pDMS197 (ATCC® 87694)

Applications: counterselection levansucrasesuicide vectorvector permitting allelic exchange of cloned inserts  /  Depositors: DM Schifferli

Designations pDMS197
Permits and Restrictions

View Permits

Depositors DM Schifferli
Biosafety Level 1
Distribution host: Escherichia coli SM10 lambda pir
Vector Information
Size (kb): 6.5000000000000000
Intact vector size: 6.500
Type of vector: plasmid
Cloning sites: BamHI XbaI KpnI SacI SmaI
Polylinker sites: BamHI HindIII XbaI KpnI SphI EcoRV SacI SmaI EcoRI
Construction: pRE107, tetR (pBR322)
Host range: broad host range; Escherichia coli
Features (with orientation and position when available):
MCS: BamHI...EcoRI, ->
marker(s): sacB1, ->
restriction site: EcoRI HindIII, ->
replicon: conditional R6K ori
replicon: oriT
marker(s): tetR, ->
Vector: pDMS197 (plasmid)
Construction: pRE107, tetR (pBR322)
Construct size (kb): 6.5
Features: marker(s): sacB1
marker(s): tetR
replicon: conditional R6K ori
replicon: oriT
MCS: BamHI...EcoRI
restriction site: EcoRI HindIII
counterselection levansucrase
suicide vector
vector permitting allelic exchange of cloned inserts
Restriction digests of the clone give the following sizes (kb): XbaI-- 6.55; KpnI-- 6.55; HindIII-- 2.9, 2.2, 1.4.
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.
Media ATCC® Medium 1631: LB medium (ATCC medium 1065) with 10 mcg/ml tetracycline
Growth Conditions
Temperature: 37.0°C

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

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