LOCUS       sequence-366218-        7407 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tier-1 vector encoding PhCMV-driven tetracycline-dependent VPR-based
            transactiavtor (PhCMV-TetR-VPR-pA)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7407)
  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 7407)
  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: pAB303-Tier1-PhCMV-TetR_VPR
FEATURES             Location/Qualifiers
     source          1..7407
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(58..78)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        63..392
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      243..378
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     305..324
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             459..1253
                     /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(513..532)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1123..1142
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    1427..1548
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1464..1483)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1518..1537
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1597..1613)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1597..1613)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1610..1632)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1621..1637
                     /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(1645..1651,1652..1669,1670..1675))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1690..1711
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1828..1845)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1999..2587)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2079..2098)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2758..3549,3550..3618))
                     /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     3381..3400
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3619..3723)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        3794..4173
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4174..4377
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4328..4348
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4422..4441
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4422..4440
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             4493..5110
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="SRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVK
                     NKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRP
                     TEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTD
                     SMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(4563..4582)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             5132..5281
                     /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             5306..5326
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5756..6112
                     /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             6131..6697
                     /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(6752..6769)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6758..6982
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      join(7028..7407,1..49)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7115..7134)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7325..7346
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga
       61 atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
      121 gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca
      181 gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc
      241 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
      301 tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag
      361 gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt
      421 tcggatctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc
      481 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga
      541 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt
      601 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat
      661 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg
      721 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
      781 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc
      841 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga
      901 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag
      961 ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc
     1021 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
     1081 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata
     1141 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg
     1201 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac
     1261 tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc
     1321 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     1381 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc
     1441 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     1501 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat
     1561 accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     1621 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     1681 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     1741 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     1801 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     1861 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     1921 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     1981 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     2041 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     2101 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     2161 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     2221 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     2281 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     2341 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     2401 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     2461 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     2521 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     2581 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     2641 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     2701 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     2761 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     2821 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     2881 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     2941 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     3001 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     3061 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     3121 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     3181 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     3241 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     3301 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     3361 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     3421 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     3481 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     3541 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     3601 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     3661 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     3721 gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc tccacgcgtg
     3781 gtaccctcga gttgacattg attattgagt agttattaat agtaatcaat tacggggtca
     3841 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
     3901 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
     3961 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
     4021 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
     4081 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
     4141 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
     4201 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
     4261 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
     4321 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc
     4381 tggctaacta gagaacccac tgcttactgg cttatcgaaa ttaatacgac tcactatagg
     4441 gagacccaag ctgttcgaag cggaattcac catgactagt ggtggttctg gttccagatt
     4501 agataaaagt aaagtgatta acagcgcatt agagctgctt aatgaggtcg gaatcgaagg
     4561 tttaacaacc cgtaaactcg cccagaagct aggtgtagag cagcctacat tgtattggca
     4621 tgtaaaaaat aagcgggctt tgctcgacgc cttagccatt gagatgttag ataggcacca
     4681 tactcacttt tgccctttag aaggggaaag ctggcaagat tttttacgta ataacgctaa
     4741 aagttttaga tgtgctttac taagtcatcg cgatggagca aaagtacatt taggtacacg
     4801 gcctacagaa aaacagtatg aaactctcga aaatcaatta gcctttttat gccaacaagg
     4861 tttttcacta gagaatgcat tatatgcact cagcgctgtg gggcatttta ctttaggttg
     4921 cgtattggaa gatcaagagc atcaagtcgc taaagaagaa agggaaacac ctactactga
     4981 tagtatgccg ccattattac gacaagctat cgaattattt gatcaccaag gtgcagagcc
     5041 agccttctta ttcggccttg aattgatcat atgcggatta gaaaaacaac ttaaatgtga
     5101 aagtgggtcc gctagtggtt ccggacgggc tgacgcattg gacgattttg atctggatat
     5161 gctgggaagt gacgccctcg atgattttga ccttgacatg cttggttcgg atgcccttga
     5221 tgactttgac ctcgacatgc tcggcagtga cgcccttgat gatttcgacc tggacatgct
     5281 gattaactct agaagttccg gatctccgaa aaagaaacgc aaagttggta gccagtacct
     5341 gcccgacacc gacgaccggc accggatcga ggaaaagcgg aagcggacct acgagacatt
     5401 caagagcatc atgaagaagt cccccttcag cggccccacc gaccctagac ctccacctag
     5461 aagaatcgcc gtgcccagca gatccagcgc cagcgtgcca aaacctgccc cccagcctta
     5521 ccccttcacc agcagcctga gcaccatcaa ctacgacgag ttccctacca tggtgttccc
     5581 cagcggccag atctctcagg cctctgctct ggctccagcc cctcctcagg tgctgcctca
     5641 ggctcctgct cctgcaccag ctccagccat ggtgtctgca ctggctcagg caccagcacc
     5701 cgtgcctgtg ctggctcctg gacctccaca ggctgtggct ccaccagccc ctaaacctac
     5761 acaggccggc gagggcacac tgtctgaagc tctgctgcag ctgcagttcg acgacgagga
     5821 tctgggagcc ctgctgggaa acagcaccga tcctgccgtg ttcaccgacc tggccagcgt
     5881 ggacaacagc gagttccagc agctgctgaa ccagggcatc cctgtggccc ctcacaccac
     5941 cgagcccatg ctgatggaat accccgaggc catcacccgg ctcgtgacag gcgctcagag
     6001 gcctcctgat ccagctcctg cccctctggg agcaccaggc ctgcctaatg gactgctgtc
     6061 tggcgacgag gacttcagct ctatcgccga tatggatttc tcagccttgc tgggctctgg
     6121 cagcggcagc cgggattcca gggaagggat gtttttgccg aagcctgagg ccggctccgc
     6181 tattagtgac gtgtttgagg gccgcgaggt gtgccagcca aaacgaatcc ggccatttca
     6241 tcctccagga agtccatggg ccaaccgccc actccccgcc agcctcgcac caacaccaac
     6301 cggtccagta catgagccag tcgggtcact gaccccggca ccagtccctc agccactgga
     6361 tccagcgccc gcagtgactc ccgaggccag tcacctgttg gaggatcccg atgaagagac
     6421 gagccaggct gtcaaagccc ttcgggagat ggccgatact gtgattcccc agaaggaaga
     6481 ggctgcaatc tgtggccaaa tggacctttc ccatccgccc ccaaggggcc atctggatga
     6541 gctgacaacc acacttgagt ccatgaccga ggatctgaac ctggactcac ccctgacccc
     6601 ggaattgaac gagattctgg ataccttcct gaacgacgag tgcctcttgc atgccatgca
     6661 tatcagcaca ggactgtcca tcttcgacac atctctggct agcggatcca ccggtgtcta
     6721 gaaagctttg aggccggcct gcagggatca gcctcgactg tgccttctag ttgccagcca
     6781 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     6841 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     6901 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct
     6961 ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctctaggggg
     7021 tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     7081 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     7141 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     7201 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     7261 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     7321 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     7381 gatttataag ggattttgcc gatttcg
//
