Part:BBa_K3760003

T7 promoter + hACE2 target RNA coding sequence

A T7 promoter is added to 5' end of hACE2 gene （human angiotensin converting enzyme 2) This part is the template for RNA in vitro synthesis. The product will be the mRNA of hACE2 used in verification of RfxCas13d 's cleavage activity in virto.

Result

The double-stranded DNA (dsDNA) carrying T7 promoter sequence were prepared by PCR. Then the transcription reaction was carried out with DNA templates using the T7 High-Efficiency Transcription Kit (TransGen Biotech) at 37°C (1 h). The RNA was purified by the Easy Pure RNA Purification Kit (TransGen Biotech) and quantified with Nanodrop.Then purified RNA’s quality was assayed by nondenaturing agarose gel electrophoresis.

Figure 1 Purified products of in virto transcription on nondenaturing agarose gel.

In addition, we tested the targeting effect of sgRNA by in vitro enzyme activity assay. When RfxCas13d is activated by sgRNA and will cut reporter. The reporter will emit green fluorescence. The dynamic changes of fluorescence were observed using a qPCR instrument (ABI, USB). As shown, RfxCas13d cleaved fluorescent reporter showed considerable activity in the presence of sgRNA.

Figure 3 Cleavage activity of RfxCas13d in virto.(a)The fluorescence signals of target RNA(A,B,C) and control(D,E,F,G) were detected on real-time PCR.(b) The cleavage activity of RfxCas13d were tested with ssRNA reporter. Control: Without target RNA. Error bars represent the means ± SD from three replicates (n = 3)

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 525
  Illegal BamHI site found at 1090
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 2442
  Illegal SapI site found at 28
  Illegal SapI site found at 315