| 质粒名称: | pAAV-Ef1a-DIO hChR2(E123T/T159C)-mCherry |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 35510 |
| 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: | Stratagene |
| 载体抗性: | 氨苄青霉素 |
| 空载体大小: | 5605 bp |
| 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 |
| 物种来源: | 莱茵衣藻 |
| 插入大小: | 1646 bp |
| 基因突变: | E123T and T159C |
| 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(E123T/T159C)-mCherry was a gift from Karl Deisseroth (Addgene plasmid # 35510 ; http://n2t.net/addgene:35510 ; RRID:Addgene_35510) |
| 参考文献部分: | Principles for applying optogenetic tools derived from direct comparative analysis of microbial opsins. Mattis J, Tye KM, Ferenczi EA, Ramakrishnan C, O'Shea DJ, Prakash R, Gunaydin LA, Hyun M, Fenno LE, Gradinaru V, Yizhar O, Deisseroth K. Nat Methods. 2011 Dec 18;9(2):159-72. doi: 10.1038/nmeth.1808. 10.1038/nmeth.1808 PubMed 22179551 |
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质粒图谱.png)
载体简介
载体序列
LOCUS pAAV-Ef1a-DIO hChR2(E123T/T159C)-mCherry 7241 bp ds-DNA circular SYN 18-JUN-2019
DEFINITION AAV expression of Ef1a-driven, cre-dependent, hChR2 variant for
ultrafast optogenetic control..
ACCESSION .
VERSION .
KEYWORDS pAAV-Ef1a-DIO hChR2(E123T/T159C)-mCherry
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7241)
AUTHORS Mattis J, Tye KM, Ferenczi EA, Ramakrishnan C, O'Shea DJ, Prakash R,
Gunaydin LA, Hyun M, Fenno LE, Gradinaru V, Yizhar O, Deisseroth K
TITLE Principles for applying optogenetic tools derived from direct
comparative analysis of microbial opsins.
JOURNAL Nat Methods. 2011 Dec 18;9(2):159-72. doi: 10.1038/nmeth.1808.
PUBMED 22179551
REFERENCE 2 (bases 1 to 7241)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Jun 18, 2019 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..7241
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind 32..51
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
repeat_region 193..322
/label=AAV2 ITR (alternate)
/note="Functional equivalent of wild-type AAV2 ITR"
promoter 423..1601
/label=EF-1-alpha promoter
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
intron 654..1592
/label=EF-1-alpha intron A
/note="intron upstream of the start codon of human
EF-1-alpha"
primer_bind 1549..1569
/label=EF1a-F
/note="Human elongation factor-1a promoter, forward primer"
protein_bind 1632..1665
/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 1716..1749
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
CDS complement(1761..2468)
/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(2039..2058)
/label=mCherry-F
/note="mCherry, forward primer"
primer_bind 2308..2326
/label=mCherry-R
/note="mCherry, reverse primer"
regulatory 3404..3413
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
protein_bind complement(3418..3451)
/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(3502..3535)
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
misc_feature 3560..4148
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(3613..3633)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(4031..4042)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
polyA_signal 4180..4656
/label=hGH poly(A) signal
/note="human growth hormone polyadenylation signal"
primer_bind complement(4379..4398)
/label=hGH-PA-R
/note="Human growth hormone terminator, reverse primer"
repeat_region 4696..4836
/label=AAV2 ITR
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
repeat_region 4696..4825
/label=AAV2 ITR
rep_origin 4911..5366
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(4998..5017)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 5208..5229
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(5383..5402)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 5502..5524
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(5562..5580)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 5648..5752
/gene="bla"
/label=AmpR promoter
CDS 5753..6613
/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(5971..5990)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 6784..131
