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Thank you very much for your help.

Would the only problem with cloning in the PTD after the MCS (or rather cloning it somewhere in the MCS) be the loss of restriction sites (assuming you wanted to avoid removing the PTD)? It wouldn't affect the creation of a fusion protein?

Good to know. I have another question, all that needs to be done to make a PTD fusion plasmid is to clone in the PTD sequence somewhere in the MCS and then clone in a gene of interest downstream of that (presumably the closer the better) right? I think it's pretty obvious that it should work if the cloning is done right but I just want to check.

Right right. For some reason I had confused methionine for a stop codon. I think that the paper was assuming that whatever gene was being cloned in either had a stop codon or a promoter in it's 5' UTR, so I think as long as it doesn't have either of those between the PTD and the gene of interest it should be fine (ie if you were to use only the ORF of a gene). However I'm wondering if the choice of restriction enzymes used to clone in a gene could cause problems. You don't think that any extra base pairs in between the PTD and the cloned in gene would cause a problem if they are only there because the restriction site didn't immediately follow the PTD? In that second link there are a couple of plasmid maps. In the first map it shows the MCS immediately following the PTD, which is TAT in those maps, but I'm going to guess that you don't need to use BamHI as one of your restriction sites since in the second plasmid map they put a tag in between the PTD and MCS. I guess what I'm asking is whether or not base pairs in between the PTD and cloning site of a gene would prevent the two from fusing as a protein, assuming those particular base pairs aren't part of any UTR and don't code for a stop codon.

Hi, I'm reading a couple of papers about making transducible proteins (proteins that can pass through the cell membrane) and it seems the way to do this is to make a fusion by attaching a protein transduction domain (PTD) onto whatever protein your interested in making transducible. Now it seems this is done by cloning the gene(s) needed to make your protein of interest into a plasmid that already contains a PTD. I'm just wondering how the PTD plasmid is made in the first place. It seems like what you have to do is somewhere in between your promoter site and your multiple cloning site you have to insert a 6 histidine sequence, followed by your PTD sequence, and then a glycine residue. After this your multiple cloning site should follow. Now in one of the papers below it says you should delete the 5' UTR sequence from whatever gene your cloning in. However does this mean that the start codon from the gene of interest should also be deleted? It makes sense if that is the case since I could see how that would fuse a protein to the PTD. Anyways the papers I used to come to this conclusion are below and it would be nice if someone can verify/refute my interpretation of them. http://www.esi2.us.es/~ramonrd/english/ptd4_techinfo2.pdf http://cmm.ucsd.edu/Lab_Pages/dowdy/tat_mta.pdf Also should mention that those papers only talk about using TAT as a PTD however I'm just talking about the general case where any given PTD is used.