The cloning system based on RNAi GG vector construct using type IIS restriction enzyme BsaI is deviced.

Vector containing a Pdk intron region; form a hairpin loop structure, two copies of the ccdB gene flanked by BsaI restriction sites. For SaLac1 gene insertion to the RNAi-GG vector, the ccdB genes are removed with BsaI; identifying sequences upstream and downstream of the pdk intron. Subsequently; adaptor sequences specific to the RNAi-GG vector are then added to Salac gene, enabling its insertion in both sense and antisense directions on either side of the Pdk intron region. The resulting hairpin loop incorporates Salac; triggers the SaLac silencing through RNA interference. (DNA)本载体包含一段Pdk内含子区域（Pdk intron region），可形成发夹环结构；该区域两侧侧翼带有被BsaI限制性酶切位点（BsaI restriction sites）包裹的两段ccdB基因（ccdB gene）。若需将Salac1基因插入RNAi-GG载体（RNAi-GG vector），可通过BsaI酶切移除ccdB基因，并鉴定pdk内含子的上下游序列。随后，为Salac基因添加适配于RNAi-GG载体的特异性接头序列，使其可在Pdk内含子区域两侧以正义及反义方向插入。最终构建的发夹环结构将携带Salac基因，通过RNA干扰（RNA interference）介导SaLac基因的沉默。（DNA）