Part:BBa_K4058016
CLE18 multiplex gRNA (CLE18 sgRNAs A, B, C, D, E)
This sequence contains all five of the 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 CLE18 guide A, B, C, D, and E. This construct contained a copy of each sequence in order to increase the number of potential sites for the dCas9-VP64 construct to bind. The multiplex was inserted into the pHSN6A01 CRISPRa plasmid via golden gate 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 the CLE18 multiplex.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 9
Illegal BsaI.rc site found at 2897
| None |