LOCUS       sequence-322476-        6379 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Constitutive expression of VPR-FRB (NES) under the CMV promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6379)
  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 6379)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: pPD881 VPR-FRB (NES)
FEATURES             Location/Qualifiers
     source          1..6379
                     /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"
     CDS             1660..2016
                     /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             2035..2604
                     /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(3009..3026)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3015..3239
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3285..3713
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3372..3391)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3582..3603
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3736..3932
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3783..3918
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3845..3864
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    4052..4173
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4089..4108)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4143..4162
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4222..4238)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4222..4238)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4235..4257)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4246..4262
                     /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(4270..4276,4277..4294,4295..4300))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4315..4336
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4453..4470)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4624..5212)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4704..4723)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5383..6174,6175..6243))
                     /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     6006..6025
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6244..6348)
                     /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 cggatctgtt ggtagccagt acctgcccga caccgacgac
     1261 cggcaccgga tcgaggaaaa gcggaagcgg acctacgaga cattcaagag catcatgaag
     1321 aagtccccct tcagcggccc caccgaccct agacctccac ctagaagaat cgccgtgccc
     1381 agcagatcca gcgccagcgt gccaaaacct gccccccagc cttacccctt caccagcagc
     1441 ctgagcacca tcaactacga cgagttccct accatggtgt tccccagcgg ccagatctct
     1501 caggcctctg ctctggctcc agcccctcct caggtgctgc ctcaggctcc tgctcctgca
     1561 ccagctccag ccatggtgtc tgcactggct caggcaccag cacccgtgcc tgtgctggct
     1621 cctggacctc cacaggctgt ggctccacca gcccctaaac ctacacaggc cggcgagggc
     1681 acactgtctg aagctctgct gcagctgcag ttcgacgacg aggatctggg agccctgctg
     1741 ggaaacagca ccgatcctgc cgtgttcacc gacctggcca gcgtggacaa cagcgagttc
     1801 cagcagctgc tgaaccaggg catccctgtg gcccctcaca ccaccgagcc catgctgatg
     1861 gaataccccg aggccatcac ccggctcgtg acaggcgctc agaggcctcc tgatccagct
     1921 cctgcccctc tgggagcacc aggcctgcct aatggactgc tgtctggcga cgaggacttc
     1981 agctctatcg ccgatatgga tttctcagcc ttgctgggct ctggcagcgg cagccgggat
     2041 tccagggaag ggatgttttt gccgaagcct gaggccggct ccgctattag tgacgtgttt
     2101 gagggccgcg aggtgtgcca gccaaaacga atccggccat ttcatcctcc aggaagtcca
     2161 tgggccaacc gcccactccc cgccagcctc gcaccaacac caaccggtcc agtacatgag
     2221 ccagtcgggt cactgacccc ggcaccagtc cctcagccac tggatccagc gcccgcagtg
     2281 actcccgagg ccagtcacct gttggaggat cccgatgaag agacgagcca ggctgtcaaa
     2341 gcccttcggg agatggccga tactgtgatt ccccagaagg aagaggctgc aatctgtggc
     2401 caaatggacc tttcccatcc gcccccaagg ggccatctgg atgagctgac aaccacactt
     2461 gagtccatga ccgaggatct gaacctggac tcacccctga ccccggaatt gaacgagatt
     2521 ctggatacct tcctgaacga cgagtgcctc ttgcatgcca tgcatatcag cacaggactg
     2581 tccatcttcg acacatctct gttttctggt tctggttctg gttcaggatc cggtggtggc
     2641 gggtctggag gaggaggtag tggtggaggt ggctctggta ccatgcttga gatgtggcat
     2701 gaaggcctgg aagaggcatc tcgtttgtac tttggggaaa ggaacgtgaa aggcatgttt
     2761 gaggtgctgg agcccttgca tgctatgatg gaacggggac cccagactct gaaggaaaca
     2821 tcctttaatc aggcctatgg tcgagattta atggaggccc aagagtggtg caggaagtac
     2881 atgaaatcag ggaatgtcaa ggacctcctc caagcctggg acctctatta tcatgtgttc
     2941 cgacgaatct caaagactag atgagcggcc gctcgagtct agagggcccg tttaaacccg
     3001 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     3061 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     3121 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     3181 caagggggag gattgggaag tcaatagcag gcatgctggg gatgcggtgg gctctatggc
     3241 ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg
     3301 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     3361 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     3421 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     3481 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     3541 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     3601 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     3661 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg
     3721 tggaatgtgt gtcagttagg ctcaattagt cagcaaccat agtcccgccc ctaactccgc
     3781 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     3841 tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag
     3901 gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt
     3961 tcggaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg
     4021 ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta
     4081 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     4141 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag
     4201 ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     4261 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     4321 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     4381 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     4441 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     4501 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     4561 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     4621 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     4681 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     4741 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     4801 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     4861 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     4921 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     4981 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     5041 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     5101 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     5161 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     5221 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     5281 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     5341 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     5401 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     5461 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     5521 gcgagatcca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     5581 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     5641 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     5701 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     5761 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     5821 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     5881 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     5941 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     6001 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     6061 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     6121 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     6181 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     6241 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     6301 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     6361 aaaagtgcca cctgacgtc
//
