S. cerevisiae/E. coli marker swap vectors (ATCC® 87561)

Depositors: FR Cross, DJ Stillman

Permits and Restrictions
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Designations S. cerevisiae/E. coli marker swap vectors
Depositors FR Cross, DJ Stillman
Applications This is a set of 15 marker swap vectors, each containing a gene disruption conversion cassette for conversion between standard markers used for transformation selection in Saccharomyces cerevisiae.  The vectors are useful in changing markers for gene disruptions or for changing markers on plasmids.

To convert the host phenotype from the existing yeast auxotrophic marker to a new marker (eg. HIS3 → LEU2), transform with the restriction enzyme digested vector (eg. ApaI+PstI digested pHL3) and select for the appropriate phenotype (for pHL3, Leu+).

Some combinations of marker swap plasmids and target locus may result in relatively high reversion rates.  In most, but not all cases, the frequencies of successful convertants are greater than 30%.  When swapping markers on an episomal plasmid, appropriate phenotype may result from loss of the plasmid unless a second selectable or scorable marker is used to ensure plasmid maintenance.
Vector ATCC® Catalog No. 87542
Designation: pHL3 Other name(s): HIS3 → LEU2 converter Excise insert: ApaI+PstI
Size of construct (kb): 7.7
Markers: cmlR, LEU2
Growth temperature: 37°C
Growth medium: LB medium + 25 µg/mL chloramphenicol

NOTES:
Restriction digests of the clone give the following sizes (kb): BglII--4.4, 3.1;EcoRI--7.6; HindIII--4.1, 3.5.
– ATCC staff
To convert the host phenotype from HIS3 to LEU2, transform with the ApaI+PstI digested vector and select for Leu+ transformants. Vector was constructed by replacing an internal BglII fragment of HIS3 with a 2.7 kb BglII fragment containing the LEU2 coding sequence. LEU2 and HIS3 are in the same orientation.
- Yeast 13: 647-653, 1997



ATCC® Catalog No. 87543
Designation: pHT6
Other name(s): HIS3 → TRP1 converter
Excise insert: SmaI+XhoI
Size of construct (kb): 7.0
Markers: cmlR, kanR, TRP1
Growth temperature: 37°C
Growth medium: LB + kanamycin (50 µg/mL) + chloramphenicol (25 µg/mL)

NOTES:
Restriction digests of the clone give the following sizes (kb): HindIII/PstI--5.5, 1.0, 0.6; HindIII--7.2; HindIII/SalI--5.1, 2.2.
- ATCC staff
To convert the host phenotype from HIS3 to TRP1, transform with the SmaI+XhoI digested vector and select for Trp+ transformants. Vector was constructed by replacing an internal HindIII fragment of HIS3 with a SmaI fragment containing the TPR1 and kanR coding sequences. TRP1 and HIS3 are in the same orientation.
- Yeast 13: 647-653, 1997



ATCC® Catalog No. 87544
Designation: pHU10
Other name(s): HIS3 → URA3 converter
Excise insert: SmaI+XhoI
Size of construct (kb): 6.6
Markers: cmlR, kanR, URA3
Growth temperature: 37°C
Growth medium: LB + kanamycin (50 µg/mL) + chloramphenicol (25 µg/mL)

NOTES: Restriction digests of the clone give the following sizes (kb): HindIII--6.5; HindIII/PstI--5.0, 1.1, 0.5.
- ATCC staff
To convert the host phenotype from HIS3 to URA3, transform with the SmaI+XhoI digested vector and select for Ura+ transformants. Vector was constructed by replacing an internal HindIII fragment of HIS3 with a SmaI fragment containing the URA3 and kanR coding sequences. URA3 and HIS3 are in the opposite orientation.
- Yeast 13: 647-653, 1997



