I looked at some sequences, and I assume you are talking about pGAPZalpha B plasmid. The map I have shows a box around several bases of the PstI site, which is an indication that these bases are only present in the B form, not the A or C forms. THis changes the relationship of the reading frames between the alpha secretion sequence and the myc epitope. That isa a feature of any set of plasmids with an A, B and C version. It is up to you to decide which to use, if you need the PstI site and this reeading frame, it is the best for you. If the reading frame is off, you could use one of the others in the set, or you have to pick the correct enzymes for cloning, as the cleavage sites each have their own relationship to the reading frame, and possibly add a base or two to bring things into frame without disrupting your coding sequences. THe PstI site is there, not missing unless you remove it. With careful design, and altering sequence by PCR or mutagenesis if necessary, you can clone any coding sequence you wnat in frame with such a plasmid.

In order to solve both problems it would perhaps be useful to perform site-directed mutagenesis. The first step would be to alter the stop codon by switching a base pair. For example, if the stop codon is TGA, you could change it to GGA which encodes for glycine. The second step would be to introduce or delete an extra nucleotide in order to bring everything into the same reading frame.

I looked at some sequences, and I assume you are talking about pGAPZalpha B plasmid. The map I have shows a box around several bases of the PstI site, which is an indication that these bases are only present in the B form, not the A or C forms. THis changes the relationship of the reading frames between the alpha secretion sequence and the myc epitope. That isa a feature of any set of plasmids with an A, B and C version. It is up to you to decide which to use, if you need the PstI site and this reeading frame, it is the best for you. If the reading frame is off, you could use one of the others in the set, or you have to pick the correct enzymes for cloning, as the cleavage sites each have their own relationship to the reading frame, and possibly add a base or two to bring things into frame without disrupting your coding sequences. THe PstI site is there, not missing unless you remove it. With careful design, and altering sequence by PCR or mutagenesis if necessary, you can clone any coding sequence you wnat in frame with such a plasmid.

Have you considered using Gibson (or SLIC) cloning to get the precise sequence without requiring restriction enzymes? We've had tremendous success using SLIC and have not needed restrictions enzymes for cloning in a number of years. NEB has a kit for Gibson assembly.

Just insert or delete as many bases (1 or 2) to get back in them in frame. Point mutation is quite straight forward, requires generally 10-16 PCR cycles and DpnI to degrade the methylated template that was amplified in E. coli, and voila! Check the Agilent (formerly Stratagene) Quickchange II protocol but you don't need to buy their stuff, just buy pfy turbo as polymerase and DpnI from NEB for example. Good luck!

why u dont go for gateway system (TOPO) , which requires no restriction sites. more reliable and easy to handle.