Part:BBa_K4343118

hisG-URA-hisG

URA gene is a commonly used screening marker in Y. lipolytica. Only strains with URA3 gene can grow in the medium without uracil. Since the Y. lipolytica strains we used were nutrition-deficient (uracil and leucine double deficient) and all plasmids we constructed (except BBa_K4343069) contained URA3gene, it could be verified whether the genome have been successfully edited. HisG tag were utilized in combination with URA3 gene to recycle this screening marker for further use in the same strain. To be more specific, 5-fluoroorotic acid (5-FOA) could be converted into a toxic form (e.g., 5-fluorouracil) by functional OMP decarboxylase encoded by URA3 gene, thus making yeast cells unable to grow. To survive the toxicity, yeast would miss the URA3 gene by HigG tag through negative screening. In this circumstance, URA deficient strain allows for the next transformation.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 1920
  Illegal XhoI site found at 1953
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 2108
  Illegal AgeI site found at 97
  Illegal AgeI site found at 458
  Illegal AgeI site found at 3161
  Illegal AgeI site found at 3522
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1
  Illegal BsaI site found at 638
  Illegal BsaI site found at 3702
  Illegal SapI.rc site found at 363
  Illegal SapI.rc site found at 3427