I will be working on a project in which I will inducibly express a gene of interest. We are using a Tet On system, in which I will clone my gene of interest into a vector expressing mCherry under a Tet responsive promoter. mCherry is fused to an Internal Ribosome Entry Site (IRES2), downstream of which is the multiple cloning site. Once my gene is inserted, it will allow translation of both proteins from a single transcript, in a Tet inducible fashion. I have picked restriction enzymes that will cut such that the natural ATG of my gene is in-frame with the ATG at the end of the IRES2 (see attachment 3
. Attachments 1+2
for vector info).However, I noticed that there will be a stop codon between the IRES2 ATG and the ATG of our gene. I am wondering if this will
decrease expression levels of our gene, since the ribosome will begin translation at the ATG of IRES2, and then fall off at the stop codon before the ATG of our gene.
I realise this is a difficult question to answer, and I am contacting the manufacturer. I just wanted to see if anyone had any input.
Cloning downstream of IRES2
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