Part:BBa_K3271023

His3::NatMX-EX-RFP-SP pSB1K3 Type IIS Assembly

RFP is flanked by a prefix and a suffix at the 5' and 3' ends, respectively. It is also flanked by Type IIS BsaI restriction sites along with their corresponding promoter and terminator fusion sites on 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 (for example: traditional endonucleases that target the prefix and the suffix; type IIS assembly using BsaI; or another method). 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 at 3414
  Illegal suffix found at 3470
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 3414
  Illegal SpeI site found at 3471
  Illegal PstI site found at 3485
  Illegal NotI site found at 3420
  Illegal NotI site found at 3478
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 3414
  Illegal XhoI site found at 1026
  Illegal XhoI site found at 2052
  Illegal XhoI site found at 3461
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found at 3414
  Illegal suffix found at 3471
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found at 3414
  Plasmid lacks a suffix.
  Illegal XbaI site found at 3429
  Illegal SpeI site found at 3471
  Illegal PstI site found at 3485
  Illegal NgoMIV site found at 2945
  Illegal NgoMIV site found at 3034
  Illegal AgeI site found at 2663
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BsaI site found at 3436
  Illegal BsaI.rc site found at 3464