LOCUS       sequence-322419-        5014 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive expression of VP16-FRB (no NLS) under the CMV 
            promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5014)
  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 5014)
  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: pPD682 VP16-FRB (no NLS)
FEATURES             Location/Qualifiers
     source          1..5014
                     /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"
     primer_bind     complement(1644..1661)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1650..1874
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1920..2348
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2007..2026)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2217..2238
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2371..2567
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      2418..2553
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2480..2499
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    2687..2808
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2724..2743)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2778..2797
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2857..2873)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2857..2873)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2870..2892)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2881..2897
                     /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(2905..2911,2912..2929,2930..2935))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2950..2971
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3088..3105)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3259..3847)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3339..3358)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4018..4809,4810..4878))
                     /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     4641..4660
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4879..4983)
                     /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 ttcccgtcga cggtggactc
     1021 caagccccac caaccgatgt ttctttaggt gatgagttac atttggacgg tgaggacgta
     1081 gctatggcac atgcagatgc cttagacgat tttgacttgg atatgttagg agatggagat
     1141 tcaccaggac ctggtttcac accacacgat tcagctccat acggtgcttt ggacatggcc
     1201 gatttcgaat ttgaacagat gtttactgat gccttgggta tagacgaata cggtggtgga
     1261 ggatccggtg gtggcgggtc tggaggagga ggtagtggtg gaggtggctc tggtaccatg
     1321 cttgagatgt ggcatgaagg cctggaagag gcatctcgtt tgtactttgg ggaaaggaac
     1381 gtgaaaggca tgtttgaggt gctggagccc ttgcatgcta tgatggaacg gggaccccag
     1441 actctgaagg aaacatcctt taatcaggcc tatggtcgag atttaatgga ggcccaagag
     1501 tggtgcagga agtacatgaa atcagggaat gtcaaggacc tcctccaagc ctgggacctc
     1561 tattatcatg tgttccgacg aatctcaaag actagatgag cggccgctcg agtctagagg
     1621 gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     1681 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     1741 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     1801 ggtggggcag gacagcaagg gggaggattg ggaagtcaat agcaggcatg ctggggatgc
     1861 ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca
     1921 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     1981 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     2041 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     2101 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     2161 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     2221 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     2281 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     2341 cgcgaattaa ttctgtggaa tgtgtgtcag ttaggctcaa ttagtcagca accatagtcc
     2401 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     2461 atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat
     2521 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag
     2581 cttgtatatc cattttcgga ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     2641 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc
     2701 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     2761 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat
     2821 accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     2881 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     2941 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     3001 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     3061 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     3121 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     3181 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     3241 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     3301 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     3361 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     3421 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     3481 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     3541 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     3601 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     3661 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     3721 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     3781 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     3841 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     3901 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     3961 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     4021 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     4081 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     4141 tgctgcaatg ataccgcgag atccacgctc accggctcca gatttatcag caataaacca
     4201 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     4261 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     4321 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     4381 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     4441 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     4501 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     4561 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     4621 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     4681 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     4741 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     4801 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     4861 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     4921 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     4981 gcgcacattt ccccgaaaag tgccacctga cgtc
//
