LOCUS       sequence-450283-        5995 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Validates the expression and detection of epitope-tagged proteins of
            interest in Drosophila cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5995)
  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 5995)
  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: pAHW/Joseph2-Multitagged Plasmid
FEATURES             Location/Qualifiers
     source          1..5995
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      2226..2235
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2265..2354
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     protein_bind    2376..2400
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2415..2504
                     /codon_start=1
                     /product="3 tandem Myc epitope tags"
                     /label=3xMyc
                     /translation="EQKLISEEDLEQKLISEEDLEQKLISEEDL"
     CDS             2535..3248
                     /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             2535..3248
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGQKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFYKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKMEYNYNSHNVYIMADKPKNGIKV
                     NFKIRHNIKDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMILLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(2565..2593)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     CDS             3279..3350
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
     CDS             3381..3422
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             3423..3464
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             3465..3506
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             3537..3554
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    complement(3559..3658)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3675..3694)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3676..3694)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3699..3715)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3699..3715)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3817..3836)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3828..4637
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(3895..3914)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4784..5372
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5273..5292
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5526..5543
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(5702..5729)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 attttcggac tgtccgcttt gatttcagtt tcttggctta ttcaaaaagc aaagtaaagc
       61 caaaaaagcg agatggcaat accaaatgcg gcaaaacggt agtggaagga aaggggtgcg
      121 gggcagcgga aggaagggtg gggcggggcg tggcgggggc tgtgggtggg ggcggcggca
      181 mccgncggtg gggccacccc cttgaccctb tctcttttaa acatctccca gcccacgaga
      241 ccygcgracm actcgtatgc ccgcttytkc tttaaaaaaa aaaaaataaa aacccacaca
      301 caccatctga tgatactgat gatattttaa gatgatgcca gmcmaaaagg tttrawtttt
      361 tgcktttttt gccgamcgcr ktgmwtgtgm atnttttttt tttttgngaa tnattnaatt
      421 tttsggacnt gyccgttttk rtttyanwtt ttttggntnt tattnnaaaa aagaaaagan
      481 aaagcnaaaa aaggggagan ggnaaatmcm aaagggngga aaargnggtg ngtgganagg
      541 raagnggggg ngngggggcr ggggarggag ggggggggsg gggcgtggcg gggtctgtgg
      601 ctgggcgcga cgtcaccgac gttggagcca ctcctttgac catgtgtgcg tgtgtgtatt
      661 attcgtgtct cgccactcgc cggttgtttt tttcttttta tgctgcgctc tctctagcgc
      721 catctcgctt acgcatgctc aacgcaccgc atgttgccgt ttccttttat gcgtcatttt
      781 ggctcgaaat aggcaattat ttaaacaaag attagtcaac gaaaacgcta aaataaataa
      841 gtctacaata tggttactta ttgccatgtg tgtgcagcca acgatagcaa caaaagcaac
      901 aacacaggtg gctttccctc tttcactttt tgtttgcaag ccgcgtgcga gcaagacggc
      961 acgaccggca aacgcaatta cgctgacaaa gagcagacga agttttggcg aaaaacatca
     1021 aggcgcctga tacgaatgca tttgcaataa caattgcgat atttaatatt gtttatgaag
     1081 ctgtttgact tcaaaacaca caaaaaaaaa aataaaacaa attatttgaa agagaattag
     1141 gaatcggacg cttatcgtta gggtaacaac aagaaatgct tactgagtca cagcctctgg
     1201 aaaactgccg caagccagag agagagagaa aaagagggag agcagcttag accgcatgtg
     1261 cttgtgtgtg aggcgtctct ctcttcgtct ctgttgcgca aacgcataga ctgcactgaa
     1321 aaaatcgatt acctattttt tatgaatgaa tatttgcact attactattc aaaactatta
     1381 agatagcaat cacattcaat agccaaatac tataccacct gagcgatgca acgaaatgat
     1441 caatttgagc aaaaatgctg catatttagg acggcatcat tatagaaatg cttcttgctg
     1501 tgtacttttc tctcgtctgg cagctgtttc gccgttattg ttaaaaccgg cttaagttag
     1561 gtgtgttttc tacgactagt gaatgcccta ctagaagatg tgtgttgcac aaaatgtccc
     1621 tggaataacc aatttgaagt gcagatagca gtaaacgtaa gctaatatga atattattta
     1681 actgtaatgt tttaatatcg ctggacatta ctaataaacc cactataaac acatgtacat
     1741 atgtatgttt tggcatacaa tgagtagttg gggaaaaaat gtgtaaaagc accgtgacca
     1801 tcacagcata aagataacca gctgaagtat cgaatatgag taacccccaa attgaatcac
