Cloning of SNAP-tag Fusions in pSNAP-tag(m) (N9172)
Cloning by PCR
To subclone the gene of interest into pSNAP-tag(m) fused to the N-terminus of the SNAP-tag use the available restriction sites ClaI , EcoRV (blunt) and EcoRI which are located upstream of the SNAP-tag.
To subclone the gene of interest into pSNAP-tag(m) fused to the C-Terminus of the SNAP-tag use the available restriction sites downstream of the SNAP-tag: SbfI , AscI , BamHI , XhoI and NotI .
Note: When fusing the gene of interest to the C-Terminus of the SNAP-tag, note that there is a stop codon between the XhoI and NotI sites, so SbfI, AscI, BamHI or XhoI must be used as the 5´ cloning site for your insert.
Primer Design and Cloning Considerations:
- Design your PCR primers to include a sufficient overlap with the sequence of the gene you want to amplify.
- You may also want to include a Stop codon at the C-Terminus of the fusion (in front of the downstream cloning site) in order to terminate translation at this position.
- For fusions upstream of the SNAP-tag, ensure that a start codon is included. The addition of a Kozak sequence (e.g. GCCRCCATG, where the start codon is underlined) will increase the translation efficiency.
- In general, any linker peptide between the proteins should be kept short to avoid degradation by proteases. If required, specific protease cleavage sites can be introduced into the linker peptide.
- Care should be taken to design the cloning so that the fusion partners in the resulting construct are in frame.
- Perform the PCR reaction and subsequent cloning steps according to established protocols for molecular biology.
- After subcloning the gene of interest into pSNAP-tag(m) as a fusion with the snap26m gene, the resulting plasmid can be used for stable or transient expression of the SNAP-tag fusion proteins in a suitable cell line.
- NEB 10-beta Competent E. coli (High Efficiency) (NEB #C3019) is recommended for propagating and subcloning this vector, unless the ClaI site will be used. The restriction endonucleases ClaI (NEB #R0197 ) is methylation sensitive. When using the ClaI site, dam-/dcm- Competent E. coli (NEB #C2925) is recommended.
- Direct Cloning
Direct cloning can also be used to make fusions with the SNAP-tag. This is only possible if the fusion partner has compatible sites adjacent to the gene of interest.
Care should be taken to design the cloning so that the fusion partners in the resulting construct are in frame.
Note: When fusing the gene of interest to the C-terminus of the SNAP-tag, note that there is a stop codon between the XhoI and NotI sites, so SbfI, AscI, BamHI or XhoI must be used as the 5´ cloning site for your insert.
If subcloning of your gene of interest with the SNAP-tag does not work, reconfirm all the cloning steps (primer design, choice of restriction site, etc.). If all steps are confirmed as being correct, then re-try the cloning using different restriction sites. Be sure to include a positive control for the ligation reaction.
Alternatively try to subclone the SNAP-tag gene into an expression vector already containing your gene of interest.