agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg
     6781 gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac
     6841 aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgtac gggccagata
     6901 tacgc
//
