LOCUS       sequence-322420-        4960 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive expression of VP64-FRB (no NLS) under the CMV 
            promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4960)
  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 4960)
  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: pPD683 VP64-FRB (no NLS)
FEATURES             Location/Qualifiers
     source          1..4960
                     /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             1012..1161
                     /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(1590..1607)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1596..1820
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1866..2294
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1953..1972)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2163..2184
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2317..2513
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      2364..2499
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2426..2445
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    2633..2754
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2670..2689)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2724..2743
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2803..2819)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2803..2819)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2816..2838)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2827..2843
                     /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(2851..2857,2858..2875,2876..2881))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2896..2917
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3034..3051)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3205..3793)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3285..3304)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3964..4755,4756..4824))
                     /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     4587..4606
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4825..4929)
                     /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 tggtagtggt atccatggcg ttcccggatc agacgcattg
     1021 gacgattttg atctggatat gctgggaagt gacgccctcg atgattttga ccttgacatg
     1081 cttggttcgg atgcccttga tgactttgac ctcgacatgc tcggcagtga cgcccttgat
     1141 gatttcgacc tggacatgct gattaactct agaagttccg gatcttctgg ttctggttct
     1201 ggttcaggat ccggtggtgg cgggtctgga ggaggaggta gtggtggagg tggctctggt
     1261 accatgcttg agatgtggca tgaaggcctg gaagaggcat ctcgtttgta ctttggggaa
     1321 aggaacgtga aaggcatgtt tgaggtgctg gagcccttgc atgctatgat ggaacgggga
     1381 ccccagactc tgaaggaaac atcctttaat caggcctatg gtcgagattt aatggaggcc
     1441 caagagtggt gcaggaagta catgaaatca gggaatgtca aggacctcct ccaagcctgg
     1501 gacctctatt atcatgtgtt ccgacgaatc tcaaagacta gatgagcggc cgctcgagtc
     1561 tagagggccc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc
     1621 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     1681 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     1741 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gtcaatagca ggcatgctgg
     1801 ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta
     1861 tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     1921 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     1981 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     2041 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     2101 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     2161 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     2221 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     2281 atttaacgcg aattaattct gtggaatgtg tgtcagttag gctcaattag tcagcaacca
     2341 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     2401 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg
     2461 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc
     2521 cgggagcttg tatatccatt ttcggaggtt gggcttcgga atcgttttcc gggacgccgg
     2581 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt
     2641 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     2701 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     2761 ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt
     2821 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     2881 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     2941 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     3001 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     3061 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     3121 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     3181 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     3241 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     3301 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     3361 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     3421 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     3481 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     3541 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     3601 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     3661 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     3721 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     3781 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     3841 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     3901 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     3961 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     4021 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     4081 ccccagtgct gcaatgatac cgcgagatcc acgctcaccg gctccagatt tatcagcaat
     4141 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     4201 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     4261 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     4321 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     4381 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     4441 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     4501 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     4561 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     4621 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     4681 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     4741 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     4801 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     4861 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     4921 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
//
