Imagine this:
PromoterCMV-GFP-PromoterEF1a-RFP-Terminator SV40
(please don't ask why I want to express GFP and RFP at the same time :D It's an example)
Wiill this construct work like this? In theory, the CMV promoter transcript will be GFP plus a long 3'UTR (containing the EF1a and RFP mRNA)
The EF1a transcript will be just RFP.
I need high level expression of both genes. Will it work like this?
Most important, will the RFP work for sure?
I am asking because there is no other option (re-synthesizing the vector would be like 3000$)
Hello Andreas,
I have used construct with Two promoters and one terminators, and most importantly both gene express well and I got sufficient amount of protein. Only the difference in expression of two genes is, the second gene i.e. RFP here will express more than GFP theoretically. As RFP has its own one promoter and another promoter from GFP, which is also used to express RFP. But, GFP has only one promoter. So, overall I think both of your gene express well in optimized conditions.
Hi Simran,
The GFP will have a stop codon of course.
Shiv,
In eukaryotes you'll need one promoter per construct, and translation starts at the first ATG, so I would assume that the first promoter only expresses GFP and leaves the rest as junk RNA.
Best,
Andreas
as you are saying that the first one contain Stop codon it is the possibility that the second protein will synthesize or not . and if it is synthesize it 'll be in low concentration.
if you would have IRES sites just before the second protein your protein will theoretically synthesized.
all the best
Hi!
no, each gene has it's own promoter, so both should be expressed.
But one of them may be silenced due to leaky terminat9on or whatever?
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