Efficiency of cloning for two or three inserts.

Restriction-ligations were performed from 5 to 30 minutes in Promega ligation buffer, and followed by digestion and heat inactivation. Cloning for two or three inserts was performed in duplicate.*The zero minute time point was performed without adding BsaI to the restriction-ligation mix.

本实验在普洛麦格（Promega）连接缓冲液中开展限制性连接反应，反应时长设置为5至30分钟，随后依次进行酶切与热灭活处理。针对2个或3个插入片段的克隆实验均以平行重复形式开展。*零分钟时间点的反应体系未向限制性连接混合液中添加BsaI核酸内切酶。