我们所展示的质粒图谱主要是从文献和开放数据库中收集而来，主要是为了方便研究工作，其中一小部分质粒进行了质量控制，可供科学家使用。
所有的产品都严格仅供科学研究使用，不能应用于临床试验，包括人体摄入、注射或外用的药理学用途。
确保质粒的关键元件正确，但是我们并不能保证实验结果。页面展示的图谱序列为理论序列，可能与测序结果不一致，请自行比对后确定是否满足要求。(如果测过序，本页面一般会提供下载)
开放数据库中的大多数载体序列都没有被完全测序。如果实际序列与参考序列的相似度超过99%，则将其视为正确。
由于科学研究是在探索未知，具有很大的不确定性，在任何情况下，我们都不承担超出质粒本身的额外经济损失或责任。
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.
- 载体名称:
- pAAV-hSyn-DIO-hM3D(Gq)-mCherry
- 载体抗性:
- Ampicillin
- 载体长度:
- 7315 bp
- 载体类型:
- AAV
- 载体来源:
- Bryan Roth
- 筛选标记:
- mCherry
- 拷贝数:
- Low copy number
- 启动子:
- hSyn
- 感受态:
- Stbl3
- 培养温度:
- 37℃
pAAV-hSyn-DIO-hM3D(Gq)-mCherry 载体载体图谱
质粒操作方法
1. 发货形式：质粒干粉(常温运输，存于-20度，请务必先转化提质粒后使用)
2. 收到质粒干粉后请先5000rpm离心1min，再加入20μl ddH2O溶解质粒;（质粒复测的浓度有时候与标称值差距较大，这可能是因为冻干质粒在管中的位置、复溶效率、测量偏差以及管壁的吸附导致，因此建议先转化提质粒后再使用）
3. 取1支100μl 感受态于冰上解冻10min，加入2μl质粒，再冰浴30min后，42℃热激60s，不要搅动，再冰浴2min；
4. 加入900μl无抗的LB液体培养基，180rpm震荡37℃培养45min (30℃培养1-1.5小时);
5. 6000rpm离心5min，仅留100μl上清液重悬细菌沉淀，并涂布至目标质粒抗性的LB平板上;
6. 将平板倒置37℃培养14h，如果要求是30℃则培养20h; (菌落过多则将质粒稀释后再转化；没有菌落则加入10μl质粒转化；建议不要直接转表达感受态， 要先转克隆感受态，重提质粒后再导入表达感受态);
7. 挑取单菌落至LB液体培养基中，加入对应抗生素，220rpm震荡培养14h，根据实验需要和质粒提取试剂盒说明书提取质粒。
pAAV-hSyn-DIO-hM3D(Gq)-mCherry 载体载体序列
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"