LOCUS       sequence-322058-        4975 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive expression of VP64-FRB under the CMV promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4975)
  AUTHORS   Donahue PS, Draut JW, Muldoon JJ, Edelstein HI, Bagheri N, Leonard 
            JN
  TITLE     The COMET toolkit for composing customizable genetic programs in 
            mammalian cells.
  JOURNAL   Nat Commun. 2020 Feb 7;11(1):779. doi: 10.1038/s41467-019-14147-5.
  PUBMED    32034124
REFERENCE   2  (bases 1 to 4975)
  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 
            Commun."; date: "2020-02-7"; volume: "11(1):779. doi"; pages: " 
            10.1038/s41467-019-14147-5"
COMMENT     Sequence Label: pPD354 VP64-FRB (NLS)
FEATURES             Location/Qualifiers
     source          1..4975
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      901..910
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             910..975
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             982..1002
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1027..1176
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     primer_bind     complement(1605..1622)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1611..1835
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1881..2309
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1968..1987)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2178..2199
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2332..2528
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      2379..2514
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2441..2460
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    2648..2769
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2685..2704)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2739..2758
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2818..2834)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2818..2834)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2831..2853)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2842..2858
                     /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(2866..2872,2873..2890,2891..2896))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2911..2932
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3049..3066)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3220..3808)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3300..3319)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3979..4770,4771..4839))
                     /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     4602..4621
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4840..4944)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagtcccaa gctggctagc
      901 gccaccatgg attataagga tcacgatggc gactacaagg accacgatat tgactacaaa
      961 gacgatgacg ataaaatggc tcctaagaaa aagcgcaaag tcggtatcca tggcgttccc
     1021 ggatcagacg cattggacga ttttgatctg gatatgctgg gaagtgacgc cctcgatgat
     1081 tttgaccttg acatgcttgg ttcggatgcc cttgatgact ttgacctcga catgctcggc
     1141 agtgacgccc ttgatgattt cgacctggac atgctgatta actctagaag ttccggatct
     1201 tctggttctg gttctggttc aggatccggt ggtggcgggt ctggaggagg aggtagtggt
     1261 ggaggtggct ctggtaccat gcttgagatg tggcatgaag gcctggaaga ggcatctcgt
     1321 ttgtactttg gggaaaggaa cgtgaaaggc atgtttgagg tgctggagcc cttgcatgct
     1381 atgatggaac ggggacccca gactctgaag gaaacatcct ttaatcaggc ctatggtcga
     1441 gatttaatgg aggcccaaga gtggtgcagg aagtacatga aatcagggaa tgtcaaggac
     1501 ctcctccaag cctgggacct ctattatcat gtgttccgac gaatctcaaa gactagatga
     1561 gcggccgctc gagtctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc
     1621 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     1681 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     1741 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagtcaa
     1801 tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg
     1861 gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     1921 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     1981 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     2041 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     2101 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     2161 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     2221 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     2281 gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca gttaggctca
     2341 attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca
     2401 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg
     2461 ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct
     2521 tttgcaaaaa gctcccggga gcttgtatat ccattttcgg aggttgggct tcggaatcgt
     2581 tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc
     2641 ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     2701 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     2761 tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc
     2821 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     2881 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     2941 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     3001 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     3061 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     3121 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     3181 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     3241 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     3301 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     3361 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     3421 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     3481 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     3541 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     3601 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     3661 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     3721 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     3781 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     3841 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     3901 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     3961 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     4021 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     4081 gggcttacca tctggcccca gtgctgcaat gataccgcga gatccacgct caccggctcc
     4141 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     4201 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     4261 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     4321 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     4381 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     4441 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     4501 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     4561 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     4621 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     4681 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     4741 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     4801 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     4861 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     4921 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtc
//