ATCC® Catalog No. 87545
Designation: pLH7
Other name(s): LEU2→ HIS3 converter
Excise insert: XhoI+HpaI
Size of construct (kb): 8.7
Markers: cmlR, kanR, HIS3
Growth temperature: 37°C
Growth medium: LB + kanamycin (50 µg/mL) + chloramphenicol (25 µg/mL)

NOTES:
Restriction digests of the clone give the following sizes (kb): BamHI/EcoRI--6.6, 2.0; XhoI--8.7.
- ATCC staff
To convert the host phenotype from LEU2 to HIS3, transform with the XhoI+HpaI digested vector and select for His+ transformants. Vector was constructed by replacing an internal EcoRV fragment of LEU2 with a SmaI fragment containing the HIS3 and kanR coding sequences. HIS3 and LEU2 are in the same orientation.
- Yeast 13: 647-653, 1997



ATCC® Catalog No. 87546
Designation: pLT11
Other name(s): LEU2 → TRP1 converter
Excise insert: XhoI+HpaI
Size of construct (kb): 10.5
Markers: cmlR, ampR, TRP1
Growth temperature: 37°C
Growth medium: LB + ampicillin (50 µg/mL) + chloramphenicol (20 µg/mL)

NOTES:
Restriction digests of the clone give the following sizes (kb): BamHI--8.4, 2.1; BamHI/XhoI--4.9, 3.3, 2.2.
- ATCC staff
To convert the host phenotype from LEU2 to TRP1, transform with the XhoI+HpaI digested vector and select for Trp+ transformants. Vector was constructed by replacing an internal EcoRI-EcoRV fragment of LEU2 with an XhoI-EcoRI fragment containing the TRP1 and ampR coding sequences.
- Yeast 13: 647-653, 1997



ATCC® Catalog No. 87547
Designation: pLU12
Other name(s): LEU2 → URA3 converter
Excise insert: XhoI+HpaI
Size of construct (kb): 8.5
Markers: cmlR, kanR, URA3
Growth temperature: 37°C
Growth medium: LB + kanamycin (50 µg/mL) + chloramphenicol (25 µg/mL)

NOTES:
Restriction digests of the clone give the following sizes (kb): BamHI--8.8; BamHI/XhoI--5.5, 3.2; EcoRI/HindIII--7.5, 1.2; HpaI/XhoI--4.6, 4.2.
- ATCC staff
To convert the host phenotype from LEU2 to URA3, transform with the XhoI+HpaI digested vector and select for Ura+ transformants. Vector was constructed by replacing an internal EcoRV fragment of LEU2 with a SmaI fragment containing the URA3 and kanR coding sequences. URA3 and LEU2 are in the opposite orientation.
- Yeast 13: 647-653, 1997



ATCC® Catalog No. 87548
Designation: pTH4
Other name(s): TRP1 → HIS3
Excise insert: XhoI+EcoRI
Size of construct (kb): 6.7
Markers: cmlR, kanR, HIS3
Growth temperature: 37°C
Growth medium: LB + kanamycin (50 µg/mL) plus chloramphenicol (25 µg/mL)

NOTES:
Restriction digests of the clone give the following sizes (kb): EcoRI--6.7; HindIII--4.8, 1.9; EcoRI/XhoI--3.5, 3.3.
- ATCC staff
To convert the host phenotype from TRP1 to HIS3, transform with the XhoI+EcoRI digested vector and select for His+ transformants. Vector was constructed by replacing an internal EcoRV fragment of TRP1 with a SmaI fragment containing the HIS3 and kanR coding sequences. HIS3 and TRP1 are in the same orientation.
- Yeast 13: 647-653, 1997



ATCC® Catalog No. 87549
Designation: pTL7
Other name(s): TRP→ LEU2 converter
Excise insert: XhoI+SmaI
Size of construct (kb): 7.8
Markers: cmlR, kanR, LEU2
Growth temperature: 37°C
Medium composition: LB + kanamycin (50 µg/mL) + chloramphenicol (25 µg/mL)

