Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V013790 | pKD4-2×ME-pTRC-mCherry-Mob | In stock, 1 week for quality controls |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
- Vector Name:
- pKD4-2×ME-pTRC-mCherry-Mob
- Antibiotic Resistance:
- Ampicillin, Kanamycin
- Length:
- 5834 bp
- Type:
- Gene knockout
- Replication origin:
- R6K γ ori
- Host:
- E. coli
- Copy Number:
- Low
- Promoter:
- trc
- Growth Strain(s):
- S17-1λpir
- Growth Temperature:
- 37℃
pKD4-2×ME-pTRC-mCherry-Mob 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.
pKD4-2×ME-pTRC-mCherry-Mob vector Sequence
LOCUS 62056_13325 5834 bp DNA circular SYN 01-JAN-1980
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5834)
AUTHORS .
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..5834
/mol_type="other DNA"
/organism="synthetic DNA construct"
misc_feature 21..39
/label=Tn5 ME
/note="hyperactive mosaic end for Tn5 transposase
recognition (Reznikoff et al., 2004)"
CDS 478..1269
/codon_start=1
/label=NeoR/KanR
/note="aminoglycoside phosphotransferase"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
protein_bind complement(1463..1496)
/label=FRT (minimal)
/note="supports FLP-mediated excision but not integration
(Turan and Bode, 2011)"
CDS complement(1524..2231)
/codon_start=1
/label=mCherry
/note="monomeric derivative of DsRed fluorescent protein
(Shaner et al., 2004)"
/translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
EGRHSTGGMDELYK"
protein_bind complement(2278..2294)
/label=lac operator
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
promoter complement(2302..2331)
/label=trc promoter
/note="strong E. coli promoter; hybrid between the trp and
lac UV5 promoters"
misc_feature complement(2344..2362)
/label=Tn5 ME
/note="hyperactive mosaic end for Tn5 transposase
recognition (Reznikoff et al., 2004)"
rep_origin complement(2375..2758)
/direction=LEFT
/label=R6K gamma ori
/note="gamma replication origin from E. coli plasmid R6K;
requires the R6K initiator protein pi for replication"
CDS complement(2831..3688)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(3689..3793)
/label=AmpR promoter
CDS complement(4415..4783)
/codon_start=1
/label=traJ
/note="oriT-recognizing protein"
/translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
KQDELGKVMMGVVRPRAEP"
oriT complement(4816..4925)
/direction=LEFT
/label=oriT
/note="incP origin of transfer"
terminator 5602..5688
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 5780..5807
/label=rrnB T2 terminator
/note="transcription terminator T2 from the E. coli rrnB
gene"