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pET-50b(+)

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At a Glance

pET-50b(+), image provided by Novagen

Type

Cloning, Expression

Origin of Replication

pBR322
f1

Copy #

~40 copies per cell

Markers

Kanamycin

Link to Sequence

Novagen

Features

T7 Promoter
N Terminal His Tag
Nus Tag
lacI
C Terminal S Tag

Accession Number




Notes

The pET-50b(+) vector is designed for cloning and high-level expression of target proteins fused with the 495 aa NusA (Nus•Tag™) protein that is cleavable with the human rhinovirus (HRV) 3C protease[1]. Cloning sites are also available for producing fusion proteins containing two cleavable N-terminal His•Tag® sequences for detection and purification. The additional N-terminal His•Tag sequence significantly enhances purification yields when utilizing His•Mag™ Agarose Beads for high throughput purification[2]. In addition, this plasmid contains a strong T7lac promoter, optimized RBS, the coding sequence for the HRV 3C protease cleavage site (LeuGluValLeuPheGlnGlyPro) for N-terminal fusion tag removal and a multiple cloning site that encodes restriction enzyme sites. An optional C-terminal S•Tag™ coding sequence is compatible with purification, detection, and quantification[3].

History

You can help EcoliWiki by writing about the history of this plasmid.

Sources

Novagen

References

  1. Walker, PA et al. (1994) Efficient and rapid affinity purification of proteins using recombinant fusion proteases. Biotechnology (N.Y.) 12 601-5 PubMed EcoliWiki page
  2. Novy, R. and Drott, D. (2002) inNovations 14, 12-13.
  3. Kim, JS & Raines, RT (1993) Ribonuclease S-peptide as a carrier in fusion proteins. Protein Sci. 2 348-56 PubMed EcoliWiki page

