Part:BBa_K3271018

Ade4::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 Ade4 locus from S. cerevisiae, respectively. To target a DNA part of interest to the Ade4 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 Ade4 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 Ade4 locus.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found in sequence at 2290
  Illegal suffix found in sequence at 2346
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 2290
  Illegal SpeI site found at 2347
  Illegal PstI site found at 2361
  Illegal NotI site found at 2296
  Illegal NotI site found at 2354
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 2290
  Illegal XhoI site found at 1026
  Illegal XhoI site found at 2052
  Illegal XhoI site found at 2337
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 2290
  Illegal suffix found in sequence at 2347
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found in sequence at 2290
  Illegal XbaI site found at 2305
  Illegal SpeI site found at 2347
  Illegal PstI site found at 2361
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 2312
  Illegal BsaI.rc site found at 2340