LOCUS       sequence-322424-        6337 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive expression of VPR-FRB (no NLS) under the CMV promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6337)
  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 6337)
  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: pPD684 VPR-FRB (no NLS)
FEATURES             Location/Qualifiers
     source          1..6337
                     /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"
     CDS             1618..1974
                     /codon_start=1
                     /product="transcriptional activation domain of human RelA, 
                     also known as p65 (O'Shea and Perkins, 2008)"
                     /label=RelA (p65) AD
                     /translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS
                     EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD
                     EDFSSIADMDFSALL"
     CDS             1993..2562
                     /codon_start=1
                     /product="transcriptional activation domain from the human 
                     herpesvirus 4 (Epstein-Barr virus) replication and 
                     transcription activator Rta/BRLF1 (Hardwick et al., 1992; 
                     Chavez et al., 2015)"
                     /label=Rta AD
                     /translation="RDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWAN
                     RPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKAL
                     REMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEIL
                     DTFLNDECLLHAMHISTGLSIFDTSLF"
     primer_bind     complement(2967..2984)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2973..3197
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3243..3671
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3330..3349)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3540..3561
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3694..3890
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3741..3876
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3803..3822
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    4010..4131
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4047..4066)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4101..4120
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4180..4196)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4180..4196)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4193..4215)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4204..4220
                     /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(4228..4234,4235..4252,4253..4258))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4273..4294
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4411..4428)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4582..5170)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4662..4681)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5341..6132,6133..6201))
                     /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     5964..5983
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6202..6306)
                     /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 gatctgttgg tagccagtac
     1201 ctgcccgaca ccgacgaccg gcaccggatc gaggaaaagc ggaagcggac ctacgagaca
     1261 ttcaagagca tcatgaagaa gtcccccttc agcggcccca ccgaccctag acctccacct
     1321 agaagaatcg ccgtgcccag cagatccagc gccagcgtgc caaaacctgc cccccagcct
     1381 taccccttca ccagcagcct gagcaccatc aactacgacg agttccctac catggtgttc
     1441 cccagcggcc agatctctca ggcctctgct ctggctccag cccctcctca ggtgctgcct
     1501 caggctcctg ctcctgcacc agctccagcc atggtgtctg cactggctca ggcaccagca
     1561 cccgtgcctg tgctggctcc tggacctcca caggctgtgg ctccaccagc ccctaaacct
     1621 acacaggccg gcgagggcac actgtctgaa gctctgctgc agctgcagtt cgacgacgag
     1681 gatctgggag ccctgctggg aaacagcacc gatcctgccg tgttcaccga cctggccagc
     1741 gtggacaaca gcgagttcca gcagctgctg aaccagggca tccctgtggc ccctcacacc
     1801 accgagccca tgctgatgga ataccccgag gccatcaccc ggctcgtgac aggcgctcag
     1861 aggcctcctg atccagctcc tgcccctctg ggagcaccag gcctgcctaa tggactgctg
     1921 tctggcgacg aggacttcag ctctatcgcc gatatggatt tctcagcctt gctgggctct
     1981 ggcagcggca gccgggattc cagggaaggg atgtttttgc cgaagcctga ggccggctcc
     2041 gctattagtg acgtgtttga gggccgcgag gtgtgccagc caaaacgaat ccggccattt
     2101 catcctccag gaagtccatg ggccaaccgc ccactccccg ccagcctcgc accaacacca
     2161 accggtccag tacatgagcc agtcgggtca ctgaccccgg caccagtccc tcagccactg
     2221 gatccagcgc ccgcagtgac tcccgaggcc agtcacctgt tggaggatcc cgatgaagag
     2281 acgagccagg ctgtcaaagc ccttcgggag atggccgata ctgtgattcc ccagaaggaa
     2341 gaggctgcaa tctgtggcca aatggacctt tcccatccgc ccccaagggg ccatctggat
     2401 gagctgacaa ccacacttga gtccatgacc gaggatctga acctggactc acccctgacc
     2461 ccggaattga acgagattct ggataccttc ctgaacgacg agtgcctctt gcatgccatg
     2521 catatcagca caggactgtc catcttcgac acatctctgt tttctggttc tggttctggt
     2581 tcaggatccg gtggtggcgg gtctggagga ggaggtagtg gtggaggtgg ctctggtacc
     2641 atgcttgaga tgtggcatga aggcctggaa gaggcatctc gtttgtactt tggggaaagg
     2701 aacgtgaaag gcatgtttga ggtgctggag cccttgcatg ctatgatgga acggggaccc
     2761 cagactctga aggaaacatc ctttaatcag gcctatggtc gagatttaat ggaggcccaa
     2821 gagtggtgca ggaagtacat gaaatcaggg aatgtcaagg acctcctcca agcctgggac
     2881 ctctattatc atgtgttccg acgaatctca aagactagat gagcggccgc tcgagtctag
     2941 agggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
     3001 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     3061 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     3121 tggggtgggg caggacagca agggggagga ttgggaagtc aatagcaggc atgctgggga
     3181 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc
     3241 ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     3301 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     3361 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     3421 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     3481 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     3541 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     3601 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     3661 taacgcgaat taattctgtg gaatgtgtgt cagttaggct caattagtca gcaaccatag
     3721 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     3781 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc
     3841 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg
     3901 gagcttgtat atccattttc ggaggttggg cttcggaatc gttttccggg acgccggctg
     3961 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat
     4021 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     4081 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     4141 tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     4201 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     4261 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     4321 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     4381 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     4441 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     4501 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     4561 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     4621 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     4681 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     4741 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     4801 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     4861 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     4921 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     4981 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     5041 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     5101 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     5161 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     5221 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     5281 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     5341 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     5401 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     5461 cagtgctgca atgataccgc gagatccacg ctcaccggct ccagatttat cagcaataaa
     5521 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     5581 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     5641 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     5701 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     5761 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     5821 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     5881 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     5941 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     6001 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     6061 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     6121 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     6181 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     6241 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     6301 tccgcgcaca tttccccgaa aagtgccacc tgacgtc
//
