| 质粒名称: | pMK288 (mAID-Bsr) |
|---|---|
| 出品公司： | addgene |
| 目录编号: | 72826 |
| 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: | 5051 bp |
| 载体类型: | 哺乳细胞表达 |
| 筛选标记: | Blasticidin |
| 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: | AID-Bsr |
| 物种来源: | H. sapiens (human), M. musculus (mouse), R. norvegicus (rat), G. gallus (chicken |
| 插入大小: | 2176 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 (未破坏) |
| 3’克隆位点: | BamHI (未破坏) |
| 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: | pMK288 (mAID-Bsr) was a gift from Masato Kanemaki (Addgene plasmid # 72826 ; http://n2t.net/addgene:72826 ; RRID:Addgene_72826) |
| 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 270 |
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
质粒图谱.png)
载体简介
载体序列
LOCUS pMK288 (mAID-Bsr) 5051 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION C-terminal tagging with mAID using CRISPR/Cas9.
ACCESSION .
VERSION .
KEYWORDS pMK288 (mAID-Bsr)
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5051)
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 5051)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..5051
/organism="synthetic DNA construct"
/mol_type="other DNA"
rep_origin 4..459
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(91..110)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 301..322
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind 585..607
/label=M13/pUC Forward
/note="In lacZ gene"
primer_bind 599..616
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind 600..616
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 626..645
/label=T7
/note="T7 promoter, forward primer"
promoter 626..644
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 681..884
/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"
polyA_signal 1005..1126
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(1042..1061)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 1096..1115
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
protein_bind complement(1157..1190)
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
promoter 1251..1750
/label=PGK promoter
/note="mouse phosphoglycerate kinase 1 promoter"
primer_bind complement(1286..1309)
/label=MSCV-rev
/note="Murine stem cell virus, reverse primer"
primer_bind 1683..1702
/label=mPGK-F
/note="Mouse PGK promoter, forward primer"
protein_bind complement(2781..2814)
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
primer_bind complement(2863..2883)
/label=T3
/note="T3 promoter, forward primer"
promoter complement(2863..2881)
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind complement(2902..2918)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(2902..2918)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(2915..2937)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 2926..2942
/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(2950..2980)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 2995..3016
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(3133..3150)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(3304..3892)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(3384..3403)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(4063..4923)
/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 4686..4705
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(4924..5028)
/gene="bla"
/label=AmpR promoter
ORIGIN
1 ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
61 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
121 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
181 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
241 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
301 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
361 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
421 ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc cattcgccat tcaggctgcg
481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggagctccc
661 cgggatccgg tgcaggcgcc aaggagaaga gtgcttgtcc taaagatcca gccaaacctc
721 cggccaaggc acaagttgtg ggatggccac cggtgagatc ataccggaag aacgtgatgg
781 tttcctgcca aaaatcaagc ggtggcccgg aggcggcggc gttcgtgaag gtatcaatgg
841 acggagcacc gtacttgagg aaaatcgatt tgaggatgta taaataatct agataactga
901 tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc
961 tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag
1021 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
1081 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttaacgc gtcgatcata
1141 ttcaataacc cttaatataa cttcgtataa tgtatgctat acgaagttat taggtctgaa
1201 gaggagttta cgtccagcca agcttaggat ctcgacctcg aaattctacc gggtagggga
1261 ggcgcttttc ccaaggcagt ctggagcatg cgctttagca gccccgctgg gcacttggcg
1321 ctacacaagt ggcctctggc ctcgcacaca ttccacatcc accggtaggc gccaaccgac
1381 tccgttcttt ggtggcccct tcgcgccacc ttctactcct cccctagtca ggaagttccc
1441 ccccgccccg cagctcgcgt cgtgcaggac gtgacaaatg gaagtagcac gtctcactag
1501 tctcgtgcag atggacagca ccgctgagca atggaagcgg gtaggccttt ggggcagcgg
1561 ccaatagcag ctttgctcct tcgctttctg ggctcagagg ctgggaaggg gtgggtccgg
1621 gggcgggctc aggggcgggc tcaggggcgg ggcgggcgcc cgaaggtcct ccggaggccc
1681 ggcattctgc acgcttcaaa agcgcacgtc tgccgcgctg ttctcctctt cctcatctcc
1741 gggcctttcg acctgcatcc atctagatct cgagcagctg aagcttaccg ctagcatgaa
1801 aacatttaac atttctcaac aagatctaga attagtagaa gtagcgacag agaagattac
1861 aatgctttat gaggataata aacatcatgt gggagcggca attcgtacga aaacaggaga
1921 aatcatttcg gcagtacata ttgaagcgta tataggacga gtaactgttt gtgcagaagc
1981 cattgcgatt ggtagtgcag tttcgaatgg acaaaaggat tttgacacga ttgtagctgt
2041 tagacaccct tattctgacg aagtagatag aagtattcga gtggtaagtc cttgtggtat
2101 gtgtagggag ttgatttcag actatgcacc agattgtttt gtgttaatag aaatgaatgg
2161 caagttagtc aaaactacga ttgaagaact cattccactc aaatataccc gaaattaagc
2221 tagccgcccg ccccacgacc cgcagcgccc gaccgaaagg agcgcacgac cccatgcatc
2281 gatgatatca gatccccggg atgcagaaat tgatgatcta ttaaacaata aagatgtcca
2341 ctaaaatgga agtttttcct gtcatacttt gttaagaagg gtgagaacag agtacctaca
2401 ttttgaatgg aaggattgga gctacggggg tgggggtggg gtgggattag ataaatgcct
2461 gctctttact gaaggctctt tactattgct ttatgataat gtttcatagt tggatatcat
2521 aatttaaaca agcaaaacca aattaagggc cagctcattc ctcccactca tgatctatag
2581 atctatagat ctctcgtggg atcattgttt ttctcttgat tcccactttg tggttctaag
2641 tactgtggtt tccaaatgtg tcagtttcat agcctgaaga acgagatcag cagcctctgt
2701 tccacataca cttcattctc agtattgttt tgccaagttc taattccatc agaagctggt
2761 cgagatccgg aacccttaat ataacttcgt ataatgtatg ctatacgaag ttattaggtc
2821 cctcgaagag gttcactagg atccggtacc cagcttttgt tccctttagt gagggttaat
2881 tgcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
2941 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
3001 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
3061 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
3121 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
3181 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
3241 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
3301 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
3361 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
3421 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
3481 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
3541 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
3601 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
3661 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
3721 gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt
3781 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
3841 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
3901 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
3961 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
4021 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
4081 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
4141 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
4201 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
4261 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
4321 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
4381 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
4441 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
4501 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
4561 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
4621 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
4681 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
4741 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
4801 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
4861 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
4921 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
4981 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
5041 aaaagtgcca c
//
