Distribution host: Escherichia coli FD 27747 (ATCC 35673)

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

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

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.

Temperature: 37°C

component of:ATCC 87472