pET-52 3c/LIC
At a Glance
Image of pET-52 3c/LIC. Novagen
<protect>
| Type |
Cloning |
|---|---|
| Origin of Replication |
f1 |
| Copy # |
~40 |
| Markers |
Ampicillin |
| Link to Sequence | |
| Features |
T7 promoter |
| Accession Number |
|
| edit table |
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Notes
Ligation-independent cloning (LIC) was developed for directional cloning of PCR products without restriction enzyme digestion or ligation reactions[1][2] The LIC method takes advantage of the 3′ → 5′ exonuclease activity of T4 DNA polymerase to create very specific 12 to 15 nucleotide single stranded overhangs in the vector and the insert.The pET-52b(+) 3C/LIC vector carries an N-terminal Strep•Tag II coding sequence [3] followed by a recognition site for the human rhinovirus (HRV) 3C protease. This protease is highly specific for cleavage of the sequence LEVLFQ↓GP [4], and is active at low temperatures [3]. The 3C/LIC cloning site is followed by an optional thrombin recognition site and a C-terminal His•Tag coding sequence.
History
The pET-52 3C/LIC vector is a 3C/LIC version of pET-52b(+).
Sources
References
See Help:References for how to manage references in EcoliWiki.
- ↑ Aslanidis, C & de Jong, PJ (1990) Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res. 18 6069-74 PubMed
- ↑ Haun, RS & Moss, J (1992) Ligation-independent cloning of glutathione S-transferase fusion genes for expression in Escherichia coli. Gene 112 37-43 PubMed
- ↑ 3.0 3.1 Wang, QM et al. (1997) A continuous colorimetric assay for rhinovirus-14 3C protease using peptide p-nitroanilides as substrates. Anal. Biochem. 252 238-45 PubMed
- ↑ Cordingley, MG et al. (1989) Cleavage of small peptides in vitro by human rhinovirus 14 3C protease expressed in Escherichia coli. J. Virol. 63 5037-45 PubMed
Sequence
1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
61 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
241 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
301 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
361 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
421 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttctg gcggcacgat
481 ggcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
541 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
601 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
661 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
721 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
781 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
841 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
901 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
961 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
1021 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
1081 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
1141 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
1201 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
1261 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
1321 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
1381 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
1441 atactcttcc tttttcaatc atgattgaag catttatcag ggttattgtc tcatgagcgg
1501 atacatattt gaatgtattt agaaaaataa acaaataggt catgaccaaa atcccttaac
1561 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
1621 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
1681 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
1741 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
1801 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
1861 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
1921 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
1981 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
2041 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
2101 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
2161 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
2221 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
2281 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
2341 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
2401 attttctcct tacgcatctg tgcggtattt cacaccgcat atatggtgca ctctcagtac
2461 aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg
2521 gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
2581 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
2641 ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg
2701 tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga
2761 agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg
2821 gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa
2881 cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt
2941 tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt
3001 caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct
3061 gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac
3121 gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag
3181 cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc
3241 cgccagccta gccgggtcct caacgacagg agcacgatca tgctagtcat gccccgcgcc
3301 caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc
3361 taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
3421 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
3481 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
3541 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
3601 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc
3661 gtatcccact accgagatgt ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
3721 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
3781 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
3841 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
3901 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
3961 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
4021 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
4081 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
4141 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
4201 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
4261 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
4321 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
4381 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
4441 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact
4501 ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg
4561 gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga
4621 ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc
4681 ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt
4741 ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg
4801 tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc gatctcgatc
4861 ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag
4921 aaataatttt gtttaacttt aagaaggaga tataccatgg caagctggag ccacccgcag
4981 ttcgaaaagg gtgcacttga agtcctcttt cagggacccg ggtaccagga tcctgtacaa
5041 gtcgacgcgg ccgcagagct cgctctggtg ccacgcggta gttccgctca tcaccaccat
5101 catcaccatc accaccacta attaacctag gctgctgcca ccgctgagca ataactagca
5161 taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata
5221 tccggat
