Part:BBa_K1744000
araC-PBAD-vcrx028-ampR
Part Description
This part is designed for clean deletion recombineering experiments. It has an arabinose induced toxin for negative selection and an ampicillin resistance gene for positive selection. This part is designed to be amplified using primer with homology to the targeted region and then integrated in that region using lambda red recombineering systems available commercially such as the heat-inducible pSIM plasmid family. The toxin vcrx028 is toxic for the cell. To repress its expression you must put glucose in the medium (we use 5%w/v). To activate the arabinose killswitch, we use 1 %w/v arabinose in the medium. To do the clean deletion (without DNA scar in the sequence) through recombineering, you must first insert the cassette in the genome by recombination, causing the deletion, and select the recombinants with ampicillin without forgetting to use a medium containing glucose to avoid unwanted toxin expression. Then you have to make the cassette pop out by recombineering using a fusion PCR of both adjacent regions to the one you want to delete and counterselect the cells that did not lose the part with arabinose (triggering the killswitch).
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 1619
Illegal PstI site found at 1631
Illegal PstI site found at 2708 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1227
Illegal PstI site found at 1631
Illegal PstI site found at 2708 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1166
Illegal BamHI site found at 1613 - 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 1619
Illegal PstI site found at 1631
Illegal PstI site found at 2708 - 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 1619
Illegal PstI site found at 1631
Illegal PstI site found at 2708
Illegal AgeI site found at 1001 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1815
Illegal BsaI site found at 2882
Illegal SapI site found at 983
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