| 质粒名称: | pcDNA3 APEX2-NES |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 49386 |
| 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: | -- |
| 载体抗性: | 氨苄青霉素 |
| 空载体大小: | 5385 bp |
| 完整质粒大小: | 6198 bp |
| 载体修饰: | -- |
| 载体类型: | 哺乳细胞表达 |
| 筛选标记: | -- |
| 高拷贝/低拷贝: | -- |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | Flag - APEX2 - NES |
| 物种来源: | soybean |
| 插入大小: | 813 bp |
| 基因突变: | K14D, W41F, E112K, A134P on soybean APX; improved for proteomics and EM applications |
| GenBank编号 | -- |
| 启动子 | CMV |
| 标签/融合蛋白: | Flag（插入N端子） |
| 克隆方法: | 限制性内切酶 |
| 5' 克隆位点: | NotI(未破坏) |
| 3' 克隆位点: | XhoI (未破坏) |
| 5' 测序引物 | CMVf |
| 3' 测序引物 | -- |
|
文献引用方法 |
|
| 材料和方法部分: | pcDNA3 APEX2-NES was a gift from Alice Ting (Addgene plasmid # 49386 ; http://n2t.net/addgene:49386 ; RRID:Addgene_49386) |
| 参考文献部分: | Directed evolution of APEX2 for electron microscopy and proximity labeling. Lam SS, Martell JD, Kamer KJ, Deerinck TJ, Ellisman MH, Mootha VK, Ting AY. Nat Methods. 2014 Nov 24. doi: 10.1038/nmeth.3179. 10.1038/nmeth.3179 PubMed 25419960 |
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
载体简介
载体序列
LOCUS pcDNA3 APEX2-NES 6197 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION Expresses APEX2 in the cytosol of mammalian cells.
ACCESSION .
VERSION .
KEYWORDS pcDNA3 APEX2-NES
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6197)
AUTHORS Lam SS, Martell JD, Kamer KJ, Deerinck TJ, Ellisman MH, Mootha VK,
Ting AY
TITLE Directed evolution of APEX2 for electron microscopy and proximity
labeling.
JOURNAL Nat Methods. 2014 Nov 24. doi: 10.1038/nmeth.3179.
PUBMED 25419960
REFERENCE 2 (bases 1 to 6197)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..6197
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 115..494
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 495..698
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 649..669
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
primer_bind 743..762
/label=T7
/note="T7 promoter, forward primer"
promoter 743..761
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
regulatory 781..790
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 790..813
/codon_start=1
/product="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
/label=FLAG
/translation="DYKDDDDK"
CDS 814..1560
/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 1567..1593
/codon_start=1
/product="nuclear export signal from the HIV Rev protein
(Fischer et al., 1995)"
/label=NES
/translation="LPPLERLTL"
promoter complement(1630..1648)
/label=SP6 promoter
/note="promoter for bacteriophage SP6 RNA polymerase"
primer_bind complement(1631..1648)
/label=SP6
/note="SP6 promoter, forward primer"
primer_bind complement(1668..1685)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 1674..1898
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 1944..2372
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(2031..2050)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 2241..2262
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(2381..2401)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
promoter 2386..2715
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 2566..2701
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 2628..2647
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
CDS 2782..3576
/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(2836..2855)
/label=Neo-R
/note="Neomycin resistance gene, reverse primer"
primer_bind 3446..3465
/label=Neo-F
/note="Neomycin resistance gene, forward primer"
polyA_signal 3750..3871
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(3787..3806)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 3841..3860
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(3920..3936)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(3920..3936)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(3933..3955)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 3944..3960
/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(3968..3998)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 4013..4034
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(4151..4168)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(4322..4910)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(4402..4421)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(5081..5941)
/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 5704..5723
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(5942..6046)
/gene="bla"
/label=AmpR promoter
primer_bind complement(6121..6140)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
ORIGIN
1 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
61 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
121 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
181 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
241 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
301 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
361 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
421 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
481 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
541 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
601 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
661 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
721 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgcggcc
781 gccaccatgg actacaagga tgacgacgat aagggaaagt cttacccaac tgtgagtgct
841 gattaccagg acgccgttga gaaggcgaag aagaagctca gaggcttcat cgctgagaag
901 agatgcgctc ctctaatgct ccgtttggca ttccactctg ctggaacctt tgacaagggc
961 acgaagaccg gtggaccctt cggaaccatc aagcaccctg ccgaactggc tcacagcgct
1021 aacaacggtc ttgacatcgc tgttaggctt ttggagccac tcaaggcgga gttccctatt
1081 ttgagctacg ccgatttcta ccagttggct ggcgttgttg ccgttgaggt cacgggtgga
1141 cctaaggttc cattccaccc tggaagagag gacaagcctg agccaccacc agagggtcgc
1201 ttgcccgatc ccactaaggg ttctgaccat ttgagagatg tgtttggcaa agctatgggg
1261 cttactgacc aagatatcgt tgctctatct gggggtcaca ctattggagc tgcacacaag
1321 gagcgttctg gatttgaggg tccctggacc tctaatcctc ttattttcga caactcatac
1381 ttcacggagt tgttgagtgg tgagaaggaa ggtctccttc agctaccttc tgacaaggct
1441 cttttgtctg accctgtatt ccgccctctc gttgacaaat atgcagcgga cgaagatgcc
1501 ttctttgctg attacgctga ggctcaccaa aagctttccg agcttgggtt tgctgatgcc
1561 ctgcagctgc ctcccctgga gcgcctgacc ctggactaat agctcgagca tgcatctaga
1621 gggccctatt ctatagtgtc acctaaatgc tagagctcgc tgatcagcct cgactgtgcc
1681 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
1741 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
1801 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
1861 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
1921 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
1981 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
2041 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
2101 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
2161 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
2221 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
2281 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
2341 tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg
2401 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
2461 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
2521 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
2581 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
2641 gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
2701 ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag
2761 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc
2821 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
2881 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
2941 tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg
3001 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
3061 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
3121 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
3181 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
3241 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
3301 ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
3361 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
3421 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
3481 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
3541 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga
3601 ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg
3661 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
3721 atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca
3781 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
3841 gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct
3901 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
3961 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
4021 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
4081 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
4141 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
4201 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
4261 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
4321 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
4381 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
4441 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
4501 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
4561 ccaagctggg ctgtgtgcac aaaccccccg ttcagcccga ccgctgcgcc ttatccggta
4621 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
4681 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
4741 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
4801 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
4861 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
4921 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
4981 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
5041 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
5101 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
5161 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
5221 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
5281 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
5341 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
5401 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
5461 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
5521 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
5581 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
5641 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
5701 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
5761 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
5821 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
5881 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
5941 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
6001 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
6061 aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag
6121 tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga
6181 ggtcgctgag tagtgcg
//
