**3. Donor DNA plasmid for homology directed gene knock-in**

The knock-in of a relatively short gene fragment (up to ~1.5kb) can be achieved by MMEJ or- SSA with a linearized donor DNA fragment containing short (about 20–60bp) homologous- DNA regions, called homology arms [42, 43, 45–47]. The knock-in efficiency decreases as the- size of the insert DNA increases [48]. In the case of the insertion of a long DNA fragment (more- than 1.5kb), the knock-in efficiency increased as the length of the homology arms increased,- up to about 1500 bp [48]. Thus, long homology arms (more than 500bp) are usually used for- the knock-in of such a long DNA fragment [45, 49–52]. In this case, the knock-in is mediated- by HR.-The knock-in efficiency by HR is low [53], and accordingly, a selectable marker is introduced into the donor DNA plasmid for screening the knock-in clones in some cases [49–52].- Therefore, the selectable donor DNA plasmid for the HR-mediated gene knock-in contains left- and right arms, the inserted gene of interest, and a selectable marker gene with promoter and transcription terminator regions. An example of a donor DNA plasmid is shown in **Figure 3**  [52]. In our donor DNA plasmid, the left and right arms contain the promoter and transcription terminator regions of the inserted gene of interest, respectively. However, the native transcription terminator region is separated from the gene of interest by the neighboring selectable

**Figure 3.** A donor DNA plasmid for HR-mediated gene knock-in.-

marker gene. Therefore, the SV40 transcription terminator sequence is also placed just downstream of the gene of interest. Thus, the structure of the donor DNA plasmid is complicated,- as it contains multiple inserted DNA regions. It is difficult to construct such a complicated- plasmid by using classical restriction enzyme-mediated cloning methods. In some cases, the- donor DNA plasmids were constructed by a site-specific recombinational cloning method- named In-Fusion technology [54], provided by Clontech [42, 55].-
