LOCUS       sequence-375606-        6683 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of EK0493 with frameshift mutation (EGFP is 
            frameshifted)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6683)
  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 6683)
  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: EK0545
            SFFV-mCherry-SGc(GAPDH.s1)cSG-fs(EGFP)-...
FEATURES             Location/Qualifiers
     source          1..6683
                     /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             1350..1415
                     /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             1564..1629
                     /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(1636..1638,1639..2349)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             2436..2465
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(2521..2538)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2527..2751
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2923..2942)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3074..3121
                     /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             3129..4151
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3129..4151
                     /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    4281..4402
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4318..4337)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4372..4391
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4451..4467)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4451..4467)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4464..4486)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4475..4491
                     /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(4499..4505,4506..4523,4524..4529))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4544..4565
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4682..4699)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4853..5441)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4933..4952)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5612..6403,6404..6472))
                     /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     6235..6254
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6473..6577)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6652..6671)
                     /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 caagagcgac gcgttggaag
     1321 ccagacacaa acagaaaatt gtggctccgg tgaaacagac tttgaatttt gaccttctga
     1381 agctggcggg agacgtcgag tccaaccccg gtcccggtac cggatcctca aggggtctac
     1441 atggcaactg tgaggagggg agattcagtg tggtagggga ctgagtgtgg cagggactcc
     1501 ccagcagtga gggtctctac cggtactagt gaggcacggc ataagcaaaa gatcgtagcc
     1561 ccagtaaagc aaacactcaa cttcgatcta ctcaaactcg caggtgatgt ggaatctaat
     1621 ccaggaccct cgagtgtctc aaagggcgag gaacttttta cgggcgttgt tccgatcctc
     1681 gtcgaattgg acggggacgt taatgggcat aaattctcag ttagtggtga gggtgagggg
     1741 gacgcgacat atgggaaact tacgctgaag ttcatatgta ccaccggaaa attgccagtg
     1801 ccgtggccta ctctcgtcac caccctgaca tatggtgttc agtgttttag ccggtatcca
     1861 gaccatatga aacagcacga tttttttaaa tccgctatgc ctgaaggtta cgtacaggag
     1921 cgaacgatat tttttaaaga cgacggaaac tacaaaacta gagccgaggt aaaattcgag
     1981 ggtgacactc tcgtgaaccg gatcgaactg aagggcatag attttaagga ggatggcaat
     2041 atactgggcc acaagctcga atacaactat aattctcata atgtctatat catggcggac
     2101 aaacagaaaa acggcatcaa ggtaaacttt aaaatccgac acaatattga ggacggctct
     2161 gtccagcttg cggaccatta ccaacagaac acgccgatag gggatggtcc agtacttctc
     2221 cctgataatc actatttgtc tactcagtca gctcttagca aggacccaaa cgaaaaacgg
     2281 gatcacatgg tccttcttga gttcgtcact gcggctggga taacactcgg gatggacgaa
     2341 ctgtataagt aggccggagg gcccacgtac accatcaggg tacgtctcag acaccatcag
     2401 ggtctgtctg gtacagcatc agcgtaccat ggcccgaaca aaaactcatc tcagaagagg
     2461 atctgaatag cgccgtcgac catcatcata atcatcattg agtttaaacc cgctgatcag
     2521 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     2581 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     2641 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     2701 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gagatctatc
     2761 gatgttctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct
     2821 ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     2881 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     2941 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     3001 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     3061 gttcacgtac ctagaagttc ctattccgaa gttcctattc tctagaaagt ataggaactt
     3121 ccttggccaa aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt
     3181 tcgacagcgt ctccgacctg atgcagctct cggagggcga agaatctcgt gctttcagct
     3241 tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca
     3301 aagatcgtta tgtttatcgg cactttgcat cggccgcgct cccgattccg gaagtgcttg
     3361 acattgggga attcagcgag agcctgacct attgcatctc ccgccgtgca cagggtgtca
     3421 cgttgcaaga cctgcctgaa accgaactgc ccgctgttct gcagccggtc gcggaggcca
     3481 tggatgcgat cgctgcggcc gatcttagcc agacgagcgg gttcggccca ttcggaccgc
     3541 aaggaatcgg tcaatacact acatggcgtg atttcatatg cgcgattgct gatccccatg
     3601 tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg
     3661 atgagctgat gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt
     3721 tcggctccaa caatgtcctg acggacaatg gccgcataac agcggtcatt gactggagcg
     3781 aggcgatgtt cggggattcc caatacgagg tcgccaacat cttcttctgg aggccgtggt
     3841 tggcttgtat ggagcagcag acgcgctact tcgagcggag gcatccggag cttgcaggat
     3901 cgccgcggct ccgggcgtat atgctccgca ttggtcttga ccaactctat cagagcttgg
     3961 ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg atgcgacgca atcgtccgat
     4021 ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg
     4081 atggctgtgt agaagtactc gccgatagtg gaaaccgacg ccccagcact cgtccgaggg
     4141 caaaggaata gcacgtacta cgagatttcg attccaccgc cgccttctat gaaaggttgg
     4201 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     4261 tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca
     4321 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     4381 ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg
     4441 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     4501 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     4561 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     4621 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     4681 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     4741 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     4801 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     4861 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     4921 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     4981 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     5041 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     5101 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     5161 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     5221 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     5281 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     5341 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     5401 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     5461 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     5521 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     5581 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     5641 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     5701 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     5761 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     5821 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     5881 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     5941 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     6001 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     6061 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     6121 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     6181 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     6241 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     6301 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     6361 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     6421 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     6481 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     6541 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     6601 ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca gtacaatctg
     6661 ctctgatgcc gcatagttaa gcc
//
