Difference between revisions of "Part:BBa K1150049"
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__NOTOC__
 
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<partinfo>BBa_K1150049 short</partinfo>
 
<partinfo>BBa_K1150049 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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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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Behind a strong CMV promoter a [https://parts.igem.org/Part:BBa_K1150016 HA-tag], a [https://parts.igem.org/Part:BBa_K1150010 NLS] and [https://parts.igem.org/Part:BBa_K1150000 dCas9] in a shortened version followed by another NLS and a [https://parts.igem.org/Part:BBa_K1150012 BGH terminator] were assembled via the iGEM cloning method.  
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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.  
  
  
 
===Usage and Biology===
 
===Usage and Biology===
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This part can be used in the uniCAS Toolkit of team Freiburg as a shorter version of Cas9.  
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This part can be used in the uniCAS Toolkit of team Freiburg as a shorter version of dCas9.
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Truncation 3: 1131bp of the anterior part of Cas9 are missing
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Truncation 3: 1131bp of the anterior part of dCas9 are missing.
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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_K1150049 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|]]

Latest revision as of 23:36, 4 October 2013

Truncated CMV dCas9 Device #3

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 dCas9.

Truncation 3: 1131bp of the anterior part of dCas9 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
  • 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