1.For each mutation site, please enter the upstream sequence of the mutation site into the left text box, enter the sequence after mutation of the mutation site into the middle text box, and enter the downstream sequence of the mutation site into the right text box. The directions are all from 5' to 3', just enter the sequence of sense strand.

2.Click‘Generate Amplification Primers (Partially reverse complemented)’button, and the amplification primers of this mutation site are showed in the lower text box (the lowercase sequence in the primer is the mutated sequence).

3.Click the‘Empty’button to continue designing amplification primer pair of another mutation site until all of the mutation sites have been designed.

Note:

1.A mutation site refers to a position of less than 50 bp in the target DNA sequence, which may be one base or multiple bases.

2.The sequence after mutation of the mutation site being inputted into the middle text box should be as short as possible, which can significantly shorten the length of the amplification primer. For single-base mutation, please just enter one base. For continuous or discontinuous multi-base mutation, e.g., it is required that the ACT bases in the NNNaNNNcNNtNNNN sequence will be mutated to G, then please input gNNNgNNg into the middle text box.

3.If no sequence is inputted in the middle text box, seamless splicing of two sequences in the left text box and the right text box can be realized, which can be used for deletion of a particular sequence in vector.

4.Please ensure that: the sequence in left text box sequence + the sequence in middle text box + the sequence in right text box = complete sequence after mutation! Otherwise, errors such as base deletion or redundancy may occur after the mutation is completed.

5.Not applicable to primer design for single-point mutation.