| 质粒名称: | pCMV-lyso-pHluorin |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 70113 |
| 存储实验室: |
Christian Rosenmund
|
|
载体骨架基本信息 |
|
| 载体名称: | pCMV |
| 载体出品公司: | -- |
| 空载体大小: | 3878 bp |
| 完整质粒大小: | 5336 bp |
| 载体修饰: | -- |
| 载体类型: | 哺乳动物表达 |
| 筛选标记: | 新霉素（G418筛选） |
| 载体抗性: | 卡那霉素，50μg/mL |
| 生长温度: | 37 ℃ |
| 宿主菌: | XL10 Gold |
| 拷贝数: | -- |
|
插入基因信息 |
|
| 插入基因名称: | lyso-pHluorin |
| 物种来源: | M. musculus (mouse); Aequorea victoriais |
| 插入大小: | 3492 bp |
| 基因突变: | -- |
| GenBank编号 | 12512; KT880226 |
| 启动子: | CMV |
| 标签/融合蛋白: | pH氟化物 HA标签（插入件上的C端子） |
| 克隆方法: | 限制性内切酶 |
| 5’克隆位点: | NheI(未破坏) |
| 3’克隆位点: | PacI(未破坏) |
| 5’测序引物 | CGCAAATGGGCGGTAGGCGTG |
| 3’测序引物 | TTGTGAAATTTGTGATGCTATTGC |
|
文献引用方法 |
|
| 材料和方法部分: | pCMV-lyso-pHluorin was a gift from Christian Rosenmund (Addgene plasmid # 70113 ; http://n2t.net/addgene:70113 ; RRID:Addgene_70113) |
| 参考文献部分: | Optogenetic acidification of synaptic vesicles and lysosomes. Rost BR, Schneider F, Grauel MK, Wozny C, G Bentz C, Blessing A, Rosenmund T, Jentsch TJ, Schmitz D, Hegemann P, Rosenmund C. Nat Neurosci. 2015 Nov 9. doi: 10.1038/nn.4161. 10.1038/nn.4161 PubMed 26551543 |
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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 pCMV-lyso-pHluorin 5336 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Expression of lyso-pHluorin in mammalian cells for optical readout
of lysosomal acidification.
ACCESSION .
VERSION .
KEYWORDS pCMV-lyso-pHluorin
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5336)
AUTHORS Rost BR, Schneider F, Grauel MK, Wozny C, G Bentz C, Blessing A,
Rosenmund T, Jentsch TJ, Schmitz D, Hegemann P, Rosenmund C
TITLE Optogenetic acidification of synaptic vesicles and lysosomes.
JOURNAL Nat Neurosci. 2015 Nov 9. doi: 10.1038/nn.4161.
PUBMED 26551543
REFERENCE 2 (bases 1 to 5336)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..5336
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 61..364
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 365..568
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 519..539
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
regulatory 738..747
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 744..1460
/codon_start=1
/product="pH-sensitive mutant of green fluorescent protein
(Ng et al., 2002)"
/label=superecliptic pHluorin
/note="excited strongly by blue light at neutral pH but
minimally fluorescent at acidic pH"
/translation="MGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNDHQVYIMADKQKNGIK
ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLFTTSTLSKDPNEKRDHMVLL
EFVTAAGITHGMDELYK"
primer_bind complement(777..805)
/label=GFP-R
/note="GFP, reverse primer. Does NOT anneal to EGFP"
primer_bind 1400..1420
/label=GFP-F
/note="GFP, forward primer. Does NOT anneal to EGFP"
CDS 2064..2090
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/label=HA
/translation="YPYDVPDYA"
polyA_signal 2124..2245
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(2161..2180)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 2215..2234
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
rep_origin complement(2252..2707)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(2389..2410)
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind 2601..2620
/label=F1ori-R
/note="F1 origin, reverse primer"
promoter 2734..2838
/gene="bla"
/label=AmpR promoter
promoter 2840..3197
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
primer_bind complement(2863..2883)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
rep_origin 3048..3183
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 3110..3129
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
CDS 3232..4026
/codon_start=1
/gene="aph(3')-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/label=NeoR/KanR
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
primer_bind complement(3286..3305)
/label=Neo-R
/note="Neomycin resistance gene, reverse primer"
primer_bind 3896..3915
/label=Neo-F
/note="Neomycin resistance gene, forward primer"
primer_bind complement(4214..4233)
/label=TK-pA-R
/note="Thymidine kinase polyA, reverse primer"
polyA_signal 4258..4305
/label=HSV TK poly(A) signal
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
rep_origin 4634..5222
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 5123..5142
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
ORIGIN
1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcacca
601 tggcggtgga aggaggaatg aagtgtgtca agtttttgct ctacgttctc ctgctggcct
661 tctgcgcctg tgcagtggga ttgatcgcca ttggtgtagc ggttcaggtt gtcttgaagc
721 aggccgaagc ggatctagcc accatgggaa gtaaaggaga agaacttttc actggagttg
781 tcccaattct tgttgaatta gatggtgatg ttaatgggca caaattttct gtcagtggag
841 agggtgaagg tgatgcaaca tacggaaaac ttacccttaa atttatttgc actactggaa
