| 质粒名称: | pAAV-Ef1a-DIO hChR2(C128S/D156A)-mCherry |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 35504 |
| 存储实验室: | Karl Deisseroth |
|
载体骨架基本信息 |
|
| 载体名称: | pAAV |
| 载体出品公司: | -- |
| 载体抗性: | 氨苄青霉素 |
| 空载体大小: | -- |
| 完整质粒大小: | -- |
| 载体修饰: | 添加EF1a启动子、Lox位点和WPRE |
| 载体类型: | 哺乳细胞表达,AAV |
| 筛选标记: | -- |
| 高拷贝/低拷贝: | 高拷贝 |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | hChR2(C128S/D156A)-mCherry,SSFO-mCherry |
| 物种来源: | 莱茵衣藻 |
| 插入大小: | 1646 bp |
| 基因突变: | C128S and D156A |
| GenBank编号 | -- |
| 启动子 | Ef1a |
| 标签/融合蛋白: | mCherry（插入件上的C端子） |
| 克隆方法: | 限制性内切酶 |
| 5' 克隆位点: | AscI (未破坏) |
| 3' 克隆位点: | NheI(未破坏) |
| 5' 测序引物 | CACCCACACAAAGGAAAAGGGCC |
| 3' 测序引物 | GCAATAGCATGATACAAAGG |
|
文献引用方法 |
|
| 材料和方法部分: | pAAV-Ef1a-DIO hChR2(C128S/D156A)-mCherry was a gift from Karl Deisseroth (Addgene plasmid # 35504 ; http://n2t.net/addgene:35504 ; RRID:Addgene_35504) |
| 参考文献部分: | Neocortical excitation/inhibition balance in information processing and social dysfunction. Yizhar O, Fenno LE, Prigge M, Schneider F, Davidson TJ, O'Shea DJ, Sohal VS, Goshen I, Finkelstein J, Paz JT, Stehfest K, Fudim R, Ramakrishnan C, Huguenard JR, Hegemann P, Deisseroth K. Nature. 2011 Jul 27;477(7363):171-8. doi: 10.1038/nature10360. 10.1038/nature10360 PubMed 21796121 |
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pAAV-Ef1a-DIO hChR2(C128S/D156A)-mCherry载体质粒基本信息
pAAV-Ef1a-DIO hChR2(C128S/D156A)-mCherry质粒图谱载体图谱和pAAV-Ef1a-DIO hChR2(C128S/D156A)-mCherry载体序列质粒序列多克隆位点信息
-mCherry.png)
pAAV-Ef1a-DIO hChR2(C128S/D156A)-mCherry质粒载体简介
pAAV-Ef1a-DIO hChR2(C128S/D156A)-mCherry质粒序列载体序列
LOCUS pAAV-Ef1a-DIO hChR2(C128S/D156A)-mCherry 7243 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS pAAV-Ef1a-DIO hChR2(C128S/D156A)-mCherry
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7243)
AUTHORS Yizhar O, Fenno LE, Prigge M, Schneider F, Davidson TJ, O'Shea DJ,
Sohal VS, Goshen I, Finkelstein J, Paz JT, Stehfest K, Fudim R,
Ramakrishnan C, Huguenard JR, Hegemann P, Deisseroth K
TITLE Neocortical excitation/inhibition balance in information processing
and social dysfunction.
JOURNAL Nature. 2011 Jul 27;477(7363):171-8. doi: 10.1038/nature10360.
