Difference between revisions of "Part:BBa K3764001:Design"

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We design the cassette for TA cloning in such a way that the distance between the two Eam1105I (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 blue-white screening procedure will recognize these clones as empty (blue). 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.
 
We design the cassette for TA cloning in such a way that the distance between the two Eam1105I (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 blue-white screening procedure will recognize these clones as empty (blue). 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.
  
−
We located a flexible linker between the T-vector module and the mRFP1 CDS (https://pubmed.ncbi.nlm.nih.gov/10404163/).
+
We located a flexible linker between the T-vector module and the mRFP1 CDS (https://pubmed.ncbi.nlm.nih.gov/10404163/). The EcoRI recognition site found inside the linker's sequence was mutagenized.
  
−
The AhdI restriction site in CDS of mRFP1 was removed via the introduction of a silent mutation D59D.
+
The AhdI restriction site in the CDS of mRFP1 was removed via the introduction of a silent mutation D59D.
  
 
===Source===
 
===Source===
  
 
A modified version of part BBa_K3764000.
 
A modified version of part BBa_K3764000.
 +
Flexible linker - (https://pubmed.ncbi.nlm.nih.gov/10404163/).
 +
mRFP1 - E1010.
  
 
===References===
 
===References===

Latest revision as of 15:17, 21 October 2021


Module for generation of a T-vector with mRFP1 based selection


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 43
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 51
    Illegal BamHI site found at 160
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 750
    Illegal AgeI site found at 862
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

We design the cassette for TA cloning in such a way that the distance between the two Eam1105I (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 blue-white screening procedure will recognize these clones as empty (blue). 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.

We located a flexible linker between the T-vector module and the mRFP1 CDS (https://pubmed.ncbi.nlm.nih.gov/10404163/). The EcoRI recognition site found inside the linker's sequence was mutagenized.

The AhdI restriction site in the CDS of mRFP1 was removed via the introduction of a silent mutation D59D.

Source

A modified version of part BBa_K3764000. Flexible linker - (https://pubmed.ncbi.nlm.nih.gov/10404163/). mRFP1 - E1010.

References