The commonly used CAG promoter is usually labeled as three parts, take pCAGGS-mCherry (https://www.addgene.org/41583/) for example.
CMV enhancer + chicken beta-actin promoter + chimeric intron
(The chimeric intron is a chimera between introns from chicken β-actin and rabbit β-globin.)
My question is where is the transcription start site following the CAG promoter. Is the chimeric intron part of the transcript?
There is actually ATG inside the chimeric intron. If the transcription starts inside chimeric intron, there might be additional sequence added at the N-terminus of the gene under the control of the CAG promoter. This could be a potential problem.
I do not have an answer but actually face the same question. In my case, the ATG inside the chimeric intron is unfortunately in-frame with my insert, thus there is a contiguous ORF from the chimeric intron's ATG all the way through my desired product. So I'm also wondering if I might look at a potentially altered N-term.
Does somebody have an idea whether the chimeric intron ATG can lead to undesiredly translated product when it gives rise to an ORF (or is even contiguous with the ORF of the insert)?
Hi Dr. Xia and Dr. Seitter, I also am running into this question--did either of you find that there was an N-terminal addition to your insert when using a CAG expression vector? Thanks, Emily
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