Part:BBa_J61026
[FRT][R6K]
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
This part belongs to a family useful for the construction of transposons and knockin cassettes.
FRT The FRT sequence is the recombination substrate for Flp recombinase. Like the Cre/Lox system, sequences flanked by FRT sites oriented in the same direction get excised by expression of Flp. Cassettes flanked by FRT allow the introduction of selectable markers into the genome followed by subsequent removal by the recombinase.
Tn5 The two Tn5 elements are the substrates for the Tnp transposase in both orientations. Sequences flanked by the two Tn5 elements (in opposing orientation) are mobilizable elements. In the presence of Tnp, they are excised from their plasmid and introduced randomly into the genome or plasmids present in the cell.
Tnp The gene for a high-activity mutant of Tn5 transposase.
OriTr Special strains of E. coli with remnants of the RP4 plasmid, including Rlambda (part ) and BW20767, can transfer plasmids with oriTr (part J01003) to diverse species of bacteria.
OriTf Special strains of E. coli with remanants of the F plasmid, such as J23055, can transfer plasmids with oriTf (part J01002) to other E. coli.
R6K A conditional origin of transfer requiring the pir gene expressed in trans.
Construction of Transposon Plasmids
Transposition mutagenesis is usually carried out by mating a donor strain containing a transposon on a plasmid into a recipient strain. The plasmid with the transposon and transposase transfers to the recipient strain, the transposon is excised from the plasmid, and inserts randomly into the genome. A selectable marker in the transposon and in the recipient strain allows selection of cells receiving the transposon over both the donor and recipient strain.
Construction of the transposon donor strain requires a conjugative donor strain harboring a plasmid with the following elements:
- A conjugative origin of transfer
- A conditional origin of replication, usually R6K, so that the plasmids cannot replicate within the recipient strain (so no second origin of replication)
- A transposon containing a selectable marker flanked by Tn5 ends
- The transposase gene
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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