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 Vector Map
Plasmid Resuspension 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 ℃.
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"
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