pNKY85 (ATCC® 87625)

Applications: contains sequence 3-isopropylmalate dehydrogenase beta-isopropylmalate dehydrogenasecontains sequence ATP phosphoribosyltransferasemarker deletion vector 3-isopropylmalate dehydrogenase beta-isopropylmalate dehydrogenaseproduces protein uridine monophosphate synthetase UMP synthase, orotate phosphoribosyltransferase, orotidine 5'-phosphate decarboxylase, orotate phosphoribosyltransferase 1  /  Depositors: E Alani

Permits and Restrictions

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Designations pNKY85
Depositors E Alani
Biosafety Level 1
Vector Information
Size (kb): 9.6000003814697270
Vector: pNKY85 (plasmid)
Construction: pNKY51, pBR322, LEU2
Marker(s):URA3,ampR
Construct size (kb): 9.600000381469727
Features: marker(s): URA3
marker(s): ampR
replicon: pMB1
restriction site: BglII
coding sequence: 3' LEU2
coding sequence: 5' LEU2
coding sequence: hisG
Applications
contains sequence 3-isopropylmalate dehydrogenase beta-isopropylmalate dehydrogenase
contains sequence ATP phosphoribosyltransferase
marker deletion vector 3-isopropylmalate dehydrogenase beta-isopropylmalate dehydrogenase
produces protein uridine monophosphate synthetase UMP synthase, orotate phosphoribosyltransferase, orotidine 5'-phosphate decarboxylase, orotate phosphoribosyltransferase 1
Comments
Restriction digests of the clone give the following sizes (kb): PstI--7.1, 2.5; XhoI--9.6.
Restriction digests of the clone give the following sizes (kb): BglII--6.0, 3.6; PstI--7.0, 2.5.
The two step selection process requires a ura3 transformation host (this host can be created using pJL164 (ATCC 87471)). After transformation with the BglII digested plasmid, URA3 integrants are selected on ura- plates.
The deletion strain is then recovered by selection on 5-FOA plates (loss of URA3 marker by a homologous recombination event between the two hisG repeats).
E. coli containing this plasmid should be grown on media lacking pyrimidines to select for URA3-containing cells.
This deleter vector is used to create yeast strains with a leu2 auxotrophic marker deletion.
The 6.1 kb BglII insert contains two direct repeats of the Salmonella hisG gene flanking URA3 plus LEU2 sequences flanking the hisG-URA3-hisG sequence.
The plasmid was constructed by inserting the 3.8 kb BamHI-BglII hisG-URA3-hisG fragment into the modified EcoRI site within the LEU2 gene of a pBR322 derived vector.
Media ATCC® Medium 2057: M9 salts with supplements
Growth Conditions
Temperature: 37.0°C
References

Alani E, et al. A method for gene disruption that allows repeated use of URA3 selection in the construction of multiply disrupted yeast strains. Genetics 116: 541-545, 1987. PubMed: 3305158

Jef D Boeke, personal communication

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