PUBMED 21796121
REFERENCE 2 (bases 1 to 7243)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..7243
/organism="synthetic DNA construct"
/mol_type="other DNA"
repeat_region 61..190
/label=AAV2 ITR (alternate)
/note="Functional equivalent of wild-type AAV2 ITR"
promoter 291..1469
/label=EF-1-alpha promoter
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
intron 522..1460
/label=EF-1-alpha intron A
/note="intron upstream of the start codon of human
EF-1-alpha"
primer_bind 1417..1437
/label=EF1a-F
/note="Human elongation factor-1a promoter, forward primer"
protein_bind 1500..1533
/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 1584..1617
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
CDS complement(1629..2336)
/codon_start=1
/product="monomeric derivative of DsRed fluorescent protein
(Shaner et al., 2004)"
/label=mCherry
/note="mammalian codon-optimized"
/translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
GRHSTGGMDELYK"
primer_bind complement(1907..1926)
/label=mCherry-F
/note="mCherry, forward primer"
primer_bind 2176..2194
/label=mCherry-R
/note="mCherry, reverse primer"
regulatory 3272..3281
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
protein_bind complement(3288..3321)
/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(3372..3405)
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
misc_feature 3430..4018
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(3483..3503)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(3901..3912)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
polyA_signal 4050..4526
/label=hGH poly(A) signal
/note="human growth hormone polyadenylation signal"
primer_bind complement(4249..4268)
/label=hGH-PA-R
/note="Human growth hormone terminator, reverse primer"
repeat_region 4566..4706
/label=AAV2 ITR
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
repeat_region 4566..4695
/label=AAV2 ITR
rep_origin 4781..5236
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(4868..4887)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 5078..5099
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(5253..5272)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 5372..5394
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(5432..5450)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 5518..5622
/gene="bla"
/label=AmpR promoter
CDS 5623..6483
/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(5841..5860)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 6654..7242
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 7143..7162
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
ORIGIN
1 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
61 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
121 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
181 aggggttcct gcggccgcac gcgtaagctt tgcaaagatg gataaagttt taaacagaga
241 ggaatctttg cagctaatgg accttctagg tcttgaaagg agtgggaatt ggctccggtg
301 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg
361 gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt
421 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg
481 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt
541 cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct
601 ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga
661 ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct
721 ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt
781 ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc
841 ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg
901 acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac
961 cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc
1021 cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag
1081 cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct
1141 cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg
1201 tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga
1261 gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc
1321 acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg
1381 aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa
1441 gtttttttct tccatttcag gtgtcgtgag gtaccggatc ctctagagtc gactccggaa
1501 taacttcgta taggatactt tatacgaagt tatgcagaat ggtagctgga ttgtagctgc
1561 tattagcaat atgaaacctc ttaataactt cgtatagcat acattatacg aagttatggc
1621 gcgccctatt acttgtacag ctcgtccatg ccgccggtgg agtggcggcc ctcggcgcgt
1681 tcgtactgtt ccacgatggt gtagtcctcg ttgtgggagg tgatgtccaa cttgatgttg
1741 acgttgtagg cgccgggcag ctgcacgggc ttcttggcct tgtaggtggt cttgacctca
1801 gcgtcgtagt ggccgccgtc cttcagcttc agcctctgct tgatctcgcc cttcagggcg
1861 ccgtcctcgg ggtacatccg ctcggaggag gcctcccagc ccatggtctt cttctgcatt
1921 acggggccgt cggaggggaa gttggtgccg cgcagcttca ccttgtagat gaactcgccg
1981 tcctgcaggg aggagtcctg ggtcacggtc accacgccgc cgtcctcgaa gttcatcacg
2041 cgctcccact tgaagccctc ggggaaggac agcttcaagt agtcggggat gtcggcgggg
2101 tgcttcacgt aggccttgga gccgtacatg aactgagggg acaggatgtc ccaggcgaag
2161 ggcagggggc cacccttggt caccttcagc ttggcggtct gggtgccctc gtaggggcgg
2221 ccctcgccct cgccctcgat ctcgaactcg tggccgttca cggagccctc catgtgcacc
2281 ttgaagcgca tgaactcctt gatgatggcc atgttatcct cctcgccctt gctcacggcg
2341 gccgctggca cggctccggc ctcggcttcg tcttcgacga gagtctcgac ctcgatctcc
