| 质粒名称: | pAAV-hSyn-SOUL-P2A-tdTomato |
|---|---|
| 出品公司： | addgene |
| 目录编号: | 153019 |
| 存储实验室: | Guoping Feng |
|
载体骨架基本信息 |
|
| 载体名称: | pAAV |
| 完整质粒大小: | 7050 bp |
| 载体类型: | 哺乳细胞、AAV表达 |
| 载体抗性: | 氨苄霉素 |
| 生长温度: | 37 ℃ |
| 宿主菌: | DH5a |
| 拷贝数: | 高拷贝 |
|
插入基因信息 |
|
| 插入基因名称: | SOUL /Step-function opsin with ultra-high light sensitivity / ChR2(C128S/D156A/T159C) /SSFO(T159C) |
| 插入大小: | 930bp |
| 基因突变: | C128S/D156A/T159C |
| 标签/融合蛋白: | P2A-tdTomato (C terminal on insert) |
|
文献引用方法 |
|
| 材料和方法部分: | pAAV-hSyn-SOUL-P2A-tdTomato was a gift from Guoping Feng (Addgene plasmid # 153019 ; http://n2t.net/addgene:153019 ; RRID:Addgene_153019) |
| 参考文献部分: | An Ultra-Sensitive Step-Function Opsin for Minimally Invasive Optogenetic Stimulation in Mice and Macaques. Gong X, Mendoza-Halliday D, Ting JT, Kaiser T, Sun X, Bastos AM, Wimmer RD, Guo B, Chen Q, Zhou Y, Pruner M, Wu CW, Park D, Deisseroth K, Barak B, Boyden ES, Miller EK, Halassa MM, Fu Z, Bi G, Desimone R, Feng G. Neuron. 2020 Jul 8;107(1):197. doi: 10.1016/j.neuron.2020.06.018. 10.1016/j.neuron.2020.06.018 PubMed 32645306 |
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载体基本信息
载体图谱质粒图谱和多克隆位点信息
载体简介
载体序列
LOCUS pAAV-hSyn-SOUL-P2A-tdTomato 7050 bp ds-DNA circular SYN 13-MAY-2021
DEFINITION Step-function opsin with ultra-high light sensitivity (SOUL).
Capable to be used for noninvasive stimulation in mice and minimally
invasive stimulation in macaque..
ACCESSION .
VERSION .
KEYWORDS pAAV-hSyn-SOUL-P2A-tdTomato
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7050)
AUTHORS Gong X, Mendoza-Halliday D, Ting JT, Kaiser T, Sun X, Bastos AM,
Wimmer RD, Guo B, Chen Q, Zhou Y, Pruner M, Wu CW, Park D,
Deisseroth K, Barak B, Boyden ES, Miller EK, Halassa MM, Fu Z, Bi G,
Desimone R, Feng G
TITLE An Ultra-Sensitive Step-Function Opsin for Minimally Invasive
Optogenetic Stimulation in Mice and Macaques.
JOURNAL Neuron. 2020 Apr 14. pii: S0896-6273(20)30239-7. doi:
10.1016/j.neuron.2020.03.032.
