What can I do if my gene of interest has a SapI and I want to make an exact fusion in pTXB1 or pTYB1?
For cloning in to pTXB1 you can use the SpeI site in the Mxe intein. So your 3’ primer will be designed as the reverse complement to: 15-18 bp target gene – TGC….MxeI intein ACTAGT NNNNNN where NNNNNN is any 6 nt to allow for efficient cleavage of your PCR product. The TGC is the first cysteine of the Mxe intein and ACTAGT is the SpeI site. You can cut the vector and insert with NdeI/SpeI and you will regenerate the intein sequence getting an exact fusion. There could be a two-step procedure for cloning a gene containing a single internal SapI site into pTYB1 vector. First, the 5′ fragment of the target gene (NdeI in the forward primer – internal SapI site sequence plus Not or XhoI in the reverse primer) is cloned into pTYB1. This results in a NdeI-SapI-Xho-SapI-intein-CBD fusion. Please remember that you can use SapI for directional cloning in most cases where the three-nt overhangs generated at the two SapI sites are not the same. The second PCR uses one forward primer containing t
