| 质粒名称: | pAAV-EF1a-DIO-hM3D(Gq)-mCherry |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 50460 |
| 存储实验室: | Bryan Roth |
|
载体骨架基本信息 |
|
| 载体名称: | pAAV |
| 载体抗性: | 氨苄青霉素 |
| 空载体大小: | 4818 bp |
| 完整质粒大小: | 8105 bp |
| 载体类型: | AAV |
| 高拷贝/低拷贝: | 低拷贝 |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | hM3D(Gq)-mCherry |
| 物种来源: | H. sapiens (human) |
| 插入大小: | 2508 bp |
| 基因突变: | See supplemental documents for DREADD mutations |
| 启动子 | EF1a |
| 标签/融合蛋白: |
HA（插入件上的N端子）
mCherry（插入件上的C端子）
|
| 克隆方法: | 限制性内切酶 |
| 5' 克隆位点: | Asc I(未破坏) |
| 3' 克隆位点: | Nhe I (未破坏) |
| 5' 测序引物 | TTTTTGAGTTTGGATCTTGG |
| 3' 测序引物 | GCATTAAAGCAGCGTATCCACATAGC |
|
文献引用方法 |
|
| 材料和方法部分: | pAAV-EF1a-DIO-hM3D(Gq)-mCherry was a gift from Bryan Roth (Addgene plasmid # 50460 ; http://n2t.net/addgene:50460 ; RRID:Addgene_50460) |
买家导航
载体基本信息
载体图谱质粒图谱和多克隆位点信息
-mCherry质粒图.png)
载体简介
载体序列
LOCUS pAAV-EF1a-DIO-hM3D(Gq)-mCherry 8095 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION Double floxed Gq-coupled hM3D DREADD fused with mCherry under the
control of EF1a promoter .
ACCESSION .
VERSION .
KEYWORDS pAAV-EF1a-DIO-hM3D(Gq)-mCherry
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8095)
AUTHORS Bryan Roth
TITLE Roth lab DREADDs
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 8095)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..8095
/organism="synthetic DNA construct"
/mol_type="other DNA"
repeat_region 1..130
/label=AAV2 ITR (alternate)
/note="Functional equivalent of wild-type AAV2 ITR"
promoter 231..1409
/label=EF-1-alpha promoter
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
intron 462..1400
/label=EF-1-alpha intron A
/note="intron upstream of the start codon of human
EF-1-alpha"
primer_bind 1357..1377
/label=EF1a-F
/note="Human elongation factor-1a promoter, forward primer"
protein_bind 1440..1473
/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."
protein_bind 1524..1557
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
CDS complement(1566..2276)
/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(1844..1863)
/label=mCherry-F
/note="mCherry, forward primer"
primer_bind 2113..2131
/label=mCherry-R
/note="mCherry, reverse primer"
regulatory 2273..2282
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
protein_bind complement(4080..4113)
/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."
