Difference between revisions of "Part:BBa K1150033:Design"

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===Design Notes===
 
===Design Notes===
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Behind a constitutive active CMV promoter we cloned a acGFP , a NLS and an BGH-terminator  for bright green fluorescence in the nucleus of mammalian cells.  
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All templates were received from AG Weber, BIOSS. The different parts of this device were amplified by PCR with primer with overlaps and via a four-fragment Gibson assembly ligated together. By this overlap a NLS behind the acGFP was introduced to ensure florescence in the nucleus. To be able to easily exchange the promoter of the construct a NheI cutting site was introduced in front of the CMV promoter and a SacII cutting site behind the promoter. Behind the acGFP two cuttingsites, SalI and BamHI, were established to be able to insert a DNA cutting recognition site here. This leads to the possibility to separate the nls from the gfp leading to fluorescence of the cytosol of mammalian cells. Behind the bgh terminator HindIII and KpnI cuttingsites were introduced to substitute a multiple cloning site found in most commercial available plasmids.
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===Source===
 
===Source===

Latest revision as of 08:12, 24 September 2013

Constitutive GFP Reporter


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 6
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1422
    Illegal BamHI site found at 1379
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Behind a constitutive active CMV promoter we cloned a acGFP , a NLS and an BGH-terminator for bright green fluorescence in the nucleus of mammalian cells. All templates were received from AG Weber, BIOSS. The different parts of this device were amplified by PCR with primer with overlaps and via a four-fragment Gibson assembly ligated together. By this overlap a NLS behind the acGFP was introduced to ensure florescence in the nucleus. To be able to easily exchange the promoter of the construct a NheI cutting site was introduced in front of the CMV promoter and a SacII cutting site behind the promoter. Behind the acGFP two cuttingsites, SalI and BamHI, were established to be able to insert a DNA cutting recognition site here. This leads to the possibility to separate the nls from the gfp leading to fluorescence of the cytosol of mammalian cells. Behind the bgh terminator HindIII and KpnI cuttingsites were introduced to substitute a multiple cloning site found in most commercial available plasmids.

Source

All templates for PCR were received from AG Weber, BIOSS

References