1.Enter the complete sequence of inserts into the text box (non-ATCG characters will be automatically filtered).

2.Select the‘Add restriction sites at ends’option. If you select‘Yes’, please choose the restriction enzyme from selection list.

3.Click the‘Generate CE Entry Amplification Primer’button. The complete forward and reverse amplification primers of the insert are showed in the lower left text box. Each primer contains a recombinant sequence (lowercase) and a specific amplification sequence (uppercase).

4.Click the‘Empty’button to continue designing the CE Entry cloning primers for other inserts.

Note: Some of restriction enzyme have degenerate recognition sites. For restriction enzyme design scheme, the degenerate base may appear in the cleavage sites of primer sequence, and the degenerate base should be replaced as follows:

M=A or C；R=A or G；W=A or T

S=C or G；Y=C or T；K=G or T

V=A or C or G；H=A or C or T

D=A or G or T；B=C or G or T

N=A or G or C or T