Difference between revisions of "Part:BBa K1150063"
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{| style="color:black" cellpadding="6" cellspacing="1" border="2" align="right"
 
{| style="color:black" cellpadding="6" cellspacing="1" border="2" align="right"
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! colspan="2" style="background:#FFBF00;"|HA-NLS-Cas9-Linker
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! colspan="2" style="background:#FFBF00;"|HA-NLS-dCas9-Linker
 
|-
 
|-
 
|'''Function'''
 
|'''Function'''
 
|A new effector can be fused to
 
|A new effector can be fused to
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this Cas9 intermediate part
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this dCas9 intermediate part
 
|-
 
|-
 
|'''Use in'''
 
|'''Use in'''
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|-
 
|-
 
|'''RFC standard'''
 
|'''RFC standard'''
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|Front [https://parts.igem.org/Help:Assembly_standard_10 RFC 10] & back [https://parts.igem.org/Help:Assembly_standard_25 RFC 25]
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|[https://parts.igem.org/Help:Assembly_standard_25 RFC 25]
 
|-
 
|-
 
|'''Backbone'''
 
|'''Backbone'''
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|}
 
|}
  
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==Biology and Usage==
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This intermediate part can be used to engineer your own dCas9 fusion proteins. [http://2013.igem.org/Team:Freiburg Freiburg 2013] already fused VP16, KRAB, G9a, UVR8, PIF6 and CRY2 to dCas9. With the HA tag you can verify via wester blot that your fusion protein is expressed or you can purify the protein with affinity chromatography. The NLS makes sure your fusion protein is transported into the nucleus of mammalian cells. The short 3 amino acids linker will make a connection beween dCas9 and your effector domain.
  
 +
==Usage==
 +
 +
At the front of this intermediate part you can ligate any favored promoter (Freiburg 2013 used SV40 and CMV promoters). At the back of this part you can fuse an effector domain without frameshift by using the RFC 25 standard.
  
 
<span class='h3bb'>
 
<span class='h3bb'>

Latest revision as of 23:21, 4 October 2013

HA-NLS-Cas9-Linker 3 AA

.

HA-NLS-dCas9-Linker
Function A new effector can be fused to

this dCas9 intermediate part

Use in Mammalians
RFC standard RFC 25
Backbone pSB1C3
Submitted by [http://2013.igem.org/Team:Freiburg Freiburg 2013]

This intermediate part can be used to engineer your own dCas9 fusion proteins. [http://2013.igem.org/Team:Freiburg Freiburg 2013] already fused VP16, KRAB, G9a, UVR8, PIF6 and CRY2 to dCas9. With the HA tag you can verify via wester blot that your fusion protein is expressed or you can purify the protein with affinity chromatography. The NLS makes sure your fusion protein is transported into the nucleus of mammalian cells. The short 3 amino acids linker will make a connection beween dCas9 and your effector domain.

Usage

At the front of this intermediate part you can ligate any favored promoter (Freiburg 2013 used SV40 and CMV promoters). At the back of this part you can fuse an effector domain without frameshift by using the RFC 25 standard.

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 312
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Literature