**6. Advantages of MultiSite Gateway technology for donor DNA plasmid construction**

MultiSite Gateway technology is suitable for the construction of a complicated donor DNA plasmid, for several reasons. In this method, all parts of the complicated donor DNA are first cloned into entry vectors, before their assembly. The cloned parts are easily verified by DNA sequencing. The verified entry clones can be used as parts for constructing other donor DNAs. For example, the entry clone containing the required part for expressing the neomycin resistance gene can be reused for constructing donor DNA plasmids targeting the other genes. For generating knock-in cells, other antibiotic (such as blasticidin, puromycin, hygromycin, and zeocin) resistance genes are sometimes required as the selection markers. In this case, if the DNA fragments required for expressing the other antibiotic resistance genes are cloned into the entry vector, we can substitute the neomycin resistance gene of the donor DNA plasmid in **Figure 3** with the other antibiotic genes very easily (**Figure 5**). One of the purposes of generating knock-in cells is to elucidate the function and regulation of the gene product of interest, by expressing the mutants of the gene related to genetic diseases or altered protein modification sites. By using the MultiSite Gateway cloning method, the donor DNA plasmids containing a variety of mutant genes can be easily constructed by simply substituting only the entry clones containing the mutant genes of interest (**Figure 5**). For these reasons, MultiSite Gateway cloning is a convenient and useful method for constructing the complicated donor DNA plasmids.-
