I am trying to design a plasmid for a 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?