PUBMED 32353253
REFERENCE 2 (bases 1 to 7050)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..7050
/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 183..630
/label=hSyn promoter
/note="human synapsin I promoter; confers neuron-specific
expression (Kugler et al., 2003)"
regulatory 678..687
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 684..1610
/codon_start=1
/product="H134R mutant of an N-terminal fragment of
channelrhodopsin-2 from the alga Chlamydomonas reinhardtii
(Nagel et al., 2005)"
/label=ChR2(H134R)
/note="blue light-activated cation channel"
/translation="MDYGGALSAVGRELLFVTNPVVVNGSVLVPEDQCYCAGWIESRGT
NGAQTASNVLQWLAAGFSILLLMFYAYQTWKSTCGWEEIYVCAIEMVKVILEFFFEFKN
PSMLYLATGHRVQWLRYAEWLLTSPVILIHLSNLTGLSNDYSRRTMGLLVSAIGCIVWG
ATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVS
WGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDI
RKTTKLNIGGTEIEVETLVEDEAEAGAV"
CDS 1629..1685
/codon_start=1
/product="2A peptide from porcine teschovirus-1
polyprotein"
/label=P2A
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="ATNFSLLKQAGDVEENPGP"
CDS 1686..3116
/codon_start=1
/product="tandem dimeric (pseudo-monomeric) derivative of
DsRed (Shaner et al., 2004)"
/label=tdTomato
/note="mammalian codon-optimized"
/translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
HLFLGHGIGSTGSGSSGTASSEDNNMAVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPY
EGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVM
NFEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGV
LKGEIHQALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYE
RSEGRHHLFLYGMDELYK"
misc_feature 3177..3765
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(3230..3250)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(3648..3659)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
polyA_signal 3797..4273
/label=hGH poly(A) signal
/note="human growth hormone polyadenylation signal"
primer_bind complement(3996..4015)
/label=hGH-PA-R
/note="Human growth hormone terminator, reverse primer"
repeat_region 4313..4453
/label=AAV2 ITR
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
repeat_region 4313..4442
/label=AAV2 ITR
rep_origin 4528..4983
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(4615..4634)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 4825..4846
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(5000..5019)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 5119..5141
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(5179..5197)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 5265..5369
/gene="bla"
/label=AmpR promoter
CDS 5370..6230
/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(5588..5607)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 6401..6989
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 6890..6909
/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 gcgtttaatt aagtgtctag actgcagagg gccctgcgta
181 tgagtgcaag tgggttttag gaccaggatg aggcggggtg ggggtgccta cctgacgacc
241 gaccccgacc cactggacaa gcacccaacc cccattcccc aaattgcgca tcccctatca
301 gagaggggga ggggaaacag gatgcggcga ggcgcgtgcg cactgccagc ttcagcaccg
361 cggacagtgc cttcgccccc gcctggcggc gcgcgccacc gccgcctcag cactgaaggc
421 gcgctgacgt cactcgccgg tcccccgcaa actccccttc ccggccacct tggtcgcgtc
481 cgcgccgccg ccggcccagc cggaccgcac cacgcgaggc gcgagatagg ggggcacggg
541 cgcgaccatc tgcgctgcgg cgccggcgac tcagcgctgc ctcagtctgc ggtgggcagc
601 ggaggagtcg tgtcgtgcct gagagcgcag tcgagaaacc ggctagagga tccttcgaaa
661 ccggtgctag cgcggccgcc accatggact atggcggcgc tttgtctgcc gtcggacgcg
721 aacttttgtt cgttactaat cctgtggtgg tgaacgggtc cgtcctggtc cctgaggatc
781 aatgttactg tgccggatgg attgaatctc gcggcacgaa cggcgctcag accgcgtcaa
841 atgtcctgca gtggcttgca gcaggattca gcattttgct gctgatgttc tatgcctacc
901 aaacctggaa atctacatgc ggctgggagg agatctatgt gtgcgccatt gaaatggtta
961 aggtgattct cgagttcttt tttgagttta agaatccctc tatgctctac cttgccacag
1021 gacaccgggt gcagtggctg cgctatgcag agtggctgct cacttctcct gtcatcctta
1081 tccacctgag caacctcacc ggcctgagca acgactacag caggagaacc atgggactcc
1141 ttgtctcagc catcgggtgt atcgtgtggg gggctaccag cgccatggca accggctatg
1201 ttaaagtcat cttcttttgt cttggattgt gctatggcgc gaacacattt tttcacgccg
1261 ccaaagcata tatcgagggt tatcatactg tgccaaaggg tcggtgccgc caggtcgtga
1321 ccggcatggc atggctgttt ttcgtgagct ggggtatgtt cccaattctc ttcattttgg
1381 ggcccgaagg ttttggcgtc ctgagcgtct atggctccac cgtaggtcac acgattattg
1441 atctgatgag taaaaattgt tgggggttgt tgggacacta cctgcgcgtc ctgatccacg
1501 agcacatatt gattcacgga gatatccgca aaaccaccaa actgaacatc ggcggaacgg
1561 agatcgaggt cgagactctc gtcgaagacg aagccgaggc cggagccgtg ccagttaacg
1621 gaagcggagc cactaacttc tccctgttga aacaagcagg ggatgtcgaa gagaatcccg
1681 ggccaatggt gagcaagggc gaggaggtca tcaaagagtt catgcgcttc aaggtgcgca
1741 tggagggctc catgaacggc cacgagttcg agatcgaggg cgagggcgag ggccgcccct
1801 acgagggcac ccagaccgcc aagctgaagg tgaccaaggg cggccccctg cccttcgcct
1861 gggacatcct gtccccccag ttcatgtacg gctccaaggc gtacgtgaag caccccgccg
1921 acatccccga ttacaagaag ctgtccttcc ccgagggctt caagtgggag cgcgtgatga
1981 acttcgagga cggcggtctg gtgaccgtga cccaggactc ctccctgcag gacggcacgc
2041 tgatctacaa ggtgaagatg cgcggcacca acttcccccc cgacggcccc gtaatgcaga
2101 agaagaccat gggctgggag gcctccaccg agcgcctgta cccccgcgac ggcgtgctga
