Gel Electrophoresis and DNA Sequencing Protocol

To separate the indicated DNA fragments, the prepared PCR products were added to a 1% agarose gel with Golden View™ (TaKaRa) and electrophoresed with DL2000 DNA marker (TaKaRa) at 120 V for approximately 25 min, as we reported (20 (link)). PCR band products were visualized using a Gel Doc™ EZ imaging system (Bio-Rad, CA). Positive PCR products were purified (MiniBEST Agarose Gel DNA Extraction Kit, TaKaRa), tailed with “A”-overhang tails (DNA A-Tailing Kit, TaKaRa), and cloned into the pMD19-T vector (TaKaRa). Positive recombinant plasmids were sequenced by Ruibiotech Co., Ltd. (Beijing, China). Nucleotide sequences were blasted against sequences deposited in GenBank using the basic local alignment search tool, nt (BLASTn)¹ for similarity analysis.