I have constructed a Kanamycin cassette with flanking 300 bp forward and reverse sequences (Fwflanking-Kan-Rvflanking) of a target gene which I want to knock-out. For some unknown reasons, the linear product is not able to generate a knock-out by double recombination. Is it possible to clone only the forward flanking (or reverse flanking) sequence, which is NOT internal to the target gene, and use it for a single cross-over? Will it still inactivate the gene?
A single cross-over with only one of the flanking region would not lead to knockout but only to integration of the constructed cassette. For Knockout to occur, the second flanking fragment is required which would result in homologous recombination and replacement of the region with the kanamycin cassette
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