DNA

Part:BBa_K2100003

Designed by: Kathleen Brandes   Group: iGEM16_MIT   (2016-10-10)


pENTR hEF1a

The pENTR hEF1a part is an entry vector containing the human elongation factor 1 alpha (hEF1a) promoter, a constituitive mammalian promoter.

We have characterized the hEF1a promoter in all four cell lines we experimented on in the duration of our project using the following fluorescent tags: eYFP, mKate, and tagBFP.

Sequence and Features

Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 6
    Illegal EcoRI site found at 1210
    Illegal PstI site found at 337
    Illegal PstI site found at 842
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 6
    Illegal EcoRI site found at 1210
    Illegal PstI site found at 337
    Illegal PstI site found at 842
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 6
    Illegal EcoRI site found at 1210
    Illegal BglII site found at 591
    Illegal BamHI site found at 1197
    Illegal XhoI site found at 990
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 6
    Illegal EcoRI site found at 1210
    Illegal PstI site found at 337
    Illegal PstI site found at 842
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 6
    Illegal EcoRI site found at 1210
    Illegal PstI site found at 337
    Illegal PstI site found at 842
    Illegal NgoMIV site found at 725
    Illegal AgeI site found at 103
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal EcoRI site found at 6
    Illegal EcoRI site found at 1210
    Illegal PstI site found at 337
    Illegal PstI site found at 842

The MIT iGEM team used the hEF1a promoter to constitutively express fluorescent proteins as transfection markers in our experiments in HEK293, tHESC, MCF-7, and ISH cells. The amount of fluorescent units observed is an indication of the number of plasmids a particular cell uptakes. The graph below shows two single color controls from one of our experiments. We transfected one well of HEK293 cells with hEF1a-mKate and another with hEF1a-eYFP. The cells that uptook the hEF1a-mKate plasmids showed an increase in only the red fluorescent output, while the cells that uptook the hEF1a-eYFP plasmids showed a clear increase in only the yellow fluorescent output.

T--MIT--hEF1a.png



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Categories
//awards/part_collection/2016
Parameters
None