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pESC-LEU vector (V010321)

Price Information

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V010321 pESC-LEU In stock, instant shipping

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

Basic Vector Information

pESC-LEU is a yeast episomal vector with a LEU2 marker, for galactose-regulated expression and tagging of up to two genes.

Vector Name:
pESC-LEU
Antibiotic Resistance:
Ampicillin
Length:
7746 bp
Type:
Yeast Plasmids
Replication origin:
ori
Host:
Yeast
Source/Author:
Agilent Technologies
Copy Number:
High copy number
Promoter:
GAL1,10
Growth Strain(s):
Stbl3
Growth Temperature:
37℃

pESC-LEU vector Map

Copy Sequence View Map
pESC-LEU7746 bp3006009001200150018002100240027003000330036003900420045004800510054005700600063006600690072007500LEU2LEU2 promoterf1 oriADH1 terminatorFLAGGAL1,10 promoterT7 promoterMycCYC1 terminatororiAmpRAmpR promoter2u ori

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

  • Ma R, Su P, Ma Q, Guo J, Chen S, Jin B, Zhang H, Tang J, Zhou T, Xiao C, Cui G, Huang L. Identification of (-)-bornyl diphosphate synthase from Blumea balsamifera and its application for (-)-borneol biosynthesis in Saccharomyces cerevisiae. Synth Syst Biotechnol. 2021 Dec 11;7(1):490-497.

pESC-LEU vector Sequence

LOCUS       Exported                7746 bp DNA     circular SYN 02-SEP-2024
DEFINITION  Cloning vector pESC-LEU, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7746)
  AUTHORS   Lu Q.
  TITLE     pESC-LEU
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7746)
  AUTHORS   Lu Q.
  TITLE     Direct Submission
  JOURNAL   Submitted (08-MAY-1998) Technical Services, Stratagene, 11011 N. 
            Torrey Pines Rd., La Jolla, CA 92037, USA
REFERENCE   3  (bases 1 to 7746)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7746)
  TITLE     Direct Submission
REFERENCE   5  (bases 1 to 7746)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
            (08-MAY-1998) Technical Services, Stratagene, 11011 N. Torrey Pines
            Rd., La Jolla, CA 92037, USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
COMMENT     SGRef: number: 4; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7746
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(666..1757)
                     /codon_start=1
                     /label=LEU2
                     /note="3-isopropylmalate dehydrogenase, required for
                     leucine biosynthesis"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     promoter        complement(1758..2165)
                     /label=LEU2 promoter
     rep_origin      complement(2466..2921)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     terminator      complement(3000..3165)
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae
                     alcohol dehydrogenase 1 (ADH1) gene"
     CDS             complement(3313..3336)
                     /codon_start=1
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
                     /translation="DYKDDDDK"
     promoter        3383..4047
                     /label=GAL1,10 promoter
                     /note="divergent inducible promoter, regulated by Gal4"
     promoter        4058..4076
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             4094..4123
                     /codon_start=1
                     /label=Myc
                     /note="Myc (human c-Myc proto-oncogene) epitope tag"
                     /translation="EQKLISEEDL"
     terminator      4153..4342
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     rep_origin      complement(4585..5173)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5347..6204)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(6205..6309)
                     /label=AmpR promoter
     rep_origin      complement(6336..7678)
                     /direction=LEFT
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"