Part:BBa_K2927051
pHMT-LbCas12a plasmid (LbCas12a protein purification)
T7 promoter can produce high yields in the transcription of E. coli when T7 RNA polymerase is present. The sequence we designed is for Cas12a protein expression and purification. Therefore, E.coli BL21 was used for producing our Cas12a protein. 10 times histidine and MBP are used for protein purification, and the TEV cutting site lets Cas12a protein separate from the composition (10X His-MBP-TEV site-Cas12a). 10 times histidine has a strong affinity with nickel ion, and the nickel ion is bind with nickel column. Therefore, we can separate the composition from the solution by nickel ion after breaking bacteria. In the second step of protein purification, the TEV enzyme cut the TEV cutting site for separate Cas12a protein from the composition. Next, we will get Cas12a protein from solution after gathering 10X His- MBP on nickel column.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 4921
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 453
Illegal BglII site found at 2600
Illegal BglII site found at 3337 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 4251
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 151
Illegal BsaI.rc site found at 2664
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