LOCUS       sequence-443765-        5380 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses 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 5380)
  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 5380)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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(△1-64)
FEATURES             Location/Qualifiers
     source          1..5380
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1052..1507
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1139..1158)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1349..1370
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1533..1637
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1638..1706,1707..2498)
                     /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     complement(1856..1875)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2669..3257
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3158..3177
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3410..3427
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3544..3565
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3580..3585,3586..3603,3604..3610)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3618..3634
                     /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)."
     primer_bind     3623..3645
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3642..3658
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3642..3658
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     3677..3697
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        3679..3697
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     polyA_signal    3810..3944
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3847..3866)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3901..3920
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3949..3968
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3949..3967
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(4456..4893)
                     /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"
     CDS             complement(join(4918..5238,5239..5241))
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNK
                     PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL
                     LKLE"
     regulatory      5238..5247
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(5257..5283)
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     promoter        complement(5324..5342)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(5325..5342)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     promoter        complement(join(5377..5380,1..981))
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 cctaaacgag cattgcttat atagacctcc cattaggcac gcctaccgcc catttacgtc
       61 aatggaacgc ccatttgcgt cattgcccct ccccattgac gtcaatgggg atgtacttgg
      121 cagccatcgc gggccattta ccgccattga cgtcaatggg agtactgcca atgtaccctg
      181 gcgtacttcc aatagtaatg tacttgccaa gttactatta atagatattg atgtactgcc
      241 aagtgggcca tttaccgtca ttgacgtcaa tagggggcgt gagaacggat atgaatgggc
      301 aatgagccat cccattgacg tcaatggtgg gtggtcctat tgacgtcaat gggcattgag
      361 ccaggcgggc catttaccgt aattgacgtc aatgggggag gcgccatata cgtcaatagg
      421 accgcccata tgacgtcaat aggaaagacc atatatagag accattgacg tcaatggggg
      481 agtggctatg ggcggtatta ggaagcccca tatatggtat atgggaccgc ccattgggag
      541 gggctatcta cgtcaatagg aaaacccata tatggaatac tatatggcat agggccaata
      601 catagtattg aacctggcca atagccatat tggcataggg ccatattgga tattgcctat
      661 atattgatcc tggcatatag ccaatatggc cgccattatt ggcaccatgc caattcaata
      721 tggcggactt ggcacagggc caagacccct cctcaaactt ggcaccgtgc caagtagacc
      781 cctccccagt tggcacagtg ccaggtccgc catattgaat tggcatggtg ccaagtccgc
      841 catattgaat tggctggggt ccagatccac catattgaat tggcacgagt ccatatacgc
      901 catattgaat tggcacaggt ccagatccac catattgaat tggcatgagt ccatatacgc
      961 catattgaat tggctgtcga ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc
     1021 caacagttgc gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg
     1081 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     1141 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     1201 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     1261 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     1321 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     1381 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     1441 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt
     1501 acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     1561 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     1621 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     1681 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     1741 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     1801 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     1861 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     1921 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     1981 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     2041 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     2101 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     2161 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     2221 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     2281 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     2341 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     2401 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     2461 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     2521 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     2581 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     2641 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc ataatctgct
     2701 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     2761 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     2821 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     2881 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     2941 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     3001 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     3061 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     3121 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     3181 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     3241 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     3301 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     3361 ccgcctttga gtgagctgat accgctcccg cagccgaacg accgagcgca gcgagtcagt
     3421 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
     3481 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
     3541 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgctcccggc
     3601 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca
     3661 tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg gtaccgggcc
     3721 caatgcattg gcgccgcgga cgcgaattaa aaaacctccc acacctcccc ctgaacctga
     3781 aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca
     3841 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     3901 gtggtttgtc caaactcatc aatgtatctt atcatgtctg gatctacgta atacgactca
     3961 ctatagggag gctagctatc tagactagga ggagtctgta cagggagagg gtggcggtgg
     4021 gtagaggtga tgagaatagc tcctctctgt gtatggagaa ggtgggcagc tttcatagtt
     4081 ctcctctgct gacaagtatt ggcttcgggg tgtgggaggt gggggcacag gttctgaatc
     4141 atagttcaag tcactggtat agcccttggc tgttgccact gaggtcattc tccgactagg
     4201 agcatagtca ctgtcacaaa catctgtgct gcagggtgtg gtggggggtg caaagtgccg
     4261 gtagctatat ggcctgtagc tgtatgacct atgagtggaa ggactgtttg aagaatatcc
     4321 aaattccata gtgtaatgtg atcgctctgt ggctggggat ggtggagggt tcaaaattgc
     4381 agggaagtaa gtgcctttgg tgcttgaaga actacttgat gatgctcctg taacatgggc
     4441 tcggtcatag catgcaaagt aattttcgtc gttcgctgcc tcagcaatct gaaaggctgt
     4501 ctttttgatc aggcagaccg cctcacgctc agcggcgcca tgaagacgtt cggtgccatc
     4561 cagctgcacg ccaatgaagt actggacgtc gcccttataa tcgcgcatcg gttgcaggtg
     4621 gaacacgttc cagaacttct tgccgctctt ggtataattg atcagctgaa cggtcacttc
     4681 agtctggttg tcaatagcat ctctgatttt ccgaacagtg gcacggtctg tctcgggtcc
     4741 ttgcagaaaa cggcaattgc gccccaggat ctcttcccga gaatattctg tcagttgcag
     4801 gaaggaatcg ctcgcaaaaa tgattgggtt gtcaggcagt ctcggatcag taatcacgaa
     4861 atttttctcg atccgttcca gtgtggttgc cagaaactcc atgagctcgc cgctgccttc
     4921 cagttttaga agctccacat cgaagacgag agtggcatgt ggtgggatga tgcctgggtg
     4981 cccagtggca ccataggcat aatctggaga tatagtcagt ttggctctct gacccacact
     5041 catctgggca accccttctt cccagcctcg gatcacctcc tgcttgccta gcataaactt
     5101 aaagggcttg tttctgtccc gggaggaatc aaatttcttt ccatcttcaa gcatcccggt
     5161 gtagtgcacc acgcaggtct ggccgcgctt ggggaaggtg cgcccgtctc ctggggagat
     5221 ggtttccacc tgcactccca tggtggcgaa ttcctcgaga gtaagacgtt caagaggagg
     5281 aagttgaagc atggatcctg caaaaagaac aagtagcttg tattctatag tgtcacctaa
     5341 atcaagcttg ctccgacgtc cccaggcaga atggcggttc
//
