The rtTA(alt) driver encodes a conditional transcriptional activator that contains a DNA-binding domain derived from the tetracycline repressor (tetR) gene of the transposon Tn10-encoded operon of Escherichia coli fused to the transcriptional activation domain from the Herpes simplex virus Type 1 VP16 gene. The tetR DNA-binding domain can bind specifically to the tetO DNA sequence and thus tetO and rtTA(alt) form a conditional binary expression system that can be used to control the spatial and temporal expression of a gene of interest: a transgene or modified endogenous locus carrying the target gene of interest downstream of tetO sequences is combined with a second transgene or modified endogenous locus encoding the rtTA(alt) driver. The tetR DNA-binding domain in the rtTA(alt) driver has been mutated such that binding to tetO, and subsequent activation of transcription only occurs in the presence of tetracycline or related derivatives. Two additional modifications have been made in the tetR sequence of rtTA(alt) compared to the rtTA driver: a cryptic splicing site that created an intron between amino acids 8 and 144 has been removed and the codon usage has been optimized for mammalian cells (FBrf0138521).