| 质粒名称: | pRK5-HA-Ubiquitin-K48R |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 17604 |
| 存储实验室: | Ted Dawson |
|
载体骨架基本信息 |
|
| 载体名称: | pRK5-HA |
| 空载体大小: | 4800 bp |
| 完整质粒大小: | 4989 bp |
| 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 / Ub |
| 物种来源: | H. sapiens (human) |
| 插入大小: | 220 bp |
| 基因突变: | K48R (expected to disrupt G76-K48 ubiquitin chain assembly) |
| GenBank编号 | JN247409 |
| 启动子: | CMV |
| 标签/融合蛋白: | HA (N terminal on backbone) |
| 克隆方法: | 限制性内切酶 |
| 5’克隆位点: | SalI(未破坏) |
| 3’克隆位点: | NotI (未破坏) |
| 5’测序引物 | SP6 |
|
文献引用方法 |
|
| 材料和方法部分: |
pRK5-HA-Ubiquitin-K48R was a gift from Ted Dawson (Addgene plasmid # 17604 ; http://n2t.net/addgene:17604 ; RRID:Addgene_17604)
|
| 参考文献部分: | Parkin mediates nonclassical, proteasomal-independent ubiquitination of synphilin-1: implications for Lewy body formation. Lim KL, Chew KC, Tan JM, Wang C, Chung KK, Zhang Y, Tanaka Y, Smith W, Engelender S, Ross CA, Dawson VL, Dawson TM. J Neurosci. 2005 Feb 23. 25(8):2002-9. 10.1523/JNEUROSCI.4474-04.2005 PubMed 15728840 |
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 pRK5-HA-Ubiquitin-K48R 4996 bp ds-DNA circular SYN 18-JAN-2022
DEFINITION Mammalian expression of HA tagged ubiquitin K48R.
ACCESSION .
VERSION .
KEYWORDS pRK5-HA-Ubiquitin-K48R
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4996)
AUTHORS Lim KL, Chew KC, Tan JM, Wang C, Chung KK, Zhang Y, Tanaka Y, Smith
W, Engelender S, Ross CA, Dawson VL, Dawson TM
TITLE Parkin mediates nonclassical, proteasomal-independent ubiquitination
of synphilin-1: implications for Lewy body formation.
JOURNAL J Neurosci. 2005 Feb 23. 25(8):2002-9.
PUBMED 15728840
REFERENCE 2 (bases 1 to 4996)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Jan 18, 2022 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..4996
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind complement(2..21)
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
primer_bind 248..268
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
polyA_signal 360..494
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(384..403)
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind 438..457
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
regulatory 498..507
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS complement(544..771)
/codon_start=1
/product="human ubiquitin variant in which Lys-48 has been
mutated to arginine"
/label=ubiquitin-K48R
/translation="MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF
AGRQLEDGRTLSDYNIQKESTLHLVLRLRGG"
CDS complement(802..828)
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/label=HA
/translation="YPYDVPDYA"
regulatory 831..840
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
primer_bind complement(925..944)
/label=pMT2-F
/note="Synthetic intron, forward primer"
promoter complement(957..975)
/label=SP6 promoter
/note="promoter for bacteriophage SP6 RNA polymerase"
primer_bind complement(958..975)
/label=SP6
/note="SP6 promoter, forward primer"
primer_bind complement(1185..1209)
/label=LNCX
/note="Human CMV promoter, forward primer"
promoter complement(1206..1409)
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind complement(1235..1255)
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
enhancer 1410..1789
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
primer_bind complement(1818..1834)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(1818..1834)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(1831..1853)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 1842..1858
/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(1866..1896)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 1911..1932
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(2049..2066)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(2220..2808)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(2300..2319)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(2979..3839)
/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 3602..3621
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(3840..3944)
/gene="bla"
/label=AmpR promoter
primer_bind 4012..4030
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
primer_bind complement(4068..4090)
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind 4190..4209
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
rep_origin complement(4226..4681)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(4363..4384)
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind 4575..4594
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 4879..4901
/label=M13/pUC Forward
/note="In lacZ gene"
primer_bind 4893..4910
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind 4894..4910
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
promoter complement(4930..291)
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 4944..83
/label=SV40 ori
/note="SV40 origin of replication"
ORIGIN
1 gcctcggcct ctgcataaat aaaaaaaatt agtcagccat ggggcggaga atgggcggaa
61 ctgggcggag ttaggggcgg gatgggcgga gttaggggcg ggactatggt tgctgactaa
121 ttgagatgca tgctttgcat acttctgcct gctggggagc ctggggactt tccacacctg
