IRES sequence - pIRES2-EGFP doen't work (Jul/31/2003 )
You definitely need to include a start codon for your second gene. Gaining similar expression levels from your IREs-dependent gene is unlikely. In most cases, the expression is going to be less, however there are of course many variables to protein expression levels dependent on what you are trying to express. I would recommend to pIRES2 vectors, they work well, however there are still nuiances to using this system, some of which is still not well understood.
Tap14, when I construct one gene of interest into MCSA site of the pIRES vector(BD,Clontech;Catalog No. 631605) and insert another gene into MCSB, can I contain the stop coden at the end of the first gene and will this cause any problem for getting a complete mRNA for the downstream cDNA?
You need the stop codon for the first gene, otherwise you will continue translating into the IRES element and have a screwed up C-terminus. Just make sure there is no polyadenylation signal included because that will terminate the transcript. Good Luck!
Hello,tap14! Thank you for your answers .Now I want to insert a gene which contain the start codon and the stop codon into MCSA site (between XhoI and EcoRI site),should I concern the triplet codon sequence?By the way ,the start codon is just after the XhoI site,will the gene be transcripted and translated from the first start codon correctly?
