A putative RBS can be cloned into the ClaI site using a double stranded oligonucleotide having a GC overhang at the 3' end. The vector allows testing of various oligonucleotide sequences as suitable ribosome binding sites (RBS) for expression of the gene in prokaryotic cells. The plasmid construct lacks a 5' leader sequence and RBS. The optimal RBS may vary from protein to protein. To facilitate testing other proteins, the insert coding sequence can be replaced by other foreign gene sequences, using ClaI and BamHI. The foreign gene must provide its own ATG initiation codon. Recommendation for verification: BamHI+ClaI, BglI, PstI. |
