sentence: pGex4T1_PkBE was cloned as BamHI / EcoRI fragment that was amplified with PkBssRI_WT_for_Bam and T3XL ( PkBssRI_WT_for_Bam TATGGATCCCTGCCGGCGCGCATTCCC , T3XL CGAAATTAACCCTCACTAAAGGGA ) into the corresponding sites of pGex4T1.The eight candidate Nmo sites ( Ser / Thr followed by Pro ) were mutated to Ala as follows : first , using pBS2SKP_Sple_cloneable [ 28 ] as template , ‘ mega primers ’ were amplified with primers Pk_B / Emut_for_3_BamHI and PK_BE_Mut rev 1 , and Pk_BE_Mut for2 and PK_BE_Mut rev 2 , respectively .