Just do it manually. 15-20 bases left and right of the site where you want to introduce the mutation.

Alternatively, if you have unique or usable restriction sites nearby, design primers which do anneal for about 15 to 18 bases before the site to be mutated, amplify with a high amount of primer (do touch down and few cycles) and insert the PCR product.

Alternatively, when you have flanking RE sites, you also may use short pieces of synthetic DNA.

You can try SPRINP mutagenesis protocol or Primer3 plus .

best wishes

Hi,

You can see the description by clicking the "?Help" button or use the PrimerX: Primer Design Based on DNA Sequence, which is another online tool for mutagenic primer design (see also the Documentation section for more details and information).