Hi,
I want to delete multiple regions from a viral vector to reduce the size of the same. What I have in mind is homologous recombination but that would need me to swap it with another DNA fragment or perform multiple rounds of PCR. Has anyone removed multiple fragments of DNA from a vector which are at different positions? Is there a quick and efficient way to do it?
I would recommend Gibson assembly. You can use restriction enzymes to remove a large section of the vector, and then use PCR amplicons to add back in any sections of the vector that you want to keep. NEB's manual on Gibson assembly is a good place to start.
Just be aware that some viruses need a genome of a specific size range to package efficiently. Also have you considered a helper plasmid approach where you only have the origin of replication and packaging signal on your small plasmid of interest?
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