     1861 atgccgcaac tgataggacc catggaagta cactcttcat ggcgatatac aagacacaca
     1921 caagcacgaa cacccagttg cggaggaaat tctccgtaaa tgaaaaccca atcggcgaac
     1981 aattcatacc catatatggt aaaagttttg aacgcgactt gagagcggag agcattgcgg
     2041 ctgataaggt tttagcgcta agcgggcttt ataaaacggg ctgcgggacc agttttcata
     2101 tcactaccgt ttgagttctt gtgctgtgtg gatactcctc ccgacacaaa gccgctccat
     2161 cagccagcag tcgtctaatc cagagacccc ggatccagat atcgaggcct gtctagagaa
     2221 gctccgccac catggatctc caccgcggtg gaggccgcat cttttaccca tacgatgttc
     2281 ctgactatgc gggctatccc tatgacgtcc cggactatgc aggatcctat ccatatgacg
     2341 ttccagatta cgctgctcat ggcggacacc ggtatacaag tttgtacaaa aaagcaggct
     2401 tcggcagcgg aagcgagcaa aagctcatct cggaggaaga tctagaacag aagctaatat
     2461 cagaggagga cctagagcaa aaacttattt cggaagaaga tctgggcagc ggatccggtt
     2521 cgggaagcgg tagtatgagt aaaggagaag aacttttcac tggagttgtc ccaattcttg
     2581 ttgaattaga tggcgatgtt aatgggcaaa aattctctgt cagtggagag ggtgaaggtg
     2641 atgcaacata cggaaaactt acccttaaat ttatttgcac tactgggaag ctacctgttc
     2701 catggccaac acttgtcact actttgactt atggtgttca atgcttttca agatacccag
     2761 atcatatgaa acagcatgac tttttcaaga gtgccatgcc cgaaggttat gtacaggaaa
     2821 gaactatatt ttacaaagat gacgggaact acaagacacg tgctgaagtc aagtttgaag
     2881 gtgataccct tgttaataga atcgagttaa aaggtattga ttttaaagaa gatggaaaca
     2941 ttcttggaca caaaatggaa tacaactata actcacataa tgtatacatc atggcagaca
     3001 aaccaaagaa tggaatcaaa gttaacttca aaattagaca caacattaaa gatggaagcg
     3061 ttcaattagc agaccattat caacaaaata ctccaattgg cgatggccct gtccttttac
     3121 cagacaacca ttacctgtcc acacaatctg ccctttccaa agatcccaac gaaaagagag
     3181 atcacatgat ccttcttgag tttgtaacag ctgctgggat tacacatggc atggatgaac
     3241 tatacaaagg tagtggttcc ggtagcggca gtgggagtga ttataaagac gatgatgaca
     3301 aggactataa ggacgacgac gacaaggatt acaaggatga tgacgacaaa ggctccggtt
     3361 cggggtcggg aagcggtagt ggtaaaccga ttcccaatcc ccttcttggt cttgattcta
     3421 caggcaaacc catcccgaac ccactcctcg gccttgactc cacaggcaag cctattccaa
     3481 atcctctact aggcctcgat tctacgggta gcggttcggg atccggaagt ggaagccacc
     3541 atcatcatca ccattaggac ccagctttct tgtacaaagt tggcattata agaaagcatt
     3601 gcttatcaat ttgttgcaac gaacaggtca ctatcagtca aaataaaatc attatttgcc
     3661 atccagctga tatcccctat agtgagtcgt attacatggt catagctgtt tcctggcagc
     3721 tctggcccgt gtctcaaaat ctctgatgtt acattgcaca agataaaaat atatcatcat
     3781 gaacaataaa actgtctgct tacataaaca gtaatacaag gggtgttatg agccatattc
     3841 aacgggaaac gtcgaggccg cgattaaatt ccaacatgga tgctgattta tatgggtata
     3901 aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctatcgcttg tatgggaagc
     3961 ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat gatgttacag
     4021 atgagatggt cagactaaac tggctgacgg aatttatgcc tcttccgacc atcaagcatt
     4081 ttatccgtac tcctgatgat gcatggttac tcaccactgc gatccccgga aaaacagcat
     4141 tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt
     4201 tcctgcgccg gttgcattcg attcctgttt gtaattgtcc ttttaacagc gatcgcgtat
     4261 ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg agtgattttg
     4321 atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcat aaacttttgc
     4381 cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac cttatttttg
     4441 acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca gaccgatacc
     4501 aggatcttgc catcctatgg aactgcctcg gtgagttttc tccttcatta cagaaacggc
     4561 tttttcaaaa atatggtatt gataatcctg atatgaataa attgcagttt catttgatgc
     4621 tcgatgagtt tttctaatca gaattggtta attggttgta acactggcag agcattacgc
     4681 tgacttgacg ggacggcgca agctcatgac caaaatccct taacgtgagt tacgcgtcgt
     4741 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     4801 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     4861 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     4921 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     4981 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     5041 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     5101 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     5161 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     5221 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     5281 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     5341 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     5401 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     5461 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     5521 agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc
     5581 ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg
     5641 gcagtgagcg caacgcaatt aatacgcgta ccgctagcca ggaagagttt gtagaaacgc
     5701 aaaaaggcca tccgtcagga tggccttctg cttagtttga tgcctggcag tttatggcgg
     5761 gcggcctgcc cccccccccc ccggccgktg cttcwcaamn cttcaaawcc cccccccgcg
     5821 ggkttttyct wctccygrga gsrkyswycy mcccancaaa maacaaawan aaaaaaaaav
     5881 smcrrnscty csymcyncga gscyttygtt tttttttttn tyctkgyagt ttcctmcyct
     5941 cccgstwacs cyagcatggg tgtttttccm gtycccmccg tkktaaaaaa ccggc
//
