Cloning by PCR
The protein of interest can be expressed with the SNAP-tag (20 kDa) as either an N- or a C-terminal fusion.
For fusion to the C-terminus of the SNAP-tag, subclone your gene of interest into the 3´ MCS of the pSNAP-tag(T7)-2 Vector using the available restriction sites SbfI , PstI , BamHI , XmaI , SmaI , XhoI and PacI which are located upstream of the stop codon, or NotI downstream of the stop codon in 3´MCS. Include a stop codon where necessary. To subclone the gene of interest into pSNAP-tag(T7)-2 fused to the N-terminus of the SNAP-tag use the available restriction sites NdeI, NheI, NcoI, EcoRV, HindIII, AgeI and EcoRI which are located upstream of the SNAP-tag.
Primer Design and Cloning Hints:
- Design your PCR primers to include a sufficient overlap (15–20 bp) with the sequence of the gene you want to amplify.
- Care should be taken to design the cloning so that the fusion partners in the resulting construct are in frame.
- Adapt the flanking regions to the cloning destination in pSNAP-tag(T7)-2. If you add an upstream SbfI and a downstream XbaI site in the primers, cloning downstream of SNAP26b in pSNAP-tag(T7)-2 should be straightforward.
- For fusions to the C-terminus of the SNAP-tag 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.
- After subcloning the gene of interest into pSNAP-tag(T7)-2 as a fusion with the SNAP26b gene, the resulting plasmid can be used for expression of the SNAP-tag fusion proteins in a suitable E. coli host strain.
- 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.
- Perform the PCR reaction and subsequent cloning steps according to established protocols for molecular biology.
Direct cloning can also be used to make fusions with the SNAP-tag. This is possible if the gene of interest is flanked by sites compatible with the polylinker in pSNAP-tag(T7)-2.
The sequence of the SNAP26b
gene is flanked by a number of restriction sites which can be found on the plasmid map.
If subcloning of the SNAP-tag with your gene of interest does not work, reconfirm all the cloning steps (primer design, choice of restriction site, etc.) If all steps are confirmed as being correct, then try the cloning using different restriction sites. Be sure to include a positive and negative control for the ligation reaction.
Alternatively, subclone the SNAP-tag gene into an expression vector already containing your gene of interest.