DNA

Part:BBa_K2429063

Designed by: Nia Myrie   Group: iGEM17_MIT   (2017-10-25)
Revision as of 18:12, 25 October 2017 by Nmyrie (Talk | contribs)

mKate FF4 Guide Family Member Parts K2429063 through K2429068 and parts K2429083 through K2429093 are a family of guide sequences used by either a dCas13a variant or Ms2 to target the intron of the mKate FF4 reporters (BBa_K2429033 and BBa_K2429049). These guides lie downstream of a U6 promoter, which is a constitutive promoter typically used for the production of guide RNA.

Our team used multiple guide sequences to "tile" along the reporter’s intron to determine what part of the intron is best to target when trying to control its splicing efficiency. Based on the level of fluorescence associated with each guide, we can compare the results to see whether a specific targeted sequence is integral in the process of splicing, and whether the binding of the RNA binding protein to said sequence changes the splicing efficiency.

Tiling.PNG

The guides shift by a few base pairs so we can make more precise conclusions about what intronic sequences are important for splicing.


Parts K-63 through K-68 lead to guide RNA sequences that serve as a guide for the dCas13a protein.

Lsh_FF4_guides.png


Parts K-83 through K-87 as well as K-93 lead to the production of antisense oligonucleotides (ASOs). Parts K-88 through K-92 consist of the same ASO sequences corresponding to the same order (i.e. K-83 sequence is used in K-88); however, they have an additional portion of nucleotides that will form a hairpin loop which Ms2 can bind. The addition of oligonucleotides without hairpins loops serves as a control to test whether it’s the guide sequence or the protein that is actually affecting splicing.

ASO_FF4_guides.png


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 6
    Illegal EcoRI site found at 322
    Illegal EcoRI site found at 4697
    Illegal XbaI site found at 30
    Illegal PstI site found at 2205
    Illegal PstI site found at 2574
    Illegal PstI site found at 3303
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 6
    Illegal EcoRI site found at 322
    Illegal EcoRI site found at 4697
    Illegal PstI site found at 2205
    Illegal PstI site found at 2574
    Illegal PstI site found at 3303
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 6
    Illegal EcoRI site found at 322
    Illegal EcoRI site found at 4697
    Illegal BglII site found at 949
    Illegal BglII site found at 1573
    Illegal BglII site found at 1969
    Illegal BglII site found at 2260
    Illegal BglII site found at 2350
    Illegal BglII site found at 3511
    Illegal BglII site found at 3598
    Illegal BamHI site found at 339
    Illegal BamHI site found at 4593
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 6
    Illegal EcoRI site found at 322
    Illegal EcoRI site found at 4697
    Illegal XbaI site found at 30
    Illegal PstI site found at 2205
    Illegal PstI site found at 2574
    Illegal PstI site found at 3303
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 6
    Illegal EcoRI site found at 322
    Illegal EcoRI site found at 4697
    Illegal XbaI site found at 30
    Illegal PstI site found at 2205
    Illegal PstI site found at 2574
    Illegal PstI site found at 3303
    Illegal NgoMIV site found at 4550
    Illegal NgoMIV site found at 4569
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 935
    Illegal SapI.rc site found at 1041
    Illegal SapI.rc site found at 1911
    Illegal SapI.rc site found at 2745


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