LOCUS       sequence-436960-        5792 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GFP11x7-P2A-NES-FKBP-iLID-LRP6c(△1-64) component in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5792)
  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 5792)
  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:: GFP11x7-P2A-NES-FKBP-iLID-LRP6c(△1-64)
FEATURES             Location/Qualifiers
     source          1..5792
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..978
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
     CDS             1015..1440
                     /codon_start=1
                     /product="seven tandem copies of the 11th beta-strand of 
                     superfolder GFP"
                     /label=GFP11x7
                     /note="each GFP11 associates with GFP(1-10), consisting of 
                     the first 10 beta-strands, to reconstitute fluorescent GFP 
                     (Kamiyama et al., 2016)"
                     /translation="RDHMVLHEYVNAAGITGGSGGRDHMVLHEYVNAAGITGGSGGRDH
                     MVLHEYVNAAGITGGSGGRDHMVLHEYVNAAGITGGSGGRDHMVLHEYVNAAGITGGSG
                     GRDHMVLHEYVNAAGITGGSGGRDHMVLHEYVNAAGIT"
     CDS             1450..1506
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1513..1539
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     CDS             join(1540..1542,1543..1863)
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNK
                     PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL
                     LKLE"
     CDS             1888..2325
                     /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(2799..2818)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2800..2818)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    2823..2957
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2847..2866)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2901..2920
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3070..3090)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3070..3088)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3109..3125)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3109..3125)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3122..3144)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3133..3149
                     /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(3157..3163,3164..3181,3182..3187))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3202..3223
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3340..3357)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3510..4098)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3590..3609)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4269..5060,5061..5129))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLYNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4892..4911
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5130..5234)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5260..5715)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5397..5418)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5609..5628
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gccaattcaa tatggcgtat atggactcat gccaattcaa tatggtggat ctggacctgt
       61 gccaattcaa tatggcgtat atggactcgt gccaattcaa tatggtggat ctggacccca
      121 gccaattcaa tatggcggac ttggcaccat gccaattcaa tatggcggac ctggcactgt
      181 gccaactggg gaggggtcta cttggcacgg tgccaagttt gaggaggggt cttggccctg
      241 tgccaagtcc gccatattga attggcatgg tgccaataat ggcggccata ttggctatat
      301 gccaggatca atatataggc aatatccaat atggccctat gccaatatgg ctattggcca
      361 ggttcaatac tatgtattgg ccctatgcca tatagtattc catatatggg ttttcctatt
      421 gacgtagata gcccctccca atgggcggtc ccatatacca tatatggggc ttcctaatac
      481 cgcccatagc cactccccca ttgacgtcaa tggtctctat atatggtctt tcctattgac
      541 gtcatatggg cggtcctatt gacgtatatg gcgcctcccc cattgacgtc aattacggta
      601 aatggcccgc ctggctcaat gcccattgac gtcaatagga ccacccacca ttgacgtcaa
      661 tgggatggct cattgcccat tcatatccgt tctcacgccc cctattgacg tcaatgacgg
      721 taaatggccc acttggcagt acatcaatat ctattaatag taacttggca agtacattac
      781 tattggaagt acgccagggt acattggcag tactcccatt gacgtcaatg gcggtaaatg
      841 gcccgcgatg gctgccaagt acatccccat tgacgtcaat ggggaggggc aatgacgcaa
      901 atgggcgttc cattgacgta aatgggcggt aggcgtgcct aatgggaggt ctatataagc
      961 aatgctcgtt tagggaaccg ccattctgcc tggggacgtc ggagcaagct tatgcgtgac
     1021 cacatggtcc ttcatgagta tgtaaatgct gctgggatta caggtggctc tggaggtaga
     1081 gatcatatgg ttctccacga atacgttaac gccgcaggca tcactggcgg tagtggagga
     1141 cgcgaccata tggtactaca tgaatatgtc aatgcagccg gaataaccgg agggtccgga
     1201 ggccgggatc acatggtgct gcatgagtat gtgaacgcgg cgggtataac tggtgggtcg
     1261 ggcggacgag accatatggt gcttcacgaa tacgtaaacg cagctggcat tactggcgga
     1321 tcaggtggca gggatcacat ggtactccat gagtacgtga acgctgctgg aatcacaggc
     1381 ggtagcggcg gtcgggacca tatggtcctg cacgaatatg tcaatgctgc cggtatcacc
