Amplify the insert gene fragment by PCR with primers including 21 nt of overlapping sequence with the target gene.

Linearize the backbone with restriction enzymes (NEB).

Treat linearized backbone with quick CIP (NEB).

Run PCR products and linearized backbone in an agarose gel to confirm the size.

Purify the DNA from gel using a Gel extraction kit (Bio Basic).

Ligate the linearized backbone and the insert with the T4 ligase (NEB).

Transform the ligation products into home made competent cells.

Perform colony PCR to screen for colons that with inserted gene.

Sequence to verify that the inserted gene is correct.