Price Information
| Cat No. | Plasmid Name | Availability | Buy one, get one free! (?) |
|---|---|---|---|
| V004786 | pME-LifeAct-GFP | In stock, instant shipping |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
- Vector Name:
- pME-LifeAct-GFP
- Antibiotic Resistance:
- Kanamycin
- Length:
- 3342 bp
- Type:
- Cloning vector
- Replication origin:
- ori
- Source/Author:
- Hall TE, Currie PD.
- Growth Strain(s):
- GT115
pME-LifeAct-GFP vector Map
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.
pME-LifeAct-GFP vector Sequence
LOCUS 40924_30500 3342 bp DNA circular SYN 18-DEC-2018
DEFINITION Cloning vector pME-LifeAct-GFP, complete sequence.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 3342)
AUTHORS Hall TE, Currie PD.
TITLE Direct Submission
JOURNAL Submitted (27-SEP-2011) Australian Regenerative Medicine Institute,
Monash University, Building 75, Melbourne, VIC 3800, Australia
REFERENCE 2 (bases 1 to 3342)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 3342)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Submitted
(27-SEP-2011) Australian Regenerative Medicine Institute, Monash
University, Building 75, Melbourne, VIC 3800, Australia"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..3342
/mol_type="other DNA"
/organism="synthetic DNA construct"
terminator complement(268..295)
/label=rrnB T2 terminator
/note="transcription terminator T2 from the E. coli rrnB
gene"
terminator complement(387..473)
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
primer_bind 537..553
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 569..668
/label=attL1
/note="recombination site for the Gateway(R) LR reaction"
misc_feature 674..745
/label=LifeAct 5-prime tag
/note="LifeAct 5-prime tag"
CDS 674..724
/codon_start=1
/product="peptide that binds filamentous actin (Riedl et
al., 2008)"
/label=Lifeact
/note="first 17 amino acids of budding yeast Abp140"
/translation="MGVADLIKKFESISKEE"
CDS 746..1462
/codon_start=1
/label=EGFP
/note="enhanced GFP"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
protein_bind complement(1467..1566)
/label=attL2
/note="recombination site for the Gateway(R) LR reaction"
promoter complement(1584..1602)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(1607..1623)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
CDS 1736..2542
/codon_start=1
/label=KanR
/note="aminoglycoside phosphotransferase"
/translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
rep_origin 2692..3280
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"