LOCUS       sequence-365008-        7398 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tier-1 vector encoding PhCMV-driven tetracycline-activated VPR-based
            transactiavtor (PhCMV-rTetR-VPR-pA)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7398)
  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 7398)
  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: pAB301-Tier1-PhCMV-rTetR_VPR
FEATURES             Location/Qualifiers
     source          1..7398
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        99..478
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        479..682
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     633..653
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     727..746
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        727..745
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1428..1577
                     /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             1602..1622
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2052..2408
                     /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             2427..2993
                     /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(3048..3065)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3054..3278
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3324..3752
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3411..3430)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3621..3642
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3761..3781)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3766..4095
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3946..4081
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4008..4027
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4162..4956
                     /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(4216..4235)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4826..4845
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5130..5251
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5167..5186)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5221..5240
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5300..5316)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5300..5316)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5313..5335)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5324..5340
                     /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(5348..5354,5355..5372,5373..5378))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5393..5414
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5531..5548)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5702..6290)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5782..5801)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6461..7252,7253..7321))
                     /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     7084..7103
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7322..7398,1..28))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg
       61 acggatcggg agatctccac gcgtggtacc ctcgagttga cattgattat tgagtagtta
      121 ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac
      181 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc
      241 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt
      301 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac
      361 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac
      421 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
      481 gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc
      541 aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt
      601 tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg
      661 ggaggtctat ataagcagag ctctctggct aactagagaa cccactgctt actggcttat
      721 cgaaattaat acgactcact atagggagac ccaagctgtt cgaagcggaa ttcaccatga
      781 ctagtggatc aagactggac aagagcaaag tcataaactc tgctctggaa ttactcaatg
      841 gagtcggtat cgaaggcctg acgacaagga aactcgctca aaagctggga gttgagcagc
      901 ctaccctgta ctggcacgtg aagaacaagc gggccctgct cgatgccctg ccaatcgaga
      961 tgctggacag gcatcatacc cactcctgcc ccctggaagg cgagtcatgg caagactttc
     1021 tgcggaacaa cgccaagtca taccgctgtg ctctcctctc acatcgcgac ggggctaaag
     1081 tgcatctcgg cacccgccca acagagaaac agtacgaaac cctggaaaat cagctcgcgt
     1141 tcctgtgtca gcaaggcttc tccctggaga acgcactgta cgctctgtcc gccgtgggcc
     1201 actttacact gggctgcgta ttggaggaac aggagcatca agtagcaaaa gaggaaagag
     1261 agacacctac caccgattct atgcccccac ttctgaaaca agcaattgag ctgttcgacc
     1321 ggcagggagc cgaacctgcc ttccttttcg gcctggaact aatcatatgt ggcctggaga
     1381 aacagctaaa gtgcgaaagc ggcggggcta gtggttccgg acgggctgac gcattggacg
     1441 attttgatct ggatatgctg ggaagtgacg ccctcgatga ttttgacctt gacatgcttg
     1501 gttcggatgc ccttgatgac tttgacctcg acatgctcgg cagtgacgcc cttgatgatt
     1561 tcgacctgga catgctgatt aactctagaa gttccggatc tccgaaaaag aaacgcaaag
     1621 ttggtagcca gtacctgccc gacaccgacg accggcaccg gatcgaggaa aagcggaagc
     1681 ggacctacga gacattcaag agcatcatga agaagtcccc cttcagcggc cccaccgacc
     1741 ctagacctcc acctagaaga atcgccgtgc ccagcagatc cagcgccagc gtgccaaaac
     1801 ctgcccccca gccttacccc ttcaccagca gcctgagcac catcaactac gacgagttcc
     1861 ctaccatggt gttccccagc ggccagatct ctcaggcctc tgctctggct ccagcccctc
     1921 ctcaggtgct gcctcaggct cctgctcctg caccagctcc agccatggtg tctgcactgg
     1981 ctcaggcacc agcacccgtg cctgtgctgg ctcctggacc tccacaggct gtggctccac
     2041 cagcccctaa acctacacag gccggcgagg gcacactgtc tgaagctctg ctgcagctgc
     2101 agttcgacga cgaggatctg ggagccctgc tgggaaacag caccgatcct gccgtgttca
     2161 ccgacctggc cagcgtggac aacagcgagt tccagcagct gctgaaccag ggcatccctg
     2221 tggcccctca caccaccgag cccatgctga tggaataccc cgaggccatc acccggctcg
     2281 tgacaggcgc tcagaggcct cctgatccag ctcctgcccc tctgggagca ccaggcctgc
     2341 ctaatggact gctgtctggc gacgaggact tcagctctat cgccgatatg gatttctcag
     2401 ccttgctggg ctctggcagc ggcagccggg attccaggga agggatgttt ttgccgaagc
     2461 ctgaggccgg ctccgctatt agtgacgtgt ttgagggccg cgaggtgtgc cagccaaaac
     2521 gaatccggcc atttcatcct ccaggaagtc catgggccaa ccgcccactc cccgccagcc
     2581 tcgcaccaac accaaccggt ccagtacatg agccagtcgg gtcactgacc ccggcaccag
     2641 tccctcagcc actggatcca gcgcccgcag tgactcccga ggccagtcac ctgttggagg
     2701 atcccgatga agagacgagc caggctgtca aagcccttcg ggagatggcc gatactgtga
     2761 ttccccagaa ggaagaggct gcaatctgtg gccaaatgga cctttcccat ccgcccccaa
     2821 ggggccatct ggatgagctg acaaccacac ttgagtccat gaccgaggat ctgaacctgg
     2881 actcacccct gaccccggaa ttgaacgaga ttctggatac cttcctgaac gacgagtgcc
     2941 tcttgcatgc catgcatatc agcacaggac tgtccatctt cgacacatct ctggctagcg
     3001 gatccaccgg tgtctagaaa gctttgaggc cggcctgcag ggatcagcct cgactgtgcc
     3061 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     3121 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     3181 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     3241 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     3301 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     3361 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     3421 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     3481 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     3541 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     3601 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     3661 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     3721 tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg
     3781 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     3841 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     3901 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
     3961 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     4021 gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     4081 ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag
     4141 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc
     4201 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     4261 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     4321 tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg
     4381 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     4441 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
     4501 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     4561 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     4621 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     4681 ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     4741 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     4801 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     4861 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     4921 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga
     4981 ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg
     5041 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
     5101 atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca
     5161 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     5221 gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct
     5281 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     5341 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     5401 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     5461 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     5521 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     5581 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     5641 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     5701 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     5761 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     5821 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     5881 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     5941 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     6001 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     6061 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     6121 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     6181 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     6241 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     6301 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     6361 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     6421 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     6481 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     6541 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     6601 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     6661 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     6721 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     6781 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     6841 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     6901 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     6961 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     7021 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     7081 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     7141 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     7201 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     7261 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     7321 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     7381 acatatttga atgtattt
//
