pNK2885 (ATCC® 77341)

Organism: Escherichia coli (Migula) Castellani and Chalmers  /  Clone Type: Clone  /  Depositors: N Kleckner

Permits and Restrictions

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Designations pNK2885
Species Escherichia coli (Migula) Castellani and Chalmers
Depositors N Kleckner
Applications
produces protein transposase, Tn10, ats1 ats2 (ATS)
Vector
Construct size (kb): 8.100000381469727
Insert
DNA: genomic
Insert lengths(kb): 6.0
Gene product: transposase, Tn10, ats1 ats2 (ATS) [URA3]
Target Gene: transposase, Tn10, ats1 ats2 (ATS)
Insert Size (kb) 6.0
Media Medium 1227: LB Medium (ATCC medium 1065) with 50 mcg/ml ampicillin
Biosafety Level 1
Comments
Restriction digests of the clone give the following sizes (kb): EcoRI--6.0, 2.1; HindIII--4.9, 1.1, 1.0, 0.72; PstI--4.1, 4.0.
This cassette is inserted into the HindIII site (original nt 2272) of the ATS transposase-containing EcoRI fragment of pNK2881, ATCC 77351.
Construct useful for insertional mutagenesis in E. coli and Saccharomyces cerevisiae.
pBR322 was modified by deletion of bp 75-2352 and destruction (by filling in) of the HindIII site.
Contains the ats1 ats2 transposase gene that permits relaxed insertion specificity (altered target specificity, ATS). Expression is regulated by the Ptac promoter, inducible by IPTG.
The transposase segment extends from IS10R to the EcoRI site at nt 3140 of Tn10, with a deletion of nucleotides 1329-1942 to remove the transposase binding site.
Contains a 3.4 kb mini-Tn10 cassette conferring kanamycin resistance and encoding URA3, bounded by inverted repeats of the outermost 70 bp of IS10R and embedded in 40 bp of lambdacI terminating in HindIII sites.
Transcription from the Ptac promoter is in a direction opposite to that of amp.
The order of the major features in this plasmid is: EcoRI - Ptac - ATS transposase - mini-Tn10 URA3 kan - EcoRI - pMB1 ori - ampR.
Classification Enterobacteriaceae, Escherichia
References

Kleckner N, et al. Uses of transposons with emphasis on Tn10. Methods Enzymol. 204: 139-180, 1991. PubMed: 1658561

Riles L, et al. Physical maps of the six smallest chromosomes of Saccharomyces cerevisiae at a resolution of 2.6-kilobase pairs. Genetics 134: 81-150, 1993. PubMed: 8514151

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