Part:BBa_K3271024

Gal4::His3-EX-RFP-SP pSB1K3 Type IIS Assembly

RFP is flanked by a prefix and a suffix at the 5' and 3' ends, respectively. It is also flanked by Type IIS BsaI restriction sites along with their corresponding promoter and terminator fusion sites on 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 (for example: traditional endonucleases that target the prefix and the suffix; type IIS assembly using BsaI; or another method). 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 at 3304
  Illegal suffix found at 3360
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 3304
  Illegal NheI site found at 2982
  Illegal SpeI site found at 3361
  Illegal PstI site found at 3375
  Illegal NotI site found at 3310
  Illegal NotI site found at 3368
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  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
  Illegal XhoI site found at 3351
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found at 3304
  Illegal suffix found at 3361
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found at 3304
  Plasmid lacks a suffix.
  Illegal XbaI site found at 3319
  Illegal SpeI site found at 3361
  Illegal PstI site found at 3375
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BsaI site found at 3326
  Illegal BsaI.rc site found at 3354