LOCUS       sequence-436961-        5572 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses NES-FKBP-iLID-LRP6c component in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5572)
  AUTHORS   Lee S, Cui M, Lee D, Han K, Sun W, Lee D
  TITLE     A rationally designed optochemogenetic switch for activating 
            canonical Wnt signaling.
  JOURNAL   iScience. 2023 Feb 19;26(3):106233. doi: 10.1016/j.isci.2023.106233.
            eCollection 2023 Mar 17.
  PUBMED    36915690
REFERENCE   2  (bases 1 to 5572)
  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: "iScience.";
            date: "2023-02-19"; volume: "26(3):106233. doi"; pages: " 
            10.1016/j.isci.2023.106233. eCollection 2023 Mar 17"
COMMENT     Sequence Label: pCMV:: NES-FKBP-iLID-LRP6c
FEATURES             Location/Qualifiers
     source          1..5572
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1007..1025
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1007..1024
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             1066..1092
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     regulatory      1102..1111
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1108..1110,1111..1431)
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNK
                     PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL
                     LKLE"
     CDS             1456..1893
                     /codon_start=1
                     /product="improved light-inducible dimer when used with 
                     SspB nano or SspB micro (Guntas et al., 2015)"
                     /label=iLID
                     /translation="LATTLERIEKNFVITDPRLPDNPIIFASDSFLQLTEYSREEILGR
                     NCRFLQGPETDRATVRKIRDAIDNQTEVTVQLINYTKSGKKFWNVFHLQPMRDYKGDVQ
                     YFIGVQLDGTERLHGAAEREAVCLIKKTAFQIAEAANDENYF"
     primer_bind     complement(2573..2592)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2574..2592)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    2597..2731
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2621..2640)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2675..2694
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2844..2864)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2844..2862)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2883..2899)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2883..2899)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2896..2918)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2907..2923
                     /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(2931..2937,2938..2955,2956..2961))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2976..2997
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3114..3131)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3284..3872)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3364..3383)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4043..4834,4835..4903))
                     /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     4666..4685
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4904..5008)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5034..5489)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5171..5192)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5383..5402
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        join(5567..5572,1..972)
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 tcaatatggc gtatatggac tcatgccaat tcaatatggt ggatctggac ctgtgccaat
       61 tcaatatggc gtatatggac tcgtgccaat tcaatatggt ggatctggac cccagccaat
      121 tcaatatggc ggacttggca ccatgccaat tcaatatggc ggacctggca ctgtgccaac
      181 tggggagggg tctacttggc acggtgccaa gtttgaggag gggtcttggc cctgtgccaa
      241 gtccgccata ttgaattggc atggtgccaa taatggcggc catattggct atatgccagg
      301 atcaatatat aggcaatatc caatatggcc ctatgccaat atggctattg gccaggttca
      361 atactatgta ttggccctat gccatatagt attccatata tgggttttcc tattgacgta
      421 gatagcccct cccaatgggc ggtcccatat accatatatg gggcttccta ataccgccca
      481 tagccactcc cccattgacg tcaatggtct ctatatatgg tctttcctat tgacgtcata
      541 tgggcggtcc tattgacgta tatggcgcct cccccattga cgtcaattac ggtaaatggc
      601 ccgcctggct caatgcccat tgacgtcaat aggaccaccc accattgacg tcaatgggat
      661 ggctcattgc ccattcatat ccgttctcac gccccctatt gacgtcaatg acggtaaatg
      721 gcccacttgg cagtacatca atatctatta atagtaactt ggcaagtaca ttactattgg
      781 aagtacgcca gggtacattg gcagtactcc cattgacgtc aatggcggta aatggcccgc
      841 gatggctgcc aagtacatcc ccattgacgt caatggggag gggcaatgac gcaaatgggc
      901 gttccattga cgtaaatggg cggtaggcgt gcctaatggg aggtctatat aagcaatgct
      961 cgtttaggga accgccattc tgcctgggga cgtcggagca agcttgattt aggtgacact
     1021 atagaataca agctacttgt tctttttgca ggatccatgc ttcaacttcc tcctcttgaa
     1081 cgtcttactc tcgaggaatt cgccaccatg ggagtgcagg tggaaaccat ctccccagga
     1141 gacgggcgca ccttccccaa gcgcggccag acctgcgtgg tgcactacac cgggatgctt
