Part:BBa_K3271014

Ade4::Ura3-EX-RFP-SP pSB1K3

RFP is flanked by a prefix and a suffix at the 5' and 3' ends, respectively. Between the upstream homology and the prefix is the Ura3 auxotrophic selection marker. 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. 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 along with Ura3 auxotrophic selection marker 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 Ura-.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found in sequence at 3530
  Illegal suffix found in sequence at 4621
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 3530
  Illegal SpeI site found at 4622
  Illegal PstI site found at 4636
  Illegal NotI site found at 3536
  Illegal NotI site found at 4629
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 3530
  Illegal XhoI site found at 1026
  Illegal XhoI site found at 2052
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 3530
  Illegal suffix found in sequence at 4622
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found in sequence at 3530
  Illegal XbaI site found at 3545
  Illegal SpeI site found at 4622
  Illegal PstI site found at 4636
  Illegal AgeI site found at 4332
  Illegal AgeI site found at 4444
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 3212
  Illegal SapI.rc site found at 3059