[Babcock_Hall]

Hello Everyone,
 
http://www.gelifesci...em_handbook.pdf
 
We are trying to understand a plasmid we were sent, and my knowledge of cloning is out of date.  The plasmid consists of the phosphatase of interest (of known sequence) cloned into a vector designed to make glutathione S-transferase (GST) fusion proteins, namely pGEX-4T-1, and there is a site for thrombin cleavage.  After the Arg-Gly thrombin site are six nucleotides that would express a serene and a proline residue.  The next nucleotides begin a restriction site for EcoRI, GAATTC.  The paper we are following indicates that EcoRI and XhoI were the enzymes used to clone the gene of interest.
 
"Collectively, the pGEX vectors provide all three translational reading frames beginning with the EcoRI restriction site (Fig 1.1). pGEX-1λT, pGEX-6P-1, pGEX-4T-1, and pGEX-5X-1 can directly accept and express cDNA inserts isolated from λgt11 libraries."  Based upon this passage from the handbook above, I would offer a guess that the phosphatase gene was cloned out of λgt11, but I don't know this with certainty.  What I don't know is what lies between the EcoRI site and the beginning of the phosphatase gene.  Is there any way to make an intelligent guess about this?  is there any resource that describes cloning from lambda-gt11 into pGEX-4T-1?
 
We have the mass of the fusion protein, and we would like to compare the predicted versus the theoretical mass to verify the existence of the thrombin cleavage site.  Thanks for any help or suggestions.

[Yggdrasil]

Probably the best way to assess this would be to sequence your plasmid (in general, it is good practice to perform sequencing on any plasmid you receive from an external source to verify that it is correct.  Researchers can often have hundred of different plasmids that are very similar and sometimes they can send you the wrong one, especially if the person who made those plasmids is no longer in the lab).

Based on the map of the pGEX-4T-1 plasmid, you can probably perform sequencing with the M13_pUC-rev primer, and it should give you the sequence of the GST plus the linker between GST and the phosphatase.  If your phosphatase sequence is short enough (<~700 bp) you could probably also try the sequencing with the pGEX3 primer.  Or you could design a sequencing primer within the GST coding region that would be able to give you more of the phosphatase sequence.