Plasmid

Part:BBa_K4058021

Designed by: Sarah Cumberland   Group: iGEM21_Guelph   (2021-10-20)


CLE18 sgRNA D in pHSN6A01

This plasmid contains a dCas9 region fused to a VP64 transcriptional activation domain. It also contains a sgRNA sequence which directs the CRISPRa system to bind to CLE18’s promoter.

This plasmid is one of five CLE18 CRISPRa plasmids that were assembled. Five target sequences were chosen from the promoter region of CLE18 in order to test for sgRNA binding efficiency. These sequences were termed CLE18 guide A, B, C, D, and E. Each sgRNA was ordered as two complementary single stranded oligos that when combined into one tube, formed a single double stranded oligo with a 4bp overhang on either side. These 4bp overhangs were designed to be complementary to our BsaI-digested vector pHSN6A01, which allowed for Golden Gate cloning to be used to piece the sgRNA and the rest of the vector together. Each plasmid was then transformed into E. coli DH5 alpha and plated on LB media with kanamycin. The presence of the modified pHSN6A01 plasmid in E. coli colonies was confirmed via PCR. Each plasmid was subsequently transformed into Agrobacterium tumefaciens, plated on LB media with kanamycin and gentamicin, and the presence of the plasmid was once again confirmed via PCR. Next, Agrobacterium tumefaciens cells containing the plasmid were floral dipped into Arabidopsis thaliana plants. Seeds produced by the floral dipped plants were screened for the presence of the plasmid by plating on MS media with hygromycin. Screening revealed no positive transformants from the transformation using Agrobacterium tumefaciens containing CLE18 sgRNA D. However, two positive transformants were identified from the seeds transformed using Agrobacterium tumefaciens containing CLE18 sgRNA B.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 7833
    Illegal XbaI site found at 2961
    Illegal SpeI site found at 9996
    Illegal PstI site found at 2032
    Illegal PstI site found at 3326
    Illegal PstI site found at 3872
    Illegal PstI site found at 5294
    Illegal PstI site found at 5498
    Illegal PstI site found at 6740
    Illegal PstI site found at 12904
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 7833
    Illegal NheI site found at 4538
    Illegal SpeI site found at 9996
    Illegal PstI site found at 2032
    Illegal PstI site found at 3326
    Illegal PstI site found at 3872
    Illegal PstI site found at 5294
    Illegal PstI site found at 5498
    Illegal PstI site found at 6740
    Illegal PstI site found at 12904
    Illegal NotI site found at 2953
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 7833
    Illegal BglII site found at 4128
    Illegal BglII site found at 4818
    Illegal BamHI site found at 2930
    Illegal BamHI site found at 4422
    Illegal BamHI site found at 6460
    Illegal BamHI site found at 7557
    Illegal XhoI site found at 2922
    Illegal XhoI site found at 3746
    Illegal XhoI site found at 4928
    Illegal XhoI site found at 6092
    Illegal XhoI site found at 8657
    Illegal XhoI site found at 9751
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 7833
    Illegal XbaI site found at 2961
    Illegal SpeI site found at 9996
    Illegal PstI site found at 2032
    Illegal PstI site found at 3326
    Illegal PstI site found at 3872
    Illegal PstI site found at 5294
    Illegal PstI site found at 5498
    Illegal PstI site found at 6740
    Illegal PstI site found at 12904
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 7833
    Illegal XbaI site found at 2961
    Illegal SpeI site found at 9996
    Illegal PstI site found at 2032
    Illegal PstI site found at 3326
    Illegal PstI site found at 3872
    Illegal PstI site found at 5294
    Illegal PstI site found at 5498
    Illegal PstI site found at 6740
    Illegal PstI site found at 12904
    Illegal NgoMIV site found at 1457
    Illegal NgoMIV site found at 4690
    Illegal NgoMIV site found at 10733
    Illegal AgeI site found at 9030
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1746
    Illegal BsaI.rc site found at 533
    Illegal SapI site found at 3664
    Illegal SapI.rc site found at 5131


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