LOCUS       sequence-415569-        4769 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses NLS-FRB-VP16 component in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4769)
  AUTHORS   Cui M, Lee S, Ban SH, Ryu JR, Shen M, Yang SH, Kim JY, Choi SK, Han 
            J, Kim Y, Han K, Lee D, Sun W, Kwon HB, Lee D
  TITLE     A single-component, light-assisted uncaging switch for 
            endoproteolytic release.
  JOURNAL   Nat Chem Biol. 2024 Mar;20(3):353-364. doi: 
            10.1038/s41589-023-01480-6. Epub 2023 Nov 16.
  PUBMED    37973890
REFERENCE   2  (bases 1 to 4769)
  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"; doi: 
            "10.1038/s41589-023-01480-6"; journalName: "Nat Chem Biol"; date: 
            "2024-03"; volume: "20"; issue: "3"; pages: "353-364"
COMMENT     Sequence Label: pCMV-NLS-FRB-VP16
FEATURES             Location/Qualifiers
     source          1..4769
                     /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"
     promoter        1014..1032
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1014..1031
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             1054..1074
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1096..1374
                     /codon_start=1
                     /product="T2098L mutant of FKBP-rapamycin binding domain of
                     human FRAP"
                     /label=FRB*
                     /note="binds synthetic ligands such as AP21967"
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK"
     CDS             1531..1764
                     /codon_start=1
                     /gene="UL48"
                     /product="transcriptional activation domain of herpes 
                     simplex virus protein VP16 (Triezenberg et al., 1988; 
                     Cousens et al., 1989)"
                     /label=VP16 AD
                     /translation="APPTDVSLGDELHLDGEDVAMAHADALDDFDLDMLGDGDSPGPGF
                     TPHDSAPYGALDMADFEFEQMFTDALGIDEYGG"
     primer_bind     complement(1776..1795)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1777..1795)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    1800..1934
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1824..1843)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1878..1897
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2047..2067)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2047..2065)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2086..2102)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2086..2102)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2099..2121)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2110..2126
                     /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(2134..2140,2141..2158,2159..2164))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2179..2200
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2317..2334)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2487..3075)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2567..2586)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3246..4037,4038..4106))
                     /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     3869..3888
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4107..4211)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4237..4692)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     4586..4605
                     /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 ttcatttagg
     1021 tgacactata gaatacaagc tacttgttct atgcctaaaa agaagcgtaa agtcggagga
     1081 tccatggctt ctcgaatcct ctggcatgag atgtggcatg aaggcctgga agaggcatct
     1141 cgtttgtact ttggggaaag gaacgtgaaa ggcatgtttg aggtgctgga gcccttgcat
     1201 gctatgatgg aacggggccc ccagactctg aaggaaacat cctttaatca ggcctatggt
     1261 cgagatttaa tggaggccca agagtggtgc aggaagtaca tgaaatcagg gaatgtcaag
     1321 gacctcctcc aagcctggga cctctattat catgtgttcc gacgaatctc aaagactagt
     1381 gcgtacagcc gcgcgcgtac gaaaaacaat tacgggtcta ccatcgaggg cctgctcgat
     1441 ctcccggacg acgacgcccc cgaagaggcg gggctggcgg ctccgcgcct gtcctttctc
     1501 cccgcgggac acacgcgcag actatcgacg gcccccccga ccgatgtcag cctgggggac
     1561 gagctccact tagacggcga ggacgtggcg atggcgcatg ccgacgcgct agacgatttc
     1621 gatctggaca tgttggggga cggggattcc ccgggtccgg gatttacccc ccacgactcc
     1681 gccccctacg gcgctctgga tatggccgac ttcgagtttg agcagatgtt taccgatgcc
     1741 cttggaattg acgagtacgg tgggtaggct agcctcccta tagtgagtcg tattacgtag
     1801 atccagacat gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa
     1861 aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct
     1921 gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg
     1981 tgtgggaggt tttttaattc gcgtccgcgg cgccaatgca ttgggcccgg tacccagctt
     2041 ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc
     2101 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggga gcataaagtg
     2161 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     2221 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     2281 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     2341 ggtcattcgg ctgcgggagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     2401 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     2461 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     2521 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     2581 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     2641 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     2701 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     2761 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     2821 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     2881 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     2941 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     3001 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     3061 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     3121 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     3181 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     3241 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     3301 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     3361 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     3421 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     3481 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     3541 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     3601 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     3661 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     3721 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     3781 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     3841 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     3901 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     3961 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     4021 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     4081 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     4141 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     4201 ggggttccgc gcacatttcc ccgaaaagtg ccacctaaat tgtaagcgtt aatattttgt
     4261 taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg
     4321 gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccggttt
     4381 ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct
     4441 atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt
     4501 gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa
     4561 agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc
     4621 tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc
     4681 tacagggcgc gtcccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc
     4741 gggcctcttc gctattacgc cagtcgacc
//
