sentence: The SixA ( H8A ) - TEV - ( His ) expression plasmid pJS70 was constructed with a three-piece assembly ; the vector backbone was amplified from pJS65 using primers pBR322-seq-Rev-comp / pET-seq-F-comp , the 5 ’ end of sixA was amplified from pJS67 with primers pBR322-seq-Rev / SixA-H8A-R1 , and the 3 ’ end of sixA was amplified from pJS67 with primers SixA-H8A-F2 / pET-seq-F.The insertions in all engineered plasmids were verified to be correct by DNA sequencing .