LOCUS       sequence-450280-        6294 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Validates the expression and detection of epitope-tagged proteins of
            interest in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6294)
  AUTHORS   Robinson E, Alonso EB, A Waters J, Bileckyj C, D House C, A Johnston
            C, M Cripps R
  TITLE     pJoseph2: a family of plasmids as positive controls for bacterial 
            protein expression, transfections, and western blots.
  JOURNAL   Biotechniques. 2024;76(7):299-309. doi: 
            10.1080/07366205.2024.2343609. Epub 2024 May 12.
  PUBMED    39185782
REFERENCE   2  (bases 1 to 6294)
  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: 
            "Biotechniques."; date: "2024"; volume: "76(7):299-309. doi"; pages:
            " 10.1080/07366205.2024.2343609. Epub 2024 May 12"
COMMENT     Sequence Label: pMH-HA/Joseph2-Multitagged Plasmid
FEATURES             Location/Qualifiers
     source          1..6294
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      179..314
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     241..260
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             354..1148
                     /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="MVEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(408..427)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1018..1037
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    1322..1443
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1359..1378)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1413..1432
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1476..1493)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1647..2235)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1727..1746)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2406..3197,3198..3266))
                     /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     3029..3048
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3267..3371)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        3436..3812
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3814..4017
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3968..3988
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      4074..4083
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4101..4127
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4128..4154
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4173..4193
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     protein_bind    4194..4218
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             4233..4322
                     /codon_start=1
                     /product="3 tandem Myc epitope tags"
                     /label=3xMyc
                     /translation="EQKLISEEDLEQKLISEEDLEQKLISEEDL"
     CDS             4353..5066
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MSKGEELFTGVVPILVELDGDVNGQKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFYKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKMEYNYNSHNVYIMADKPKNGIKV
                     NFKIRHNIKDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMILLE
                     FVTAAGITHGMDELYK"
     CDS             4353..5066
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGQKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFYKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKMEYNYNSHNVYIMADKPKNGIKV
                     NFKIRHNIKDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMILLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(4383..4411)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     CDS             5097..5168
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
     CDS             5199..5240
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             5241..5282
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             5283..5324
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             5355..5372
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    complement(5377..5401)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             5429..5449
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5453..5473
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5477..5497
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5510..5539
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(5575..5592)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5581..5805
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      5851..6279
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5938..5957)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6148..6169
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(join(6288..6294,1..14))
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        join(6293..6294,1..328)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
ORIGIN
        1 gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
       61 gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag
      121 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
      181 cccgccccta actccgccca gttccgccca ttctccgccc ctaggctgac taattttttt
      241 tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg
      301 cttttttgga ggcctaggct tttgcaaaaa gctccccccc cgggaggtcc acaatggttg
      361 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
      421 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
      481 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctccaggacg
      541 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg
      601 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
      661 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
      721 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
      781 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
      841 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgtatgccc gacggcgagg
      901 atctcgtcgt gactcatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
      961 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
     1021 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
     1081 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
     1141 tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc
     1201 acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg
     1261 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc
     1321 caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     1381 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     1441 ttatcatgtc tgtataccgt cgatctttcc gcttcctcgc tcactgactc gctgcgctcg
     1501 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     1561 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     1621 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     1681 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     1741 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     1801 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     1861 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgta tgcacgaacc ccccgttcag
     1921 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     1981 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     2041 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     2101 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     2161 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     2221 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     2281 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     2341 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     2401 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     2461 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     2521 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     2581 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     2641 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     2701 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     2761 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     2821 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     2881 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     2941 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     3001 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     3061 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     3121 agatccagtt cgatgtaacc cactcgggca cccaactgat cttcagcatc ttttactttc
     3181 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     3241 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     3301 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     3361 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcagatcgac ggatcgggag
     3421 atcggtaccg aattcacatt gattattgag tagttattaa tagtaatcaa ttacggggtc
     3481 attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc
     3541 tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt
     3601 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca
     3661 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg
     3721 taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca
     3781 gtacatctac ggttagtcat cgctattacc atagtgatgc ggttttggca gtacatcaat
     3841 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
     3901 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
     3961 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctttc
     4021 tggctaacta gagaacccac tgcttactgg cacgtggaaa ttaatacgac gtggccacca
     4081 tggcccaggt gcagctgcag tacccatacg acgtgccaga ctacgcatac ccatacgacg
     4141 tgccagacta cgcactgcag gtcgacctcg aggaaaacct gtattttcag ggcacaagtt
     4201 tgtacaaaaa agcaggcttc ggcagcggaa gcgagcaaaa gctcatctcg gaggaagatc
     4261 tagaacagaa gctaatatca gaggaggacc tagagcaaaa acttatttcg gaagaagatc
     4321 tgggcagcgg atccggttcg ggaagcggta gtatgagtaa aggagaagaa cttttcactg
     4381 gagttgtccc aattcttgtt gaattagatg gcgatgttaa tgggcaaaaa ttctctgtca
     4441 gtggagaggg tgaaggtgat gcaacatacg gaaaacttac ccttaaattt atttgcacta
     4501 ctgggaagct acctgttcca tggccaacac ttgtcactac tttgacttat ggtgttcaat
     4561 gcttttcaag atacccagat catatgaaac agcatgactt tttcaagagt gccatgcccg
     4621 aaggttatgt acaggaaaga actatatttt acaaagatga cgggaactac aagacacgtg
     4681 ctgaagtcaa gtttgaaggt gatacccttg ttaatagaat cgagttaaaa ggtattgatt
     4741 ttaaagaaga tggaaacatt cttggacaca aaatggaata caactataac tcacataatg
     4801 tatacatcat ggcagacaaa ccaaagaatg gaatcaaagt taacttcaaa attagacaca
     4861 acattaaaga tggaagcgtt caattagcag accattatca acaaaatact ccaattggcg
     4921 atggccctgt ccttttacca gacaaccatt acctgtccac acaatctgcc ctttccaaag
     4981 atcccaacga aaagagagat cacatgatcc ttcttgagtt tgtaacagct gctgggatta
     5041 cacatggcat ggatgaacta tacaaaggta gtggttccgg tagcggcagt gggagtgatt
     5101 ataaagacga tgatgacaag gactataagg acgacgacga caaggattac aaggatgatg
     5161 acgacaaagg ctccggttcg gggtcgggaa gcggtagtgg taaaccgatt cccaatcccc
     5221 ttcttggtct tgattctaca ggcaaaccca tcccgaaccc actcctcggc cttgactcca
     5281 caggcaagcc tattccaaat cctctactag gcctcgattc tacgggtagc ggttcgggat
     5341 ccggaagtgg aagccaccat catcatcacc attaggaccc agctttcttg tacaaagtgg
     5401 tctcgagatc aaacgggcgg ccgcagatcc aaaaaagaag agaaaggtag atccaaaaaa
     5461 gaagagaaag gtagatccaa aaaagaagag aaaggtagat acggccgcag aacaaaaact
     5521 catctcagaa gaggatctga atggggccgc atagtctaga agctcgctga tcagcctcga
     5581 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc
     5641 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc
     5701 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt
     5761 gggaagacaa tagcaggcat gctggggatg gcccgggctc tatggcttct gaggcggaaa
     5821 gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg
     5881 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     5941 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     6001 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctctcc cccaaaaaac
     6061 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     6121 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     6181 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     6241 taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga atgt
//
