Transcription factors (TFs) and their target promoters are central to synthetic biology. By arranging these components into complex regulatory networks, synthetic biologists have been able to create a wide variety of phenotypes, including bistable switches, oscillators, and logic gates. However, transcription factors do not instantaneously regulate downstream targets. After the gene encoding a TF is turned on, it must first be transcribed, the transcripts must be translated, and sufficient TF must accumulate in order to bind operator sites of the target promoter. The time to complete this process, here called the “transcriptional delay,” is a critical aspect in the design of dynamic regulatory networks, yet it remains poorly characterized. In this work, I measured the delay of two TFs in Escherichia coli, which are commonly used in synthetic biology: the activator AraC and the repressor LacI. I found that the delay can range from a few to tens of minutes, and are affected by the expression rate of the TF. The single-cell data also shows that the variability of the delay increases with its mean. To validate these time measurements, I constructed a two-step genetic cascade, and showed that the timing of the full cascade can be predicted from those of its constituent steps. These results demonstrate the timescale of transcriptional regulation in living cells, which is important for understanding the dynamics of synthetic transcriptional gene circuits.