NOTES:
Restriction digests of the clone give the following sizes (kb): EcoRI--6.2, 1.7; HindIII--7.8; SmaI/XhoI--4.4, 3.3.
- ATCC staff
To convert the host phenotype from TRP1 to LEU2, transform with the XhoI+SmaI digested vector and select for Leu+ transformants. Vector was constructed by replacing an internal EcoRV fragment of TRP1 with a SmaI fragment containing the LEU2 and kanR coding sequences. LEU2 and TRP1 are in the same orientation.
- Yeast 13: 647-653, 1997



ATCC® Catalog No. 87550
Designation: pTU10
Other name(s): TRP1→ URA3 converter
Excise insert: XhoI+EcoRI
Size of construct (kb): 6.6
Markers: cmlR, kanR, URA3
Growth temperature: 37°C
Medium composition: LB + kanamycin (50 µg/mL) plus chloramphenicol (25 µg/mL)

NOTES:
Restriction digests of the clone give the following sizes (kb): EcoRI--6.6; HindIII--5.0, 1.6; EcoRI/XhoI--3.4 3.2.
- ATCC staff
To convert the host phenotype from TRP1 to URA3, transform with the XhoI+EcoRI digested vector and select for Ura+ transformants. Vector was constructed by replacing an internal EcoRV fragment of TRP1 with a SmaI fragment containing the URA3 and kanR coding sequences. URA3 and TRP1 are in the opposite orientation.
- Yeast 13: 647-653, 1997



ATCC® Catalog No. 87551
Designation: pUH7
Other name(s): URA3 → HIS3 converter
Excise insert: SmaI
Size of construct (kb): 6.5
Markers: ampR, kanR, HIS3
Growth temperature: 37°C
Medium composition: LB medium + 50 µg/mL ampicillin and 20 µg/mL kanamycin

NOTES:
Restriction digests of the clone give the following sizes (kb): EcoRI--6.4; SmaI--3.5, 2.8; XbaI--3.6, 2.7.
- ATCC staff
To convert the host phenotype from URA3 to HIS3, transform with the SmaI digested vector and select for His+ transformants. Vector was constructed by replacing an internal StuI fragment of URA3 with a SmaI fragment containing the HIS3 and kanR coding sequences. HIS3 and URA3 are probably in the opposite orientation.
- Yeast 13: 647-653, 1997



ATCC® Catalog No. 87552
Designation: pUL9
Other name(s):URA3 → LEU2 converter
Excise insert: SmaI
Size of construct (kb): 7.6
Markers: ampR, kanR, LEU2
Growth temperature: 37°C
Growth medium: LB medium + 50 µg/mL ampicillin and 20 µg/mL kanamycin

NOTES:
Restriction digests of the clone give the following sizes (kb): SmaI--4.3, 3.2; EcoRI--4.5, 2.9.
- ATCC staff
To convert the host phenotype from URA3 to LEU2, transform with the SmaI digested vector and select for Leu+ transformants. Vector was constructed by replacing an internal StuI fragment of URA3 with a SmaI fragment containing the LEU2 and kanR coding sequences. LEU2 and URA3 are in the same orientation.
- Yeast 13: 647-653, 1997



ATCC® Catalog No. 87553
Designation: pUT11
Other name(s): URA3 → TRP1 converter
Excise insert: SmaI
Size of construct (kb): 6.7
Markers: ampR, kanR, TRP1
Growth temperature: 37°C
Growth medium: LB medium + 50 µg/mL ampicillin and 20 µg/mL kanamycin

NOTES:
Restriction digests of the clone give the following sizes (kb): HindIII--4.6, 2.0; SmaI--3.8, 2.7.
- ATCC staff
To convert the host phenotype from URA3 to TRP1, transform with the SmaI digested vector and select for Trp+ transformants. Vector was constructed by replacing an internal StuI fragment of URA3 with a SmaI fragment containing the TRP1 and kanR coding sequences. TRP1 and URA3 are in the same orientation.
- Yeast 13: 647-653, 1997



