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 ATCC® Medium 1227: LB Medium (ATCC medium 1065) with 50 mcg/ml ampicillin
Biosafety Level 1

Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.

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