LOCUS       sequence-450284-        4149 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Validates the expression and detection of epitope-tagged proteins of
            interest in bacterial cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4149)
  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 4149)
  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: pEXP1/Joseph2-Multitagged Plasmid
FEATURES             Location/Qualifiers
     source          1..4149
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     252..269
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     354..373
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        354..372
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             404..426
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             446..463
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             467..499
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(503..511,512..526)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     protein_bind    536..560
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             578..658
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             689..778
                     /codon_start=1
                     /product="3 tandem Myc epitope tags"
                     /label=3xMyc
                     /translation="EQKLISEEDLEQKLISEEDLEQKLISEEDL"
     CDS             809..1522
                     /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             809..1522
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGQKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFYKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKMEYNYNSHNVYIMADKPKNGIKV
                     NFKIRHNIKDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMILLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(839..867)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     CDS             1553..1624
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
     CDS             1655..1696
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1697..1738
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1739..1780
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     protein_bind    complement(1797..1821)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(1884..1902)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1898..1945
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2042..2497
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2129..2148)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2339..2360
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2523..2627
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2628..2696,2697..3488)
                     /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     complement(2846..2865)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(3659..4149,1..98)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     join(4148..4149,1..18)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
       61 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
      121 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
      181 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
      241 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc
      301 ctctccccgc gcgttggccg attcattaat gcaggatctc gatcccgcga aattaatacg
      361 actcactata gggagaccac aacggtttcc ctctagaaat aattttgttt aactttaaga
      421 aggagatata catatgcggg gttctcatca tcatcatcat catggtatgg ctagcatgac
      481 tggtggacag caaatgggtc gggatctgta cgacgatgac gataaggatc atcaaacaag
      541 tttgtacaaa aaagcaggct tcagtggaag cggttgctat ccctacgatg tccctgacta
      601 cgcctaccca tacgacgttc cggattatgc ttacccgtat gatgtgccag actacgcggg
      661 aagtggctcc ggatcgggca gcggaagcga gcaaaagctc atctcggagg aagatctaga
      721 acagaagcta atatcagagg aggacctaga gcaaaaactt atttcggaag aagatctggg
      781 cagcggatcc ggttcgggaa gcggtagtat gagtaaagga gaagaacttt tcactggagt
      841 tgtcccaatt cttgttgaat tagatggcga tgttaatggg caaaaattct ctgtcagtgg
      901 agagggtgaa ggtgatgcaa catacggaaa acttaccctt aaatttattt gcactactgg
      961 gaagctacct gttccatggc caacacttgt cactactttg acttatggtg ttcaatgctt
     1021 ttcaagatac ccagatcata tgaaacagca tgactttttc aagagtgcca tgcccgaagg
     1081 ttatgtacag gaaagaacta tattttacaa agatgacggg aactacaaga cacgtgctga
     1141 agtcaagttt gaaggtgata cccttgttaa tagaatcgag ttaaaaggta ttgattttaa
     1201 agaagatgga aacattcttg gacacaaaat ggaatacaac tataactcac ataatgtata
     1261 catcatggca gacaaaccaa agaatggaat caaagttaac ttcaaaatta gacacaacat
     1321 taaagatgga agcgttcaat tagcagacca ttatcaacaa aatactccaa ttggcgatgg
     1381 ccctgtcctt ttaccagaca accattacct gtccacacaa tctgcccttt ccaaggatcc
     1441 caacgaaaag agagatcaca tgatccttct tgagtttgta acagctgctg ggattacaca
     1501 tggcatggat gaactataca aaggtagtgg ttccggtagc ggcagtggga gtgattataa
     1561 agacgatgat gacaaggact ataaggacga cgacgacaag gattacaagg atgatgacga
     1621 caaaggctcc ggttcggggt cgggaagcgg tagtggtaaa ccgattccca atccccttct
     1681 tggtcttgat tctacaggca aacccatccc gaacccactc ctcggccttg actccacagg
     1741 caagcctatt ccaaatcctc tactaggcct cgattctacg ggtagcggtt cgtaggaccc
     1801 agctttcttg tacaaagtgg ttcgatcgaa gcttgatccg gctgctaaca aagcccgaaa
     1861 ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc
     1921 taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat ctggcgtaat
     1981 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
     2041 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     2101 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     2161 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     2221 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     2281 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     2341 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     2401 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     2461 aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg
     2521 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     2581 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     2641 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     2701 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     2761 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     2821 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     2881 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     2941 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     3001 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     3061 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     3121 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     3181 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     3241 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     3301 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     3361 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     3421 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     3481 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     3541 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     3601 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     3661 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     3721 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     3781 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     3841 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     3901 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     3961 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     4021 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     4081 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     4141 ttccagggg
//
