Part:BBa_K4058033
CLE18 multiplex gRNA in pHSN6A01
This plasmid contains a dCas9 region fused to a VP64 transcriptional activation domain. It also contains a multiplex gRNA sequence which directs the CRISPRa system to bind to five regions in CLE18’s promoter.
This plasmid contains all five CLE18 CRISPRa gRNAs that were designed for this project. Five target sequences were chosen from the promoter region of CLE18 in order to test for sgRNA binding efficiency. These sequences were termed ICE2 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 multiplex gRNA.
Sequence and Features
- 10INCOMPATIBLE 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 - 12INCOMPATIBLE 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 - 21INCOMPATIBLE 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 - 23INCOMPATIBLE 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 - 25INCOMPATIBLE 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 - 1000INCOMPATIBLE 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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