Figure 4. PCR cloning primer design software interface.

(1) Upstream portion of the vector, up to the cloning position. (2) Additional sequence to include between the vector and the 5’ end of the target gene, eg protease cleavage site. (3) Sequence of the target, encoded by the template. (4) Additional sequence to include between the 3’ end of the target gene, eg protease cleavage site. (5) Downstream portion of the vector. (6) Resulting sequence, i.e. (1)+(2)+(3)+(4)+(5). (7) Output window containing information about primer design and primers to be ordered. pv5 is the primer annealing to the 5′ end of the vector, pi5 is primer annealing to the 5′ end of the insert, pv3 and pi3 are primers annealing to the 3′ end of the vector and the insert, correspondingly. Several versions of primers to be ordered are designed, see Figure 5 for more detailed.