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

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.

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

Shipped freeze-dried
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