181 gttgctgact aattgagatg catgctttgc atacttctgc ctgctgggga gcctggggac
241 tttccacacc ctaactgaca cacattccac agctggttct ttccgcctca gaaggtacct
301 aaccaagttc ctctttcaga ggttatttca ggccatggtg ctgcgccgaa ttaattcccg
361 atccagacat gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa
421 aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct
481 gcaataaaca agttgggcca tggcggccaa gctggggatc cttacttact tagcggccgc
541 tcaaccacct ctgagacgga ggaccaggtg cagggtggac tccttctgaa tgttgtaatc
601 agacagcgtt ctaccgtcct cgagctgcct accggcaaag atcaatcttt gttgatcagg
661 tggaatgcct tccttgtctt gaattttagc cttaacgttt tcgatggtat cggatggttc
721 aacttctaga gttatggttt taccggttaa cgtcttgacg aagatctgca tggtcgaccc
781 accaccacca ccattgagat ctgcgtagtc aggcacatca taggggtagc ccatggtggc
841 gcgaatggcc ttggtgaaga gaccgactgt cggttcggga attcaatcga tagaaccgag
901 gtgcagttgg acctgggagt ggacacctgt ggagagaaag gcaaagtgga tgttattcta
961 tagtgtcacc taaatcgtat gtgtatgata cataaggtta tgtattaatt gtagccgcgt
1021 tctaacgaag ccaagggggt gggcctatag actctatagg cggtacttac gtcactcttg
1081 gcacggggaa tccgcgttcc aatgcaccgt tcccggccgc ggaggctgga tcggtcccgg
1141 tgtcttctat ggaggtcaaa acagcgtgga tggcgtctcc aggcgatctg acggttcact
1201 aaacgagctc tgcttatata gacctcccac cgtacacgcc taccgcccat ttgcgtcaat
1261 ggggcggagt tgttacgaca ttttggaaag tcccgttgat tttggtgcca aaacaaactc
1321 ccattgacgt caatggggtg gagacttgga aatccccgtg agtcaaaccg ctatccacgc
1381 ccattgatgt actgccaaaa ccgcatcacc atggtaatag cgatgactaa tacgtagatg
1441 tactgccaag taggaaagtc ccataaggtc atgtactggg cataatgcca ggcgggccat
1501 ttaccgtcat tgacgtcaat agggggcgta cttggcatat gatacacttg atgtactgcc
1561 aagtgggcag tttaccgtaa atactccacc cattgacgtc aatggaaagt ccctattggc
1621 gttactatgg gaacatacgt cattattgac gtcaatgggc gggggtcgtt gggcggtcag
1681 ccaggcgggc catttaccgt aagttatgta acgcggaact ccatatatgg gctatgaact
1741 aatgaccccg taattgatta ctattaataa ctagtcaata atcaatgtcg ggcgagctcg
1801 aattaattcg taatcatgtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
1861 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
1921 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
1981 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
2041 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
2101 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
2161 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
2221 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
2281 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
2341 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
2401 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
2461 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
2521 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
2581 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
2641 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
2701 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
2761 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
2821 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
2881 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
2941 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
3001 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
3061 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
3121 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
3181 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
3241 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
3301 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
3361 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
3421 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
3481 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
3541 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
3601 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
3661 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
3721 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
3781 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
3841 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
3901 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
3961 gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt
4021 atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg
4081 cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt
4141 cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag
4201 cagattgtac tgagagtgca ccataaaatt gtaaacgtta atattttgtt aaaattcgcg
4261 ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct
4321 tataaattaa aagaatagcc cgagataggg ttgagtgttg ttccagtttg gaacaagagt
4381 ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat
4441 ggcccactac gtgaaccatc acccaaatca agttttttgg ggtcgaggtg ccgtaaagca
4501 ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac
4561 gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta
4621 gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg
4681 tactatggtt gctttgacgt atgcggtgtg aaataccgca cagatgcgta aggagaaaat
4741 accgcatcag gcgccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc
4801 gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt
4861 gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt gccaagctgt
4921 taacagcttt ttgcaaaagc ctaggcctcc aaaaaagcct cctcactact tctggaatag
4981 ctcagaggcc gaggcg
//
