pET-50b(+)
At a Glance
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Type |
Cloning, Expression |
---|---|
Origin of Replication |
pBR322 |
Copy # |
~40 copies per cell |
Markers |
Kanamycin |
Link to Sequence | |
Features |
T7 Promoter |
Accession Number |
|
edit table |
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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
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Sources
References
- ↑ Walker, PA et al. (1994) Efficient and rapid affinity purification of proteins using recombinant fusion proteases. Biotechnology (N.Y.) 12 601-5 PubMed
- ↑ Novy, R. and Drott, D. (2002) inNovations 14, 12-13.
- ↑ Kim, JS & Raines, RT (1993) Ribonuclease S-peptide as a carrier in fusion proteins. Protein Sci. 2 348-56 PubMed
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