pNKY1009 (ATCC® 87624)

Applications: contains sequence ATP phosphoribosyltransferasecontains sequence phosphoribosylanthranilate isomerasemarker deletion vector phosphoribosylanthranilate isomeraseproduces 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 pNKY1009
Depositors E Alani
Biosafety Level 1
Host
Distribution host: Escherichia coli FD 27747 (ATCC 35673)
Vector Information
Size (kb): 9.60
DESCRIPTION OF VECTOR:
Intact vector size: 9.600
Type of vector: plasmid
Cloning sites:
Polylinker sites:
Other unique sites: PvuII
Construction: YRp7, pNKY51
Host range: Saccharomyces cerevisiaeCandida robusta; Escherichia coli
Features (with orientation and position when available):
restriction site: EcoRI
coding sequence: 3' TRP1, <-
coding sequence: hisG, ->
marker(s): URA3, ->
coding sequence: hisG, ->
coding sequence: 5' TRP1, <-
restriction site: BglII
coding sequence: ROP, ->
replicon: pMB1
marker(s): ampR, <-
replicon: ARS1, ->
Vector: pNKY1009 (plasmid)
Construction: YRp7, pNKY51
Marker(s):URA3,ampR
Construct size (kb): 9.60
Features: marker(s): URA3
marker(s): ampR
replicon: ARS1
replicon: pMB1
restriction site: BglII
restriction site: EcoRI
coding sequence: 3' TRP1
coding sequence: 5' TRP1
coding sequence: ROP
coding sequence: hisG
Applications
contains sequence ATP phosphoribosyltransferase
contains sequence phosphoribosylanthranilate isomerase
marker deletion vector phosphoribosylanthranilate isomerase
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): BglII--9.6; EcoRI--5.2, 4.4; BglII/EcoRI--4.6, 4.4, 0.6.
The two step selection process requires a ura3 transformation host (this host can be created using pJL164 (ATCC 87471)). After transformation with the EcoRI/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 trp1 auxotrophic marker deletion.
The 4.6 kb EcoRI/BglII insert contains two direct repeats of the Salmonella hisG gene flanking URA3 plus TRP1 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 EcoRV site within the TRP1 gene of YEp7.
Media Medium 2057: M9 salts with supplements
Growth Conditions
Temperature: 37°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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component of:ATCC 87472
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