Part:BBa_K3271015

His3::NatMX-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. Between the upstream homology and the prefix is the NatMX6 selection marker which confers resistance to nourseothricin in S. cerevisiae. 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 and plate on nourseothricin selection. 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 3414
  Illegal suffix found in sequence at 4505
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 3414
  Illegal SpeI site found at 4506
  Illegal PstI site found at 4520
  Illegal NotI site found at 3420
  Illegal NotI site found at 4513
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 3414
  Illegal XhoI site found at 1026
  Illegal XhoI site found at 2052
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 3414
  Illegal suffix found in sequence at 4506
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found in sequence at 3414
  Illegal XbaI site found at 3429
  Illegal SpeI site found at 4506
  Illegal PstI site found at 4520
  Illegal NgoMIV site found at 2945
  Illegal NgoMIV site found at 3034
  Illegal AgeI site found at 2663
  Illegal AgeI site found at 4216
  Illegal AgeI site found at 4328
  
• 1000
  COMPATIBLE WITH RFC[1000]