Part:BBa_K2876013
pd sgRNA
This part is a pyogenes dcas9 sgRNA targeting GFP.
Our project builds on a dual-dCas9 system by implementing a transcriptional-based reporter system by bridging a dual-dCas9 system with the prokaryotic two-hybrid (P2H) system. In our system the proximity of dCas9 binding brings together a larger protein complex (adapted from the P2H system) and initiates transcription of red fluorescent protein (RFP).
We are adapting a prokaryotic two-hybrid system and combining it with dCas9 in order to selectively detect DNA sequences for disease diagnostics. In order to ensure specificity of sgRNA to dCas9 binding, we used two different dCas9 proteins derived from streptococcus pyogenes and streptococcus aureus bacteria. The pyogenes dCas9 was fused via a tri-alanine linker to a lambda repressor that binds the pOL-62 promoter region 35 base pairs upstream of the RFP initiation site. The aureus dCas9 was fused via a tri-alanine linker to the alpha-subunit of RNA polymerase, which in turn recruits polymerase for transcription. The two sgRNA sequences are produced separately and bind selectively to the dCas9 molecules and target DNA, thus bringing together the entire complex and activating RFP.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 74
Illegal SpeI site found at 61 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 38
Illegal NheI site found at 224
Illegal NheI site found at 247
Illegal SpeI site found at 61 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 74
Illegal SpeI site found at 61 - 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 74
Illegal SpeI site found at 61 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 403
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