Difference between revisions of "Part:BBa K5490034"
 
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Vector of BBa_K5490017
 
Vector of BBa_K5490017
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This backbone contains a cmv promoter/enhancer in its prefix and a polyA tail in its suffix
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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
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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 vector 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
 
Partial Digestion Cloning

Latest revision as of 22:23, 28 September 2024


pCMV-FLAG-TRIM32

Vector of BBa_K5490017


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 vector 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.