ATCC® Catalog No. 87557
Designation: M2371
Other name(s): HIS3 → ADE2 converter
Excise insert: PstI+ SacI
Size of construct (kb): 7.734
Markers: ampR ADE2
Growth temperature: 37°C
Growth medium: LB Medium + 50 µg/mL ampicillin

NOTES:
Restriction digests of the clone give the following sizes (kb): BamHI--3.7, 2.7, 0.7, 0.6; PvuI--6.8, 0.9; HindIII--3.9, 1.6, 1.2, 1.0.
- ATCC staff
To convert the host phenotype from HIS3 to ADE2, transform with the PstI+SacI digested vector (4.99 kb) and select for Ade+ transformants. Vector was constructed by replacing an internal MscI-NsiI fragment of HIS3 with a 3.7 kb NotI (blunt)-PstI fragment containing the ADE2 coding sequence. ADE2 and HIS3 are in the opposite orientation.
- Yeast 13: 647-653, 1997



ATCC® Catalog No. 87558
Designation: M3499
Other name(s):URA3 → ADE2 converter
Excise insert: PstI
Size of construct (kb): 7.426
Markers: ampR ADE2
Growth temperature: 37°C
Growth medium: LB Medium + 50 µg/mL ampicillin

NOTES:
Restriction digests of the clone give the following sizes (kb): HindIII--2.5, 1.3, 1.1 (doublet), 1.0; BamHI--6.6, 0.5; PvuI--6.2, 0.8.
- ATCC staff
To convert the host phenotype from URA3 to ADE2, transform with the PstI digested vector (4.45 kb) and select for Ade+ transformants. Vector was constructed by replacing an internal StuI-EcoRV fragment of URA3 with a 3.7 kb BamHI fragment containing the ADE2 coding sequence. ADE2 and URA3 are in the same orientation.
- Yeast 13: 647-653, 1997



ATCC® Catalog No. 87559
Designation: M2660
Other name(s): URA3 → LYS2
Excise insert: HindIII
Size of construct (kb): 8.308
Markers: ampR LYS2
Growth temperature: 37°C
Growth medium: LB Medium + 50 µg/mL ampicillin

NOTES:
Restriction digests of the clone give the following sizes (kb): BglII--4.3, 4.1; HindIII--5.6, 2.8; HpaI--8.5; PvuI--5.1, 2.4, 0.9.
- ATCC staff
To inactivate URA3, transform with HindIII-digested plasmid (5.57 kb) and select for Lys+. Constructed by replacing an internal EcoRV-PstI fragment of URA3 with a 4.6 kb PvuII-PstI fragment containing LYS2. URA3 and LYS2 are in the same orientation.
- Yeast 13: 647-653, 1997
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Biosafety Level 1
Shipping Information Frozen glycerol of Escherichia coli HB101 stocks containing the plasmids arrayed on a microtiter plate
Comments Some combinations of marker swap plasmids and target locus may result in relatively high reversion rates. In most but not all cases the frequencies of successful convertants are greater than 30%.

A set of marker swap vectors, containing gene disruptions for standard markers such as his3::LEU2, useful in changing markers in the S. cerevisiae host chromosome or on plasmids.

The set allows interconversion among all six of the common markers HIS3, LEU2, TRP1, URA3, ADE2, and LYS2.

To convert the host phenotype from the existing yeast auxotrophic marker to a new marker (eg. HIS3 → LEU2), transform with the restriction enzyme digested vector (eg. ApaI+PstI digested pHL3) and select for the appropriate phenotype (for pHL3, Leu+).

When swapping markers on an episomal plasmid, appropriate phenotype may result from loss of the plasmid unless a second selectable or scorable marker is used to ensure plasmid maintenance.
References

Cross FR. 'Marker swap' plasmids: convenient tools for budding yeast molecular genetics. Yeast 13: 647-653, 1997. PubMed: 9200814