Part:BBa_K2933171

Tac promoter+RBS a+Linker g+GST+Linker e+JOHN-1

This part consists of Tac promoter,RBS and protein coding sequence (GST+Linker e+JOHN-1),and the biological module can be built into E.coli for protein expression.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 870
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 870
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 870
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 870
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 181
  Illegal SapI.rc site found at 1394
  

Molecular cloning

First, we used the vector pGEX-6p-1 to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.

Figure 1. Left: The PCR result of JOHN-1. Right: 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.