I was wondering if anyone has ever seen large deletions (~40-50 bp) occur when using phusion polymerase to clone a gene. I have recently tried using Circular Polymerase Extension cloning to clone a 200 bp insert into a 6.5kb plasmid and obtained two separate clones with 40bp+ deletions in the center of the insert.
Phusion has a 5'->3' exonuclease activity, but I didn't think it could cut in the center of a sequence. While our inserts were created with oligo synthesis, it is my understanding that such large deletions are rare with oligo synthesis. If you have heard of this happening please let me know.