Part:BBa_K3271022

Ade4::Ura3-EX-RFP-SP pSB1K3 Type IIS

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 at 3530
  Illegal suffix found at 3586
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 3530
  Illegal SpeI site found at 3587
  Illegal PstI site found at 3601
  Illegal NotI site found at 3536
  Illegal NotI site found at 3594
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 3530
  Illegal XhoI site found at 1026
  Illegal XhoI site found at 2052
  Illegal XhoI site found at 3577
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found at 3530
  Illegal suffix found at 3587
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found at 3530
  Plasmid lacks a suffix.
  Illegal XbaI site found at 3545
  Illegal SpeI site found at 3587
  Illegal PstI site found at 3601
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BsaI site found at 3212
  Illegal BsaI site found at 3552
  Illegal BsaI.rc site found at 3580
  Illegal SapI.rc site found at 3059