Plasmid

Part:BBa_K1391016

Designed by: Jing Wei "Raymond" Liu   Group: iGEM14_MIT   (2014-10-17)
Revision as of 21:49, 1 November 2014 by Krbrink (Talk | contribs)

pENTR_hEF1a

Strong constitutive promoter for mammalian gene transcription. This part is a repeat of BBa_K779200 .

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 7
    Illegal EcoRI site found at 1211
    Illegal PstI site found at 338
    Illegal PstI site found at 843
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 7
    Illegal EcoRI site found at 1211
    Illegal PstI site found at 338
    Illegal PstI site found at 843
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 7
    Illegal EcoRI site found at 1211
    Illegal BglII site found at 592
    Illegal BamHI site found at 1198
    Illegal XhoI site found at 991
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 7
    Illegal EcoRI site found at 1211
    Illegal PstI site found at 338
    Illegal PstI site found at 843
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 7
    Illegal EcoRI site found at 1211
    Illegal PstI site found at 338
    Illegal PstI site found at 843
    Illegal NgoMIV site found at 726
    Illegal AgeI site found at 104
  • 1000
    COMPATIBLE WITH RFC[1000]



To test our miRNA’s effectiveness at down-regulating BACE1 expression we transfected separate HEK293 cultures with either (1) BACE1 under constitutive expression by the Hef1a promoter or (2) with BACE1 under constitutive expression by the Hef1a promoter and with a miRNA-generating vector inducibly regulated by the TRE promoter. We performed these transfections in duplicate—once with native BACE1, and then once with eYFP-tagged BACE1. There were two variants of this eYFP-tagged BACE1 construct—one with eYFP linked to the N-terminus of Bace1 and another with eYFP on the C-terminus. We created these two variants after considering the possibility that linking eYFP to either terminus of Bace1 might interfere with the peptide’s native structure or function.

After transfection and Doxycycline-induction of our miRNA-generating vectors, we used flow cytometry to verify that the miRNA-generating vectors were being expressed. Proper splicing of the mature miRNA out of the expression vector leaves an intact mKate coding sequence in the vector, so emission of red fluorescence from our transfected HEK293 indicates proper miRNA processing.

miRNA1

miRNA2

miRNA3

miRNA4

Fig. 1. Determining whether miRNA's 1-4 are successfully processed. The data represent transfection with Hef1a:BACE1, Hef1a:tagBFP, and each of the listed miRNA's. Expression of each miRNA was induced by treatment with 1uM Doxycycline.

[edit]
Categories
Parameters
None