2401 gttccgccga tgttcagttt ggtggttttg cggatatctc cgtgaatcaa tatgtgctcg
2461 tggatcagga cgcgcaggta gtgtcccaac aacccccaac aatttttact catcagatca
2521 ataatcgtgt gacctacggt ggagccatag acgctcagga cgccaaaacc ttcgggcccc
2581 aaaatgaaga gaattgggaa cataccccag ctcacgaaaa acagccatgc catgccggtc
2641 acgacctggc ggcaccgacc ctttggcaca gtatgataac cctcgatata tgctttggcg
2701 gcgtgaaaaa atgtgttcgc gccatagcac aatccaagac aaaagaagat gactttaaca
2761 tagccggttg ccatggcgct ggtagccccc cacacgatag tcccgatggc tgagacaagg
2821 agtcccatgg ttctcctgct gtagtcgttg ctcaggccgg tgaggttgct caggtggata
2881 aggatgacag gagaagtgag cagccactct gcatagcgca gccactgcac ccggtgtcct
2941 gtggcaaggt agagcataga gggattctta aactcaaaaa agaactcgag aatcacctta
3001 accatttcaa tggcgcacac atagatctcc tcccagccgc atgtagattt ccaggtttgg
3061 taggcataga acatcagcag caaaatgctg aatcctgctg caagccactg caggacattt
3121 gacgcggtct gagcgccgtt cgtgccgcga gattcaatcc atccggcaca gtaacattga
3181 tcctcaggga ccaggacgga cccgttcacc accacaggat tagtaacgaa caaaagttcg
3241 cgtccgacgg cagacaaagc gccgccatag tccatggtgg cgctagcata acttcgtata
3301 aagtatccta tacgaagtta tttgccttaa cccagaaatt atcactgtta ttctttagaa
3361 tggtgcaaag aataacttcg tataatgtat gctatacgaa gttatgaatt cgatatcaag
3421 cttatcgata atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac
3481 tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt
3541 gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat
3601 gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca
3661 acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc
3721 cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg
3781 gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct
3841 tggctgctcg cctatgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct
3901 tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt
3961 ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcat
4021 cgataccgag cgctgctcga gagatctacg ggtggcatcc ctgtgacccc tccccagtgc
4081 ctctcctggc cctggaagtt gccactccag tgcccaccag ccttgtccta ataaaattaa
4141 gttgcatcat tttgtctgac taggtgtcct tctataatat tatggggtgg aggggggtgg
4201 tatggagcaa ggggcaagtt gggaagacaa cctgtagggc ctgcggggtc tattgggaac
4261 caagctggag tgcagtggca caatcttggc tcactgcaat ctccgcctcc tgggttcaag
4321 cgattctcct gcctcagcct cccgagttgt tgggattcca ggcatgcatg accaggctca
4381 gctaattttt gtttttttgg tagagacggg gtttcaccat attggccagg ctggtctcca
4441 actcctaatc tcaggtgatc tacccacctt ggcctcccaa attgctggga ttacaggcgt
4501 gaaccactgc tcccttccct gtccttctga ttttgtaggt aaccacgtgc ggaccgagcg
4561 gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac
4621 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag
4681 cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct
4741 gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca
4801 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta
4861 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
4921 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
4981 cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt
5041 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
5101 acaacactca actctatctc gggctattct tttgatttat aagggatttt gccgatttcg
5161 gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
5221 ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
5281 agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
5341 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
5401 ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt
5461 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
5521 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
5581 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
5641 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
5701 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
5761 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
5821 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
5881 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
5941 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
6001 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
6061 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
6121 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
6181 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
6241 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
6301 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
6361 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
6421 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
6481 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
6541 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
6601 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
6661 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
6721 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
6781 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
6841 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
6901 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
6961 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
7021 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
7081 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
7141 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
7201 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaa
//
