Part:BBa_K2933272
RBS+Linker h+His+Linker f+CPS-1+T7 terminator
This part consists of RBS, protein coding sequence(His+Linker h+Sumo+Linker f+CPS-1) and T7 terminator,and the biological module can be build into E.coli for protein expression. This part can be prefaced with promoters of different strengths and types to regulate expression function.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 665
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 93
Illegal NheI site found at 1009
Illegal PstI site found at 665 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 947
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 665
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 665
Illegal AgeI site found at 566 - 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
This composite part is made up with three basic parts, the HiS tag, the thrombin restriction site and our target protein CPS-1. It encodes a protein which is CPS-1 fused with His tag. The fusion protein is about 33.2 kD. It is convenient for us to purify our target protein.
Molecular cloning
First, we used the vector pET28a to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.
Figure 1. a: The PCR result of CPS-1. b: The verification results by enzyme digestion.
After verification, it was determined that the construction is successful. We converted the plasmid to E. coli BL21(DE3) for expression and purification.
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