Hi! I'm looking for information about how to switch our current promoter(+enhacer) to another from a second plasmid we have, using restriction enzymes, for protein expression in HEK293 cells. A specific question to start with is-Do I need to switch the polyA region also? and do the amino acid around the polyA region matter (so I would have to complete them in the other plasmid) or not? Thanks!
You can get some idea by looking at PGL3 enhancer vector which is derived from pgl3 vector. PGL3 enhancer vector is there with promega.
You should be able to clone a new promoter into your plasmid without having to change the 3' UTR or poly-A addition signal. Translation should stop upstream of the poly a signal, thus there should be no amino acid coding around the poly-a signal which is generally some form of AAUAAA.
I have used several different promoters (general as well as tissue-specific) either with SV40 or BGH pA and never encountered any problems. My understanding is that you are free to mix and match you promoters and polyA signals. There are differences in the strength and directionality of polyA signals though, e.g. SV40 pA is bidirectional while BGH pA is not, in case this is important for you.
You may find the links below helpful:
http://blog.addgene.org/plasmids-101-terminators-and-polya-signals
http://info.addgene.org/download-addgenes-ebook-plasmids-101-1st-edition
Please anybody correct me in case I am wrong or have overgeneralized!
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