On inserting a CDFS into a MCS, there are sequences on either side, sometimes between the CDS and tag coding sequences. What happens to these? Also, if there is a His tag, it there typically a sequence for enzymatic removal of the tag? (I often do not see it specifically labeled as such).
Hi there,
The MCS sequences (upstream and downstream the cloning sites) are conserved in the final construct. Therefore it is essential to respect the reading frame determined by the initiation codon (usually in the upstream sequence). If in need of tag removal then you have to chose the vector carefully according to the location of the cutting site and the type of protease you are intending to use.
It really depends on the method used to insert the CDS. If you use a Gibson assembly or similar method you can design the overhanging portions of your insert to modify the existing sequences surrounding the CDS, this way you can include a cleavage site or restriction sites that you want. If using regular restriction and ligation the restriction sites used are often destroyed unless the piece being inserted restores their sequences, but the rest of the MCS and surrounding sequences including any tag should not be altered but they might not be in frame with the CDS. The insert can't have a stop codon before the tag and it also has to be checked that it doesn't change the reading frame to introduce a stop in the portion before the tag. You also need to make sure there is a stop codon at whatever point you do want it.
If there is no labeled cleavage site on the plasmid map then it probably isn't included but a good idea would be to search the plasmid using whatever program you use (I'm most familiar with APE and Snapgene) to make sure everything is in frame, starting and stopping where you want. You can then check that translated ORF for any known cleavage sites.
The MCS remaining nucleotides will still be there, but the MCS of vectors that contain a tag often times have the restriction sites in-frame with the tag, but you will have a few amino acids between your protein sequence and the tag. Many vectors commonly will have a site to remove the tag but you'd need to look at the vector beforehand to confirm. I know many GST-tagged vectors do.