Flavinkins (@Flavinkins)

Also, what you are going to do if you tried to just move a single nucleotide from a sampled (but more distantly related) template, where you used to determine the right nucleotide to change in order to remove a restriction site, synthesized and ligated the genome, and then found out that the genome failed to be rescued because of the same reason why the site you want to remove is conserved in all (more closely related in the surrounding segment) the viruses you initially used to create the consensus? You try taking a bigger segment of your more distantly related template, surrounding the site, apply it to your consensus and try again. RNA structural conservation determines nucleotide conservation in coronavirus genomes and the determinant for the conservation of a nucleotide position usually resides in the segment surrounding the nucleotide that you want to change. Swapping in the entire segment usually fixes the incompatibility issue, and the clone is then able to be rescued. With the side effect that the segment you swap out trying to fix the compatibility issue now show up on the ReCCA analysis too, with the more distantly related template now becoming the “closest related segment” at the removed site. Natural recombination, however, won’t guarantee either the transfer of only this specific spillover-irrelevant segment from that rare hypothetical “ancestor” (the template that you originally used not because of closeness or spillover potential, only out of necessity to enable cloning without breaking the consensus genome—while the whole genome of the template strain itself being also, not easy to clone due to incompatible and poorly space sites elsewhere on the genome),or that the same recombination event don’t add or remove any other sites (that would be incompatible to cloning), or that this one-out-of-50+ template strain is what that end up donating this segment prior to spillover. All these specific choices matter only when you are trying to clone a virus, not at all if a wild virus is trying to spill over into people.