T2A peptide had higher expression levels than the same genes linked by a mix of 2A sequences. It is reported that the CMV promoter is not consistently effective in ES cells [31,32,33,34], so we were interested to examine whether the apparent beneficial effect of the T2A peptide was also manifested under the control of a stronger promoter. The CAG promoter/enhancer is reported to act efficiently in ES cells [35] and we found a range of construct designs were more efficiently expressed under the control of the CAG promoter compared with CMV. Despite this increased efficiency there was still a further beneficial effect of the T2A peptide. It is been well documented that similar levels of gene expression can be achieved for all genes within a 2A polycistronic vector [4,8,11,17,18,36], therefore, it is reasonable to assume that the expression of EGFP is representative of the expression levels of all the proteins within a single expression cassette. The advantage of using a reporter like EGFP for this study is that it provides a relatively cost effective method for detecting and quantifying protein expression levels and isolation of transfected cells by FACS. There are conflicting reports on the relative cleavage efficiency of the 2A sequences. Some reports show a greater efficiency of T2A compared to F2A and E2A [19,23] and another that F2A was better than T2A and E2A [11]. In those studies GSG linkers on the N-terminus of the 2A peptides were not included in the design. This linker can facilitate almost complete cleavage of the peptides [15,37], and in this study we found that each of the three 2A peptides used had an equally high cleavage efficiency in mouse ES cells. The observation that EGFP production was the same whether it was first or last in the sequence of an expression cassette provides confirmation that in this model cleavage efficiency was not a limiting factor to efficiency of expression. 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