Part:BBa_K5490034

pCMV-FLAG-TRIM32

Vector of BBa_K5490017 This backbone contains a cmv promoter/enhancer in its prefix and a polyA tail in its suffix

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 4637
  Illegal SpeI site found at 3999
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 4637
  Illegal SpeI site found at 3999
  Illegal NotI site found at 4629
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 4637
  Illegal BglII site found at 4649
  Illegal BamHI site found at 1
  Illegal XhoI site found at 995
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 4637
  Illegal SpeI site found at 3999
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 4637
  Illegal SpeI site found at 3999
  Illegal NgoMIV site found at 3337
  Illegal AgeI site found at 553
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BsaI site found at 391
  Illegal BsaI.rc site found at 2360
  Illegal SapI site found at 1277
  

When performing immunocytochemistry on the original plasmid pCMV-MycNFAT provided by Professor Meško and her team, significant background noise was observed. In contrast, the production of NFAT was successfully detected via Western blot analysis. To address the background issue, we decided to add a FLAG tag to the N-terminus of the Myc tag through subcloning. For this purpose, we utilized the pCMV-FLAG-TRIM32 construct, which was readily available in the lab. To facilitate the subcloning process, it was necessary to remove the TRIM32 sequence downstream of the FLAG tag while extracting the NFAT insert from the original construct. We needed to identify suitable enzymes to maintain directionality and preserve the open reading frame (ORF). We developed two different strategies to achieve this outcome

Partial Digestion Cloning

Insert Preparation: We first identified a BglII site at the borders of the NFAT insert; however, an additional BglII site was present within the NFAT sequence, necessitating a partial digestion.

Vector Preparation: For the vector, we identified a BamHI site at the borders of the TRIM32 gene. BamHI is isoschizomeric to BglII, but it cuts within the TRIM32 gene, which is acceptable as we only require the backbone from this construct.