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

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.

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 ATCC® 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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