Part:BBa_K4041008
ctxA cas12a crRNA transcription device (crRNA5)
Template for T7 transcription of crRNA for Vibrio pathogenic gene marker. Need to be used with a XbaI overnight clean cut or PCR for the transcription to end at break point, to generate a short crRNA instead of transcription doesn't end. Designed on 100% conserved region.
Figure 1: Demonstration for a 2 oligo anneal system that is similar to a plasmid double stranded transcription system. The upper strand is annealed to the “T7 promoter” primer for reverse transcription. Need to be used with a T7 promoter based short RNA transcription kit, like MEGAshortscript RNA transcription kit, followed by purification, like spin columns or lithium ion precipitation.
15% UREA Page (Run in room temperature with 80V), indicating that the crRNA is successfully transcribed
crRNA used with single stranded template, showcasing that the crRNA best work with 1X NEB2.1 buffer and functions to cleave reporter very rapidly.
| The following crRNA were proved to work in this block in in Vibrio | |
| TDH forward (crRNA1) | ACTGTGAACATTAATGATAA |
| vcgC forward (crRNA3) | ATAGCACTAATGTGTCATCT |
| vcgC reverse (crRNA4, with PAM) | GCGGAGACGAGATCGCTATC |
| ctxA Forward (crRNA5) | TGATCATGCAAGAGGAACTC |
| crRNA 2 (high background) and crRNA 6 (no cleavage signal) is not functional from design, and is discarded |
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |