How should a cloning strategy for C-terminal fusions (pTXB1,3 and pTYB1,2,3,4) be designed if the gene of interest contains both NdeI and NcoI sites?
Several other restriction enzymes are compatible with NcoI (c/cATGg) and thus may be used for subcloning a target gene or ORF into pTXB3, pTYB3 or pTYB4. The ATG within the recognition sequences of these enzymes can be used for translation initiation. For example, a target gene can be amplified by PCR using a sense primer, which has a BspHI (t/cATGa) or a BspLU11I (a/cATGt) tag (depending on the restriction map of the target gene and the second codon of the ORF). Another enzyme AflIII (a/cRYGt) may also be used for cloning. Second, the NcoI site in the vector (pTYB3 or pTYB4) can be filled-in and used for ligation with a 5´ blunt end fragment starting from the second codon of the ORF. Third, other sites downstream of NdeI or NcoI sites may be chosen as 5´ cloning site. However, this would result in addition of vector-derived amino acid residues to the N-terminus of the protein of interest. Finally, an alternative strategy is to use the XbaI site, upstream of the polylinker region, as a
Related Questions
- How should a cloning strategy for C-terminal fusions (pTYB1,2,3,4 and pTXB1,3) be designed if the gene of interest contains both NdeI and NcoI sites?
- How should a cloning strategy for C-terminal fusions (pTXB1,3 and pTYB1,2,3,4) be designed if the gene of interest contains both NdeI and NcoI sites?
- How are cloning vectors and plasmids used in gene transfer experiments
