The A2UCOE lacks classic enhancer activity. (A) Illustration of the pGL-2 plasmid vector–based constructs. The pGL-2 promoter construct consists of a minimal, enhancer-less SV40 promoter driving a luciferase reporter gene. A 2.2-kb BamHI fragment consisting of a minimal A2UCOE (Figure 1A) was inserted downstream of the luciferase gene in pGL-2 promoter in both forward (pGL-2 UCOE5′) and reverse (pGL-2 UCOE3′) orientations. The pGL-2 control construct has the SV40 enhancer element inserted at the same position as the A2UCOE test fragment and acts as a positive enhancer control. (B) The 4 plasmids illustrated in panel A were used to conduct transient transfection assays in HeLa, HT1080, Jurkat, and K562 cells. Total protein cell lysates were analyzed for luciferase activity 24 hours after transfection. The mean and standard deviation of triplicate experiments for each cell line are shown. Con indicates pGL-2 control; Pro, pGL-2 promoter; A2-5′, pGL-2 A2UCOE5′; and A2-3′, pGL-2 A2UCOE3′. Note that the pGL-2 A2UCOE5′ and pGL-2 A2UCOE3′ test constructs give luciferase activities that are no higher than pGL-2 promoter in all 4 cell lines, indicating the absence of a classic enhancer function within this element.