RESUMEN
Manipulation of genes, including knock-out or knock-in, replacement of gene elements (such as promoters), fusion with a fluorescent protein gene, and construction of in situ gene reporter, is required in most of the biotechnological laboratories. The widely used gene manipulating methods based on two-step allelic exchange are cumbersome in terms of constructing plasmids, transforming and screening. In addition, the efficiency of using this method for long fragment knockout is low. To simplify the process of gene manipulation, we constructed a minimized integrative vector pln2. When a gene needs to be inactivated, an internal fragment of the target gene (non-frameshift) is cloned into the pln2 plasmid. Once the single-crossover recombination between genome and the constructed plasmid occurs, the endogenous gene is segmented by the plasmid backbone and thus inactivated. We developed a toolbox based on pln2 that can be used for different genomic operation mentioned above. With the help of this toolbox, we successfully knocked out large fragments of 20-270 kb.