2. Upstream primer design: select and copy the 5'-3' gene sequence with a forward direction of about 23bp; Add your upstream restriction site (generally 6bp) to the 5' end of this sequence, and then add two arbitrary bases before it as protective bases; The sequence is: protective base (2bp)+ upstream restriction site (6bp)+ gene initiation sequence 5'-3', and the total length of primer base is about 28-30bp.

3. Downstream primer design: complementary strand sequence is obtained by sequence complementation, the 5'-3' gene sequence is about 20bp, and downstream restriction sites and protective bases are added at the 5' end; Generally speaking, stop codons need to be added between restriction sites and genes. The sequence is: protective base (2bp)+ downstream restriction site (6bp)+ stop codon (3bp)+ initial sequence of gene complementary sequence 5'-3', and the total length of primer base is about 28-30bp.

4. Check: (1) primer length; (2) Whether the upstream restriction site is frameshifted, and if it is frameshifted, 1-2 bases should be added to restore the reading frame; (3) If the downstream stop codon is not designed, it should also be tested whether the frame shift occurs and whether the PCR can be successfully terminated; (4) Annealing temperature, the annealing temperature of a pair of primers should not be too different, otherwise the base should be adjusted to meet the requirements.