Part:BBa_K4249002

The expression cassette carrying an ADH1 promoter, NcEgt1 coding sequence and an CYC1 terminator

These two parts are the gene expression cassettes corresponding to the above two genes respectively. The expression cassettes both carrying a promoter, gene coding sequence and a terminator. In order to enable the engineered strain to produce ergothioneine in a more sustainable way, we chose the constitutive promoter to introduce the expression of the two heterologous genes. According to literature references and existing resources in our laboratory, we finally chose apply pTEF1 and pADH1 to our work, they are commonly used for metabolic engineering of yeast, more importantly, both of them could provide relatively high expression values of genes, which means we could get more production of ergothioneine in the end. Transcription terminators play a role in terminating the progress of gene transcription, and are thus essential elements in the gene expression. For both gene expression cassettes, we chose apply tCYC to our work. In addition, it should be noted that we integrated these gene expression cassettes into the yeast genome via homology repair, which means the homology arm are required. Here, these two gene expression cassettes were added with ~0.5kb homology arms on both sides when they were used as the linear integration fragments.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1461
  Illegal EcoRI site found at 2788
  Illegal XbaI site found at 2145
  Illegal XbaI site found at 3375
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1461
  Illegal EcoRI site found at 2788
  Illegal NotI site found at 3362
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1461
  Illegal EcoRI site found at 2788
  Illegal BglII site found at 1323
  Illegal BglII site found at 1809
  Illegal BglII site found at 2142
  Illegal BglII site found at 2811
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1461
  Illegal EcoRI site found at 2788
  Illegal XbaI site found at 2145
  Illegal XbaI site found at 3375
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1461
  Illegal EcoRI site found at 2788
  Illegal XbaI site found at 2145
  Illegal XbaI site found at 3375
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 90