LOCUS       sequence-322060-        6352 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive expression of VPR-FRB under the CMV promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6352)
  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 6352)
  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: pPD355 VPR-FRB (NLS)
FEATURES             Location/Qualifiers
     source          1..6352
                     /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"
     CDS             1633..1989
                     /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             2008..2577
                     /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(2982..2999)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2988..3212
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3258..3686
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3345..3364)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3555..3576
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3709..3905
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3756..3891
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3818..3837
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    4025..4146
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4062..4081)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4116..4135
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4195..4211)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4195..4211)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4208..4230)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4219..4235
                     /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(4243..4249,4250..4267,4268..4273))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4288..4309
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4426..4443)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4597..5185)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4677..4696)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5356..6147,6148..6216))
                     /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     5979..5998
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6217..6321)
                     /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 gttggtagcc agtacctgcc cgacaccgac gaccggcacc ggatcgagga aaagcggaag
     1261 cggacctacg agacattcaa gagcatcatg aagaagtccc ccttcagcgg ccccaccgac
     1321 cctagacctc cacctagaag aatcgccgtg cccagcagat ccagcgccag cgtgccaaaa
     1381 cctgcccccc agccttaccc cttcaccagc agcctgagca ccatcaacta cgacgagttc
     1441 cctaccatgg tgttccccag cggccagatc tctcaggcct ctgctctggc tccagcccct
     1501 cctcaggtgc tgcctcaggc tcctgctcct gcaccagctc cagccatggt gtctgcactg
     1561 gctcaggcac cagcacccgt gcctgtgctg gctcctggac ctccacaggc tgtggctcca
     1621 ccagccccta aacctacaca ggccggcgag ggcacactgt ctgaagctct gctgcagctg
     1681 cagttcgacg acgaggatct gggagccctg ctgggaaaca gcaccgatcc tgccgtgttc
     1741 accgacctgg ccagcgtgga caacagcgag ttccagcagc tgctgaacca gggcatccct
     1801 gtggcccctc acaccaccga gcccatgctg atggaatacc ccgaggccat cacccggctc
     1861 gtgacaggcg ctcagaggcc tcctgatcca gctcctgccc ctctgggagc accaggcctg
     1921 cctaatggac tgctgtctgg cgacgaggac ttcagctcta tcgccgatat ggatttctca
     1981 gccttgctgg gctctggcag cggcagccgg gattccaggg aagggatgtt tttgccgaag
     2041 cctgaggccg gctccgctat tagtgacgtg tttgagggcc gcgaggtgtg ccagccaaaa
     2101 cgaatccggc catttcatcc tccaggaagt ccatgggcca accgcccact ccccgccagc
     2161 ctcgcaccaa caccaaccgg tccagtacat gagccagtcg ggtcactgac cccggcacca
     2221 gtccctcagc cactggatcc agcgcccgca gtgactcccg aggccagtca cctgttggag
     2281 gatcccgatg aagagacgag ccaggctgtc aaagcccttc gggagatggc cgatactgtg
     2341 attccccaga aggaagaggc tgcaatctgt ggccaaatgg acctttccca tccgccccca
     2401 aggggccatc tggatgagct gacaaccaca cttgagtcca tgaccgagga tctgaacctg
     2461 gactcacccc tgaccccgga attgaacgag attctggata ccttcctgaa cgacgagtgc
     2521 ctcttgcatg ccatgcatat cagcacagga ctgtccatct tcgacacatc tctgttttct
     2581 ggttctggtt ctggttcagg atccggtggt ggcgggtctg gaggaggagg tagtggtgga
     2641 ggtggctctg gtaccatgct tgagatgtgg catgaaggcc tggaagaggc atctcgtttg
     2701 tactttgggg aaaggaacgt gaaaggcatg tttgaggtgc tggagccctt gcatgctatg
     2761 atggaacggg gaccccagac tctgaaggaa acatccttta atcaggccta tggtcgagat
     2821 ttaatggagg cccaagagtg gtgcaggaag tacatgaaat cagggaatgt caaggacctc
     2881 ctccaagcct gggacctcta ttatcatgtg ttccgacgaa tctcaaagac tagatgagcg
     2941 gccgctcgag tctagagggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag
     3001 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     3061 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     3121 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagtcaatag
     3181 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg
     3241 ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     3301 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     3361 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     3421 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     3481 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     3541 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     3601 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     3661 gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt aggctcaatt
     3721 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     3781 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     3841 cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     3901 gcaaaaagct cccgggagct tgtatatcca ttttcggagg ttgggcttcg gaatcgtttt
     3961 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
     4021 ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     4081 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     4141 atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata
     4201 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     4261 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     4321 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     4381 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     4441 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     4501 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     4561 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     4621 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     4681 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     4741 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     4801 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     4861 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     4921 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     4981 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     5041 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     5101 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     5161 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     5221 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     5281 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     5341 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     5401 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     5461 cttaccatct ggccccagtg ctgcaatgat accgcgagat ccacgctcac cggctccaga
     5521 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     5581 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     5641 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     5701 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     5761 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     5821 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     5881 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     5941 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     6001 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     6061 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     6121 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     6181 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     6241 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     6301 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tc
//
