Difference between revisions of "Part:BBa K1150047"
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<partinfo>BBa_K1150047 short</partinfo>
 
<partinfo>BBa_K1150047 short</partinfo>
  
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Behind a strong CMV promoter a HA-tag, a NLS and Cas9 in a shortened version followed by another NLS and a BGH terminator were assembled via the iGEM cloning method.  
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Behind a strong CMV promoter a HA-tag, a NLS and dCas9 in a shortened version followed by another NLS and a BGH terminator were assembled via the iGEM cloning method.  
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The truncation of the huge Cas9 gene was performed by PCR over the backbone (=Cas9 in pSB1C3). The part between the primer pair is not amplified. One of the primers has an overlap compatible to the first bases of the other one, so that after this PCR a one fragment Gibson assembly could be performed.  
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The truncation of the huge dCas9 gene was performed by PCR over the backbone (=dCas9 in pSB1C3). The part between the primer pair is not amplified. One of the primers has an overlap compatible to the first bases of the other one, so that after this PCR a one fragment Gibson assembly could be performed.  
  
  
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<!-- Uncomment this to enable Functional Parameter display
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===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K1150047 parameters</partinfo>
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To verify the expression of the truncated dCas9 version we performed westernblot with anti-HA antibody.
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[[File:Blots-truncation-registry1_Team_Freiburg.PNG|700px|]]

Revision as of 19:01, 3 October 2013

Truncated CMV dCas9 Device #1

Behind a strong CMV promoter a HA-tag, a NLS and dCas9 in a shortened version followed by another NLS and a BGH terminator were assembled via the iGEM cloning method. The truncation of the huge dCas9 gene was performed by PCR over the backbone (=dCas9 in pSB1C3). The part between the primer pair is not amplified. One of the primers has an overlap compatible to the first bases of the other one, so that after this PCR a one fragment Gibson assembly could be performed.


Usage and Biology

This part can be used in the uniCAS Toolkit of team Freiburg as a shorter version of Cas9. Truncation 1: here 365bp at the end of Cas9 are missing


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 576
    Illegal BglII site found at 900
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Functional Parameters

To verify the expression of the truncated dCas9 version we performed westernblot with anti-HA antibody. Blots-truncation-registry1 Team Freiburg.PNG