protein_bind complement(4164..4197)
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
misc_feature 4222..4810
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(4275..4295)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(4693..4704)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
polyA_signal 4842..5318
/label=hGH poly(A) signal
/note="human growth hormone polyadenylation signal"
primer_bind complement(5041..5060)
/label=hGH-PA-R
/note="Human growth hormone terminator, reverse primer"
repeat_region 5358..5498
/label=AAV2 ITR
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
repeat_region 5358..5487
/label=AAV2 ITR
rep_origin 5573..6028
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(5660..5679)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 5870..5891
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(6045..6064)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 6164..6186
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(6224..6242)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 6310..6414
/gene="bla"
/label=AmpR promoter
CDS 6415..7275
/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(6633..6652)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 7446..8034
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 7935..7954
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
ORIGIN
1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
121 aggggttcct gcggccgcac gcgtaagctt tgcaaagatg gataaagttt taaacagaga
181 ggaatctttg cagctaatgg accttctagg tcttgaaagg agtgggaatt ggctccggtg
241 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg
301 gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt
361 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg
421 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt
481 cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct
541 ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga
601 ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct
661 ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt
721 ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc
781 ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg
841 acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac
901 cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc
961 cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag
1021 cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct
1081 cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg
1141 tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga
1201 gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc
1261 acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg
1321 aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa
1381 gtttttttct tccatttcag gtgtcgtgag gtaccggatc ctctagagtc gactccggaa
1441 taacttcgta taggatactt tatacgaagt tatgcagaat ggtagctgga ttgtagctgc
1501 tattagcaat atgaaacctc ttaataactt cgtatagcat acattatacg aagttatggc
1561 gcgccttact tgtacagctc gtccatgccg ccggtggagt ggcggccctc ggcgcgttcg
1621 tactgttcca cgatggtgta gtcctcgttg tgggaggtga tgtccaactt gatgttgacg
1681 ttgtaggcgc cgggcagctg cacgggcttc ttggccttgt aggtggtctt gacctcagcg
1741 tcgtagtggc cgccgtcctt cagcttcagc ctctgcttga tctcgccctt cagggcgccg
1801 tcctcggggt acatccgctc ggaggaggcc tcccagccca tggtcttctt ctgcattacg
1861 gggccgtcgg aggggaagtt ggtgccgcgc agcttcacct tgtagatgaa ctcgccgtcc
1921 tgcagggagg agtcctgggt cacggtcacc acgccgccgt cctcgaagtt catcacgcgc
1981 tcccacttga agccctcggg gaaggacagc ttcaagtagt cggggatgtc ggcggggtgc
2041 ttcacgtagg ccttggagcc gtacatgaac tgaggggaca ggatgtccca ggcgaagggc
2101 agggggccac ccttggtcac cttcagcttg gcggtctggg tgccctcgta ggggcggccc
2161 tcgccctcgc cctcgatctc gaactcgtgg ccgttcacgg agccctccat gtgcaccttg
2221 aagcgcatga actccttgat gatggccatg ttatcctcct cgcccttgct caccatggtg
2281 gcgaccgggg gatccttcaa ggcctgctcg ggtgcgcgct tgtgaaaaat gaccgactgt
2341 ctctgctggt actgctgctt gcgcctcttt tttttgtcac actggcacag cagcagcatc
2401 ttgaaagtgg ttctgaatgt tttgttgcac agagcatagc acacggggtt cacggtgctg
2461 ttgatgtagc acagccagta gcccagattc caaaaggttt tgggtatgca gctgtcacaa
2521 aaggtgttca ccagaaccat gatgttgtat ggggtccaag tgatgatgaa ggcaagcaag
2581 atcgcactga gggtctgggc cgctttcttc tccttgacca gggacatcct tttccgctta
2641 gtgatctgac ttctggtctt cagagcaaac ctcttggcca gagtggcttc cttgaaggac
2701 agaggtagag tggccgtgct cttacccact gaggagttga cgtcagaagt cttagctgtg
2761 tccacggctg actctagctg gatgggaagc ttggagaagc tttttggaaa actgcctcca
2821 tcgtccacgc tcttctgggc ctgcagcttg tcggctttcc tctccaagtc caccatcccc
2881 agctcctcct caggcacctg caggttgtcc gatgagggta acttggtgga gttgaggatg
2941 gtgctgtgac ccggaagctt gagcacgatg gagtagatgg ctctcgtctc ggagccaatg
3001 tcctcctcgt cggaggaggc ggagttctcc agggaggcag cagcatcatt gttgttccaa
3061 ctgtcactgc tgctgtggtc ttggtccatc tgctcggagc tgggtttcca gctcttggtt
3121 gtgaaccaga agtggcagcg gccatacttc ctcctgttgg agcgtttcat gctttgctgt
3181 tgaagttcgt aactgctgca gcttcgagaa ctgcccgtgg ggtggacaaa gttttctgtc
3241 tctgcctctg tcccagaggc ttgcaggcca gcaagctctt tggtacgctt ttcagtttcc
3301 ttatagatcc tccagtataa aatagtcata atggtgacag gcatataaaa accagcgatg
