Hi All,
Nowadays I was trying to find a good promoter, then I see the CAG promoter, it seems good, has C( the cytomegalovirus early enhancer element,) as an enhancer, A( chicken beta-actin promoter) as promoter, but what G( splice acceptor of the rabbit beta-globin gene) doing in it?
when I look into the plasmid map which has CAG promoter, there is about ~800bp sequence between A and G, and I have no idea for the function of that sequence. I don't want to clone that long part gene into my vector because vector has a size limit for transfection and recombination, So I was thinking about only took C and A part, without the seq between AG and G part. but if that sequence is essential and important, then I have to clone it.
Did anyone know anything about that sequence and what G doing in CAG promoter?
Wow, I have the exact same question and I would really like to see Andrey Luchnik's answer, but that answer seems to be gone! Can anyone gave me the gist of what they said?
Wow, Andrey Luchnik, you really knows a lot in this field! Thank you very much!
Note that A is not only the promoter of chicken-beta-actin, but also the first exon and the first intron. This intron also apparently contains some enhancer elements. So of course you also need a splice acceptor in front of your cDNA. And the splice site of the beta-globin is used for that.
Having an intron in an expression vector is also nice in order to easily distinguish between DNA and mRNA/cDNA. Usually you just clone a cDNA-sequence into your vector and when doing expression analysis with mRNA, DNA-contamination would give the same PCR-signal. This can be avoided if your expression vector contains a little intron and Primers are used accordingly.
really good answers. There are quite a few different cag/cba promoters out there though, some shortened for virus constructs etc.
Wow, I have the exact same question and I would really like to see Andrey Luchnik's answer, but that answer seems to be gone! Can anyone gave me the gist of what they said?
Oh, I can't see the Andrey Luchnik's answer. Please anyone can repost it?
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