| 质粒名称: | APEX2-NLS |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 124617 |
| 存储实验室: | Alice Ting |
|
载体骨架基本信息 |
|
| 载体名称: | pcDNA3 |
| 载体出品公司: | -- |
| 空载体大小: | -- |
| 完整质粒大小: | -- |
| 载体修饰: | -- |
| 载体类型: | 哺乳动物表达 |
| 筛选标记: | V5 |
| 载体抗性: | 氨苄青霉素 |
| 生长温度: | 37 ℃ |
| 宿主菌: | DH5a |
| 拷贝数: | -- |
|
插入基因信息 |
|
| 插入基因名称: | APEX2-NLS |
| 物种来源: | -- |
| 插入大小: | -- |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子 | CMV |
| 标签/融合蛋白: | -- |
| 克隆方法: | Gibson克隆 |
| 5’克隆位点: | -- |
| 3’克隆位点: | -- |
| 5’测序引物 | CMV |
| 3’测序引物 | SP6 |
|
文献引用方法 |
|
| 材料和方法部分: | APEX2-NLS was a gift from Alice Ting (Addgene plasmid # 124617 ; http://n2t.net/addgene:124617 ; RRID:Addgene_124617) |
| 参考文献部分: | Live-cell mapping of organelle-associated RNAs via proximity biotinylation combined with protein-RNA crosslinking. Kaewsapsak P, Shechner DM, Mallard W, Rinn JL, Ting AY. Elife. 2017 Dec 14;6. doi: 10.7554/eLife.29224. 10.7554/eLife.29224 PubMed 29239719 |
买家导航
APEX2NLS载体基本信息
APEX2NLS载体图谱质粒图谱和多克隆位点信息
APEX2NLS载体简介
APEX2NLS载体序列
LOCUS APEX2-NLS 6275 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION proximity biotinylation.
ACCESSION .
VERSION .
KEYWORDS APEX2-NLS
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6275)
AUTHORS Kaewsapsak P, Shechner DM, Mallard W, Rinn JL, Ting AY
TITLE Live-cell mapping of organelle-associated RNAs via proximity
biotinylation combined with protein-RNA crosslinking.
JOURNAL Elife. 2017 Dec 14;6. doi: 10.7554/eLife.29224.
PUBMED 29239719
REFERENCE 2 (bases 1 to 6275)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..6275
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind complement(44..63)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
enhancer 235..614
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 615..818
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 769..789
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
primer_bind 863..882
/label=T7
/note="T7 promoter, forward primer"
promoter 863..881
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
regulatory 901..910
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 922..963
/codon_start=1
/product="epitope tag from simian virus 5"
/label=V5 tag
/translation="GKPIPNPLLGLDST"
CDS 964..1710
/codon_start=1
/product="soybean ascorbate peroxidase, engineered to serve
as a monomeric tag for electron microscopy and live-cell
proteomics (Lam et al., 2015)"
/label=APEX2
/note="more active than the original APEX due to the A134P
mutation"
/translation="GKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAGT
FDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVAV
EVTGGPKVPFHPGREDKPEPPPEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSGGHT
IGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVDK
YAADEDAFFADYAEAHQKLSELGFADA"
CDS 1729..1749
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 1753..1773
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 1777..1797
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
promoter complement(1828..1846)
/label=SP6 promoter
/note="promoter for bacteriophage SP6 RNA polymerase"
primer_bind complement(1829..1846)
/label=SP6
/note="SP6 promoter, forward primer"
primer_bind complement(1866..1883)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 1872..2096
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 2142..2570
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(2229..2248)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 2439..2460
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(2579..2599)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
promoter 2584..2913
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 2764..2899
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 2826..2845
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
CDS 2980..3774
/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
GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
primer_bind complement(3034..3053)
/label=Neo-R
/note="Neomycin resistance gene, reverse primer"
primer_bind 3644..3663
/label=Neo-F
/note="Neomycin resistance gene, forward primer"
polyA_signal 3948..4069
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(3985..4004)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 4039..4058
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(4118..4134)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(4118..4134)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(4131..4153)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 4142..4158
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
promoter complement(4166..4196)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 4211..4232
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(4349..4366)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(4520..5108)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(4600..4619)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(5279..6139)
/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 5902..5921
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(6140..6244)
/gene="bla"
/label=AmpR promoter
ORIGIN
1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgcggcc
901 gccaccatgg tgcggggttc tggcaagccc atccccaacc ccctgctggg cctggacagc
961 accggaaagt cttacccaac tgtgagtgct gattaccagg acgccgttga gaaggcgaag
1021 aagaagctca gaggcttcat cgctgagaag agatgcgctc ctctaatgct ccgtttggca
1081 ttccactctg ctggaacctt tgacaagggc acgaagaccg gtggaccctt cggaaccatc
1141 aagcaccctg ccgaactggc tcacagcgct aacaacggtc ttgacatcgc tgttaggctt
1201 ttggagccac tcaaggcgga gttccctatt ttgagctacg ccgatttcta ccagttggct
1261 ggcgttgttg ccgttgaggt cacgggtgga cctaaggttc cattccaccc tggaagagag
1321 gacaagcctg agccaccacc agagggtcgc ttgcctgatc ccactaaggg ttctgaccat
1381 ttgagagatg tgtttggcaa agctatgggg cttactgacc aagatatcgt tgctctatct
1441 gggggtcaca ctattggagc tgcacacaag gagcgttctg gatttgaggg tccctggacc
1501 tctaatcctc ttattttcga caactcatac ttcacggagt tgttgagtgg tgagaaggaa
1561 ggtctccttc agctaccttc tgacaaggct cttttgtctg accctgtatt ccgccctctc
1621 gttgataaat atgcagcgga cgaagatgcc ttctttgctg attacgctga ggctcaccaa
1681 aagctttccg agcttgggtt tgctgatgcc gaattcagca gggccgaccc caagaagaag
1741 aggaaggtgg accccaagaa gaagaggaag gtggacccca agaagaagag gaaggtgtga
1801 ctcgagcatg catctagagg gccctattct atagtgtcac ctaaatgcta gagctcgctg
1861 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
1921 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
1981 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
2041 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc
2101 tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc
2161 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
2221 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
2281 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
2341 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
2401 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
2461 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
2521 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg
2581 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
2641 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
2701 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
2761 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
2821 ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga
2881 ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt
2941 ttcggatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg
3001 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
3061 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
3121 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta
3181 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
3241 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
3301 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
3361 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
3421 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
3481 gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc
3541 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
3601 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
3661 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
3721 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga
3781 ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt
3841 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
3901 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg
3961 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
4021 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
4081 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
4141 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
4201 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
4261 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
4321 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
4381 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
4441 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
4501 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
4561 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
4621 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
4681 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
4741 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
4801 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
4861 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
4921 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
4981 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
5041 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
5101 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
5161 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
5221 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
5281 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
5341 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
5401 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
5461 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
5521 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
5581 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
5641 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
5701 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
5761 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
5821 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
5881 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
5941 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
6001 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
6061 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
6121 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
6181 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
6241 cgcgcacatt tccccgaaaa gtgccacctg acgtc
//
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