     1201 gaagatggaa agaaatttga ttcctcccgg gacagaaaca agccctttaa gtttatgcta
     1261 ggcaagcagg aggtgatccg aggctgggaa gaaggggttg cccagatgag tgtgggtcag
     1321 agagccaaac tgactatatc tccagattat gcctatggtg ccactgggca cccaggcatc
     1381 atcccaccac atgccactct cgtcttcgat gtggagcttc taaaactgga aggcagcggc
     1441 gagctcatgg agtttctggc aaccacactg gaacggatcg agaaaaattt cgtgattact
     1501 gatccgagac tgcctgacaa cccaatcatt tttgcgagcg attccttcct gcaactgaca
     1561 gaatattctc gggaagagat cctggggcgc aattgccgtt ttctgcaagg acccgagaca
     1621 gaccgtgcca ctgttcggaa aatcagagat gctattgaca accagactga agtgaccgtt
     1681 cagctgatca attataccaa gagcggcaag aagttctgga acgtgttcca cctgcaaccg
     1741 atgcgcgatt ataagggcga cgtccagtac ttcattggcg tgcagctgga tggcaccgaa
     1801 cgtcttcatg gcgccgctga gcgtgaggcg gtctgcctga tcaaaaagac agcctttcag
     1861 attgctgagg cagcgaacga cgaaaattac tttgcatgca ggatgttgtg tccacgtatg
     1921 aagggagatg gggaaactat gactaatgac tatgtagttc atggaccagc ttctgtgcct
     1981 cttggttatg tgccacaccc aagttctttg tcaggatctc ttccaggaat gtctcgaggt
     2041 aaatcaatga tcagctccct cagtatcatg gggggaagca gtggaccccc ctatgaccga
     2101 gcccatgtta caggagcatc atcaagtagt tcttcaagca ccaaaggcac ttacttccct
     2161 gcaattttga accctccacc atccccagcc acagagcgat cacattacac tatggaattt
     2221 ggatattctt caaacagtcc ttccactcat aggtcataca gctacaggcc atatagctac
     2281 cggcactttg caccccccac cacaccctgc agcacagatg tttgtgacag tgactatgct
     2341 cctagtcgga gaatgacctc agtggcaaca gccaagggct ataccagtga cttgaactat
     2401 gattcagaac ctgtgccccc acctcccaca ccccgaagcc aatacttgtc agcagaggag
     2461 aactatgaaa gctgcccacc ttctccatac acagagagga gctattctca tcacctctac
     2521 ccaccgccac cctctccctg tacagactcc tcctagtcta gatagctagc ctccctatag
     2581 tgagtcgtat tacgtagatc cagacatgat aagatacatt gatgagtttg gacaaaccac
     2641 aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt
     2701 tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt
     2761 tcaggttcag ggggaggtgt gggaggtttt ttaattcgcg tccgcggcgc caatgcattg
     2821 ggcccggtac ccagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca
     2881 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     2941 gccgggagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     3001 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     3061 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     3121 actgactcgc tgcgctcggt cgttcggctg cgggagcggt atcagctcac tcaaaggcgg
     3181 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     3241 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     3301 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     3361 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     3421 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     3481 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     3541 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     3601 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     3661 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     3721 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     3781 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     3841 agcagattat gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     3901 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     3961 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     4021 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     4081 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     4141 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     4201 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     4261 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     4321 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     4381 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     4441 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     4501 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     4561 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     4621 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     4681 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     4741 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     4801 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     4861 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     4921 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     4981 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt
     5041 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
     5101 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
     5161 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
     5221 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag
     5281 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
     5341 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
     5401 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
     5461 cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca ttcaggctgc gcaactgttg
     5521 ggaagggcga tcggtgcggg cctcttcgct attacgccag tcgacagcca at
//
