From 2007.igem.org

Firstly, we extend both sides of the conservative region for transcriptional initiation of PlacUV5[], including -35 box，-10 box and +1 starting point, with two non-sense sequence selected from random groups. The product is named as P_template1 as it is the template for the promoter family. These two non-sense sequence have three main characters:

1. They will never include the restriction enzyme cutting sites that will be involved in the whole study;
2. They will never include the recognition sites of RNA Polymerases and those of either of the two repressors;
3. They will never present in complicated structures.

Secondly, another group of primers, of which the elongation region at 5’ end may contain a unique operator sequence or each, is applied at both ends of P_template1, equipping us with an according group of promoters with complete structures. These promoters can include variant operator sequences at different position in flank of the conservative region.

Then the promoter fragments are digested with XbaI and BamHI and cloned into repression-reporter plasmid, which contains lacZ alpha fragment and gfp under the promoter insertion site.