Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V011837 | pDEST15 | In stock, instant shipping |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
Gateway destination vector for inducible high-level expression of N-terminally GST-tagged proteins in bacterial cells. BL21-AI expresses T7 polymerase induced by arabinose and recognizes the T7 promoter, so expression of the protein must be induced by adding arabinose to the medium.
- Vector Name:
- pDEST15
- Antibiotic Resistance:
- Ampicillin
- Length:
- 7012 bp
- Type:
- Gateway Cloning Vectors
- Replication origin:
- ori
- Source/Author:
- Invitrogen (Life Technologies)
- Copy Number:
- High copy number
- Promoter:
- T7
- Growth Strain(s):
- DB3.1
pDEST15 vector 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.
References
- Gnidehou S, Gerbaud P, Ducarme G, Ferreira F, Badet J, Malassiné A, Evain-Brion D, Frendo JL. Expression in Escherichia coli and purification of human recombinant connexin-43, a four-pass transmembrane protein. Protein Expr Purif. 2011 Aug;78(2):174-80. doi: 10.1016/j.pep.2011.04.018. Epub 2011 May 4. PMID: 21558005.
pDEST15 vector Sequence
LOCUS 40924_14330 7012 bp DNA circular SYN 01-JAN-1980
DEFINITION Gateway(R) destination vector for inducible high-level expression of
N-terminally GST-tagged proteins in bacterial cells.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7012)
AUTHORS Invitrogen (Life Technologies)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 7012)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..7012
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 25..43
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
RBS 75..97
/label=RBS
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 105..758
/codon_start=1
/label=GST
/note="glutathione S-transferase from Schistosoma
japonicum"
/translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
protein_bind 792..916
/label=attR1
/note="recombination site for the Gateway(R) LR reaction"
promoter 941..971
/label=lac UV5 promoter
/note="E. coli lac promoter with an 'up' mutation"
CDS 1025..1702
/codon_start=1
/label=CmR
/note="chloramphenicol acetyltransferase"
/translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
DPAY"
CDS 2025..2327
/codon_start=1
/label=ccdB
/note="CcdB, a bacterial toxin that poisons DNA gyrase"
/translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
VPRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
protein_bind complement(2371..2495)
/label=attR2
/note="recombination site for the Gateway(R) LR reaction"
terminator 2573..2620
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
promoter complement(2986..3014)
/label=tet promoter
/note="E. coli promoter for tetracycline efflux protein
gene"
promoter 3127..3231
/label=AmpR promoter
CDS 3232..4089
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 4263..4851
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
misc_feature complement(5037..5179)
/label=bom
/note="basis of mobility region from pBR322"
CDS complement(5284..5472)
/codon_start=1
/label=rop
/note="Rop protein, which maintains plasmids at low copy
number"
/translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"