Difference between revisions of "Part:BBa K3219000"

Revision as of 08:11, 6 October 2019

dCas9-GFP

dCas9 enzyme is also known as a catalytically dead Cas9 enzyme. Different from traditional CRISPR Cas9 enzymes, dCas9 lacks endonuclease activity. It does not cleave DNA. Instead, with the help of a guide RNA, it specifically binds to the target, usually 20 -30 bp, and blocks transcript elongation by RNA polymerase.

In this part, a GFP is added to the C-terminus of the dCas9, connected using an SGAAAAGGS linker. The GFP is added so that the expression of both proteins could be checked easier.

This sequence can be used by cloning it into a desired vector with a promoter.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 1837
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 4116
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 644

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K3219000 short</partinfo>
-This part consists of the sequence of dead Cas9-GFP complex connected using linker 2A, a 7x His-tag and a ribosome binding site. A dead Cas9 enzyme (dCas9) is a mutation of the CRISPR Cas9 enzyme that lacks endonuclease activity. With the help of a guide RNA, the dCas9 specifically binds to the target DNA and blocks transcript elongation by RNA polymerase, and thus repress the expression of the target gene. The GFP is added for easier selection of positive colonies and to indicate that the dCas9-enzyme has been expressed. Between the dCas9 and GF, we added a linker 2A, which is a self-cleaving linker. This allows the GFP and the dCas9 to fold independently. The 7x His-Tag is for protein purification.
+dCas9 enzyme is also known as a catalytically dead Cas9 enzyme. Different from traditional CRISPR Cas9 enzymes, dCas9 lacks endonuclease activity. It does not cleave DNA. Instead, with the help of a guide RNA, it specifically binds to the target, usually 20 -30 bp, and blocks transcript elongation by RNA polymerase.
+In this part, a GFP is added to the C-terminus of the dCas9, connected using an SGAAAAGGS linker. The GFP is added so that the expression of both proteins could be checked easier.
 This sequence can be used by cloning it into a desired vector with a promoter.