pET-32 Ek/LIC
At a Glance
[[Image:#PET32EkLIC.jpg|right|thumb|300px|Image provided by Novagen]]
| Type |
Cloning/Expression[1] |
|---|---|
| Origin of Replication |
pBR322, f1 |
| Copy # |
~40 |
| Markers |
Ampicillin |
| Link to Sequence | |
| Features |
T7 Promoter |
| Accession Number |
|
| edit table |
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
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- ↑ 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
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