Part:BBa_K3221103

GAL1 and T7 promoter, coding sequence of CNS1 and CNS2 with 6�His tag and CYC terminator.

CNS1 and CNS2 are two enzymes responsible for the synthesis of COR(cordycepin). The two enzymes were found by CAS Key Laboratory of Insect Developmental and Evolutionary Biology. It was described that Cns2 catalyzes 3'-AMP to 2'-C-3'-dA and then CNS1 catalyzes 2'-C-3'-dA into COR. So this part is able to express enzymes needed for catalyze 3'-AMP into COR. The two enzymes are able to be expressed together as a fusion protein in this part.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 4045
  Illegal SpeI site found at 1266
  Illegal SpeI site found at 1795
  Illegal SpeI site found at 2449
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal SpeI site found at 1266
  Illegal SpeI site found at 1795
  Illegal SpeI site found at 2449
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1843
  Illegal BglII site found at 2430
  Illegal BglII site found at 2848
  Illegal BamHI site found at 2932
  Illegal XhoI site found at 4039
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 4045
  Illegal SpeI site found at 1266
  Illegal SpeI site found at 1795
  Illegal SpeI site found at 2449
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 4045
  Illegal SpeI site found at 1266
  Illegal SpeI site found at 1795
  Illegal SpeI site found at 2449
  Illegal AgeI site found at 150
  Illegal AgeI site found at 1909
  Illegal AgeI site found at 4108
  
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology:

Cns1 and Cns2 are two enzymes responsible for the synthesis of COR(cordycepin) in the medicinal fungus Cordyceps militari. The two enzymes were found by CAS Key Laboratory of Insect Developmental and Evolutionary Biology. It was described that Cns2 catalyzes 3'-AMP to 2'-C-3'-dA and then Cns1 catalyzes 2'-C-3'-dA into COR. So, this part is able to express enzymes needed for catalyze 3'-AMP into COR. We want to demonstrate that express these two enzymes together can improve the efficiency of the reaction, so we constructed this part. The two enzymes are able to be expressed together as a fusion protein in this part. There are only one GAL1 promoter upstream the fusion protein and one CYC1 terminator upstream the fusion protein. A V5 tag and a 6xHis tag are remained for purification at the end of fusion protein.

Results:

Plasmid construction:

We construct plasmid pYES2-cns1-linker-cns2 by double enzyme digestion and ligation from constructed plasmid pYES2-cns1-cns2. Then we transformed the constructed plasmid into E.coli DH5α for amplification. Plasmid extraction, double enzyme digestion confirmation and sequencing were carried then to verify the correct ligation(Figure.1.).