LOCUS       sequence-375602-        5992 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of the sequence matching the 54 extra bases in EK0362 in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5992)
  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 5992)
  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: EK0425 CMV-TO-BFPmut-stop-Bdnf.t+54@202 (FLP-IN)
FEATURES             Location/Qualifiers
     source          1..5992
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        91..470
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        471..674
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     625..645
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    676..694
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    697..715
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     725..749
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      841..850
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             847..1557
                     /codon_start=1
                     /product="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /label=mTagBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRND
                     MALKLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHAVAV
                     ARYCDLPSKLGHKLN"
     CDS             1576..1602
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1681..1710
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(1766..1783)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1772..1996
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2168..2187)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2319..2366
                     /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             2374..3396
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2374..3396
                     /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    3526..3647
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3563..3582)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3617..3636
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3696..3712)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3696..3712)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3709..3731)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3720..3736
                     /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(3744..3750,3751..3768,3769..3774))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3789..3810
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3927..3944)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4098..4686)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4178..4197)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4857..5648,5649..5717))
                     /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     5480..5499
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5718..5822)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5897..5916)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc
       61 gctgcttcgt acgggccaga tatacgcgtt gacattgatt attgactagt tattaatagt
      121 aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta
      181 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga
      241 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt
      301 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta
      361 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg
      421 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt
      481 tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc
      541 accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat
      601 gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct
      661 atataagcag agctctccct atcagtgata gagatctccc tatcagtgat agagatcgtc
      721 gacgagctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc tgttttgacc
      781 tccatagaag acaccgggac cgatccagcc tccggactct agcgtttaaa cttaagcttc
      841 gccaccatgg tgtctaaggg cgaagagctg attaaggaga acatgcacat gaagctgtac
      901 atggagggca ccgtggacaa ccatcacttc aagtgcacat ccgagggcga aggcaagccc
      961 tacgagggca cccagaccat gagaatcaag gtggtcgagg gcggccctct ccccttcgcc
     1021 ttcgacatcc tggctactag cttcctctac ggcagcaaga ccttcatcaa ccacacccag
     1081 ggcatccccg acttcttcaa gcagtccttc cctgagggct tcacatggga gagagtcacc
     1141 acatacgagg acgggggcgt gctgaccgct acccaggaca ccagcctcca ggacggctgc
     1201 ctcatctaca acgtcaagat cagaggggtg aacttcacat ccaacggccc tgtgatgcag
     1261 aagaaaacac tcggctggga ggccttcacc gaaacgctgt accccgctga cggcggcctg
     1321 gaaggcagaa acgacatggc cctgaagctc gtgggcggga gccatctgat cgcaaacgcc
     1381 aagaccacat atagatccaa gaaacccgct aagaacctca agatgcctgg cgtctactat
     1441 gtggactaca gactggaaag aatcaaggag gccaacaacg aaacctacgt cgagcagcac
     1501 gcggtggcag tggccagata ctgcgacctc cctagcaaac tggggcacaa acttaattga
     1561 agctagctgg ctggataccc atacgatgtt ccagattacg ctctcgagac cggtagtctg
     1621 cattacattc ctcgataatg ttgtggtttg ttgccgttgc caagaattgg atccgggccc
     1681 gaacaaaaac tcatctcaga agaggatctg aatagcgccg tcgaccatca tcataatcat
     1741 cattgagttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     1801 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     1861 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     1921 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     1981 gcggtgggct ctatggagat ctatcgatgt tctggggatg cggtgggctc tatggcttct
     2041 gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca
     2101 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     2161 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     2221 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     2281 cccaaaaaac ttgattaggg tgatggttca cgtacctaga agttcctatt ccgaagttcc
     2341 tattctctag aaagtatagg aacttccttg gccaaaaagc ctgaactcac cgcgacgtct
     2401 gtcgagaagt ttctgatcga aaagttcgac agcgtctccg acctgatgca gctctcggag
     2461 ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt cctgcgggta
     2521 aatagctgcg ccgatggttt ctacaaagat cgttatgttt atcggcactt tgcatcggcc
     2581 gcgctcccga ttccggaagt gcttgacatt ggggaattca gcgagagcct gacctattgc
     2641 atctcccgcc gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga actgcccgct
     2701 gttctgcagc cggtcgcgga ggccatggat gcgatcgctg cggccgatct tagccagacg
     2761 agcgggttcg gcccattcgg accgcaagga atcggtcaat acactacatg gcgtgatttc
     2821 atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc
     2881 agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga ctgccccgaa
     2941 gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga caatggccgc
     3001 ataacagcgg tcattgactg gagcgaggcg atgttcgggg attcccaata cgaggtcgcc
     3061 aacatcttct tctggaggcc gtggttggct tgtatggagc agcagacgcg ctacttcgag
     3121 cggaggcatc cggagcttgc aggatcgccg cggctccggg cgtatatgct ccgcattggt
     3181 cttgaccaac tctatcagag cttggttgac ggcaatttcg atgatgcagc ttgggcgcag
     3241 ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg tcgggcgtac acaaatcgcc
     3301 cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag tactcgccga tagtggaaac
     3361 cgacgcccca gcactcgtcc gagggcaaag gaatagcacg tactacgaga tttcgattcc
     3421 accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg
     3481 atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca
     3541 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     3601 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata
     3661 ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     3721 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     3781 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     3841 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     3901 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     3961 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     4021 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     4081 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     4141 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     4201 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     4261 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     4321 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     4381 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     4441 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     4501 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     4561 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     4621 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     4681 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     4741 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     4801 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     4861 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     4921 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     4981 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     5041 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     5101 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     5161 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     5221 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     5281 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     5341 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     5401 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     5461 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     5521 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     5581 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     5641 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     5701 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     5761 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     5821 cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatct cccgatcccc
     5881 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc
     5941 ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa aatttaagct ac
//
