pAAV-hSyn-DIO-hM3D(Gq)-mCherry vector (V017491)

Price Information

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V017491 pAAV-hSyn-DIO-hM3D(Gq)-mCherry In stock, instant shipping

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Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.

Basic Vector Information

Double floxed Gq-coupled hM3D DREADD fused with mCherry under the control of human synapsin promoter. When the AAV-hM3Dq-mCherry was stereotaxically injected into the ARC of AgRP-Ires-cre mice, mCherry was detected exclusively in the ARC, in a pattern consistent with AgRP neurons. Importantly, no expression was detected following injection into non–Cre-expressing mice. Furthermore, mCherry was restricted to AgRP–Cre-expressing cells.

Vector Name:
pAAV-hSyn-DIO-hM3D(Gq)-mCherry
Antibiotic Resistance:
Ampicillin
Length:
7315 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-hM3D(Gq)-mCherry vector Map

pAAV-hSyn-DIO-hM3D(Gq)-mCherry7315 bp300600900120015001800210024002700300033003600390042004500480051005400570060006300660069007200AAV2 ITR (alternate)hSyn promoterlox2272loxPmCherryMuscarinic acetylcholine receptor M3lox2272loxPWPREhGH poly(A) signalAAV2 ITRf1 oripRS-markerpGEX 3'pBRforEcoAmpR promoterAmpRori

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-hM3D(Gq)-mCherry vector Sequence

Copy Sequence

Download GenBank File(.gb)

LOCUS       Exported                7315 bp DNA     circular SYN 29-MAY-2024
DEFINITION  Double floxed Gq-coupled hM3D DREADD fused with mCherry under the 
            control of human synapsin promoter..
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-hSyn-DIO-hM3D(Gq)-mCherry
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7315)
  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 7315)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 7315)
  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..7315
                     /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..3287)
                     /codon_start=1
                     /label=Muscarinic acetylcholine receptor M3
                     /translation="MTLHNNSTTSPLFPNISSSWIHSPSDAGLPPGTVTHFGSYNVSRA
                     AGNFSSPDGTTDDPLGGHTVWQVVFIAFLTGILALVTIIGNILVIVSFKVNKQLKTVNN
                     YFLLSLACADLIIGVISMNLFTTYIIMNRWALGNLACDLWLAIDCVASNASVMNLLVIS
                     FDRYFSITRPLTYRAKRTTKRAGVMIGLAWVISFVLWAPAILFWQYFVGKRTVPPGECF
                     IQFLSEPTITFGTAIAGFYMPVTIMTILYWRIYKETEKRTKELAGLQASGTEAETENFV
                     HPTGSSRSCSSYELQQQSMKRSNRRKYGRCHFWFTTKSWKPSSEQMDQDHSSSDSWNNN
                     DAAASLENSASSDEEDIGSETRAIYSIVLKLPGHSTILNSTKLPSSDNLQVPEEELGMV
                     DLERKADKLQAQKSVDDGGSFPKSFSKLPIQLESAVDTAKTSDVNSSVGKSTATLPLSF
                     KEATLAKRFALKTRSQITKRKRMSLVKEKKAAQTLSAILLAFIITWTPYNIMVLVNTFC
                     DSCIPKTFWNLGYWLCYINSTVNPVCYALCNKTFRTTFKMLLLCQCDKKKRRKQQYQQR
                     QSVIFHKRAPEQAL"
     protein_bind    complement(3300..3333)
                     /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(3384..3417)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     misc_feature    3442..4030
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional
                     regulatory element"
     primer_bind     complement(3495..3515)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3913..3924)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4062..4538
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4261..4280)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4578..4718
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4578..4707
                     /label=AAV2 ITR
     rep_origin      4793..5248
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4880..4899)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5090..5111
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5265..5284)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
     primer_bind     5384..5406
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5444..5462)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward
                     primer"
     promoter        5530..5634
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5635..6495
                     /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(5853..5872)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6666..7254
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7155..7174
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"