Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V001744 | pTriEx-mCherry-PA-Rac1-T17N | 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:
- pTriEx-mCherry-PA-Rac1-T17N
- Antibiotic Resistance:
- Ampicillin
- Length:
- 6745 bp
- Type:
- Mammalian Expression, Bacterial Expression, Insect
- Replication origin:
- ori
- Copy Number:
- High Copy
- Promoter:
- CMV
- Cloning Method:
- Restriction Enzyme
- 5' Primer:
- pTriExUP
pTriEx-mCherry-PA-Rac1-T17N 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.
pTriEx-mCherry-PA-Rac1-T17N vector Sequence
LOCUS 40924_44242 6745 bp DNA circular SYN 13-MAY-2021
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6745)
AUTHORS Wu YI, Frey D, Lungu OI, Jaehrig A, Schlichting I, Kuhlman B, Hahn
KM
TITLE A genetically encoded photoactivatable Rac controls the motility of
living cells.
JOURNAL Nature. 2009 Sep 3. 461(7260):104-8.
PUBMED 19693014
REFERENCE 2 (bases 1 to 6745)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 6745)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nature.
2009 Sep 3. 461(7260):104-8."
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..6745
/mol_type="other DNA"
/organism="synthetic DNA construct"
enhancer 25..328
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 330..529
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 735..757
/label=pCAGGS-5
/note="Chimeric intron in CAG promoter, forward primer"
primer_bind 819..838
/label=pCAG-F
/note="Rabbit beta-globin intron, for pCAG plasmids,
forward primer"
promoter 880..898
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 899..923
/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 939..1048
/label=p10 promoter
/note="baculovirus promoter for expression in insect cells"
CDS 1069..1086
/codon_start=1
/label=6xHis
/note="6xHis affinity tag"
/translation="HHHHHH"
CDS 1093..1797
/codon_start=1
/label=mCherry
/note="monomeric derivative of DsRed fluorescent protein
(Shaner et al., 2004)"
/translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
GRHSTGGMDELYK"
CDS 2227..2799
/codon_start=1
/label=Rac1 (T17N)
/note="dominant negative mutant of human Rac family small
GTPase 1"
/translation="ELIKCVVVGDGAVGKNCLLISYTTNAFPGEYIPTVFDNYSANVMV
DGKPVNLGLWDTAGQEDYDRLRPLSYPQTDVFLICFSLVSPASFENVRAKWYPEVRHHC
PNTPIILVGTKLDLRDDKDTIEKLKEKKLTPITYPQGLAMAKEIGAVKYLECSALTQRG
LKTVFDEAIRAVLCPPPVKKRKRKCLLL"
CDS 2837..2869
/codon_start=1
/label=HSV tag
/note="HSV (herpes simplex virus) epitope tag"
/translation="QPELAPEDPED"
CDS 2876..2899
/codon_start=1
/label=8xHis
/note="8xHis affinity tag"
/translation="HHHHHHHH"
primer_bind complement(2989..3008)
/label=Bglob-pA-R
/note="Rabbit beta-globin polyA region, reverse primer"
polyA_signal 3054..3109
/label=beta-globin poly(A) signal
/note="rabbit beta-globin polyadenylation signal (Gil and
Proudfoot, 1987)"
primer_bind complement(3108..3127)
/label=rbglobpA-R
/note="Rabbit beta-globin polyA, reverse primer. Also
called rb-glob-pA-term-R"
terminator 3197..3244
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
misc_recomb 3257..3962
/label=baculovirus recombination region (ORF1629)
/note="contains part of ORF1629"
protein_bind complement(3971..4004)
/label=loxP
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (ATGTATGC) (Shaw et al., 2021)."
rep_origin complement(4172..4759)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(4775..5632)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
misc_recomb 5875..6702
/label=baculovirus recombination region (lef2/ORF603)
/note="contains ORF603 and part of lef2"
promoter 6713..6745
/label=CMV
/note="Human cytomegalovirus immediate early
enhancer/promoter"