2161 agggcgagat ccaccaggcc ctgaagctga aggacggcgg ccactacctg gtggagttca
2221 agaccatcta catggccaag aagcccgtgc aactgcccgg ctactactac gtggacacca
2281 agctggacat cacctcccac aacgaggact acaccatcgt ggaacagtac gagcgctccg
2341 agggccgcca ccacctgttc ctggggcatg gcatcggcag caccggcagc ggcagctccg
2401 gcaccgcctc ctccgaggac aacaacatgg ccgtcatcaa agagttcatg cgcttcaagg
2461 tgcgcatgga gggctccatg aacggccacg agttcgagat cgagggcgag ggcgagggcc
2521 gcccctacga gggcacccag accgccaagc tgaaggtgac caagggcggc cccctgccct
2581 tcgcctggga catcctgtcc ccccagttca tgtacggctc caaggcgtac gtgaagcacc
2641 ccgccgacat ccccgattac aagaagctgt ccttccccga gggcttcaag tgggagcgcg
2701 tgatgaactt cgaggacggc ggtctggtga ccgtgaccca ggactcctcc ctgcaggacg
2761 gcacgctgat ctacaaggtg aagatgcgcg gcaccaactt cccccccgac ggccccgtaa
2821 tgcagaagaa gaccatgggc tgggaggcct ccaccgagcg cctgtacccc cgcgacggcg
2881 tgctgaaggg cgagatccac caggccctga agctgaagga cggcggccac tacctggtgg
2941 agttcaagac catctacatg gccaagaagc ccgtgcaact gcccggctac tactacgtgg
3001 acaccaagct ggacatcacc tcccacaacg aggactacac catcgtggaa cagtacgagc
3061 gctccgaggg ccgccaccac ctgttcctgt acggcatgga cgagctgtac aagtaaggcg
3121 cgaatcgcga gtcgacggcg cgcccctgca gggaattcga tatcaagctt atcgataatc
3181 aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt
3241 ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg
3301 ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc
3361 ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt
3421 ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg
3481 ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg
3541 gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct
3601 atgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc
3661 cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc
3721 ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcatcga taccgagcgc
3781 tgctcgagag atctacgggt ggcatccctg tgacccctcc ccagtgcctc tcctggccct
3841 ggaagttgcc actccagtgc ccaccagcct tgtcctaata aaattaagtt gcatcatttt
3901 gtctgactag gtgtccttct ataatattat ggggtggagg ggggtggtat ggagcaaggg
3961 gcaagttggg aagacaacct gtagggcctg cggggtctat tgggaaccaa gctggagtgc
4021 agtggcacaa tcttggctca ctgcaatctc cgcctcctgg gttcaagcga ttctcctgcc
4081 tcagcctccc gagttgttgg gattccaggc atgcatgacc aggctcagct aatttttgtt
4141 tttttggtag agacggggtt tcaccatatt ggccaggctg gtctccaact cctaatctca
4201 ggtgatctac ccaccttggc ctcccaaatt gctgggatta caggcgtgaa ccactgctcc
4261 cttccctgtc cttctgattt tgtaggtaac cacgtgcgga ccgagcggcc gcaggaaccc
4321 ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga
4381 ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc
4441 agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
4501 caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg
4561 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg cccgctcctt
4621 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
4681 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
4741 atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
4801 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaact
4861 ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc tattggttaa
4921 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa
4981 ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
5041 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
5101 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
5161 aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
5221 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
5281 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
5341 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
5401 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
5461 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
5521 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
5581 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
5641 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
5701 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
5761 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
5821 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
5881 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
5941 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
6001 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
6061 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
6121 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
6181 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
6241 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
6301 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
6361 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
6421 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
6481 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
6541 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
6601 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
6661 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
6721 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
6781 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
6841 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
6901 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
6961 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
7021 tggccttttg ctggcctttt gctcacatgt
//
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