LOCUS       sequence-375490-        6682 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of a sensor for GAPDH 3' UTR in mammalian cells; mCherry 
            marker, EGFP output, three MS2 sequences in 3' UTR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6682)
  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 6682)
  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: EK0493 SFFV-mCherry-SGc(GAPDH.s1)cSG-EGFP-(3xM...
FEATURES             Location/Qualifiers
     source          1..6682
                     /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             1563..1628
                     /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(1635..1637,1638..2348)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             2435..2464
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(2520..2537)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2526..2750
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2922..2941)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3073..3120
                     /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             3128..4150
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3128..4150
                     /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    4280..4401
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4317..4336)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4371..4390
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4450..4466)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4450..4466)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4463..4485)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4474..4490
                     /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(4498..4504,4505..4522,4523..4528))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4543..4564
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4681..4698)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4852..5440)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4932..4951)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5611..6402,6403..6471))
                     /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     6234..6253
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6472..6576)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6651..6670)
                     /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 cggatcccaa ggggtctaca
     1441 tggcaactgt gaggagggga gattcagtgt ggtaggggac tgagtgtggc agggactccc
     1501 cagcagtgag ggtctctacc ggtactagtg aggcacggca taagcaaaag atcgtagccc
     1561 cagtaaagca aacactcaac ttcgatctac tcaaactcgc aggtgatgtg gaatctaatc
     1621 caggaccctc gagtgtctca aagggcgagg aactttttac gggcgttgtt ccgatcctcg
     1681 tcgaattgga cggggacgtt aatgggcata aattctcagt tagtggtgag ggtgaggggg
     1741 acgcgacata tgggaaactt acgctgaagt tcatatgtac caccggaaaa ttgccagtgc
     1801 cgtggcctac tctcgtcacc accctgacat atggtgttca gtgttttagc cggtatccag
     1861 accatatgaa acagcacgat ttttttaaat ccgctatgcc tgaaggttac gtacaggagc
     1921 gaacgatatt ttttaaagac gacggaaact acaaaactag agccgaggta aaattcgagg
     1981 gtgacactct cgtgaaccgg atcgaactga agggcataga ttttaaggag gatggcaata
     2041 tactgggcca caagctcgaa tacaactata attctcataa tgtctatatc atggcggaca
     2101 aacagaaaaa cggcatcaag gtaaacttta aaatccgaca caatattgag gacggctctg
     2161 tccagcttgc ggaccattac caacagaaca cgccgatagg ggatggtcca gtacttctcc
     2221 ctgataatca ctatttgtct actcagtcag ctcttagcaa ggacccaaac gaaaaacggg
     2281 atcacatggt ccttcttgag ttcgtcactg cggctgggat aacactcggg atggacgaac
     2341 tgtataagta ggccggaggg cccacgtaca ccatcagggt acgtctcaga caccatcagg
     2401 gtctgtctgg tacagcatca gcgtaccatg gcccgaacaa aaactcatct cagaagagga
     2461 tctgaatagc gccgtcgacc atcatcataa tcatcattga gtttaaaccc gctgatcagc
     2521 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     2581 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     2641 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     2701 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg agatctatcg
     2761 atgttctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc
     2821 tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     2881 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     2941 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     3001 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     3061 ttcacgtacc tagaagttcc tattccgaag ttcctattct ctagaaagta taggaacttc
     3121 cttggccaaa aagcctgaac tcaccgcgac gtctgtcgag aagtttctga tcgaaaagtt
     3181 cgacagcgtc tccgacctga tgcagctctc ggagggcgaa gaatctcgtg ctttcagctt
     3241 cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg gtttctacaa
     3301 agatcgttat gtttatcggc actttgcatc ggccgcgctc ccgattccgg aagtgcttga
     3361 cattggggaa ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac
     3421 gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg cggaggccat
     3481 ggatgcgatc gctgcggccg atcttagcca gacgagcggg ttcggcccat tcggaccgca
     3541 aggaatcggt caatacacta catggcgtga tttcatatgc gcgattgctg atccccatgt
     3601 gtatcactgg caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc aggctctcga
     3661 tgagctgatg ctttgggccg aggactgccc cgaagtccgg cacctcgtgc acgcggattt
     3721 cggctccaac aatgtcctga cggacaatgg ccgcataaca gcggtcattg actggagcga
     3781 ggcgatgttc ggggattccc aatacgaggt cgccaacatc ttcttctgga ggccgtggtt
     3841 ggcttgtatg gagcagcaga cgcgctactt cgagcggagg catccggagc ttgcaggatc
     3901 gccgcggctc cgggcgtata tgctccgcat tggtcttgac caactctatc agagcttggt
     3961 tgacggcaat ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa tcgtccgatc
     4021 cggagccggg actgtcgggc gtacacaaat cgcccgcaga agcgcggccg tctggaccga
     4081 tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc cccagcactc gtccgagggc
     4141 aaaggaatag cacgtactac gagatttcga ttccaccgcc gccttctatg aaaggttggg
     4201 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     4261 ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa
     4321 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     4381 caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc
     4441 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     4501 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     4561 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     4621 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     4681 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     4741 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     4801 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     4861 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     4921 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     4981 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     5041 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     5101 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     5161 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     5221 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     5281 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     5341 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     5401 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     5461 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     5521 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     5581 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     5641 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     5701 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     5761 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     5821 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     5881 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     5941 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     6001 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     6061 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     6121 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     6181 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     6241 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     6301 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     6361 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     6421 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     6481 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     6541 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     6601 tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag tacaatctgc
     6661 tctgatgccg catagttaag cc
//
