LOCUS       sequence-375610-        6028 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of only the trigger sequence for Bdnf.s1 (EK0301) in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6028)
  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 6028)
  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: EK0304 CMV-TO-BFPmut-stop-Bdnf.t1 (FLP-IN)
FEATURES             Location/Qualifiers
     source          1..6028
                     /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             1717..1746
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(1802..1819)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1808..2032
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2204..2223)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2355..2402
                     /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             2410..3432
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2410..3432
                     /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    3562..3683
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3599..3618)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3653..3672
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3732..3748)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3732..3748)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3745..3767)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3756..3772
                     /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(3780..3786,3787..3804,3805..3810))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3825..3846
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3963..3980)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4134..4722)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4214..4233)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4893..5684,5685..5753))
                     /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     5516..5535
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5754..5858)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5933..5952)
                     /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 cggttagttc
     1621 cacccggagt agggatggag aaaatttctt cactatccat tctggttgat aaagcgttac
     1681 atttgtatgt tgtaaagatg tttgggatcc gggcccgaac aaaaactcat ctcagaagag
     1741 gatctgaata gcgccgtcga ccatcatcat aatcatcatt gagtttaaac ccgctgatca
     1801 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     1861 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     1921 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     1981 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggagatctat
     2041 cgatgttctg gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc
     2101 tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     2161 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     2221 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     2281 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     2341 ggttcacgta cctagaagtt cctattccga agttcctatt ctctagaaag tataggaact
     2401 tccttggcca aaaagcctga actcaccgcg acgtctgtcg agaagtttct gatcgaaaag
     2461 ttcgacagcg tctccgacct gatgcagctc tcggagggcg aagaatctcg tgctttcagc
     2521 ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata gctgcgccga tggtttctac
     2581 aaagatcgtt atgtttatcg gcactttgca tcggccgcgc tcccgattcc ggaagtgctt
     2641 gacattgggg aattcagcga gagcctgacc tattgcatct cccgccgtgc acagggtgtc
     2701 acgttgcaag acctgcctga aaccgaactg cccgctgttc tgcagccggt cgcggaggcc
     2761 atggatgcga tcgctgcggc cgatcttagc cagacgagcg ggttcggccc attcggaccg
     2821 caaggaatcg gtcaatacac tacatggcgt gatttcatat gcgcgattgc tgatccccat
     2881 gtgtatcact ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc gcaggctctc
     2941 gatgagctga tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat
     3001 ttcggctcca acaatgtcct gacggacaat ggccgcataa cagcggtcat tgactggagc
     3061 gaggcgatgt tcggggattc ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg
     3121 ttggcttgta tggagcagca gacgcgctac ttcgagcgga ggcatccgga gcttgcagga
     3181 tcgccgcggc tccgggcgta tatgctccgc attggtcttg accaactcta tcagagcttg
     3241 gttgacggca atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga
     3301 tccggagccg ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc
     3361 gatggctgtg tagaagtact cgccgatagt ggaaaccgac gccccagcac tcgtccgagg
     3421 gcaaaggaat agcacgtact acgagatttc gattccaccg ccgccttcta tgaaaggttg
     3481 ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg
     3541 ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc
     3601 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     3661 tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg
     3721 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     3781 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     3841 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     3901 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     3961 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     4021 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     4081 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     4141 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     4201 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     4261 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     4321 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     4381 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     4441 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     4501 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     4561 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     4621 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     4681 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     4741 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     4801 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     4861 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     4921 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     4981 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     5041 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     5101 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     5161 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     5221 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     5281 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     5341 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     5401 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     5461 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     5521 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     5581 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     5641 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     5701 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     5761 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     5821 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     5881 cctgacgtcg acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct
     5941 gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg
     6001 agtagtgcgc gagcaaaatt taagctac
//
