LOCUS       sequence-268598-        7335 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the FRB rapamycin-inducible domain fused to the VPR 
            transcriptional activation domain in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7335)
  AUTHORS   Lebar T, Lainscek D, Merljak E, Aupic J, Jerala R
  TITLE     A tunable orthogonal coiled-coil interaction toolbox for engineering
            mammalian cells.
  JOURNAL   Nat Chem Biol. 2020 Jan 6. pii: 10.1038/s41589-019-0443-y. doi: 
            10.1038/s41589-019-0443-y.
  PUBMED    31907374
REFERENCE   2  (bases 1 to 7335)
  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 Chem 
            Biol. 2020 Jan 6. pii: 10.1038/s41589-019-0443-y. doi: 
            10.1038/s41589-019-0443-y."
COMMENT     Sequence Label: pcDNA3-His:FRB:nls:VPR
FEATURES             Location/Qualifiers
     source          1..7335
                     /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"
     CDS             953..970
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             980..1258
                     /codon_start=1
                     /product="T2098L mutant of FKBP-rapamycin binding domain of
                     human FRAP"
                     /label=FRB*
                     /note="binds synthetic ligands such as AP21967"
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK"
     CDS             1262..1282
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1307..1456
                     /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             1925..2281
                     /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             2300..2869
                     /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"
     promoter        complement(2888..2906)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2889..2906)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2926..2943)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2932..3156
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3202..3630
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3289..3308)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3499..3520
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3639..3659)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3644..3973
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3824..3959
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3886..3905
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4040..4834
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4094..4113)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4704..4723
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5008..5129
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5045..5064)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5099..5118
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5178..5194)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5178..5194)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5191..5213)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5202..5218
                     /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(5226..5232,5233..5250,5251..5256))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5271..5292
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5409..5426)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5580..6168)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5660..5679)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6339..7130,7131..7199))
                     /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     6962..6981
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7200..7304)
                     /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 ggagacccaa gcttggtacc
      901 gagctcggat ccactagtaa cggccgccag tgtgctggaa ttcgccacca tgcaccacca
      961 ccaccaccac gcttctagaa tcctctggca tgagatgtgg catgaaggcc tggaagaggc
     1021 atctcgtttg tactttgggg aaaggaacgt gaaaggcatg tttgaggtgc tggagccctt
     1081 gcatgctatg atggaacggg gcccccagac tctgaaggaa acatccttta atcaggccta
     1141 tggtcgagat ttaatggagg cccaagagtg gtgcaggaag tacatgaaat cagggaatgt
     1201 caaggacctc ctccaagcct gggacctcta ttatcatgtg ttccgacgaa tctcaaagga
     1261 tccaaaaaag aagagaaagg tagaggccag cggttcggga cgggctgacg cattggacga
     1321 ttttgatctg gatatgctgg gaagtgacgc cctcgatgat tttgaccttg acatgcttgg
     1381 ttcggatgcc cttgatgact ttgacctcga catgctcggc agtgacgccc ttgatgattt
     1441 cgacctggac atgctgatta acactagtgg cggctctggc ggcggctccg gaggctctag
     1501 ccagtacctg cccgacaccg acgaccggca ccggatcgag gaaaagcgga agcggaccta
     1561 cgagacattc aagagcatca tgaagaagtc ccccttcagc ggccccaccg accctagacc
     1621 tccacctaga agaatcgccg tgcccagcag atccagcgcc agcgtgccaa aacctgcccc
     1681 ccagccttac cccttcacca gcagcctgag caccatcaac tacgacgagt tccctaccat
     1741 ggtgttcccc agcggccaga tctctcaggc ctctgctctg gctccagccc ctcctcaggt
     1801 gctgcctcag gctcctgctc ctgcaccagc tccagccatg gtgtctgcac tggctcaggc
     1861 accagcaccc gtgcctgtgc tggctcctgg acctccacag gctgtggctc caccagcccc
     1921 taaacctaca caggccggcg agggcacact gtctgaagct ctgctgcagc tgcagttcga
     1981 cgacgaggat ctgggagccc tgctgggaaa cagcaccgat cctgccgtgt tcaccgacct
     2041 ggccagcgtg gacaacagcg agttccagca gctgctgaac cagggcatcc ctgtggcccc
     2101 tcacaccacc gagcccatgc tgatggaata ccccgaggcc atcacccggc tcgtgacagg
     2161 cgctcagagg cctcctgatc cagctcctgc ccctctggga gcaccaggcc tgcctaatgg
     2221 actgctgtct ggcgacgagg acttcagctc tatcgccgat atggatttct cagccttgct
     2281 gggctctggc agcggcagcc gggattccag ggaagggatg tttttgccga agcctgaggc
     2341 cggctccgct attagtgacg tgtttgaggg ccgcgaggtg tgccagccaa aacgaatccg
     2401 gccatttcat cctccaggaa gtccatgggc caaccgccca ctccccgcca gcctcgcacc
     2461 aacaccaacc ggtccagtac atgagccagt cgggtcactg accccggcac cagtccctca
     2521 gccactggac ccagcgcccg cagtgactcc cgaggccagt cacctgttgg aggaccccga
     2581 tgaagagacg agccaggctg tcaaagccct tcgggagatg gccgatactg tgattcccca
     2641 gaaggaagag gctgcaatct gtggccaaat ggacctttcc catccgcccc caaggggcca
     2701 tctggatgag ctgacaacca cacttgagtc catgaccgag gatctgaacc tggactcacc
     2761 cctgaccccg gaattgaacg agattctgga taccttcctg aacgacgagt gcctcttgca
     2821 tgccatgcat atcagcacag gactgtccat cttcgacaca tctctgtttt gatctagagg
     2881 gccctattct atagtgtcac ctaaatgcta gagctcgctg atcagcctcg actgtgcctt
     2941 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     3001 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     3061 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     3121 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
     3181 ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     3241 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     3301 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     3361 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     3421 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgcctt ttgacgttgg
     3481 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     3541 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     3601 agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg
     3661 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     3721 agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     3781 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
     3841 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     3901 cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     3961 aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcaagagac
     4021 aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc
     4081 ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc
     4141 cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc
     4201 cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg
     4261 cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt
     4321 gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc
     4381 catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga
     4441 ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga
     4501 tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct
     4561 caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc
     4621 gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt
     4681 ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg
     4741 cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat
     4801 cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc
     4861 gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa
     4921 aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat
     4981 ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa
     5041 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     5101 ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag
     5161 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     5221 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     5281 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     5341 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     5401 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     5461 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     5521 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     5581 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     5641 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     5701 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     5761 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     5821 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     5881 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     5941 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     6001 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     6061 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     6121 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     6181 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     6241 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     6301 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     6361 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     6421 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     6481 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     6541 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     6601 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     6661 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     6721 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     6781 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     6841 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     6901 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     6961 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     7021 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     7081 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     7141 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     7201 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     7261 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     7321 gtgccacctg acgtc
//
