pUG66 vector (Cat. No.: V002330)

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Basic Information

Note: pUG66 is a 3.58 kb E. coli-yeast shuttle vector used as a PCR template for gene knockouts in yeast. It contains an ampicillin resistance gene for bacterial selection and a loxP-flanked bleomycin resistance marker (Ble) for selection in yeast, which can be excised via Cre recombinase.

Name:: pUG66

Antibiotic Resistance:: Ampicillin

Length:: 3586 bp

Type:: PCR template vector

Replication origin:: ori

Host:: Yeast

Source/Author:: Gueldener U, Heinisch J, Koehler GJ, Voss D, Hegemann JH.

Promoter:: TEF

Growth Strain(s):: DH10B

Growth Temperature:: 37℃

$ 198.2

In stock, instant shipping

Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.

Resources

Plasmid Protocol

1. Centrifuge at 5,000×g for 5 min.

2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.

3. Close the tube and incubate for 10 minutes at room temperature.

4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.

5. Store the plasmid at -20 ℃.

6. The concentration of plasmid re-measurement sometimes differs from the nominal value, which may be due to the position of the lyophilized plasmid in the tube, the efficiency of the re-dissolution, the measurement bias, and adsorption on the wall of the tube, therefore, it is recommended to transform and extract the plasmid before using it

General Plasmid Transform Protocol

1. Take one 100μl of the competent cells and thaw it on ice for 10min, add 2μl of plasmid, then ice bath for 30min, then heat-shock it at 42℃ for 60s, do not stir, and then ice bath for 2min.

2. Add 900μl of LB liquid medium without antibiotics, and incubate at 37℃ for 45min (30℃ for 1-1.5 hours) with 180rpm shaking.

3. Centrifuge at 6000rpm for 5min, leave only 100μl of supernatant to resuspend the bacterial precipitate and spread it onto the target plasmid-resistant LB plate.

4. Invert the plate and incubate at 37℃ for 14h, or at 30℃ for 20h.

5. Pick a single colony into LB liquid medium, add the corresponding antibiotics, incubate at 220rpm for 14h, and extract the plasmid according to the experimental needs and the instructions of the plasmid extraction kit.

References

Vickers CE, Bydder SF, Zhou Y, Nielsen LK. Dual gene expression cassette vectors with antibiotic selection markers for engineering in Saccharomyces cerevisiae. Microb Cell Fact. 2013 Oct 25;12:96. doi: 10.1186/1475-2859-12-96. PMID: 24161108; PMCID: PMC4231455.

pUG66 vector (Cat. No.: V002330) Sequence

LOCUS       pUG66                   3586 bp    DNA     circular SYN 30-DEC-2025
DEFINITION  PCR template vector pUG66, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3586)
  AUTHORS   Gueldener U, Heinisch J, Koehler GJ, Voss D, Hegemann JH.
  TITLE     A second set of loxP marker cassettes for Cre-mediated multiple gene
            knockouts in budding yeast
  JOURNAL   Nucleic Acids Res. 30 (6), E23 (2002)
  PUBMED    11884642
REFERENCE   2  (bases 1 to 3586)
  AUTHORS   Gueldener U, Heinisch JH, Voss D, Koehler G, Hegemann JH.
  TITLE     Direct Submission
  JOURNAL   Submitted (23-AUG-2000) Institut fuer Mikrobiologie, 
            Heinrich-Heine-Universitaet, Universtitaetsstr. 1, Duesseldorf 
            40225, Germany
REFERENCE   3  (bases 1 to 3586)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 3586)
  TITLE     Direct Submission
REFERENCE   5  (bases 1 to 3586)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nucleic
            Acids Res."; date: "2002"; volume: "30"; issue: "6"; pages: "E23"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
            (23-AUG-2000) Institut fuer Mikrobiologie,
            Heinrich-Heine-Universitaet, Universtitaetsstr. 1, Duesseldorf
            40225, Germany"
COMMENT     SGRef: number: 3; type: "Journal Article"
COMMENT     SGRef: number: 4; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..3586
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    54..87
                     /label=loxP
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        142..485
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             486..872
                     /codon_start=1
                     /gene="ble"
                     /product="bleomycin resistance protein"
                     /label=ble
                     /protein_id="AAG34545.1"
                     /translation="MGMTDQATPNLPSRDFDPTAAFYERLGFGIVFRDAGWMILQRGDL
                     MLEFFAHPGLDPLASWFSCCLRLDDLAEFYRQCKSVGIQETSSGYPRIHAPELQEWGGT
                     MAALVDPDGTLLRLIQNELLAGIS"
     gene            486..872
                     /gene="ble"
                     /label=ble
     terminator      878..1075
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     misc_feature    1138..1171
                     /label=loxP site
                     /note="loxP site"
     protein_bind    complement(1138..1171)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     promoter        complement(1225..1243)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      complement(1501..2089)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(2263..3120)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(3121..3225)
                     /label=AmpR promoter