Part:BBa_K3971009:Design
Promoter characterization cassette for Synechococcus elongatus UTEX 2973.
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 2239
Illegal PstI site found at 362 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 2052
Illegal PstI site found at 362 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1103
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 2239
Illegal PstI site found at 362 - 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 2239
Illegal PstI site found at 362 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 152
Design Notes
In order to allow for modularity in the promoter region of the cassette, two restriction sites Nde1 (upstream) and BamH1 (downstream) flank it, so that the promoter can be easily excised out and replaced with another and the cassette can be used to characterize that promoter. Two different restriction sites were chosen in order to allow for directional cloning of the promoter. Thus any promoter that needs to be inserted into this cassette would need to be flanked by Nde1 and BamH1 in order to ligate in a directional manner.
Both ends of the composite part contain Kpn1 restriction sites since this cassette is designed to be cloned into the pSL2680 plasmid which has a Kpn1 site and a Sal1 site to allow for cloning of cassettes. Since this is a linear cassette, in order to cut it using Kpn1, whose recognition sites are at the ends of the cassette, 3 bp sequences were added to allow for efficient cleavage by Kpn1 at the ends.
Source
sYFP2 (BBa_K864100) and the terminator (BBa_B0015) are available in the iGEM distribution kit. PcpcB-m6 was generously donated by the Wangikar Lab at IIT Bombay. The restriction enzyme sites are incorporated into the cassette by adding them in primers. The homology arms for Neutral Site 1 can be amplified from the S. elongatus UTEX 2973 genome.