Abstract: Objective:This study aimed to construct a large conductance calcium activated potassium channel α (BKCa) subunit plasmid with two tags by the overlapping PCR technique to set up a steady base for future ion channel study. Methods:Based on the existing coding BKCa channel α subunit expression plasmid pcDNA3.1-hSlo, we constructed a double-tag expression plasmid, namely, pcDNA3.1-Flag-hSlo-GFP (Flag-hSlo-GFP). Results:Flag tag was inserted into the S1-S2 extracellular loop of BKCa channel α subunit, and GFP tag was connected to the C-terminus of BKCa channel α subunit. Sequence of the constructed plasmid was confirmed successful. Conclusion:The expression plasmid Flag-hSlo-GFP was constructed successfully with overlapping PCR. Overlapping PCR is a valuable method for amplifying long size genes.

Key words: large conductance calcium activated potassium channels, overlapping PCR, gene cloning