Hi all. I am currently trying to design primers to include the C-terminal HA tag in my protein. I would like to include EcoRI restriction sites at both ends (one at the start of my gene sequence and another one is at the end of the HA tag). My intention is to make it transferrable into another vector if needed in the future. Do you have suggestions on how to design primers to express a protein with the C-terminal HA tag and EcoRI restriction sites?
Thank you.
Tks for inviting. I thought you may need two enzymes. Firstly, you need a protein expression plasmid such as pCMV-HA. Then check the plasmid map and choose suitable restrictive endonuclease according to restriction sites. And then use PP6 to design the primers with ”GAATTC“ before sense primer and "TTCGAA" (HindIII for example) after anti-sense primer. Your target product and plasmid are treated with the two same enzymes and then linked together. Recombinant plasmid can express your protein.
I have succeeded to construct the primer set to amplify my gene of interest with additional HA-FLAG sequence.
Cheers.
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