I want to over express a protein in different cell lines of human. I know that we can use cDNA for it. But, I wonder is it possible to use just ORF (cDNA without UTR regions) for cloning in a vector and generation of proteins. Or do I have to use whole cDNA.
Make sure you have the required elements around your ORF: Promoter, enhancer, Kozak consensus element, ATG, Stop, polyA signal. Most of them usually are in the expression vector, maybe except the Kozak sequence, which must be in some relation to the start codon.
Only ORF without stop codon is needed for cloning in an expression vector.
Hey,
You can use ORF for protein synthesis. If you are making your protein for antibody generation (polyclonal) then you need not to amplify ORF either. Just look for peptides with a good score to generate antibody. You can use any free online tool for that purpose. And please check with your vector system, whether you have to include stop codon or not.
Cheers.
Make sure you have the required elements around your ORF: Promoter, enhancer, Kozak consensus element, ATG, Stop, polyA signal. Most of them usually are in the expression vector, maybe except the Kozak sequence, which must be in some relation to the start codon.
just ORF is OK because expression vector usually carries a strong promoter and other necessary elements for ectopic expression of a target protein.
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