Part:BBa_K1388001
aeBlue-aacC1 gene cassette for integron testing
This part is used to produce circular gene cassettes for integron testing. It contains (in order): - A spacer region designed to allow circularisation primers to anneal (this avoids secondary structure problems caused by attC, see below) - An NheI restriction site - An attC integron recombination site - An aeBlue gene (see BBa_K864401) with RBS - BamHI restriction site - An aacC1 gene with RBS (confers resistance to gentamicin).
Cassettes can be produced by using Xba/SpeI or NheI/SpeI ELAN (cutting at the prefix/suffix or and ligating each end of the part together at these points) or using Gibson assembly; Gibson circularisation can be carried out by running PCR using primers with reverse-complementary 5 prime ends and 3' ends which anneal respectively to the spacer region and the end of aacC1. Examples of circularisation primers given below:
F: 5'-GAGGTCGGACAGTATATTGAAACGTCAGGAGTCTAGATACCGAGGGACAGACTACCAACTCACA-3' R: 5'-GTATCTAGACTCCTGACGTTTCAATATACTGTCCGACCTCTTATTAGGTGGCGGTACTTGGGTCG-3'
We have validated that a synthetic cassette made from this part can be successfully integrated giving Gm resistance. The aeBlue phenotype was found not to be expressed which we believe is due to a deletion in that region of the cassette. Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 28
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1075
Illegal BamHI site found at 810 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |