I am trying to design a plasmid for our knock in experiment and wanted to clarify some technical issues.

I wanted to know exactly which components must be included in the plasmid.

I know that I have to include the 5' homology arm , insertion gene sequence, marker sequence and 3' homology arm. But, I would like to know for certain the following details:

  1. Should I take the whole sequence of the gene or do I need to remove the introns/UTR regions or any other unnecessary part of the sequence?
  2. Which sequence should I choose for gene insertion , gene sequence or mRNA transcript sequence?
  3. When designing plasmids, should I insert extra promoters and/or terminators with the insertion genes?

Additionally, can you recommend an online tool to design HDR Plasmid? I have already tried using Alt R HDR design tool and Benchling but our target genome is not available there.

  • $\begingroup$ Please clarify your specific problem or provide additional details to highlight exactly what you need. As it's currently written, it's hard to tell exactly what you're asking. $\endgroup$
    – Community Bot
    Jul 30, 2022 at 12:32
  • $\begingroup$ You would probably get better answers on the Biology forum. It would also help to mention whether you are using a commercial backbone for the plasmid or designing the whole construct from scratch. $\endgroup$
    – Cloudberry
    Aug 1, 2022 at 16:13
  • $\begingroup$ Thank you @Cloudberry for your suggesion. $\endgroup$ Aug 4, 2022 at 3:31


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