Suppose that you have an unwanted gene in a plasmid vector and you want to get rid of it in Gibson assembly. I get the whole concept behind using the primers and essentially making copies that exclude the pink region but what is the first step required to separate the plasmid and editing the plasmid?
Do you just use heat to separate the circular plasmid and then add the primer and expect a polymerase to go around the circle and make a copy without the unwanted gene? Does a circular plasmid even separate into single stranded DNA like a linear DNA?? How does the primer even access the strand in the plasmid DNA in the first place?
I haven't found a clear animation nor diagram on how this exactly works and was hoping for someone to answer this. What enzyme opens up the circular plasmid without the restriction enzyme and adds the base pairs to make copies without the unwanted gene. Please refer to the figures below for what I am trying to ask. And thank you in advance. In short how do you go from "PCR backbone" to "PCR product BB with no insert"