3361 gctgtgccaa aagtaatggt gggctcactg aggaactgaa tgaagcactc tcccggaggc
3421 acagttctct ttccaacaaa gtattgccag aacaagatgg caggagccca aaggacaaag
3481 gagatgaccc aagccagacc gatcatcaca ccggctctct ttgttgttcg tttggctcgg
3541 tacgtgagcg gcctcgtgat ggaaaagtat ctgtcaaagc tgatgaccag aagattcata
3601 acagaggcat tgctggctac gcagtcaatg gcaagccaga ggtcacaggc caagttccct
3661 aaggcccatc gattcatgat gatgtaggtc gtaaacagat tcattgaaat gaccccgata
3721 atcagatcgg cacaggccag gcttaagagg aagtagttgt tgaccgtctt cagctgcttg
3781 ttgaccttaa atgacacaat taccaggatg ttgccgatga tggtcaccaa ggccaggatg
3841 cccgttaaga aagcgatgaa gaccacttgc cagacggtat gacctcccag agggtcatcg
3901 gtggtaccgt ctggagagga gaaattgcca gctgctcgag aaacattgta gctgccgaaa
3961 tgagtgacgg ttcccggggg cagccctgca tcggaggggc tgtgtatcca ggaggagctg
4021 atgtttggaa acaaaggcga ggttgtactg ttattgtgca aggtcatggt ggcgctagca
4081 taacttcgta taaagtatcc tatacgaagt tatttgcctt aacccagaaa ttatcactgt
4141 tattctttag aatggtgcaa agaataactt cgtataatgt atgctatacg aagttatgaa
4201 ttcgatatca agcttatcga taatcaacct ctggattaca aaatttgtga aagattgact
4261 ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg
4321 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg
4381 ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg
4441 tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct cctttccggg
4501 actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc
4561 tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca
4621 tcgtcctttc cttggctgct cgcctatgtt gccacctgga ttctgcgcgg gacgtccttc
4681 tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct
4741 ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc
4801 gcctccccgc atcgataccg agcgctgctc gagagatcta cgggtggcat ccctgtgacc
4861 cctccccagt gcctctcctg gccctggaag ttgccactcc agtgcccacc agccttgtcc
4921 taataaaatt aagttgcatc attttgtctg actaggtgtc cttctataat attatggggt
4981 ggaggggggt ggtatggagc aaggggcaag ttgggaagac aacctgtagg gcctgcgggg
5041 tctattggga accaagctgg agtgcagtgg cacaatcttg gctcactgca atctccgcct
5101 cctgggttca agcgattctc ctgcctcagc ctcccgagtt gttgggattc caggcatgca
5161 tgaccaggct cagctaattt ttgttttttt ggtagagacg gggtttcacc atattggcca
5221 ggctggtctc caactcctaa tctcaggtga tctacccacc ttggcctccc aaattgctgg
5281 gattacaggc gtgaaccact gctcccttcc ctgtccttct gattttgtag gtaaccacgt
5341 gcggaccgag cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc
5401 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc
5461 ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct
5521 ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc
5581 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
5641 gccagcgcct tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
5701 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
5761 cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc
5821 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
5881 ttccaaactg gaacaacact caactctatc tcgggctatt cttttgattt ataagggatt
5941 ttgccgattt cggtctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
6001 tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat
6061 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
6121 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
6181 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta
6241 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
6301 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
6361 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
6421 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
6481 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
6541 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
6601 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
6661 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
6721 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
6781 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
6841 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
6901 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
6961 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
7021 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
7081 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
7141 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
7201 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
7261 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
7321 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
7381 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
7441 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
7501 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
7561 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
7621 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
7681 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
7741 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
7801 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
7861 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
7921 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
7981 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
8041 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgt
//
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