pcDNA 3.1 His Expression - (Oct/24/2011 )
Good to you all. I have simple questions:
1. I am working with pcDNA 3.1, like prokaryotic RBS system, eukaryotic expression plasmid requires Kozak sequence for ribosome binding, I would like to know, do we have to include the Kozak sequence with our insert? If so is there any rules in incorporating the sequence with our insert?
2. Since there are two promoter in eukaryotic plasmid, such as CMV for mammalian expression and T7 for bacterial replication, do have to check my insert start codon to be in frame with CMV start codon only or Both?
Thank you for your time and attention.
Eukaryotic translation initiation is based on a ribosomal scanning mechanism (mostly) as opposed to bacterial initiation that utilizes a ribosome binding site and positional initiation. As long as your start codon is the first one in the transcript, you shouldn't have to worry about being "in frame" since your Insert sequence will define the reading frame.
As far as the Kozak sequence, the pcDNA3.1 manual does explain this, but your insert should put the start site in the context of (G/A)NNATGG, which is the ideal start site for mammalian cells, using a G or A in the -3 position and a G in the +1 position.
Lastly, the T7 promoter is not for bacterial expression or replication, it is there for making in vitro transcripts for in vitro translation. pcDNA3.1 does not have a ribosome binding site, so there will be no protein expression in bacteria.
Best of Luck.
Thank you very much! God bless you, have nice day ahead