Difference between revisions of "Part:BBa K1699005"

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<partinfo>BBa_K1699005 short</partinfo>
 
<partinfo>BBa_K1699005 short</partinfo>
  
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gRNA for dCas9-VP64 targeting synthetic activation promoter pMLPm under U6(RNA pol III) promoter.
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gRNA for dCas9-VP64 targeting synthetic activation promoter pMLPm under U6 (RNA polymerase III) promoter. gRNA sequence is complementary to 3 different loci in the synthetic promoter pMLPm (1), and gRNA-dCas9-VP64 complex can promote transcription downstream of synthetic promoter.
  
  
  
 
===Usage and Biology===
 
===Usage and Biology===
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gRNA is part of CRISPR/Cas9 complex. guide RNA is a hundred base-long molecule with a unique two dimensional structure which binds Cas9 and guides it to a dsDNA sequence complementary to 21-22 base pairs on the 5' end of the molecule. gRNA scaffold sequence for SaCas9 was used (1).  MLP is a synthetic promoter with three complementing sites for the gRNA (2).
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Guide RNA is a hundred base-long molecule with a unique two dimensional structure which binds dCas9-VP64 and guides it to a dsDNA sequence complementary to 21-22 base pairs on the 5' end of the molecule. Upon binding to promoter, dCas9-VP64-gRNA complex will promote transcription of genes downstream of binding site. gRNA scaffold sequence for SpdCas9-VP64 was used (2).
  
 
===Characterization===
 
===Characterization===
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This part was designed as a control construct for two cancer-specific promoter-based design of CRISPR-mediated transcriptional activation. In the design, gRNA is ribozyme-flanked (to enable synthesis under RNA polymerase II promoter) and is under control of human survivin promoter.
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'''U6-gMLP''': AAV vector for the synthesis of gRNA for dCas9-VP64 targeting synthetic activation promoter pMLPm (abbreviated as gMLP) under the control of human U6 promoter (Fig. 1).
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[[Image:U6-gMLP-pAAV_Map.jpg|center|500px|thumb|'''Fig. 1'''. Plasmid map of AAV vector carrying gMLP under the control of human U6 promoter.]]
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Revision as of 11:29, 13 September 2015

gRNA for dCas9-VP64 targeting synthetic activation promoter pMLPm under U6 promoter

gRNA for dCas9-VP64 targeting synthetic activation promoter pMLPm under U6 (RNA polymerase III) promoter. gRNA sequence is complementary to 3 different loci in the synthetic promoter pMLPm (1), and gRNA-dCas9-VP64 complex can promote transcription downstream of synthetic promoter.


Usage and Biology

Guide RNA is a hundred base-long molecule with a unique two dimensional structure which binds dCas9-VP64 and guides it to a dsDNA sequence complementary to 21-22 base pairs on the 5' end of the molecule. Upon binding to promoter, dCas9-VP64-gRNA complex will promote transcription of genes downstream of binding site. gRNA scaffold sequence for SpdCas9-VP64 was used (2).

Characterization

This part was designed as a control construct for two cancer-specific promoter-based design of CRISPR-mediated transcriptional activation. In the design, gRNA is ribozyme-flanked (to enable synthesis under RNA polymerase II promoter) and is under control of human survivin promoter.

U6-gMLP: AAV vector for the synthesis of gRNA for dCas9-VP64 targeting synthetic activation promoter pMLPm (abbreviated as gMLP) under the control of human U6 promoter (Fig. 1).

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Fig. 1. Plasmid map of AAV vector carrying gMLP under the control of human U6 promoter.


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
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 250
  • 1000
    COMPATIBLE WITH RFC[1000]