Template for Building Primer Family Member
By means of PCR and molecular cloning, we come to possess the promoters that will serve as templates for the promoter family. There are several conservative sequences in the promoters, for instance, the -35 box, the -10 box, +1 start. Besides these conservative essence of all the transcriptable genes, we add two non-sense sequences, one on each side of the conservative region. The non-sense sequences were randomly produced, yet once set, they will never change again. They have three main characters:
- They will never present in complicated structures;
- They will never include the restriction enzyme cutting sites that will be involved in the whole study;
- They will never include the recognition sites of RNA Polymerases and those of either of the two repressors.
In addition, we have introduced two operators with a pair of forward and backward primers. For example, U097O26+D062O16 indicates that this part has an operator O26 on the 97th base site upstream and an operator O62 on the 62th base site downstream. At the same time, we have also intruduced two cleavage sites outside each of the two operators, respectively.
P_template1 serves as the truss for PCR cloning. By applying 7 different pair of primers, we have got 49 different promoters. Then we made them digested and loaded into a vector that has already possessed a lacZ-alpha fragment. In this way, we can read from the color of the clones on the plate whether the inserted promoter works or not.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC
- 1000Illegal BsaI.rc site found at 19