sentence: Sanger sequencing confirmed the sequence of Mir196A2_WT.In order to create the Mir196A2 variant , we used the Q5 Site-directed mutagenesis kit ( New England BioLabs E0554S ) and the pCMV-mir196A2_WT plasmid was used as a template , with the following primers F-5 ′ CAAGAAACTGtCTGAGTTACATC ′ 3 and R-5 ′ TTGCCGAGTTCAAAACTC ′ 3 .