Prok. vector x studying 5'UTR (HELP)

otello at sc2a.unige.ch otello at sc2a.unige.ch
Wed Jun 22 10:37:23 EST 1994


Dear netters,
I would like to ask your advice about a prokaryotic vector
I am looking for. I would like to test a 5'UTR sequence from chloroplast
DNA for expression in coli. Therefore, I am looking for a vector with the 
following characteristics:
-either resistance gene with a low suppression rate or Bgal or 
anything else that could give me a QUANTITATIVE measurement of the expression
rate; this should be downstream of a cloning polylinker (or suitable sites) 
designed for insertion of the foreign promoter/regulatory signal(s). I would
really like to produce a fusion protein with the reporter/marker, since
the region I'm studying goes from the promoter region to about 10-15 aa 3' 
to the ATG. The fusion protein should be stable....
-preferably low copy number vector (this is not an absolute requirement,
but it would be really helpful, since I would like to mutagenise the UTR
I'm testing and an high copy number vector may give too high a background
to notice subtle differences).
-of course, stable in coli

Some detail about the region I'm interested to study:
-about 600-700 bp (depending on what RE sites I'm going to be able to use)
-suitable sites: 5' end: EcoRI (if it is possible to include the heterologous 
promoter) or Eco47III (blunt) if not (in this case, a fusion to lac 
promoter would be great..). 3' end: StyI BamHI.

BTW: another option would be to clone the whole thing  (without promoter)
downstream of lac: is it there any low-copy number vector suitable for this?

Thanks a lot for your (precious) help.


Otello




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