pET-32 Ek/LIC
At a Glance
pET-32 Ek/LIC, Image made with PlasMapper
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| Type |
Cloning/Expression[1] |
|---|---|
| Origin of Replication |
pBR322, f1 |
| Copy # |
~40 |
| Markers |
Ampicillin |
| Link to Sequence | |
| Features |
T7 Promoter |
| Accession Number |
|
| edit table |
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Notes
The pET-32 Ek/LIC vector is an Ek/LIC version of pET-32b(+). It is designed for cloning and high-level expression of target proteins fused with the 109aa Trx•Tag thioredoxin protein[1], and His•Tag and STag coding sequences that are cleavable with enterokinase (Ek) protease.
History
Ligation-independent cloning (LIC) was developed for directional cloning of PCR products without restriction enzyme digestion or ligation reactions[2][3]. LIC 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, thus the majority of annealed products consist of the desired molecules. The annealed LIC vector and insert are transformed into competent E. coli cells, and covalent bonds are formed at the vector-insert junctions within the cell to yield circular plasmid. Directional cloning of the insert is achieved with minimal non-recombinant background, and cloning is so efficient that virtually all of the resulting colonies contain the desired recombinant.
Sources
References
See Help:References for how to manage references in EcoliWiki.
- ↑ 1.0 1.1 LaVallie, ER et al. (1993) A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm. Biotechnology (N.Y.) 11 187-93 PubMed
- ↑ Aslanidis, C & de Jong, PJ (1990) Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res. 18 6069-74 PubMed
- ↑ Haun, RS et al. (1992) Rapid, reliable ligation-independent cloning of PCR products using modified plasmid vectors. BioTechniques 13 515-8 PubMed
Sequence
You can help EcoliWiki by providing database accessions or, if none are available, the sequence of this plasmid.
1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt
181 cgacggagct cgaattcgga tccgatatcg ccatggttga ggagaagccc gggctcttgt
241 cgtcgtcatc ggtacccaga tctgggctgt ccatgtgctg gcgttcgaat ttagcagcag
301 cggtttcttt cataccagaa ccgcgtggca ccagaccaga agaatgatga tgatgatggt
361 gcatatggcc agaaccagaa ccggccaggt tagcgtcgag gaactctttc aactgacctt
421 tagacagtgc acccactttg gttgccgcca cttcaccgtt tttgaacagc agcagagtcg
481 ggataccacg gatgccatat ttcggcgcag tgccagggtt ttgatcgatg ttcagttttg
541 caacggtcag tttgccctga tattcgtcag cgatttcatc cagaatcggg gcgatcattt
601 tgcacggacc gcaccactct gcccagaaat cgacgaggat cgccccgtcc gctttgagta
661 catccgtgtc aaaactgtcg tcagtcaggt gaataatttt atcgctcata tgtatatctc
721 cttcttaaag ttaaacaaaa ttatttctag aggggaattg ttatccgctc acaattcccc
781 tatagtgagt cgtattaatt tcgcgggatc gagatcgatc tcgatcctct acgccggacg
841 catcgtggcc ggcatcaccg gcgccacagg tgcggttgct ggcgcctata tcgccgacat
901 caccgatggg gaagatcggg ctcgccactt cgggctcatg agcgcttgtt tcggcgtggg
961 tatggtggca ggccccgtgg ccgggggact gttgggcgcc atctccttgc atgcaccatt
1021 ccttgcggcg gcggtgctca acggcctcaa cctactactg ggctgcttcc taatgcagga
1081 gtcgcataag ggagagcgtc gagatcccgg acaccatcga atggcgcaaa acctttcgcg
1141 gtatggcatg atagcgcccg gaagagagtc aattcagggt ggtgaatgtg aaaccagtaa
1201 cgttatacga tgtcgcagag tatgccggtg tctcttatca gaccgtttcc cgcgtggtga
1261 accaggccag ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc
1321 tgaattacat tcccaaccgc gtggcacaac aactggcggg caaacagtcg ttgctgattg
1381 gcgttgccac ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat
1441 ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg
1501 aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta
1561 actatccgct ggatgaccag gatgccattg ctgtggaagc tgcctgcact aatgttccgg
1621 cgttatttct tgatgtctct gaccagacac ccatcaacag tattattttc tcccatgaag
1681 acggtacgcg actgggcgtg gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt
1741 tagcgggccc attaagttct gtctcggcgc gtctgcgtct ggctggctgg cataaatatc
1801 tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg cgactggagt gccatgtccg
1861 gttttcaaca aaccatgcaa atgctgaatg agggcatcgt tcccactgcg atgctggttg
1921 ccaacgatca gatggcgctg ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg
1981 gtgcggacat ctcggtagtg ggatacgacg ataccgaaga cagctcatgt tatatcccgc
