I am using a gateway cloning vector to overexpress a gene, but I want a control vector as well (without my insert). I want to cut out the ccdb gene so it wont self destruct but I dont have a vector to insert. compatible cut sites were not immediatley obvious looking at the map and looking at each restriction site is not very efficient. Is there an easy way to do this or another way to make a control vector?
I don't know the answer to this unfortunately, but if you are lucky, u can use Bsp1407I (BsrGI) (TGTACA) which is part of the gateway recombination core site so it should be flanking ccdb/cm - in case it isn't anywhere else in the vector. If just 1 additional site maybe partial digestion can work depending on position (and selection would maybe be easy because u can use ccdb susceptible cells.
In addition to the typical pairs of compatible ends like Xho1-Sal1 or BamH1-Bgl2, all blunt end restriction are of course compatible, or any pair of sites can be rendered blunt and then ligated.
Or you could just use a set of primers to amplify the plasmid section and removing the ccdB gene.
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