This part is composed of two Flipase Recognition Targets (FRT) flanking the Chloramphenicol resistance gene.
The initial part wasn't working, so we remade it from the intermediate
Intermediate for knockin or knockout cassettes with chloramphenicol selection. The cassettes can be removed by introduction of flp recombinase expressing helper plasmid pCP20 leaving behind a single FRT site scar.
Plasmid of insertion and deletion of genes
The thermosensitive origin of replication made it slow to grow
It comes from pKD46 which was modified by adding the flipase function to it
{FRT} recombination site for flp recombinase in BBb
Site for recombination by flp recombinase. Genes flanked by FRT sites (oriented in the same direction) in the genome can be excised with the introduction of flp recombinase-expressing helper plasmid pCP20. Note that the original FRT sequence contained an XbaI site that has been removed with a point mutation for compatibility with standard assembly. See Datsenko and Wanner for details of its use in markerless knockouts and knockins.
Extend ca1010F/R (73 bp, EcoRI/SpeI) Sub into pSB1A2-I13522 (EcoRI/SpeI) Product is pSB1A2-Bca1010 ---- ca1010F Forward (universal biobrick) EcoRI for FRT GGACTgaattcgcggccgcttctagag ca1010R Reverse SpeI oligo for FRT cctatactagtagaagttcctattctctaAaaagtataggaacttcctctagaagcggccgcg
Cassette for knockins or knockouts with chloramphenicol selection and a silent origin of replication. The cassettes can be removed by introduction of flp recombinase expressing helper plasmid pCP20 leaving behind a single FRT site scar. Digestion of the genomic DNA containing these cassettes followed by ligation and transformation into a pir116 or pir+ strain allows capture of the cassette as a high or medium-copy plasmid. pSB1A2-Bca9008
Intermediate for knockin or knockout cassettes with a silent origin of replication. The cassettes can be removed by introduction of flp recombinase expressing helper plasmid pCP20 leaving behind a single FRT site scar. Digestion of the genomic DNA containing these cassettes followed by ligation and transformation into a pir116 or pir+ strain allows capture of the cassette as a high or medium-copy plasmid. pSB1A2-Bca9004
Kohaku So, Takashi Hiroi, Megumi Iseki, Mami Nakatake, Ryosuke Kamei, Ryo Kariyazono
Flpe is a site-specific DNA recombinase that that recognizes FRT sequence. Flpe was derived from yeaset site-specific DNA recombinase Flp. (Buchholz, et al.,Improved properties of FLP recombinase evolved by cycling mutagenesis. Nature. Biotechnology, 16:657-662 ( 1998 )) It's internal EcoRI and SpeI site has been removed using site-directed mutagenesis.
It's internal EcoRI and SpeI site has been removed using site-directed mutagenesis.
This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: BBb Standard Description Page
Cassette for knockins or knockouts with chloramphenicol selection. The cassette can be removed by introduction of flp recombinase expressing helper plasmid pCP20 leaving behind a single FRT site scar.
site specific recombinase that recognize FRT site. FLP catalyze site specific recombination between two FRT site.
site specific recombination between two FRT site.
2??m plasmid of Saccharomyces cerevisiae.