Reduced chloramphenicol acetyltransferase activity observed with vectors containing an upstream SphI recognition sequence.

Chloramphenicol acetyltransferase (CAT) is the most commonly used reporter gene for studying the regulation of mammalian gene transcription. Some of the currently available CAT vectors contain the recognition sequence for the restriction endonuclease SphI within the multiple cloning site. This sequence introduces an ATG triplet that is out of frame with the initiation codon of the CAT gene. Transient expression of CAT fusion genes, constructed using three different cellular promoters, demonstrates that the presence of the upstream AUG triplet in the CAT transcript reduces CAT activity, presumably by interfering with the translation of the coding sequence. Deletion of the SphI site from each of the plasmids increased CAT activity between 4-fold and 5-fold. From these results, we conclude that upstream, out-of-frame ATG triplets must be avoided in order to achieve maximum expression of the reporter gene.