Hi everyone. I have recently been assigned to link a reporter gene after IRES of EMCV (EVCM-R, the name of IRES used). What I am doing is to clone an IRES out from a commercial plasmid and then link it with the report gene by restriction dig. then ligation.
Q1: It comes to my interest that there is 9 nucleotides, 3 amino acid residues following right after IRES. After this 3 amino acid met, thr, thr (referred as linker sequence below), then it comes to the sequence of the report gene. I was wondering is there any specific purpose for this linker sequence or it is just a leftover during the manufacture because I have looked into a few of the plasmids using this IRES (named as IRES2 by snapgene), this 3 amino acid residues are always found there before the report gene.
Q2: Also, I was told that this IRES has enhancer function to ensure that genes before and after the IRES can be transcripted from DNA to RNA. It is because the gene before IRES is too long and the promoter may not be able to support the transcription of such a long gene and the reporter gene as well, 5879 b.p in my case ( Gene + IRES + report gene). I was wondering is IRES bearing this ability to do so that recruit certain protein and then it interact with txn factors and stabilize it for the elongation process. Thanks
hi
َA1 - i think this work impossible by Restriction digestion with desired residue (After this 3 amino acid met, thr, thr) . Commercial vectors are designed not site for easy cutting by routine Restriction enzymes . to this way you can used gene synthesis as interest .
َA 2- IRES sequence usually reduce efficacy or recombinant gene expression .
finally you can used dual promoter vectors (such as pBudCE4.1 EF1 & CMV promoters) for reporter gene sub cloning from other commercial vectors .
good luck
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