LOCUS       sequence-375487-        6905 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of a sensor for EGFP CDS in mammalian cells; mCherry 
            marker, Cre output.
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: EK0325 SFFV-mCherry-SG(EGFP.s2)SG-Cre (FLP-IN)
FEATURES             Location/Qualifiers
     source          1..6905
                     /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"
     primer_bind     complement(1420..1441)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     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             1608..2639
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=Cre
                     /note="Cre is a site-specific recombinase from 
                     bacteriophage P1. Recombination occurs at loxP sequences."
                     /translation="MANLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKML
                     LSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGL
                     PRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAF
                     LGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVE
                     RWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQ
                     RYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLE
                     DGD"
     CDS             2658..2687
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(2743..2760)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2749..2973
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3145..3164)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3296..3343
                     /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             3351..4373
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3351..4373
                     /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    4503..4624
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4540..4559)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4594..4613
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4673..4689)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4673..4689)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4686..4708)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4697..4713
                     /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(4721..4727,4728..4745,4746..4751))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4766..4787
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4904..4921)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5075..5663)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5155..5174)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5834..6625,6626..6694))
                     /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     6457..6476
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6695..6799)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6874..6893)
                     /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 cccggcggtc acgaactcca gcaggaccat
     1441 gtgatcgcgc ttctcgttag ggtctttgct cagggcggac tgggtgctca ggtagtggtt
     1501 gtaccggtga ggcacggcat aagcaaaaga tcgtagcccc agtaaagcaa acactcaact
     1561 tcgatctact caaactcgca ggtgatgtgg aatctaatcc aggacctatg gccaatttac
     1621 tgaccgtaca ccaaaatttg cctgcattgc cggtcgatgc aacgagtgat gaggttcgca
     1681 agaacctgat ggacatgttc agggatcgcc aggcgttttc tgagcatacc tggaaaatgc
     1741 ttctgtccgt ttgccggtcg tgggcggcat ggtgcaagtt gaataaccgg aaatggtttc
     1801 ccgcagaacc tgaagatgtt cgcgattatc ttctatatct tcaggcgcgc ggtctggcag
     1861 taaaaactat ccagcaacat ttgggccagc taaacatgct tcatcgtcgg tccgggctgc
     1921 cacgaccaag tgacagcaat gctgtttcac tggttatgcg gcggatcaga aaagaaaacg
     1981 ttgatgccgg tgaacgtgca aaacaggctc tagcgttcga acgcactgat ttcgaccagg
     2041 ttcgttcact catggaaaat agcgatcgct gccaggatat acgtaatctg gcatttctgg
     2101 ggattgctta taacaccctg ttacgtatag ccgaaattgc caggatcagg gttaaagata
     2161 tctcacgtac tgacggtggg agaatgttaa tccatattgg cagaacgaaa acgctggtta
     2221 gcaccgcagg tgtagagaag gcacttagcc tgggggtaac taaactggtc gagcgatgga
     2281 tttccgtctc tggtgtagct gatgatccga ataactacct gttttgccgg gtcagaaaaa
     2341 atggtgttgc cgcgccatct gccaccagcc agctatcaac tcgcgccctg gaagggattt
     2401 ttgaagcaac tcatcgattg atttacggcg ctaaggatga ctctggtcag agatacctgg
     2461 cctggtctgg acacagtgcc cgtgtcggag ccgcgcgaga tatggcccgc gctggagttt
     2521 caataccgga gatcatgcaa gctggtggct ggaccaatgt aaatattgtc atgaactata
     2581 tccgtaacct ggatagtgaa acaggggcaa tggtgcgcct gctggaagat ggcgattagg
     2641 ccgctcgagg agggcccgaa caaaaactca tctcagaaga ggatctgaat agcgccgtcg
     2701 accatcatca taatcatcat tgagtttaaa cccgctgatc agcctcgact gtgccttcta
     2761 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     2821 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
     2881 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
     2941 gcaggcatgc tggggatgcg gtgggctcta tggagatcta tcgatgttct ggggatgcgg
     3001 tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg
     3061 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     3121 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     3181 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     3241 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt acctagaagt
     3301 tcctattccg aagttcctat tctctagaaa gtataggaac ttccttggcc aaaaagcctg
     3361 aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc
     3421 tgatgcagct ctcggagggc gaagaatctc gtgctttcag cttcgatgta ggagggcgtg
     3481 gatatgtcct gcgggtaaat agctgcgccg atggtttcta caaagatcgt tatgtttatc
     3541 ggcactttgc atcggccgcg ctcccgattc cggaagtgct tgacattggg gaattcagcg
     3601 agagcctgac ctattgcatc tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg
     3661 aaaccgaact gcccgctgtt ctgcagccgg tcgcggaggc catggatgcg atcgctgcgg
     3721 ccgatcttag ccagacgagc gggttcggcc cattcggacc gcaaggaatc ggtcaataca
     3781 ctacatggcg tgatttcata tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg
     3841 tgatggacga caccgtcagt gcgtccgtcg cgcaggctct cgatgagctg atgctttggg
     3901 ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc
     3961 tgacggacaa tggccgcata acagcggtca ttgactggag cgaggcgatg ttcggggatt
     4021 cccaatacga ggtcgccaac atcttcttct ggaggccgtg gttggcttgt atggagcagc
     4081 agacgcgcta cttcgagcgg aggcatccgg agcttgcagg atcgccgcgg ctccgggcgt
     4141 atatgctccg cattggtctt gaccaactct atcagagctt ggttgacggc aatttcgatg
     4201 atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg atccggagcc gggactgtcg
     4261 ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac cgatggctgt gtagaagtac
     4321 tcgccgatag tggaaaccga cgccccagca ctcgtccgag ggcaaaggaa tagcacgtac
     4381 tacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
     4441 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
     4501 ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     4561 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     4621 cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
     4681 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     4741 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     4801 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     4861 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     4921 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     4981 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     5041 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     5101 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     5161 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     5221 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     5281 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     5341 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     5401 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     5461 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     5521 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     5581 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     5641 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     5701 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     5761 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     5821 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     5881 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     5941 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     6001 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     6061 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     6121 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     6181 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     6241 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     6301 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     6361 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     6421 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     6481 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     6541 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     6601 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     6661 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     6721 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     6781 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg
     6841 gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     6901 aagcc
//
