Part:BBa_K4041005

TDH cas12a crRNA transcription device (crRNA1)
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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]