| 质粒名称: | pMK286 (mAID-NeoR) |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 72824 |
| 质粒用途: | C-terminal tagging with mAID using CRISPR/Cas9 |
| 存储实验室: | Masato Kanemaki |
|
载体骨架基本信息 |
|
| 载体名称: | pBluescript KS(-) |
| 空载体大小: | 2875 bp |
| 完整质粒大小: | 5394 bp |
| 载体类型: | 哺乳动物表达，CRISPR |
| 筛选标记: | 新霉素（G418） |
| 载体抗性: | 氨苄青霉素，100μg/mL |
| 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: | mAID-Neo |
| 物种来源: | H. sapiens (human), M. musculus (mouse), R. norvegicus (rat), G. gallus (chicken) |
| 插入大小: | 2519 bp |
| 标签/融合蛋白: | mAID (C terminal on backbone) |
| 克隆方法: | 限制性内切酶 |
| 5’克隆位点: | BamHI（未销毁） |
| 3’克隆位点: | BamHI（未销毁） |
| 5’测序引物 | GTAAAACGACGGCCAGT |
| 3’测序引物 | GGAAACAGCTATGACCATG |
|
文献引用方法 |
|
| 材料和方法部分: | pMK286 (mAID-NeoR) was a gift from Masato Kanemaki (Addgene plasmid # 72824 ; http://n2t.net/addgene:72824 ; RRID:Addgene_72824) |
| 参考文献部分: | Rapid Protein Depletion in Human Cells by Auxin-Inducible Degron Tagging with Short Homology Donors. Natsume T, Kiyomitsu T, Saga Y, Kanemaki MT. Cell Rep. 2016 Apr 5;15(1):210-8. doi: 10.1016/j.celrep.2016.03.001. Epub 2016 Mar 24. 10.1016/j.celrep.2016.03.001 PubMed 27052166 |
pMK286 (mAID-NeoR)
价格：元
规格： 2ug质粒（仅用于转化提取） 转染级去内毒素液体即用质粒100μg 转染级去内毒素液体即用质粒500μg 转染级去内毒素液体即用质粒1mg 更大规格-质粒构建-质粒定制-请联系我们
联系方式：I47-825O-882O
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pMK286 (mAID-NeoR)载体质粒基本信息
pMK286 (mAID-NeoR)质粒图谱载体图谱和pMK286 (mAID-NeoR)载体序列质粒序列多克隆位点信息
载体图谱.png)
pMK286 (mAID-NeoR)质粒载体简介
pMK286 (mAID-NeoR)质粒序列载体序列
LOCUS pMK286 (mAID-NeoR) 5394 bp DNA circular SYN 04-SEP-2026
DEFINITION C-terminal tagging with mAID using CRISPR/Cas9.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5394)
AUTHORS Natsume T, Kiyomitsu T, Saga Y, Kanemaki MT
TITLE Rapid Protein Depletion in Human Cells by Auxin-Inducible Degron
Tagging with Short Homology Donors.
JOURNAL Cell Rep. 2016 Apr 5;15(1):210-8. doi: 10.1016/j.celrep.2016.03.001.
Epub 2016 Mar 24.
PUBMED 27052166
REFERENCE 2 (bases 1 to 5394)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Sep 4, 2026 from SnapGene Server 8.0.1
https://www.biofeng.com
COMMENT SGRef: number: 1; type: "Journal Article"; doi:
"10.1016/j.celrep.2016.03.001"; journalName: "Cell Rep"; date:
"2016-04-5- 5"; volume: "15"; issue: "1"; pages: "210-8"
COMMENT Sequence Label: pMK286 (mAID-NeoR)
FEATURES Location/Qualifiers
source 1..5394
/mol_type="other DNA"
/organism="synthetic DNA construct"
enhancer 11..202
/label=SV40 enhancer
/note="enhancer for the SV40 early promoter (Herr, 1993)"
primer_bind 159..179
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
promoter complement(204..308)
/gene="bla"
/label=AmpR promoter
rep_origin 335..790
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
polyA_signal 797..918
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(808..827)
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind 862..881
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
CDS complement(1039..1242)
/codon_start=1
/product="minimal degron from the auxin-responsive IAA17
protein of Arabidopsis thaliana (Kubota et al., 2013)"
/label=mini-AID
/note="acts as an auxin-inducible degron (AID) in the
presence of TIR1"
/translation="KEKSACPKDPAKPPAKAQVVGWPPVRSYRKNVMVSCQKSSGGPEA
AAFVKVSMDGAPYLRKIDLRMYK"
primer_bind complement(1278..1297)
/label=T7
/note="T7 promoter, forward primer"
promoter complement(1279..1297)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(1307..1324)
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(1307..1323)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(1316..1338)
/label=M13/pUC Forward
/note="In lacZ gene"
rep_origin complement(1464..1919)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 1946..2050
/gene="bla"
/label=AmpR promoter
CDS join(2051..2119,2120..2911)
/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(2269..2288)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 3082..3670
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 3571..3590
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 3824..3841
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 3958..3979
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter join(3994..3999,4000..4017,4018..4024)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 4032..4048
/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)."
