I cloned DT_A (diphteria toxin chain A) under EF1A promoter, by substitution of the GFP cassete. According to the map everything was in frame. The plasmid also had mCherry cassete under hPGK promoter transcribing another direction. I tested the plasmid before (size 10kb) for the expression of both GFP and mcherry and everything was fine. After adding the toxin that supposed to kill the cells, nothing happened. Cytotoxicity assays were negative and mcherry was not expressed. (i'm sure the efficiency of transfection was fine) The DT_A vector was transfected after linearization and as a circular plasmid too. The DT_A gene was taken from addgene pMCS-DT_A, where DT_A served as a selection marker (negative selection).
I tried to analyze the DT_A sequence with blast, and it looked fine except several nucleotide mismatches with the wild type sequence, but i think the codon usage was different. My only concern is out of frame cloning, and presence of another promoter that was not annotated in the original plasmid upstream of the DT-A gene.