Part:BBa_K2100015

pDEST_mCherry_GG

pDEST-mCherry is a destination vector which contains the mCherry gene, a monomeric fluorescent protein which emits red light upon excitation. The construct is used as a negative selection marker to recognize colonies of bacteria which have plasmids that did not successfully undergo the LR reaction and remain as destination vectors.

MIT iGEM 2017 Contribution-- part BBa_K2429061

This is an updated version of a part developed by the previous MIT team (BBa_K2100015). The original part is a destination vector similar to the ccdB destination vector (pDEST ccdB) but instead of having the ccdB domain that leads to cell apoptosis, there is an mCherry domain that would normally make colonies red. After a successful LR reaction this mCherry domain would be substituted with the gene (s) of interest from the entry vector and when picking colonies, you would select for the white colonies instead of red (red means the destination vector in those colonies remained unaltered hence unsuccessful LR). However, what ends up happening is that there are lots of red colonies with few white colonies in between which makes picking difficult.

Our team added Sce1 restriction sites flanking the mCherry domain on the pDest, but not flanking the R restriction sites. We also added the Sce1 yeast restriction enzymes during the LR process when golden gating the entry with the destination vector. The final product was a destination vector that uses the color red to denote unsuccessful LR reaction, while white denotes disruption of the mCherry gene and a successful LR reaction is likely. The distinction between white and red, however, is much clearer than when the original part was used.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2466
  Illegal XbaI site found at 2493
  Illegal XbaI site found at 2797
  Illegal SpeI site found at 2054
  Illegal PstI site found at 1907
  Illegal PstI site found at 2505
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2466
  Illegal SpeI site found at 2054
  Illegal PstI site found at 1907
  Illegal PstI site found at 2505
  Illegal NotI site found at 971
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2466
  Illegal BamHI site found at 2487
  Illegal XhoI site found at 1031
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2466
  Illegal XbaI site found at 2493
  Illegal XbaI site found at 2797
  Illegal SpeI site found at 2054
  Illegal PstI site found at 1907
  Illegal PstI site found at 2505
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2466
  Illegal XbaI site found at 2493
  Illegal XbaI site found at 2797
  Illegal SpeI site found at 2054
  Illegal PstI site found at 1907
  Illegal PstI site found at 2505
  Illegal NgoMIV site found at 3360
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BsaI site found at 2749
  Illegal BsaI site found at 4337
  Illegal BsaI.rc site found at 2092