Sequence

       1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa 
      61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt 
     121 tgtttagcag cctaggttaa ttaagcctcg agagcagcag aagtagagct gtccatgtgc 
     181 tggcgttcga atttagcagc agcggtttct ttactaccgc gtggcaccag agcgagctct 
     241 gcggccgcaa gcttgtcgac ggacgtcggg cgcgccaagg cctgtacaga attcggatcc 
     301 tggtacccgg gtccctgaaa gaggacttca agagccgcgg agtgatggtg atggtgatga 
     361 ccagaaccac tagtcgcttc gtcaccgaac cagcaaatat tacgggcagc cataatcagt 
     421 gctccggctt tttcgtcggt caacccttcg atatcagcca gatcatcaat gccctgttcg 
     481 gcgagatctt ccagcgtaca aacgccacgg gcggccagtt tgaatgccaa atcacgatct 
     541 accccttcaa ggttcagcag atcgtcagcc ggtttgttat caccgaggct ttcttcctgg 
     601 gcctgtgcaa tggtggccag tgcattttta gcacgctcgc gcagtgcttc aacggtcggc 
     661 tcatcaaggc cttcgatttc caacagctct ttcatcggca cataggccaa ttcttccagc 
     721 gtcgagaagc cttcttctac cagaacagtc gcgaagtctt cgtcgatgtc gagatatttg 
     781 gtgaaggtgt cgatcgctgc gtgcgcttcc gcctgatgct tagcttgcag gtcgtcaacg 
     841 gtcatcacgt tgagttccca accgctcagt tgcgaagcca gacgcacgtt ctgaccgtta 
     901 cggccaatcg cctgcgccag attaccggct tcaacggcga tgtccatggt gtgtttatct 
     961 tcatccacca cgatagaagc aacgtctgcc ggtgccattg cgttaatcac gaactgcgcc 
    1021 gggttatcat cccacaggac gatatcgata cgctcgccac ccagttcagt agacaccgcc 
    1081 tgaacacgcg cgccacgcat acctacgcaa gcacctaccg gatcgatacg tttatcgttg 
    1141 gttttcaccg cgattttcgc acgagaaccc ggatcgcgag ccgctgcttt aatttcaatc 
    1201 acttcttcgc cgatttctgg cacttcaata cggaacagtt cgatcagcat ttccggcttg 
    1261 gaacgagtga cgaacagttg cgcgccacgc gcttccgggc gaacggaata gagcacgcca 
    1321 cgaacgcggt cgccagggcg gaagttttca cgcggcagca tatcttcgcg caggatcacg 
    1381 gcttcagcgt tgttgcccag atccagagag atgttgtcgc ggtttacttt tttcaccacg 
    1441 ccggtgatga tttcaccttc gtgttcacgg aactgatcaa ccaccatcgc acgttcggct 
    1501 tcacgcactt tctgcacgat aacctgtttt gccgtctggg tagtgatacg gtcaaaggta 
    1561 acagactcaa tctgatcttc aacgtaatcg cccaggttca ggctttcatc ttcataacgt 
    1621 gcggcttcaa gggtgatttc cttggtcggc tgggtgactt catcaacaac taaccagcga 
    1681 cggaaagtgt caaaatcacc gcttttgcga tcgatctgta cgcggacgtc gatctcttgt 
    1741 tcatattttt tctttgttgc tgtcgccagc gcgctttcca atgcttcgaa aatcttctcg 
    1801 cgaggtagcg ccttttcatt ggatacggct tcaactacag ccaaaatttc tttgttcata 
    1861 ctagaatggt gatggtggtg atggctgctg cccatatgta tatctccttc ttaaagttaa 
    1921 acaaaattat ttctagaggg gaattgttat ccgctcacaa ttcccctata gtgagtcgta 
    1981 ttaatttcgc gggatcgaga tcgatctcga tcctctacgc cggacgcatc gtggccggca 
    2041 tcaccggcgc cacaggtgcg gttgctggcg cctatatcgc cgacatcacc gatggggaag 
    2101 atcgggctcg ccacttcggg ctcatgagcg cttgtttcgg cgtgggtatg gtggcaggcc 
    2161 ccgtggccgg gggactgttg ggcgccatct ccttgcatgc accattcctt gcggcggcgg 
    2221 tgctcaacgg cctcaaccta ctactgggct gcttcctaat gcaggagtcg cataagggag 
    2281 agcgtcgaga tcccggacac catcgaatgg cgcaaaacct ttcgcggtat ggcatgatag 
    2341 cgcccggaag agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc 
    2401 gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca ggccagccac 
    2461 gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa ttacattccc 
    2521 aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc 
    2581 agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa 
    2641 ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg 
    2701 gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga tcattaacta tccgctggat 
    2761 gaccaggatg ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt atttcttgat 
    2821 gtctctgacc agacacccat caacagtatt attttctccc atgaagacgg tacgcgactg 
    2881 ggcgtggagc atctggtcgc attgggtcac cagcaaatcg cgctgttagc gggcccatta 
    2941 agttctgtct cggcgcgtct gcgtctggct ggctggcata aatatctcac tcgcaatcaa 
    3001 attcagccga tagcggaacg ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc 
    3061 atgcaaatgc tgaatgaggg catcgttccc actgcgatgc tggttgccaa cgatcagatg 
    3121 gcgctgggcg caatgcgcgc cattaccgag tccgggctgc gcgttggtgc ggacatctcg 
    3181 gtagtgggat acgacgatac cgaagacagc tcatgttata tcccgccgtt aaccaccatc 
    3241 aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca actctctcag 
    3301 ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc 
    3361 ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg 
    3421 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtaagtta 
    3481 gctcactcat taggcaccgg gatctcgacc gatgcccttg agagccttca acccagtcag 
    3541 ctccttccgg tgggcgcggg gcatgactag catgatcgtg ctcctgtcgt tgaggacccg 
    3601 gctaggctgg cggggttgcc ttactggtta gcagaatgaa tcaccgatac gcgagcgaac 
    3661 gtgaagcgac tgctgctgca aaacgtctgc gacctgagca acaacatgaa tggtcttcgg 
    3721 tttccgtgtt tcgtaaagtc tggaaacgcg gaagtcagcg ccctgcacca ttatgttccg 
    3781 gatctgcatc gcaggatgct gctggctacc ctgtggaaca cctacatctg tattaacgaa 
    3841 gcgctggcat tgaccctgag tgatttttct ctggtcccgc cgcatccata ccgccagttg 
    3901 tttaccctca caacgttcca gtaaccgggc atgttcatca tcagtaaccc gtatcgtgag 
    3961 catcctctct cgtttcatcg gtatcattac ccccatgaac agaaatcccc cttacacgga 
    4021 ggcatcagtg accaaacagg aaaaaaccgc ccttaacatg gcccgcttta tcagaagcca 
    4081 gacattaacg cttctggaga aactcaacga gctggacgcg gatgaacagg cagacatctg 
    4141 tgaatcgctt cacgaccacg ctgatgagct ttaccgcagc tgcctcgcgc gtttcggtga 
    4201 tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc 
    4261 ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg 
    4321 cgcagccatg acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca 
    4381 tcagagcaga ttgtactgag agtgcaccat atatgcggtg tgaaataccg cacagatgcg 
    4441 taaggagaaa ataccgcatc aggcgctctt ccgcttcctc gctcactgac tcgctgcgct 
    4501 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca 
    4561 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga 
    4621 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc 
    4681 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg 
    4741 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat 
    4801 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt 
    4861 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc 
    4921 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg 
    4981 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg 
    5041 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg 
    5101 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg 
    5161 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca 
    5221 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga 
    5281 acgaaaactc acgttaaggg attttggtca tgaacaataa aactgtctgc ttacataaac 
    5341 agtaatacaa ggggtgttat gagccatatt caacgggaaa cgtcttgctc taggccgcga 
    5401 ttaaattcca acatggatgc tgatttatat gggtataaat gggctcgcga taatgtcggg 
    5461 caatcaggtg cgacaatcta tcgattgtat gggaagcccg atgcgccaga gttgtttctg 
    5521 aaacatggca aaggtagcgt tgccaatgat gttacagatg agatggtcag actaaactgg 
    5581 ctgacggaat ttatgcctct tccgaccatc aagcatttta tccgtactcc tgatgatgca 
    5641 tggttactca ccactgcgat ccccggcaaa acagcattcc aggtattaga agaatatcct 
    5701 gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt 
    5761 cctgtttgta attgtccttt taacagtgat cgcgtatttc gtctcgctca ggcgcaatca 
    5821 cgaatgaata acggtttggt tgatgcgagt gattttgatg acgagcgtaa tggctggcct 
    5881 gttgaacaag tctggaaaga aatgcataaa cttttgccat tctcaccgga ttcagtcgtc 
    5941 actcatggtg atttctcact tgataacctt atttttgacg aggggaaatt aataggttgt 
    6001 attgatgttg gacgagtcgg aatcgcagac cgataccagg atcttgccat cctatggaac 
    6061 tgcctcggtg agttttctcc ttcattacag aaacggcttt ttcaaaaata tggtattgat 
    6121 aatcctgata tgaataaatt gcagtttcat ttgatgctcg atgagttttt ctaagaatta 
    6181 attcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg 
    6241 cacatttccc cgaaaagtgc cacctgaaat tgtaaacgtt aatattttgt taaaattcgc 
    6301 gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc 
    6361 ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag 
    6421 tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga 
    6481 tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc 
    6541 actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa 
    6601 cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt 
    6661 agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc 
    6721 gtcccattcg cca