I am using a multicistronic expression plasmid in which 4 cDNAs are separated by 3 self-cleaving 2A sequences. For one of the experimental approaches I want to replace 3 of the 4 genes with as short as possible ‘stuffers’ that maintain the reading frame. How short could such stuffers, be i.e. what is the minimal distance necessary between two 2A sequences for them to work efficiently?
Dear Hisse M van Santen,
remembering that 2A is not a "self-cleaving peptide" but instead is a sequence causing a ribosomal skip (See e.g. https://viralzone.expasy.org/914?outline=all_by_species). Thus, you need to ensure that the “2A-like”, or CHYSEL (cis-acting hydrolase element) sequence is represented and has a high activity in your expression system. Very likely you dont require much of a stuffer sequence after the Proline, but you need enough before the "apparent cleavage site" to ensure the ribosomal skip.
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