Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V000494 | ERroGFP_iE_pCDNA3 | 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:
- ERroGFP_iE_pCDNA3
- Antibiotic Resistance:
- Ampicillin
- Length:
- 6256 bp
- Type:
- Mammalian Expression
- Replication origin:
- ori
- Copy Number:
- High Copy
- Cloning Method:
- Restriction Enzyme
- 5' Primer:
- CMV forward
- 3' Primer:
- SP6
ERroGFP_iE_pCDNA3 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.
ERroGFP_iE_pCDNA3 vector Sequence
LOCUS 40924_790 6256 bp DNA circular SYN 13-MAY-2021
DEFINITION Mammalian expression vector encoding for the ER targeted and FLAGM1
tagged redox probe roGFPiE (roGFP C147, E147b, C204) .
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6256)
AUTHORS Avezov E, Cross BC, Kaminski Schierle GS, Winters M, Harding HP,
Melo EP, Kaminski CF, Ron D
TITLE Lifetime imaging of a fluorescent protein sensor reveals surprising
stability of ER thiol redox.
JOURNAL J Cell Biol. 2013 Apr 15;201(2):337-49. doi: 10.1083/jcb.201211155.
PUBMED 23589496
REFERENCE 2 (bases 1 to 6256)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 6256)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; doi: "10.1083/jcb";
journalName: "J Cell Biol"; date: "2013-04-15- 15"; volume: "201";
issue: "2"; pages: "337-49"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..6256
/mol_type="other DNA"
/organism="synthetic DNA construct"
primer_bind complement(44..63)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
enhancer 235..614
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 615..818
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 815..839
/label=LNCX
/note="Human CMV promoter, forward primer"
CDS 949..972
/codon_start=1
/label=FLAG
/note="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
/translation="DYKDDDDK"
primer_bind complement(1006..1034)
/label=GFP-R
/note="GFP, reverse primer. Does NOT anneal to EGFP"
primer_bind 1632..1652
/label=GFP-F
/note="GFP, forward primer. Does NOT anneal to EGFP"
promoter complement(1808..1826)
/label=SP6 promoter
/note="promoter for bacteriophage SP6 RNA polymerase"
polyA_signal 1852..2076
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 2122..2550
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 2564..2894
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
CDS 2961..3752
/codon_start=1
/label=NeoR/KanR
/note="aminoglycoside phosphotransferase"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
polyA_signal 3929..4062
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(4099..4115)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(4123..4139)
/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(4147..4177)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(4192..4213)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(4330..4347)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(4501..5089)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(5263..6120)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(6121..6225)
/label=AmpR promoter