Part:BBa_K3271016

Gal4::His3-EX-RFP-SP pSB1K3

RFP is flanked by a prefix and a suffix at the 5' and 3' ends, respectively. The prefix and suffix (EXSP) of the pSB1K3 backbone are flanked by the upstream and downstream homology sequences of the Gal4 locus from S. cerevisiae, respectively. Between the upstream homology and the prefix is the His3 auxotrophic selection marker. To target a DNA part of interest to the Gal4 locus in S. cerevisiae, replace the RFP casstte with the DNA of interest (the insert) within the prefix and the suffix using any method of your choice. Transform the resultant plasmid in E. coli. Successful clones are white colonies (as opposed to red). Screen for the appropriate clone and isolate the desired plasmid via miniprep plasmid extraction. Then, digest the plasmid using PmeI. This creates a DNA fragment comprised of the DNA part of interest flanked by the Gal4 locus upstream homology and the downstream homology at the 5' and 3' ends of the part, respectively. Transform the digest into S. cerevisiae using a lithium acetate protocol of choice and plate on SC His-. Homologous recombination will facilitate the insertion of the DNA part of interest in the Gal4 locus.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found in sequence at 3304
  Illegal suffix found in sequence at 4395
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 3304
  Illegal NheI site found at 2982
  Illegal SpeI site found at 4396
  Illegal PstI site found at 4410
  Illegal NotI site found at 3310
  Illegal NotI site found at 4403
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 3304
  Illegal BglII site found at 2873
  Illegal BglII site found at 2933
  Illegal XhoI site found at 1026
  Illegal XhoI site found at 2052
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 3304
  Illegal suffix found in sequence at 4396
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found in sequence at 3304
  Illegal XbaI site found at 3319
  Illegal SpeI site found at 4396
  Illegal PstI site found at 4410
  Illegal AgeI site found at 4106
  Illegal AgeI site found at 4218
  
• 1000
  COMPATIBLE WITH RFC[1000]