Size (kb): 6.0000000000000000
Vector: pRE112 (plasmid)
Construction: pRE107, cmlR (pACYC184)
Construct size (kb): 6.0
Features: marker(s): cmlR
replicon: conditional R6K ori
restriction site: EcoRI HindIII
vector permitting allelic exchange of cloned inserts
Restriction digests of the clone give the following sizes (kb): KpnI-- 6.55; XbaI-- 6.55; HindIII-- 3.2, 3.0.
The sacB1 allele is a variant of sacB with certain restriction sites removed by site directed mutagenesis. Expression of sacB confers sensitivity to sucrose.
The conditional R6K origin of replication requires that the pi protein be expressed in trans for plasmid maintenance.
One of four allelic exchange suicide vectors (ATCC 87691
- 87694) that provide both selection for chromosomal integration (ampR, cmlR, kanR or tetR) and counterselection for loss of vector DNA and the wild type allele.
Construct is suicide plasmid in any host not expressing pir.
Cloned inserts may be integrated into the host chromosome (a single recombination event) following electroporation and appropriate antibiotic selection.
Negative selection for sucrose sensitivity (sacB) selects for a second recombination event resulting in loss of vector DNA.
Edwards RA, et al. Improved allelic exchange vectors and their use to analyze 987P fimbria gene expression. Gene 207: 149-157, 1998. PubMed: 9511756
Donnenberg MS, Kaper JB. Construction of an eae deletion mutant of enteropathogenic Escherichia coli by using a positive-selection suicide vector. Infect. Immun. 59: 4310-4317, 1991. PubMed: 1937792
Blomfield IC, et al. Allelic exchange in Escherichia coli using the Bacillus subtilis sacB gene and a temperature-sensitive pSC101 replicon. Mol. Microbiol. 5: 1447-1457, 1991. PubMed: 1686293
Kolter R, et al. Trans-complementation-dependent replication of a low molecular weight origin fragment from plasmid R6K. Cell 15: 1199-1208, 1978. PubMed: 728998
McIver KS, et al. The elastase propeptide functions as an intramolecular chaperone required for elastase activity and secretion in Pseudomonas aeruginosa. Mol. Microbiol. 18: 877-889, 1995. PubMed: 8825092
Frozen glycerol stock of E. coli containing the plasmid