LOCUS       sequence-269557-        7158 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the P4 coiled coil 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 7158)
  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 7158)
  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-P4:gs:nls:VPR
FEATURES             Location/Qualifiers
     source          1..7158
                     /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             1085..1105
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1130..1279
                     /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             1748..2104
                     /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             2123..2692
                     /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(2711..2729)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2712..2729)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2749..2766)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2755..2979
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3025..3453
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3112..3131)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3322..3343
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3462..3482)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3467..3796
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3647..3782
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3709..3728
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3863..4657
                     /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(3917..3936)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4527..4546
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4831..4952
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4868..4887)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4922..4941
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5001..5017)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5001..5017)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5014..5036)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5025..5041
                     /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(5049..5055,5056..5073,5074..5079))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5094..5115
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5232..5249)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5403..5991)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5483..5502)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6162..6953,6954..7022))
                     /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     6785..6804
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7023..7127)
                     /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 tgagcccgga
      961 agataaaatt gctcagctga aacaaaaaat ccaagcgctg aaacaggaaa accagcagct
     1021 ggaagaggaa aacgccgcac tggaatatgg tggcggctct ggcggcggct ccggaggctc
     1081 tgatccaaaa aagaagagaa aggtagaggc cagcggttcg ggacgggctg acgcattgga
     1141 cgattttgat ctggatatgc tgggaagtga cgccctcgat gattttgacc ttgacatgct
     1201 tggttcggat gcccttgatg actttgacct cgacatgctc ggcagtgacg cccttgatga
     1261 tttcgacctg gacatgctga ttaacactag tggcggctct ggcggcggct ccggaggctc
     1321 tagccagtac ctgcccgaca ccgacgaccg gcaccggatc gaggaaaagc ggaagcggac
     1381 ctacgagaca ttcaagagca tcatgaagaa gtcccccttc agcggcccca ccgaccctag
     1441 acctccacct agaagaatcg ccgtgcccag cagatccagc gccagcgtgc caaaacctgc
     1501 cccccagcct taccccttca ccagcagcct gagcaccatc aactacgacg agttccctac
     1561 catggtgttc cccagcggcc agatctctca ggcctctgct ctggctccag cccctcctca
     1621 ggtgctgcct caggctcctg ctcctgcacc agctccagcc atggtgtctg cactggctca
     1681 ggcaccagca cccgtgcctg tgctggctcc tggacctcca caggctgtgg ctccaccagc
     1741 ccctaaacct acacaggccg gcgagggcac actgtctgaa gctctgctgc agctgcagtt
     1801 cgacgacgag gatctgggag ccctgctggg aaacagcacc gatcctgccg tgttcaccga
     1861 cctggccagc gtggacaaca gcgagttcca gcagctgctg aaccagggca tccctgtggc
     1921 ccctcacacc accgagccca tgctgatgga ataccccgag gccatcaccc ggctcgtgac
     1981 aggcgctcag aggcctcctg atccagctcc tgcccctctg ggagcaccag gcctgcctaa
     2041 tggactgctg tctggcgacg aggacttcag ctctatcgcc gatatggatt tctcagcctt
     2101 gctgggctct ggcagcggca gccgggattc cagggaaggg atgtttttgc cgaagcctga
     2161 ggccggctcc gctattagtg acgtgtttga gggccgcgag gtgtgccagc caaaacgaat
     2221 ccggccattt catcctccag gaagtccatg ggccaaccgc ccactccccg ccagcctcgc
     2281 accaacacca accggtccag tacatgagcc agtcgggtca ctgaccccgg caccagtccc
     2341 tcagccactg gacccagcgc ccgcagtgac tcccgaggcc agtcacctgt tggaggaccc
     2401 cgatgaagag acgagccagg ctgtcaaagc ccttcgggag atggccgata ctgtgattcc
     2461 ccagaaggaa gaggctgcaa tctgtggcca aatggacctt tcccatccgc ccccaagggg
     2521 ccatctggat gagctgacaa ccacacttga gtccatgacc gaggatctga acctggactc
     2581 acccctgacc ccggaattga acgagattct ggataccttc ctgaacgacg agtgcctctt
     2641 gcatgccatg catatcagca caggactgtc catcttcgac acatctctgt tttgatctag
     2701 agggccctat tctatagtgt cacctaaatg ctagagctcg ctgatcagcc tcgactgtgc
     2761 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag
     2821 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta
     2881 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag
     2941 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca
     3001 gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     3061 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     3121 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     3181 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     3241 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cttttgacgt
     3301 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     3361 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     3421 atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg
     3481 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     3541 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     3601 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac
     3661 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     3721 ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     3781 cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcaaga
     3841 gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc
     3901 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga
     3961 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct
     4021 gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac
     4081 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct
     4141 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt
     4201 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt
     4261 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt
     4321 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag
     4381 gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt
     4441 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg
     4501 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg
     4561 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg
     4621 catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg
     4681 accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat
     4741 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     4801 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac
     4861 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     4921 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc
     4981 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     5041 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     5101 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     5161 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     5221 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     5281 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     5341 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     5401 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     5461 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     5521 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     5581 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     5641 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     5701 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     5761 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     5821 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     5881 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     5941 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     6001 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     6061 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     6121 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     6181 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     6241 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     6301 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     6361 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     6421 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     6481 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     6541 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     6601 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     6661 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     6721 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     6781 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     6841 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     6901 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     6961 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     7021 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     7081 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     7141 aaagtgccac ctgacgtc
//
