LOCUS       sequence-364980-        6771 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tier-1 vector encoding PhCMV-driven VP64-P65-Rta expression 
            (PhCMV-VPR-pA).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6771)
  AUTHORS   Haellman V, Strittmatter T, Bertschi A, Stucheli P, Fussenegger M
  TITLE     A versatile plasmid architecture for mammalian synthetic biology 
            (VAMSyB).
  JOURNAL   Metab Eng. 2021 Jul;66:41-50. doi: 10.1016/j.ymben.2021.04.003. Epub
            2021 Apr 18.
  PUBMED    33857582
REFERENCE   2  (bases 1 to 6771)
  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: "Metab 
            Eng."; date: "2021-07"; volume: "66:41-50. doi"; pages: " 
            10.1016/j.ymben.2021.04.003. Epub 2021 Apr 18"
COMMENT     Sequence Label: pVH15-Tier1-PhCMV-VPR
FEATURES             Location/Qualifiers
     source          1..6771
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        143..522
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        523..726
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     677..697
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     771..790
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        771..789
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             845..994
                     /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             1019..1039
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1469..1825
                     /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             1844..2410
                     /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
                     DTFLNDECLLHAMHISTGLSIFDTSL"
     primer_bind     complement(2465..2482)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2471..2695
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2741..3169
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2828..2847)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3038..3059
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3178..3198)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3183..3512
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3363..3498
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3425..3444
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3579..4373
                     /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(3633..3652)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4243..4262
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4547..4668
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4584..4603)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4638..4657
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4717..4733)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4717..4733)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4730..4752)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4741..4757
                     /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(4765..4771,4772..4789,4790..4795))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4810..4831
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4948..4965)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5119..5707)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5199..5218)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5878..6669,6670..6738))
                     /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     6501..6520
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(6739..6771,1..72))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
       61 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatct
      121 ccacgcgtgg taccctcgag ttgacattga ttattgagta gttattaata gtaatcaatt
      181 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
      241 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
      301 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
      361 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
      421 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct
      481 acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag
      541 tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt
      601 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac
      661 aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc
      721 agagctctct ggctaactag agaacccact gcttactggc ttatcgaaat taatacgact
      781 cactataggg agacccaagc tgttcgaagc ggaattcacc atgactagtg gttccggacg
      841 ggctgacgca ttggacgatt ttgatctgga tatgctggga agtgacgccc tcgatgattt
      901 tgaccttgac atgcttggtt cggatgccct tgatgacttt gacctcgaca tgctcggcag
      961 tgacgccctt gatgatttcg acctggacat gctgattaac tctagaagtt ccggatctcc
     1021 gaaaaagaaa cgcaaagttg gtagccagta cctgcccgac accgacgacc ggcaccggat
     1081 cgaggaaaag cggaagcgga cctacgagac attcaagagc atcatgaaga agtccccctt
     1141 cagcggcccc accgacccta gacctccacc tagaagaatc gccgtgccca gcagatccag
     1201 cgccagcgtg ccaaaacctg ccccccagcc ttaccccttc accagcagcc tgagcaccat
     1261 caactacgac gagttcccta ccatggtgtt ccccagcggc cagatctctc aggcctctgc
     1321 tctggctcca gcccctcctc aggtgctgcc tcaggctcct gctcctgcac cagctccagc
     1381 catggtgtct gcactggctc aggcaccagc acccgtgcct gtgctggctc ctggacctcc
     1441 acaggctgtg gctccaccag cccctaaacc tacacaggcc ggcgagggca cactgtctga
     1501 agctctgctg cagctgcagt tcgacgacga ggatctggga gccctgctgg gaaacagcac
     1561 cgatcctgcc gtgttcaccg acctggccag cgtggacaac agcgagttcc agcagctgct
     1621 gaaccagggc atccctgtgg cccctcacac caccgagccc atgctgatgg aataccccga
     1681 ggccatcacc cggctcgtga caggcgctca gaggcctcct gatccagctc ctgcccctct
     1741 gggagcacca ggcctgccta atggactgct gtctggcgac gaggacttca gctctatcgc
     1801 cgatatggat ttctcagcct tgctgggctc tggcagcggc agccgggatt ccagggaagg
     1861 gatgtttttg ccgaagcctg aggccggctc cgctattagt gacgtgtttg agggccgcga
     1921 ggtgtgccag ccaaaacgaa tccggccatt tcatcctcca ggaagtccat gggccaaccg
     1981 cccactcccc gccagcctcg caccaacacc aaccggtcca gtacatgagc cagtcgggtc
     2041 actgaccccg gcaccagtcc ctcagccact ggatccagcg cccgcagtga ctcccgaggc
     2101 cagtcacctg ttggaggatc ccgatgaaga gacgagccag gctgtcaaag cccttcggga
     2161 gatggccgat actgtgattc cccagaagga agaggctgca atctgtggcc aaatggacct
     2221 ttcccatccg cccccaaggg gccatctgga tgagctgaca accacacttg agtccatgac
     2281 cgaggatctg aacctggact cacccctgac cccggaattg aacgagattc tggatacctt
     2341 cctgaacgac gagtgcctct tgcatgccat gcatatcagc acaggactgt ccatcttcga
     2401 cacatctctg gctagcggat ccaccggtgt ctagaaagct ttgaggccgg cctgcaggga
     2461 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     2521 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     2581 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     2641 ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct
     2701 gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca
     2761 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     2821 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     2881 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     2941 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     3001 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     3061 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     3121 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga
     3181 atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     3241 gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca
     3301 gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc
     3361 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     3421 tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag
     3481 gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt
     3541 tcggatctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc
     3601 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga
     3661 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt
     3721 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat
     3781 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg
     3841 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
     3901 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc
     3961 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga
     4021 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag
     4081 ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc
     4141 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
     4201 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata
     4261 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg
     4321 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac
     4381 tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc
     4441 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     4501 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc
     4561 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     4621 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat
     4681 accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     4741 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     4801 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     4861 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     4921 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     4981 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     5041 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     5101 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     5161 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     5221 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     5281 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     5341 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     5401 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     5461 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     5521 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     5581 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     5641 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     5701 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     5761 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     5821 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     5881 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     5941 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     6001 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     6061 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     6121 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     6181 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     6241 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     6301 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     6361 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     6421 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     6481 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     6541 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     6601 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     6661 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     6721 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt a
//