901 aactacctgt tccttggcca acacttgtca ctactttaac ttatggtgtt caatgctttt
961 caagataccc agatcatatg aaacggcatg actttttcaa gagtgccatg cccgaaggtt
1021 atgtacagga aagaactata tttttcaaag atgacgggaa ctacaagaca cgtgctgaag
1081 tcaagtttga aggtgatacc cttgttaata gaatcgagtt aaaaggtatt gattttaaag
1141 aagatggaaa cattcttgga cacaaattgg aatacaacta taacgatcac caggtgtaca
1201 tcatggcaga caaacaaaag aatggaatca aagctaactt caaaattaga cacaacattg
1261 aagatggagg cgttcaacta gcagaccatt atcaacaaaa tactccaatt ggcgatgggc
1321 ccgtcctttt accagacaac cattacctgt ttacaacttc tactctttcg aaagatccca
1381 acgaaaagag agaccacatg gtccttcttg agtttgtaac agctgctggg attacacatg
1441 gcatggatga actatacaaa accggtggag ctccaattac ccatgagact actgctggct
1501 cgctgttgcc tgtggtcatc attgcagtgg gtgccttcct cttcctggtg gcctttgtgg
1561 gctgctgtgg ggcctgcaag gagaactact gtctcatgat tacatttgcc atcttcctgt
1621 ctcttatcat gcttgtggag gtggctgtgg ccattgctgg ctatgtgttt agagaccagg
1681 tgaagtcaga gtttaataaa agcttccagc agcagatgca gaattacctt aaagacaaca
1741 aaacagccac tattttggac aaattgcaga aagaaaataa ctgctgtgga gcttctaact
1801 acacagactg ggaaaacatc cccggcatgg ccaaggacag agtccccgat tcttgctgca
1861 tcaacataac tgtgggctgt gggaatgatt tcaaggaatc cactatccat acccagggct
1921 gcgtggagac tatagcaata tggctaagga agaacatact gctggtggct gcagcggccc
1981 tgggcattgc ttttgtggag gtcttgggaa ttatcttctc ctgctgtctg gtgaagagta
2041 ttcgaagtgg ctatgaagta atgtacccat acgatgttcc agattacgct tagttaatta
2101 agcaagggcg aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa
2161 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
2221 tgtccaaact catcaatgta tcttaaggcg taaattgtaa gcgttaatat tttgttaaaa
2281 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa
2341 atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac
2401 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag
2461 ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt
2521 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg
2581 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca
2641 agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag
2701 ggcgcgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
2761 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
2821 tattgaaaaa ggaagagtcc tgaggcggaa agaaccagct gtggaatgtg tgtcagttag
2881 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
2941 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
3001 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
3061 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
3121 aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag
3181 gcctaggctt ttgcaaagat cgatcaagag acaggatgag gatcgtttcg catgattgaa
3241 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
3301 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
3361 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaagacgag
3421 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
3481 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
3541 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
3601 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
3661 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
3721 gggctcgcgc cagccgaact gttcgccagg ctcaaggcga gcatgcccga cggcgaggat
3781 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
3841 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
3901 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
3961 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
4021 ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac
4081 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg
4141 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccta
4201 gggggaggct aactgaaaca cggaaggaga caataccgga aggaacccgc gctatgacgg
4261 caataaaaag acagaataaa acgcacggtg ttgggtcgtt tgttcataaa cgcggggttc
4321 ggtcccaggg ctggcactct gtcgataccc caccgagacc ccattggggc caatacgccc
4381 gcgtttcttc cttttcccca ccccaccccc caagttcggg tgaaggccca gggctcgcag
4441 ccaacgtcgg ggcggcaggc cctgccatag cctcaggtta ctcatatata ctttagattg
4501 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
4561 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
4621 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
4681 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
4741 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
4801 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
4861 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
4921 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
4981 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
5041 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
5101 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
5161 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
5221 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
5281 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc atgcat
//
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