Part:BBa_K3081048
pBAD-dCas9-T7-ctrl-J23119-mRFP
Type II CRISPR/Cas9 system consists of the Cas9 nuclease and a chimeric single guide RNA (sgRNA),deriving from CRISPR/Cas9, the nuclease-deficient Cas9 (dCas9) coexpressed with a sgRNA, has been established for efficient gene repression. We use arabinose promoter to drive the expression of dCas9. For sgRNA, it is under the control of a T7 promoter, with a lactose inducible T7 polymerase integrated into the chromosome. Arabinose and IPTG are used to trigger the expression of dCas9 and sgRNA respectively.Function of the sgRNA "NT1" is to target the coding region of nontemplate strand of a fluorescent reporter gene-mRFP to repress its transcription,while "ctrl" is a sgRNA that has no binding specificity to mRFP.Since normal mRNA elongation is interrupted by occurrence of dCas9 with "NT1", fluorescence is greatly decreased as the inducer concentration increases.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1205
Illegal NheI site found at 2335
Illegal NheI site found at 5623
Illegal NheI site found at 5646 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1144
Illegal BamHI site found at 4614 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 979
Illegal AgeI site found at 6233
Illegal AgeI site found at 6345 - 1000COMPATIBLE WITH RFC[1000]
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