pET-50b(+)
At a Glance
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pET-50b(+), image provided by Novagen
<protect>
| Type |
Cloning, Expression |
|---|---|
| Origin of Replication |
pBR322 |
| Copy # |
~40 copies per cell |
| Markers |
Kanamycin |
| Link to Sequence | |
| Features |
T7 Promoter |
| Accession Number |
|
| edit table |
</protect>
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
