Part:BBa_K3271012

Ade2::KanMX-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 Ade2 locus from S. cerevisiae, respectively. Between the upstream homology and the prefix is the KanMX selection marker which confers resistance to G418 in S. cerevisiae. 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. 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 along with KanMX resistance 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 G418 selection. 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 3820
  Illegal suffix found in sequence at 4911
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 3820
  Illegal SpeI site found at 4912
  Illegal PstI site found at 4926
  Illegal NotI site found at 3826
  Illegal NotI site found at 4919
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 3820
  Illegal XhoI site found at 1126
  Illegal XhoI site found at 2152
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 3820
  Illegal suffix found in sequence at 4912
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found in sequence at 3820
  Illegal XbaI site found at 3835
  Illegal SpeI site found at 4912
  Illegal PstI site found at 4926
  Illegal AgeI site found at 4622
  Illegal AgeI site found at 4734
  
• 1000
  COMPATIBLE WITH RFC[1000]