Difference between revisions of "Part:BBa K1391031:Design"

 
 
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<p> This part was created using scarless golden gate assembly. This basic part is flanked by L1 and L2 sites and can be easily cloned into an entry vector using an LR reaction. A promoter can be easily inserted in front of this part in a one pot LR reaction with a promoter flanked by L4 and R1 sites cloned into a backbone that has a negative selection marker between R4 and R2 sites. This part adheres to RFC 65 for recombination based cloning of mammalian parts. </p>
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<p>In our miRNA-generating vector, the sequence of the premature miRNA is contained in between exons coding for mKate2. If the miRNA is processed successfully, the premature miRNA-sequence will be spliced out of the vector, leaving just an intact mKate2 coding sequence in the vector. So red fluorescence via mKate2 is our proxy for determining effective processing of our miRNA’s.
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===Design Notes===
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===Source===
 
===Source===

Latest revision as of 22:05, 1 November 2014

pENTR_miRNAG1


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 502
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 502
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 502
    Illegal XhoI site found at 398
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 502
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 502
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1024
    Illegal SapI.rc site found at 16


This part was created using scarless golden gate assembly. This basic part is flanked by L1 and L2 sites and can be easily cloned into an entry vector using an LR reaction. A promoter can be easily inserted in front of this part in a one pot LR reaction with a promoter flanked by L4 and R1 sites cloned into a backbone that has a negative selection marker between R4 and R2 sites. This part adheres to RFC 65 for recombination based cloning of mammalian parts.

In our miRNA-generating vector, the sequence of the premature miRNA is contained in between exons coding for mKate2. If the miRNA is processed successfully, the premature miRNA-sequence will be spliced out of the vector, leaving just an intact mKate2 coding sequence in the vector. So red fluorescence via mKate2 is our proxy for determining effective processing of our miRNA’s.



Source

Artificial

References