Difference between revisions of "Part:BBa K4364000:Design"

 
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===Design Notes===
 
===Design Notes===
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Be careful that other parts of your construct and/or the vector backbone do not contain AhdI recognition sites.
 
  
 +
<b>Be careful that other parts of your construct and/or the vector backbone do not contain AhdI recognition sites.</b>
  
 +
We design the cassette for TA cloning in such a way that the distance between the two AhdI recognition sites is minimal. After digestion, the fragment is way too short to be recovered efficiently by the commonly used kits. This eliminates the need for gel purification and simplifies the entire cloning procedure. Moreover, this fragment does not have stop codons in any of its 6 RFs so even if it stays and gets re-ligated in a reversed orientation, the red-white screening procedure will recognize these clones as empty (red). Last but not least, if a single T is added to the 5' ends of the used primers that will result in a stop codon formation after ligation. This improves the resolution of the screening procedure since now positive clones are extremely unlikely to develop a red color.
  
 
===Source===
 
===Source===
  
−
IDT synthesis - gBolck
+
IDT synthesis - gBolck.
 +
 
 +
This part is a modified version of part BBa_K3764000. Here we have two T7 promoters that flank the TA cloning location and a lac promoter that drives the mRFP1 expression (empty clones develop red color).
  
 
===References===
 
===References===

Latest revision as of 09:28, 12 October 2022


mRFP1-based cassette for TA cloning with dual T7 promotors


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 199
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 207
    Illegal BamHI site found at 316
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 906
    Illegal AgeI site found at 1018
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Be careful that other parts of your construct and/or the vector backbone do not contain AhdI recognition sites.

We design the cassette for TA cloning in such a way that the distance between the two AhdI recognition sites is minimal. After digestion, the fragment is way too short to be recovered efficiently by the commonly used kits. This eliminates the need for gel purification and simplifies the entire cloning procedure. Moreover, this fragment does not have stop codons in any of its 6 RFs so even if it stays and gets re-ligated in a reversed orientation, the red-white screening procedure will recognize these clones as empty (red). Last but not least, if a single T is added to the 5' ends of the used primers that will result in a stop codon formation after ligation. This improves the resolution of the screening procedure since now positive clones are extremely unlikely to develop a red color.

Source

IDT synthesis - gBolck.

This part is a modified version of part BBa_K3764000. Here we have two T7 promoters that flank the TA cloning location and a lac promoter that drives the mRFP1 expression (empty clones develop red color).

References