pSCH2002 (ATCC® 87432)

Organism: Escherichia coli (Migula) Castellani and Chalmers  /  Clone Type: Clone  /  Depositors: KJ Shaw

Permits and Restrictions

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Designations pSCH2002
GenBank Number

V00618

Species Escherichia coli (Migula) Castellani and Chalmers
Depositors KJ Shaw
Vector
Construct size (kb): 3.900000095367432
DESCRIPTION OF VECTOR COMPONENT:
Name of vector: pBluescript KS-
Intact vector size: 2.964
Type of vector: phagemid
Vector end: PstI
Vector end: PstI
Cloning sites: SacII XmaII NotI XbaI SpeI BamHI SmaI PstI EcoRI EcoRV
HindIII ClaI (SalI HincII AccI) XhoI DraII ApaI KpnI
Polylinker sites: SEE COMMENTS
Construction: pUC19
Host range: Escherichia coli
Features (with orientation and position when available):
enhancer: none
insert detection: lacZ'
marker(s): ampR
promoter: lac, T3, T7
replicon: pMB1, f1
terminator: none
Cross references:
Insert
DNA: Synthetic
DESCRIPTION OF INSERT COMPONENT:
Genome: Escherichia coli
Gene symbol: aph(3')-IIa
Gene name: aminoglycoside resistance
Contains complete coding sequence?: U
Type of DNA: unknown
Insert end: PstI
Insert end: PstI
Insert size (kb): 0.923
Cross references: DNA Seq. Acc.: V00618
Nucleotides 1-923 of the insert correspond to
nucleotides 328-1251 of V00618.
Insert lengths(kb): 0.9229999780654907
Gene product: aminoglycoside resistance [aph(3')-IIa]
Insert Size (kb) 0.923
Media Medium 1227: LB Medium (ATCC medium 1065) with 50 mcg/ml ampicillin
Biosafety Level 1
Shipping Information Distributed: freeze-dried
Comments
Restriction digests of the clone give the following sizes (kb): PstI--3.0, 1.1, 1.1; EcoRI--5.2; HindIII--5.2. The clone contains two copies of the insert.
The insert contains the following restriction sites (approximate kb from the 5' end): PvuII--0.06, 0.82; SphI--0.35; NcoI--0.38; SmaI--0.79.
A single gel purification of the PCR generated probe is necessary since flanking regions will co-amplify with the gene specific sequence. Failure to do so often results in high backgrounds and false positives with clinical E. coli strains.
A hybridization probe may be generated using the following vector specific PCR primers: modified T3 = 5'-CCCCTCACTAAAGGGAACAAAAGCTG-3' and modified T7 = 5'-CGCGTAATACGACTCACTATAGGGCGAA-3'.
The suggested PCR generated probe encodes the last 617bp of the aph(3')-IIa gene plus 290bp of the bleomycin resistance gene from Tn5. A better probe for the aph(3')-IIa gene would be the 383bp PstI/NcoI fragment.
Classification Enterobacteriaceae, Escherichia
References

Shaw KJ, et alThe application of molecular techniques for the study of aminoglycoside resistanceIn: Shaw KJ, et alMethods in molecular medicine: molecular approaches for the diagnosis and investigation of bacterial diseasesTotowa, NJHumana Presssubmitted, 1996

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