2041 cgttaaccac catcaaacag gattttcgcc tgctggggca aaccagcgtg gaccgcttgc
2101 tgcaactctc tcagggccag gcggtgaagg gcaatcagct gttgcccgtc tcactggtga
2161 aaagaaaaac caccctggcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
2221 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
2281 attaatgtaa gttagctcac tcattaggca ccgggatctc gaccgatgcc cttgagagcc
2341 ttcaacccag tcagctcctt ccggtgggcg cggggcatga ctatcgtcgc cgcacttatg
2401 actgtcttct ttatcatgca actcgtagga caggtgccgg cagcgctctg ggtcattttc
2461 ggcgaggacc gctttcgctg gagcgcgacg atgatcggcc tgtcgcttgc ggtattcgga
2521 atcttgcacg ccctcgctca agccttcgtc actggtcccg ccaccaaacg tttcggcgag
2581 aagcaggcca ttatcgccgg catggcggcc ccacgggtgc gcatgatcgt gctcctgtcg
2641 ttgaggaccc ggctaggctg gcggggttgc cttactggtt agcagaatga atcaccgata
2701 cgcgagcgaa cgtgaagcga ctgctgctgc aaaacgtctg cgacctgagc aacaacatga
2761 atggtcttcg gtttccgtgt ttcgtaaagt ctggaaacgc ggaagtcagc gccctgcacc
2821 attatgttcc ggatctgcat cgcaggatgc tgctggctac cctgtggaac acctacatct
2881 gtattaacga agcgctggca ttgaccctga gtgatttttc tctggtcccg ccgcatccat
2941 accgccagtt gtttaccctc acaacgttcc agtaaccggg catgttcatc atcagtaacc
3001 cgtatcgtga gcatcctctc tcgtttcatc ggtatcatta cccccatgaa cagaaatccc
3061 ccttacacgg aggcatcagt gaccaaacag gaaaaaaccg cccttaacat ggcccgcttt
3121 atcagaagcc agacattaac gcttctggag aaactcaacg agctggacgc ggatgaacag
3181 gcagacatct gtgaatcgct tcacgaccac gctgatgagc tttaccgcag ctgcctcgcg
3241 cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct
3301 tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc
3361 gggtgtcggg gcgcagccat gacccagtca cgtagcgata gcggagtgta tactggctta
3421 actatgcggc atcagagcag attgtactga gagtgcacca tatatgcggt gtgaaatacc
3481 gcacagatgc gtaaggagaa aataccgcat caggcgctct tccgcttcct cgctcactga
3541 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
3601 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
3661 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
3721 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
3781 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
3841 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
3901 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
3961 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
4021 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
4081 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
4141 gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
4201 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
4261 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
4321 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
4381 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
4441 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
4501 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
4561 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
4621 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
4681 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
4741 agttaatagt ttgcgcaacg ttgttgccat tgctgcaggc atcgtggtgt cacgctcgtc
4801 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
4861 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
4921 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
4981 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
5041 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
5101 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
5161 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
5221 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
5281 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
5341 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
5401 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg aaattgtaaa
5461 cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca
5521 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag
5581 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg
5641 gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt
5701 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag
5761 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc
5821 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc
5881 gcttaatgcg ccgctacagg gcgcgtccca ttcgcca