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
ORIGIN
1 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
61 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
121 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
181 ttgctcacat gtcctgcagg cagctgcgcg ctcgctcgct cactgaggcc gcccgggcgt
241 cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc
301 aactccatca ctaggggttc ctgcggccgc acgcgtaagc tttgcaaaga tggataaagt
361 tttaaacaga gaggaatctt tgcagctaat ggaccttcta ggtcttgaaa ggagtgggaa
421 ttggctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc gagaagttgg
481 ggggaggggt cggcaattga accggtgcct agagaaggtg gcgcggggta aactgggaaa
541 gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg
601 cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggtaagtg
661 ccgtgtgtgg ttcccgcggg cctggcctct ttacgggtta tggcccttgc gtgccttgaa
721 ttacttccac ctggctgcag tacgtgattc ttgatcccga gcttcgggtt ggaagtgggt
781 gggagagttc gaggccttgc gcttaaggag ccccttcgcc tcgtgcttga gttgaggcct
841 ggcctgggcg ctggggccgc cgcgtgcgaa tctggtggca ccttcgcgcc tgtctcgctg
901 ctttcgataa gtctctagcc atttaaaatt tttgatgacc tgctgcgacg ctttttttct
961 ggcaagatag tcttgtaaat gcgggccaag atctgcacac tggtatttcg gtttttgggg
1021 ccgcgggcgg cgacggggcc cgtgcgtccc agcgcacatg ttcggcgagg cggggcctgc
1081 gagcgcggcc accgagaatc ggacgggggt agtctcaagc tggccggcct gctctggtgc
1141 ctggcctcgc gccgccgtgt atcgccccgc cctgggcggc aaggctggcc cggtcggcac
1201 cagttgcgtg agcggaaaga tggccgcttc ccggccctgc tgcagggagc tcaaaatgga
1261 ggacgcggcg ctcgggagag cgggcgggtg agtcacccac acaaaggaaa agggcctttc
1321 cgtcctcagc cgtcgcttca tgtgactcca cggagtaccg ggcgccgtcc aggcacctcg
1381 attagttctc gagcttttgg agtacgtcgt ctttaggttg gggggagggg ttttatgcga
1441 tggagtttcc ccacactgag tgggtggaga ctgaagttag gccagcttgg cacttgatgt
1501 aattctcctt ggaatttgcc ctttttgagt ttggatcttg gttcattctc aagcctcaga
1561 cagtggttca aagttttttt cttccatttc aggtgtcgtg aggtaccgga tcctctagag
1621 tcgactccgg aataacttcg tataggatac tttatacgaa gttatgcaga atggtagctg
1681 gattgtagct gctattagca atatgaaacc tcttaataac ttcgtatagc atacattata
1741 cgaagttatg gcgcgcccta ttacttgtac agctcgtcca tgccgccggt ggagtggcgg
1801 ccctcggcgc gttcgtactg ttccacgatg gtgtagtcct cgttgtggga ggtgatgtcc
1861 aacttgatgt tgacgttgta ggcgccgggc agctgcacgg gcttcttggc cttgtaggtg
1921 gtcttgacct cagcgtcgta gtggccgccg tccttcagct tcagcctctg cttgatctcg
1981 cccttcaggg cgccgtcctc ggggtacatc cgctcggagg aggcctccca gcccatggtc
2041 ttcttctgca ttacggggcc gtcggagggg aagttggtgc cgcgcagctt caccttgtag
2101 atgaactcgc cgtcctgcag ggaggagtcc tgggtcacgg tcaccacgcc gccgtcctcg
2161 aagttcatca cgcgctccca cttgaagccc tcggggaagg acagcttcaa gtagtcgggg
2221 atgtcggcgg ggtgcttcac gtaggccttg gagccgtaca tgaactgagg ggacaggatg
2281 tcccaggcga agggcagggg gccacccttg gtcaccttca gcttggcggt ctgggtgccc
2341 tcgtaggggc ggccctcgcc ctcgccctcg atctcgaact cgtggccgtt cacggagccc
2401 tccatgtgca ccttgaagcg catgaactcc ttgatgatgg ccatgttatc ctcctcgccc
2461 ttgctcacgg cggccgctgg cacggctccg gcctcggctt cgtcttcgac gagagtctcg
2521 acctcgatct ccgttccgcc gatgttcagt ttggtggttt tgcggatatc tccgtgaatc
2581 aatatgtgct cgtggatcag gacgcgcagg tagtgtccca acaaccccca acaattttta
2641 ctcatcagat caataatcgt gtgacctacg gtggagccat agacgctcag gacgccaaaa
2701 ccttcgggcc ccaaaatgaa gagaattggg aacatacccc agctcacgaa aaacagccat
2761 gccatgccgg tcacgacctg gcggcaccga ccctttggca cagtatgata accctcgata
2821 tatgctttgg cggcgtgaaa aaatgtgttc gcgccatagc acaatccaag acaaaagaag
2881 atgactttaa catagccggt tgccatggcg ctggtagccc cccacacgat acacccgatg
2941 tctgagacaa ggagtcccat ggttctcctg ctgtagtcgt tgctcaggcc ggtgaggttg
3001 ctcaggtgga taaggatgac aggacaagtg agcagccagg ttgcatagcg cagccactgc
3061 acccggtgtc ctgtggcaag gtagagcata gagggattct taaactcaaa aaagaactcg
3121 agaatcacct taaccatttc aatggcgcac acatagatct cctcccagcc gcatgtagat
3181 ttccaggttt ggtaggcata gaacatcagc agcaaaatgc tgaatcctgc tgcaagccac
3241 tgcaggacat ttgacgcggt ctgagcgccg ttcgtgccgc gagattcaat ccatccggca
3301 cagtaacatt gatcctcagg gaccaggacg gacccgttca ccaccacagg attagtaacg
3361 aacaaaagtt cgcgtccgac ggcagacaaa gcgccgccat agtccatggt ggctagcata
