Part:BBa_K3271010

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 His3 locus from S. cerevisiae, respectively. To target a DNA part of interest to the His3 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 His3 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 His3 locus.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found in sequence at 2291
  Illegal suffix found in sequence at 3382
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 2291
  Illegal SpeI site found at 3383
  Illegal PstI site found at 3397
  Illegal NotI site found at 2297
  Illegal NotI site found at 3390
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 2291
  Illegal XhoI site found at 1026
  Illegal XhoI site found at 2052
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 2291
  Illegal suffix found in sequence at 3383
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found in sequence at 2291
  Illegal XbaI site found at 2306
  Illegal SpeI site found at 3383
  Illegal PstI site found at 3397
  Illegal AgeI site found at 3093
  Illegal AgeI site found at 3205
  
• 1000
  COMPATIBLE WITH RFC[1000]