Part:BBa_K4364000:Design
mRFP1-based cassette for TA cloning with dual T7 promotors
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 199
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 207
Illegal BamHI site found at 316 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 906
Illegal AgeI site found at 1018 - 1000COMPATIBLE 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).