3421 acttcgtata aagtatccta tacgaagtta tttgccttaa cccagaaatt atcactgtta
3481 ttctttagaa tggtgcaaag aataacttcg tataatgtat gctatacgaa gttatgaatt
3541 cgatatcaag cttatcgata atcaacctct ggattacaaa atttgtgaaa gattgactgg
3601 tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta
3661 tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct
3721 gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt
3781 tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac
3841 tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg
3901 ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc
3961 gtcctttcct tggctgctcg cctatgttgc cacctggatt ctgcgcggga cgtccttctg
4021 ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct
4081 gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc
4141 ctccccgcat cgataccgag cgctgctcga gagatctacg ggtggcatcc ctgtgacccc
4201 tccccagtgc ctctcctggc cctggaagtt gccactccag tgcccaccag ccttgtccta
4261 ataaaattaa gttgcatcat tttgtctgac taggtgtcct tctataatat tatggggtgg
4321 aggggggtgg tatggagcaa ggggcaagtt gggaagacaa cctgtagggc ctgcggggtc
4381 tattgggaac caagctggag tgcagtggca caatcttggc tcactgcaat ctccgcctcc
4441 tgggttcaag cgattctcct gcctcagcct cccgagttgt tgggattcca ggcatgcatg
4501 accaggctca gctaattttt gtttttttgg tagagacggg gtttcaccat attggccagg
4561 ctggtctcca actcctaatc tcaggtgatc tacccacctt ggcctcccaa attgctggga
4621 ttacaggcgt gaaccactgc tcccttccct gtccttctga ttttgtaggt aaccacgtgc
4681 ggaccgagcg gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc
4741 gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg
4801 cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc
4861 ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg
4921 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
4981 cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
5041 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
5101 gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg
5161 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
5221 ccaaactgga acaacactca actctatctc gggctattct tttgatttat aagggatttt
5281 gccgatttcg gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
5341 taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc
5401 cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
5461 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
5521 gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt
5581 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg
5641 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
5701 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
5761 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
5821 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
5881 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
5941 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
6001 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
6061 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
6121 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
6181 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
6241 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
6301 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
6361 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
6421 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
6481 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
6541 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
6601 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
6661 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
6721 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
6781 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
6841 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
6901 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
6961 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
7021 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
7081 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
7141 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
7201 agggagaaag gcggacaggt atccggtaag cggcagggtc g
//
