Part:BBa_K3271017

Ade2::EX-RFP-SP pSB1K3 Type IIS

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 Ade2 locus from S. cerevisiae, respectively. To target a DNA part of interest to the Ade2 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 Ade2 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. Homologous recombination will facilitate the insertion of the DNA part of interest in the Ade2 locus.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found in sequence at 2276
  Illegal suffix found in sequence at 2332
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 2276
  Illegal SpeI site found at 2333
  Illegal PstI site found at 2347
  Illegal NotI site found at 2282
  Illegal NotI site found at 2340
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 2276
  Illegal XhoI site found at 1012
  Illegal XhoI site found at 2038
  Illegal XhoI site found at 2323
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 2276
  Illegal suffix found in sequence at 2333
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found in sequence at 2276
  Illegal XbaI site found at 2291
  Illegal SpeI site found at 2333
  Illegal PstI site found at 2347
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 2298
  Illegal BsaI.rc site found at 2326