Phosphorylation of Ser133 in the transcription factor CREB is an important mechanism for regulating its transcriptional activity, however recent work has suggested significant roles for other regulatory inputs into CREB. To allow study of this in vivo, we have generated a Ser133 to alanine knockin mutation in the mouse CREB locus. As CREB knockout is perinatal lethal, a minigene strategy was used to allow conditional knockin of the Ser133Ala mutation in adult mice using Cre recombinase. While some expression of the mutated protein was observed prior to Cre expression, following Cre expression in either T cells or neurons only the mutated CREB protein was detected.