Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V017492 | pAAV-hSyn-DIO-hM4D(Gi)-mCherry | 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
Double floxed Gi-coupled hM4D DREADD fused with mCherry under the control of human synapsin promoter.
- Vector Name:
- pAAV-hSyn-DIO-hM4D(Gi)-mCherry
- Antibiotic Resistance:
- Ampicillin
- Length:
- 6982 bp
- Type:
- AAV
- Source/Author:
- Bryan Roth
- Selection Marker:
- mCherry
- Copy Number:
- Low copy number
- Promoter:
- hSyn
- Growth Strain(s):
- Stbl3
- Growth Temperature:
- 37℃
pAAV-hSyn-DIO-hM4D(Gi)-mCherry 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.
References
- Krashes MJ, Koda S, Ye C, Rogan SC, Adams AC, Cusher DS, Maratos-Flier E, Roth BL, Lowell BB. Rapid, reversible activation of AgRP neurons drives feeding behavior in mice. J Clin Invest. 2011 Apr;121(4):1424-8. doi: 10.1172/JCI46229. PMID: 21364278; PMCID: PMC3069789.
pAAV-hSyn-DIO-hM4D(Gi)-mCherry vector Sequence
LOCUS Exported 6982 bp DNA circular SYN 29-MAY-2024
DEFINITION Double floxed Gi-coupled hM4D DREADD fused with mCherry under the
control of human synapsin promoter..
ACCESSION .
VERSION .
KEYWORDS pAAV-hSyn-DIO-hM4D(Gi)-mCherry
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6982)
AUTHORS Krashes MJ, Koda S, Ye C, Rogan SC, Adams AC, Cusher DS,
Maratos-Flier E, Roth BL, Lowell BB
TITLE Rapid, reversible activation of AgRP neurons drives feeding behavior
in mice.
JOURNAL J Clin Invest. 2011 Apr;121(4):1424-8. doi: 10.1172/JCI46229.
PUBMED 21364278
REFERENCE 2 (bases 1 to 6982)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 6982)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; doi: "10"; journalName:
"J Clin Invest"; date: "2011-04"; volume: "121"; issue: "4"; pages:
"1424-8"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..6982
/mol_type="other DNA"
/organism="synthetic DNA construct"
repeat_region 1..130
/label=AAV2 ITR (alternate)
/note="Functional equivalent of wild-type AAV2 ITR"
promoter 175..622
/label=hSyn promoter
/note="human synapsin I promoter; confers neuron-specific
expression (Kugler et al., 2003)"
protein_bind 660..693
/label=lox2272
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GGATACTT). lox2272 sites are compatible with each
other, but incompatible with loxP or loxN sites (Lee and
Saito, 1988)."
protein_bind 744..777
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
CDS complement(786..1496)
/codon_start=1
/product="monomeric derivative of DsRed fluorescent protein
(Shaner et al., 2004)"
/label=mCherry
/note="mammalian codon-optimized"
/translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
EGRHSTGGMDELYK"
primer_bind complement(1064..1083)
/label=mCherry-F
/note="mCherry, forward primer"
primer_bind 1333..1351
/label=mCherry-R
/note="mCherry, reverse primer"
regulatory 1493..1502
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
/regulatory_class="other"
CDS complement(1518..2954)
/codon_start=1
/label=Muscarinic acetylcholine receptor M4
/translation="MANFTPVNGSSGNQSVRLVTSSSHNRYETVEMVFIATVTGSLSLV
TVVGNILVMLSIKVNRQLQTVNNYFLFSLACADLIIGAFSMNLYTVYIIKGYWPLGAVV
CDLWLALDCVVSNASVMNLLIISFDRYFCVTKPLTYPARRTTKMAGLMIAAAWVLSFVL
WAPAILFWQFVVGKRTVPDNQCFIQFLSNPAVTFGTAIAGFYLPVVIMTVLYIHISLAS
RSRVHKHRPEGPKEKKAKTLAFLKSPLMKQSVKKPPPGEAAREELRNGKLEEAPPPALP
PPPRPVADKDTSNESSSGSATQNTKERPATELSTTEATTPAMPAPPLQPRALNPASRWS
KIQIVTKQTGNECVTAIEIVPATPAGMRPAANVARKFASIARNQVRKKRQMAARERKVT
RTIFAILLAFILTWTPYNVMVLVNTFCQSCIPDTVWSIGYWLCYVNSTINPACYALCNA
TFKKTFRHLLLCQYRNIGTAR"
regulatory 2951..2960
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
/regulatory_class="other"
protein_bind complement(2967..3000)
/label=lox2272
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GGATACTT). lox2272 sites are compatible with each
other, but incompatible with loxP or loxN sites (Lee and
Saito, 1988)."
protein_bind complement(3051..3084)
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
misc_feature 3109..3697
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(3162..3182)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(3580..3591)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
polyA_signal 3729..4205
/label=hGH poly(A) signal
/note="human growth hormone polyadenylation signal"
primer_bind complement(3928..3947)
/label=hGH-PA-R
/note="Human growth hormone terminator, reverse primer"
repeat_region 4245..4385
/label=AAV2 ITR
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
repeat_region 4245..4374
/label=AAV2 ITR
rep_origin 4460..4915
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(4547..4566)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 4757..4778
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(4932..4951)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 5051..5073
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(5111..5129)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 5197..5301
/gene="bla"
/label=AmpR promoter
CDS 5302..6162
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind complement(5520..5539)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 6333..6921
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 6822..6841
/label=pBR322ori-F
/note="pBR322 origin, forward primer"