I want to design a construct and to know the expression of resistive gene by fluprescence protein. So, I need to use the P2A system for this reason. The question is, whether I should remove the stop codon TGA of resistive gene and then add the P2A sequence and the ATG for second protein or I dont need to remove the stop codon of first protien. Please let me guide by giving a satisfied explanation to this question. Thanks
Hi Zahir,
when using P2A constructs both proteins are produced in a long polypeptide that is then chopped. Because of this you should not have a STOP codon at the end of the first protein as that would prevent the translation of the P2A sequence as well as the second protein.
A different situation occurs when using IRES sequences; in this case you produce a bicistronic mRNA with two different ribosomal entry sites so in this case both sequences need to have a STOP codon.
Hope this helps,
Ezequiel
Thank you Ezequiel Marron, Could I ask you,do you have personal experience in using P2A and IRES? I think the P2A system is better than IRES, do you think so. I wanna use it in human, have you ever used it in human ESCs?
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