Dear Brett Seese,

Since the His tag, kozac sequence, and secretory tag are short sequences, it is a simple method to add a Primer and bind them using PCR.

However, how are we going to prepare the sequence of SARS-COV-2-RBD?

Although you are free to handle any nucleic acid sequence on the PC, you should comply with the law when actually conducting experiments involving nucleic acids containing the SARS-COV-2-RBD sequence. In Japan, the handling of SARS-COV-2-RBD is very strictly restricted, and gene synthesis services are not available in principle.

Best,

Narumi

@Narumi's idea is fine.

I can also add that it is better to look for a vector with these sequences included. You don't want to waste your resources and time cloning 4 inserts into your backbone while also making sure they are in frame with the promoter. I think you can find a vector with the kozac sequence and the secretion tag quite easily. Then you can design your primers for the RBD region with the his tag included and clone the His+RBD insert into that vector.

I am wary of annealing larger primer pairs containing all four sequences. Depending on the size and sequence of the final primer, the PCR amplification may not be efficient.

You can use primers to add a small tag ( e.g. His and Flag) to your gene of interest. Make sure the tag and the gene sequence are in frame If you decided to add the tag at the N-terminus.