LOCUS       sequence-375486-        6907 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of EK0209 with a second stop codon..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6907)
  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 6907)
  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: EK0544 CMV-TO-mCherry-SG(s70587-dblstop)SG-EGF...
FEATURES             Location/Qualifiers
     source          1..6907
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(81..100)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        267..646
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        647..850
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     801..821
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    852..870
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    873..891
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     901..925
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1017..1026
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1023..1730
                     /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(1168..1186)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1436..1455
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1767..1832
                     /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             1971..2036
                     /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(2037..2039,2040..2750)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             2772..2801
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(2857..2874)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2863..3087
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3259..3278)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3410..3457
                     /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             3465..4487
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3465..4487
                     /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    4617..4738
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4654..4673)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4708..4727
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4787..4803)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4787..4803)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4800..4822)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4811..4827
                     /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(4835..4841,4842..4859,4860..4865))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4880..4901
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5018..5035)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5189..5777)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5269..5288)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5948..6739,6740..6808))
                     /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     6571..6590
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(6809..6907,1..6))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat
       61 cccctatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg
      121 ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca
      181 aggcaaggct tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg
      241 cttcgtacgg gccagatata cgcgttgaca ttgattattg actagttatt aatagtaatc
      301 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
      361 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
      421 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
      481 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
      541 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
      601 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg
      661 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc
      721 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg
      781 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat
      841 aagcagagct ctccctatca gtgatagaga tctccctatc agtgatagag atcgtcgacg
      901 agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca
      961 tagaagacac cgggaccgat ccagcctccg gactctagcg tttaaactta agcttcgcca
     1021 ccatggtgag caagggcgag gaggataaca tggccatcat caaggagttc atgcgcttca
     1081 aggtgcacat ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg
     1141 gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc
     1201 ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc
     1261 accccgccga cattcccgac tacttgaagc tgtccttccc cgagggcttc aagtgggagc
     1321 gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg
     1381 acggcgagtt catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg
     1441 taatgcagaa gaagaccatg ggctgggagg cctcctccga gcggatgtac cccgaggacg
     1501 gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa ggacggcggc cactacgacg
     1561 ctgaggtcaa gaccacctac aaggccaaga agcccgtgca gctgcccggc gcctacaacg
     1621 tcaacatcaa gttggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg
     1681 aacgcgccga gggccgccac tccaccggcg gcatggacga gctgtacaag agcgaagcca
     1741 gacacaaaca gaaaattgtg gctccggtga aacagacttt gaattttgac cttctgaagc
     1801 tggcgggaga cgtcgagtcc aaccccggtc ccggatccac caagtcgaca cggatgagtg
     1861 tttccttcta tccgcgtcat tgttagtgag ccttacgtac cgatagacca ctggtgcaaa
     1921 tagagagcta taccggtgag gcacggcata agcaaaagat cgtagcccca gtaaagcaaa
     1981 cactcaactt cgatctactc aaactcgcag gtgatgtgga atctaatcca ggacctgtct
     2041 caaagggcga ggaacttttt acgggcgttg ttccgatcct cgtcgaattg gacggggacg
     2101 ttaatgggca taaattctca gttagtggtg agggtgaggg ggacgcgaca tatgggaaac
     2161 ttacgctgaa gttcatatgt accaccggaa aattgccagt gccgtggcct actctcgtca
     2221 ccaccctgac atatggtgtt cagtgtttta gccggtatcc agaccatatg aaacagcacg
     2281 atttttttaa atccgctatg cctgaaggtt acgtacagga gcgaacgata ttttttaaag
     2341 acgacggaaa ctacaaaact agagccgagg taaaattcga gggtgacact ctcgtgaacc
     2401 ggatcgaact gaagggcata gattttaagg aggatggcaa tatactgggc cacaagctcg
     2461 aatacaacta taattctcat aatgtctata tcatggcgga caaacagaaa aacggcatca
     2521 aggtaaactt taaaatccga cacaatattg aggacggctc tgtccagctt gcggaccatt
     2581 accaacagaa cacgccgata ggggatggtc cagtacttct ccctgataat cactatttgt
     2641 ctactcagtc agctcttagc aaggacccaa acgaaaaacg ggatcacatg gtccttcttg
     2701 agttcgtcac tgcggctggg ataacactcg ggatggacga actgtataag taggccgctc
     2761 gaggagggcc cgaacaaaaa ctcatctcag aagaggatct gaatagcgcc gtcgaccatc
     2821 atcataatca tcattgagtt taaacccgct gatcagcctc gactgtgcct tctagttgcc
     2881 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     2941 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     3001 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
     3061 atgctgggga tgcggtgggc tctatggaga tctatcgatg ttctggggat gcggtgggct
     3121 ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct
     3181 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     3241 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     3301 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     3361 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtacctag aagttcctat
     3421 tccgaagttc ctattctcta gaaagtatag gaacttcctt ggccaaaaag cctgaactca
     3481 ccgcgacgtc tgtcgagaag tttctgatcg aaaagttcga cagcgtctcc gacctgatgc
     3541 agctctcgga gggcgaagaa tctcgtgctt tcagcttcga tgtaggaggg cgtggatatg
     3601 tcctgcgggt aaatagctgc gccgatggtt tctacaaaga tcgttatgtt tatcggcact
     3661 ttgcatcggc cgcgctcccg attccggaag tgcttgacat tggggaattc agcgagagcc
     3721 tgacctattg catctcccgc cgtgcacagg gtgtcacgtt gcaagacctg cctgaaaccg
     3781 aactgcccgc tgttctgcag ccggtcgcgg aggccatgga tgcgatcgct gcggccgatc
     3841 ttagccagac gagcgggttc ggcccattcg gaccgcaagg aatcggtcaa tacactacat
     3901 ggcgtgattt catatgcgcg attgctgatc cccatgtgta tcactggcaa actgtgatgg
     3961 acgacaccgt cagtgcgtcc gtcgcgcagg ctctcgatga gctgatgctt tgggccgagg
     4021 actgccccga agtccggcac ctcgtgcacg cggatttcgg ctccaacaat gtcctgacgg
     4081 acaatggccg cataacagcg gtcattgact ggagcgaggc gatgttcggg gattcccaat
     4141 acgaggtcgc caacatcttc ttctggaggc cgtggttggc ttgtatggag cagcagacgc
     4201 gctacttcga gcggaggcat ccggagcttg caggatcgcc gcggctccgg gcgtatatgc
     4261 tccgcattgg tcttgaccaa ctctatcaga gcttggttga cggcaatttc gatgatgcag
     4321 cttgggcgca gggtcgatgc gacgcaatcg tccgatccgg agccgggact gtcgggcgta
     4381 cacaaatcgc ccgcagaagc gcggccgtct ggaccgatgg ctgtgtagaa gtactcgccg
     4441 atagtggaaa ccgacgcccc agcactcgtc cgagggcaaa ggaatagcac gtactacgag
     4501 atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg
     4561 ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact
     4621 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     4681 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     4741 atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc
     4801 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     4861 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     4921 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     4981 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     5041 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     5101 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     5161 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     5221 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     5281 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     5341 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     5401 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     5461 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     5521 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     5581 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     5641 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     5701 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     5761 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     5821 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     5881 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     5941 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     6001 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     6061 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     6121 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     6181 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     6241 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     6301 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     6361 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     6421 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     6481 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     6541 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     6601 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     6661 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     6721 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     6781 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     6841 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     6901 taggggt
//
