Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V012206 | pDisplay-GACh2.0 | 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:
- pDisplay-GACh2.0
- Antibiotic Resistance:
- Ampicillin
- Length:
- 7242 bp
- Type:
- Mammalian Expression
- Replication origin:
- ori
- Copy Number:
- High Copy
- Promoter:
- SV40
- Cloning Method:
- Gibson Cloning
pDisplay-GACh2.0 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.
pDisplay-GACh2.0 vector Sequence
LOCUS 40924_14715 7242 bp DNA circular SYN 13-MAY-2021
DEFINITION express the genetically-encoded fluorescent ACh indicator GACh2.0 in
mammalian cells.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7242)
AUTHORS Jing M, Zhang P, Wang G, Feng J, Mesik L, Zeng J, Jiang H, Wang S,
Looby JC, Guagliardo NA, Langma LW, Lu J, Zuo Y, Talmage DA, Role
LW, Barrett PQ, Zhang LI, Luo M, Song Y, Zhu JJ, Li Y
TITLE A genetically encoded fluorescent acetylcholine indicator for in
vitro and in vivo studies.
JOURNAL Nat Biotechnol. 2018 Sep;36(8):726-737. doi: 10.1038/nbt.4184. Epub
2018 Jul 9.
PUBMED 29985477
REFERENCE 2 (bases 1 to 7242)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 7242)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; doi: "10.1038/nbt.4184";
journalName: "Nat Biotechnol"; date: "2018-09"; volume: "36"; issue:
"8"; pages: "726-737"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..7242
/mol_type="other DNA"
/organism="synthetic DNA construct"
enhancer 10..389
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 390..593
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
promoter 638..656
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
sig_peptide 737..799
/product="leader sequence from mouse immunoglobulin kappa
light chain"
/label=Ig-kappa leader
protein_bind 800..824
/gene="mutant version of attB"
/label=attB1
/bound_moiety="BP Clonase(TM)"
/note="recombination site for the Gateway(R) BP reaction"
CDS 1697..1948
/codon_start=1
/label=VC155
/note="C-terminal fragment of mVenus for use in bimolecular
fluorescence complementation (BiFC) (Kodama and Hu, 2010)"
/translation="DKQKNGIKANFHIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNH
YLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK"
primer_bind complement(2012..2033)
/label=EGFP-N
/note="EGFP, reverse primer"
primer_bind complement(2273..2292)
/label=EXFP-R
/note="For distinguishing EGFP variants, reverse primer"
CDS 2792..2821
/codon_start=1
/label=Myc
/note="Myc (human c-Myc proto-oncogene) epitope tag"
/translation="EQKLISEEDL"
CDS 2825..2971
/codon_start=1
/label=PDGFR-beta TM domain
/note="transmembrane domain from platelet derived growth
factor receptor beta"
/translation="AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQ
KKPR"
polyA_signal 2999..3223
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin complement(3355..3943)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
polyA_signal complement(4272..4319)
/label=HSV TK poly(A) signal
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
primer_bind 4344..4363
/label=TK-pA-R
/note="Thymidine kinase polyA, reverse primer"
CDS complement(4554..5345)
/codon_start=1
/label=NeoR/KanR
/note="aminoglycoside phosphotransferase"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
promoter complement(5380..5733)
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
CDS complement(5796..6653)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(6654..6758)
/label=AmpR promoter
rep_origin 6785..7240
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"