I am thinking of cloning a four gene cluster operon and inserting into pET28a. Has anyone had success with this size product and type of gene arrangement. All four genes are subunits of a single enzyme. I am looking for some advice before embarking on this cloning adventure.
Have you had a look at the Golden Gate Technology?
http://j5.jbei.org/j5manual/pages/23.html
We have done this a few times ! We tend to favour a high-throughput approach and would try the whole operon, as well as single proteins, two proteins and three proteins- basically all combinations that can be PCred from the template. The other variable is which protein to tag, as a minimum if I was only going to PCR the whole operon I'd try two constructs, one where protein1 is tagged at the N-terminus and one where protein 4 is tagged at the C-terminus.
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