     1441 ggaagcggag ctactaactt cagcctgctg aagcaggctg gagacgtgga ggagaaccct
     1501 ggacctcttc aacttcctcc tcttgaacgt cttactctca tgggagtgca ggtggaaacc
     1561 atctccccag gagacgggcg caccttcccc aagcgcggcc agacctgcgt ggtgcactac
     1621 accgggatgc ttgaagatgg aaagaaattt gattcctccc gggacagaaa caagcccttt
     1681 aagtttatgc taggcaagca ggaggtgatc cgaggctggg aagaaggggt tgcccagatg
     1741 agtgtgggtc agagagccaa actgactata tctccagatt atgcctatgg tgccactggg
     1801 cacccaggca tcatcccacc acatgccact ctcgtcttcg atgtggagct tctaaaactg
     1861 gaaggcagcg gcgagctcat ggagtttctg gcaaccacac tggaacggat cgagaaaaat
     1921 ttcgtgatta ctgatccgag actgcctgac aacccaatca tttttgcgag cgattccttc
     1981 ctgcaactga cagaatattc tcgggaagag atcctggggc gcaattgccg ttttctgcaa
     2041 ggacccgaga cagaccgtgc cactgttcgg aaaatcagag atgctattga caaccagact
     2101 gaagtgaccg ttcagctgat caattatacc aagagcggca agaagttctg gaacgtgttc
     2161 cacctgcaac cgatgcgcga ttataagggc gacgtccagt acttcattgg cgtgcagctg
     2221 gatggcaccg aacgtcttca tggcgccgct gagcgtgagg cggtctgcct gatcaaaaag
     2281 acagcctttc agattgctga ggcagcgaac gacgaaaatt acttttatga ccgagcccat
     2341 gttacaggag catcatcaag tagttcttca agcaccaaag gcacttactt ccctgcaatt
     2401 ttgaaccctc caccatcccc agccacagag cgatcacatt acactatgga atttggatat
     2461 tcttcaaaca gtccttccac tcataggtca tacagctaca ggccatatag ctaccggcac
     2521 tttgcacccc ccaccacacc ctgcagcaca gatgtttgtg acagtgacta tgctcctagt
     2581 cggagaatga cctcagtggc aacagccaag ggctatacca gtgacttgaa ctatgattca
     2641 gaacctgtgc ccccacctcc cacaccccga agccaatact tgtcagcaga ggagaactat
     2701 gaaagctgcc caccttctcc atacacagag aggagctatt ctcatcacct ctacccaccg
     2761 ccaccctctc cctgtacaga ctcctcctag gctagcctcc ctatagtgag tcgtattacg
     2821 tagatccaga catgataaga tacattgatg agtttggaca aaccacaact agaatgcagt
     2881 gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa
     2941 gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg
     3001 aggtgtggga ggttttttaa ttcgcgtccg cggcgccaat gcattgggcc cggtacccag
     3061 cttttgttcc ctttagtgag ggttaattgc gcgcttggcg taatcatggt catagctgtt
     3121 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg ggagcataaa
     3181 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     3241 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     3301 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     3361 ctcggtcgtt cggctgcggg agcggtatca gctcactcaa aggcggtaat acggttatcc
     3421 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     3481 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     3541 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     3601 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     3661 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     3721 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     3781 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     3841 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     3901 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     3961 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     4021 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattatgcgc
     4081 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     4141 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     4201 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     4261 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     4321 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     4381 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     4441 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     4501 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     4561 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     4621 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtac
     4681 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     4741 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     4801 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     4861 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     4921 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     4981 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     5041 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     5101 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     5161 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     5221 ataggggttc cgcgcacatt tccccgaaaa gtgccaccta aattgtaagc gttaatattt
     5281 tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa
     5341 tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag
     5401 tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg
     5461 tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga
     5521 ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg
     5581 gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg
     5641 cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc
     5701 cgctacaggg cgcgtcccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg
     5761 tgcgggcctc ttcgctatta cgccagtcga ca
//