primer_bind 4037..4059
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 4056..4072
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 4056..4072
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind 4091..4111
/label=T3
/note="T3 promoter, forward primer"
promoter 4093..4111
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind 4203..4222
/label=TK-pA-R
/note="Thymidine kinase polyA, reverse primer"
CDS complement(4410..5204)
/codon_start=1
/product="aminoglycoside phosphotransferase"
/label=NeoR/KanR
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
CDS complement(4410..5204)
/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(4521..4540)
/label=Neo-F
/note="Neomycin resistance gene, forward primer"
primer_bind 5131..5150
/label=Neo-R
/note="Neomycin resistance gene, reverse primer"
promoter complement(join(5239..5394,1..202))
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 5253..5388
/label=SV40 ori
/note="SV40 origin of replication"
ORIGIN
1 agttaggggc gggactatgg ttgctgacta attgagatgc atgctttgca tacttctgcc
61 tgctggggag cctggggact ttccacacct ggttgctgac taattgagat gcatgctttg
121 catacttctg cctgctgggg agcctgggga ctttccacac cctaactgac acacattcca
181 cagctggttc tttccgcctc aggactcttc ctttttcaat attattgaag catttatcag
241 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
301 gttccgcgca catttccccg aaaagtgcca cctgacgcgc cctgtagcgg cgcattaagc
361 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
421 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
481 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
541 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
601 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
661 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
721 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
781 cttacaattt acgcgttaag atacattgat gagtttggac aaaccacaac tagaatgcag
841 tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata
901 agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg
961 gaggtgtggg aggtttttta aagcaagtaa aacctctaca aatgtggtat ggctgattat
1021 gatcagttat ctagattatt tatacatcct caaatcgatt ttcctcaagt acggtgctcc
1081 gtccattgat accttcacga acgccgccgc ctccgggcca ccgcttgatt tttggcagga
1141 aaccatcacg ttcttccggt atgatctcac cggtggccat cccacaactt gtgccttggc
1201 cggaggtttg gctggatctt taggacaagc actcttctcc ttggcgcctg caccggatcc
1261 cggggagctc caattcgccc tatagtgagt cgtattacgc gcgctcactg gccgtcgttt
1321 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc
1381 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt
1441 tgcgcagcct gaatggcgaa tggaaattgt aagcgttaat attttgttaa aattcgcgtt
1501 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
1561 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
1621 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
1681 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
1741 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
1801 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
1861 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
1921 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
1981 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
2041 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
2101 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
2161 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
2221 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
2281 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
2341 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
2401 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
2461 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
2521 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
2581 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
2641 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
2701 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
2761 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
2821 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
2881 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
2941 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
3001 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
3061 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
3121 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
3181 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
3241 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
3301 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
3361 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
3421 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
3481 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
3541 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
3601 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
3661 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
3721 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
3781 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
3841 ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
3901 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
3961 tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat
4021 gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta
4081 cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag ctgggtaccg gatccaacga
4141 cccaacaccg tgcgttttat tctgtctttt tattgccgtc atagcgcggg ttccttccgg
4201 tattgtctcc ttccgtgttt cagttagcct ccccctaggg tgggcgaaga actccagcat
4261 gagatccccg cgctggagga tcatccagcc ggcgtcccgg aaaacgattc cgaagcccaa
4321 cctttcatag aaggcggcgg tggaatcgaa atctcgtgat ggcaggttgg gcgtcgcttg
4381 gtcggtcatt tcgaacccca gagtcccgct cagaagaact cgtcaagaag gcgatagaag
4441 gcgatgcgct gcgaatcggg agcggcgata ccgtaaagca cgaggaagcg gtcagcccat
4501 tcgccgccaa gctcttcagc aatatcacgg gtagccaacg ctatgtcctg atagcggtcc
4561 gccacaccca gccggccaca gtcgatgaat ccagaaaagc ggccattttc caccatgata
4621 ttcggcaagc aggcatcgcc atgggtcacg acgagatcct cgccgtcggg catgctcgcc
4681 ttgagcctgg cgaacagttc ggctggcgcg agcccctgat gctcttcgtc cagatcatcc
4741 tgatcgacaa gaccggcttc catccgagta cgtgctcgct cgatgcgatg tttcgcttgg
4801 tggtcgaatg ggcaggtagc cggatcaagc gtatgcagcc gccgcattgc atcagccatg
4861 atggatactt tctcggcagg agcaaggtga gatgacagga gatcctgccc cggcacttcg
4921 cccaatagca gccagtccct tcccgcttca gtgacaacgt cgagcacagc tgcgcaagga
4981 acgcccgtcg tggccagcca cgatagccgc gctgcctcgt cttgcagttc attcagggca
5041 ccggacaggt cggtcttgac aaaaagaacc gggcgcccct gcgctgacag ccggaacacg
5101 gcggcatcag agcagccgat tgtctgttgt gcccagtcat agccgaatag cctctccacc
5161 caagcggccg gagaacctgc gtgcaatcca tcttgttcaa tcatgcgaaa cgatcctcat
5221 cctgtctctt gatcgatctt tgcaaaagcc taggcctcca aaaaagcctc ctcactactt
5281 ctggaatagc tcagaggccg aggcggcctc ggcctctgca taaataaaaa aaattagtca
5341 gccatggggc ggagaatggg cggaactggg cggagttagg ggcgggatgg gcgg
//
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