Composite

Part:BBa_K2927051

Designed by: KUO, PEI-CHING   Group: iGEM19_CCU_Taiwan   (2019-09-03)
Revision as of 07:28, 16 October 2019 by Harrisman (Talk | contribs)


pHMT-LbCas12a plasmid (LbCas12a protein purification)

To express and purify Cas12a protein, we combine T7 promoter, poly Histidine-tag (10XHis), Maltose binding protein (MBP), and TEV recognizing motif with Cas12 coding sequences. T7 promoter can produce high yields of protein in E. coli with T7 RNA polymerase expression. 10XHis and MBP are used for protein purification through their high affinity to Nickel (II) ion and maltose, respectively. TEV recognizing motif separate 10XHIS and MBP from Cas12a, which is used to elute untagged Cas12a protein after purification. The composition 10X His-MBP-TEV site-Cas12a is called pHMT-Cas12a.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 4921
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 453
    Illegal BglII site found at 2600
    Illegal BglII site found at 3337
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 4251
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 151
    Illegal BsaI.rc site found at 2664


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Categories
Parameters
None