| 质粒名称: | pRK5-A3-HA-ubiquitin |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 220145 |
| 存储实验室: | William Sellers |
|
载体骨架基本信息 |
|
| 载体名称: | pRK5 |
| 载体出品公司: | Clontech |
| 空载体大小: | 4800bp |
| 完整质粒大小: | 5020bp |
| 载体类型: | 哺乳细胞表达 |
| 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: | Ubiquitin C、UBC |
| 物种来源: | H. sapiens (human) |
| 启动子: | CMV |
| 标签/融合蛋白: | A3-HA (N terminal on insert) |
| 克隆方法: | Other |
| 5’测序引物 | CMV-F, SP6 |
|
文献引用方法 |
|
| 材料和方法部分: |
pRK5-A3-HA-ubiquitin was a gift from William Sellers (Addgene plasmid # 220145 ; http://n2t.net/addgene:220145 ; RRID:Addgene_220145)
|
| 参考文献部分: | Ubiquitin-specific proximity labeling for the identification of E3 ligase substrates. Huang HT, Lumpkin RJ, Tsai RW, Su S, Zhao X, Xiong Y, Chen J, Mageed N, Donovan KA, Fischer ES, Sellers WR. Nat Chem Biol. 2024 Mar 21. doi: 10.1038/s41589-024-01590-9. 10.1038/s41589-024-01590-9 PubMed 38514884 |
买家导航
pRK5-A3-HA-ubiquitin载体质粒基本信息
pRK5-A3-HA-ubiquitin质粒图谱载体图谱和pRK5-A3-HA-ubiquitin载体序列质粒序列多克隆位点信息
pRK5-A3-HA-ubiquitin质粒载体简介
pRK5-A3-HA-ubiquitin质粒序列载体序列
LOCUS pRK5-A3-HA-ubiquitin 5020 bp DNA circular SYN 14-APR-2025
DEFINITION Expresses A3-Ub for E3 substrate identification by E-STUB.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5020)
AUTHORS Huang HT, Lumpkin RJ, Tsai RW, Su S, Zhao X, Xiong Y, Chen J, Mageed
N, Donovan KA, Fischer ES, Sellers WR
TITLE Ubiquitin-specific proximity labeling for the identification of E3
ligase substrates.
JOURNAL Nat Chem Biol. 2024 Mar 21. doi: 10.1038/s41589-024-01590-9.
PUBMED 38514884
REFERENCE 2 (bases 1 to 5020)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Apr 14, 2025 from SnapGene Server 8.0.1
https://www.biofeng.com
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nat Chem
Biol. 2024 Mar 21. doi: 10.1038/s41589-024-01590-9."
COMMENT Sequence Label: pRK5-A3-HA-ubiquitin
FEATURES Location/Qualifiers
source 1..5020
/mol_type="other DNA"
/organism="synthetic DNA construct"
enhancer 1..380
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 381..584
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 535..555
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
primer_bind 581..605
/label=LNCX
/note="Human CMV promoter, forward primer"
promoter 815..833
/label=SP6 promoter
/note="promoter for bacteriophage SP6 RNA polymerase"
primer_bind 815..832
/label=SP6
/note="SP6 promoter, forward primer"
primer_bind 846..865
/label=pMT2-F
/note="Synthetic intron, forward primer"
regulatory 950..959
/label=Kozak sequence
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
/regulatory_class="other"
CDS 1010..1036
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/label=HA
/translation="YPYDVPDYA"
CDS 1043..1270
/codon_start=1
/product="human ubiquitin"
/label=ubiquitin
/translation="MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF
AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
CDS 1043..1270
/codon_start=1
/gene="Homo sapiens UBC"
/product="human ubiquitin C"
/label=ubiquitin
/translation="MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF
AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
regulatory 1307..1316
/label=Kozak sequence
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
/regulatory_class="other"
polyA_signal 1320..1454
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(1357..1376)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 1411..1430
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
promoter 1523..1880
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
enhancer 1523..1714
/label=SV40 enhancer
/note="enhancer for the SV40 early promoter (Herr, 1993)"
primer_bind complement(1546..1566)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
rep_origin 1731..1866
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind complement(1900..1917)
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(1900..1916)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(1909..1931)
/label=M13/pUC Forward
/note="In lacZ gene"
rep_origin 2129..2584
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(2216..2235)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 2426..2447
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(2601..2620)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 2720..2742
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(2780..2798)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 2866..2970
/gene="bla"
/label=AmpR promoter
CDS join(2971..3039,3040..3831)
/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(3189..3208)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 4002..4590
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 4491..4510
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 4744..4761
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 4878..4899
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter join(4914..4919,4920..4937,4938..4944)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 4952..4968
/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 4957..4979
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 4976..4992
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 4976..4992
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
ORIGIN
1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc
601 agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagacac cgggaccgat
661 ccagcctccg cggccgggaa cggtgcattg gaacgcggat tccccgtgcc aagagtgacg
721 taagtaccgc ctatagagtc tataggccca cccccttggc ttcgttagaa cgcggctaca
781 attaatacat aaccttatgt atcatacaca tacgatttag gtgacactat agaataacat
841 ccactttgcc tttctctcca caggtgtcca ctcccaggtc caactgcacc tcggttctat
901 cgattgaatt cccgaaccga cagtcggtct cttcaccaag gccattcgcg ccaccatggg
961 actgaacgac atcttcgagg ctcagaaaat cgaagggagc ggaggttcat atccgtacga
1021 tgtgccggac tatgcgggat ccatgcagat cttcgtgaag accctgactg gtaagaccat
1081 cactctcgaa gtggagccga gtgacaccat tgagaatgtc aaggcaaaga tccaagacaa
1141 ggaaggcatc cctcctgacc agcagaggtt gatctttgct gggaaacagc tggaagatgg
1201 acgcaccctg tctgactaca acatccagaa agagtccacc ctgcacctgg tcctccgtct
1261 cagaggtggt tgagcggccg ctaagtaagt aaggatcccc agcttggccg ccatggccca
1321 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
1381 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
1441 atcatgtctg gatcgggaat taattcggcg cagcaccatg gcctgaaata acctctgaaa
1501 gaggaacttg gttaggtacc ttctgaggcg gaaagaacca gctgtggaat gtgtgtcagt
1561 tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
1621 attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
1681 gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
1741 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
1801 cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg
1861 gaggcctagg cttttgcaaa aagctgttaa cagcttggca ctggccgtcg ttttacaacg
1921 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt
1981 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
2041 cctgaatggc gaatggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
2101 acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg
2161 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
2221 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
2281 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
2341 gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
2401 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
2461 cctatctcgg gctattcttt taatttataa gggattttgc cgatttcggc ctattggtta
2521 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca
2581 attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
2641 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
2701 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
2761 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
2821 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
2881 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
2941 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
3001 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
3061 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
3121 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
3181 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
3241 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
3301 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
3361 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
3421 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
3481 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
3541 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
3601 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
3661 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
3721 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
3781 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
3841 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
3901 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
3961 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
4021 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
4081 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
4141 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
4201 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
4261 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
4321 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
4381 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
4441 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
4501 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
4561 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
4621 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
4681 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
4741 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc
4801 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
4861 agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac
4921 tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga
4981 aacagctatg acatgattac gaattaattc gagctcgccc
//
