LOCUS       sequence-322425-        5002 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive expression of VP64-FRB (NES) under the CMV promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5002)
  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 5002)
  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: pPD880 VP64-FRB (NES)
FEATURES             Location/Qualifiers
     source          1..5002
                     /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             1054..1203
                     /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(1632..1649)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1638..1862
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1908..2336
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1995..2014)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2205..2226
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2359..2555
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      2406..2541
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2468..2487
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    2675..2796
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2712..2731)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2766..2785
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2845..2861)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2845..2861)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2858..2880)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2869..2885
                     /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(2893..2899,2900..2917,2918..2923))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2938..2959
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3076..3093)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3247..3835)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3327..3346)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4006..4797,4798..4866))
                     /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     4629..4648
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4867..4971)
                     /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 ataacggtat ggaggaactt tcacaggccc tggctagtag tttttcagtg
     1021 tctggtgtcg gtatccatgg cgttcccgga tcagacgcat tggacgattt tgatctggat
     1081 atgctgggaa gtgacgccct cgatgatttt gaccttgaca tgcttggttc ggatgccctt
     1141 gatgactttg acctcgacat gctcggcagt gacgcccttg atgatttcga cctggacatg
     1201 ctgattaact ctagaagttc cggatcttct ggttctggtt ctggttcagg atccggtggt
     1261 ggcgggtctg gaggaggagg tagtggtgga ggtggctctg gtaccatgct tgagatgtgg
     1321 catgaaggcc tggaagaggc atctcgtttg tactttgggg aaaggaacgt gaaaggcatg
     1381 tttgaggtgc tggagccctt gcatgctatg atggaacggg gaccccagac tctgaaggaa
     1441 acatccttta atcaggccta tggtcgagat ttaatggagg cccaagagtg gtgcaggaag
     1501 tacatgaaat cagggaatgt caaggacctc ctccaagcct gggacctcta ttatcatgtg
     1561 ttccgacgaa tctcaaagac tagatgagcg gccgctcgag tctagagggc ccgtttaaac
     1621 ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     1681 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     1741 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     1801 cagcaagggg gaggattggg aagtcaatag caggcatgct ggggatgcgg tgggctctat
     1861 ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag
     1921 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     1981 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     2041 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     2101 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     2161 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     2221 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     2281 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt
     2341 ctgtggaatg tgtgtcagtt aggctcaatt agtcagcaac catagtcccg cccctaactc
     2401 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     2461 ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt
     2521 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca
     2581 ttttcggagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg
     2641 cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg
     2701 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     2761 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc
     2821 tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     2881 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     2941 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     3001 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     3061 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     3121 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     3181 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     3241 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     3301 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     3361 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     3421 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     3481 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     3541 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     3601 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     3661 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     3721 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     3781 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     3841 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     3901 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     3961 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     4021 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     4081 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     4141 accgcgagat ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     4201 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     4261 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     4321 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     4381 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     4441 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     4501 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     4561 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     4621 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     4681 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     4741 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     4801 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     4861 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     4921 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     4981 ccgaaaagtg ccacctgacg tc
//
