| 质粒名称: | pRK5-HA-Ubiquitin-K63 |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 17606 |
| 存储实验室: | Ted Dawson |
|
载体骨架基本信息 |
|
| 载体名称: | pRK5-HA |
| 载体出品公司: | -- |
| 载体抗性: | 氨苄青霉素 |
| 空载体大小: | 4800 bp |
| 完整质粒大小: | -- |
| 载体修饰: | -- |
| 载体类型: | 哺乳细胞表达 |
| 筛选标记: | -- |
| 高拷贝/低拷贝: | 高拷贝 |
| 生长条件: | 37 ℃ |
|
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: | 仅K63，其他赖氨酸突变为精氨酸。增强G76-K63连接的多泛素化。 |
| GenBank编号 | -- |
| 启动子 | -- |
| 标签/融合蛋白: | HA（主干上的N终端） |
| 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 |
| 3' 测序引物 | -- |
|
文献引用方法 |
|
| 材料和方法部分: | pRK5-HA-Ubiquitin-K63 was a gift from Ted Dawson (Addgene plasmid # 17606 ; http://n2t.net/addgene:17606 ; RRID:Addgene_17606) |
| 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-K63 4996 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION Mammalian expression of HA tagged ubiquitin with K63 only, other
lysines mutated to arginines.
ACCESSION .
VERSION .
KEYWORDS pRK5-HA-Ubiquitin-K63
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 Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..4996
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind 94..111
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 228..249
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 264..294
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 302..318
/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 307..329
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 326..342
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 326..342
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
enhancer 371..750
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 751..954
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 905..925
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
primer_bind 951..975
/label=LNCX
/note="Human CMV promoter, forward primer"
promoter 1185..1203
/label=SP6 promoter
/note="promoter for bacteriophage SP6 RNA polymerase"
primer_bind 1185..1202
/label=SP6
/note="SP6 promoter, forward primer"
primer_bind 1216..1235
/label=pMT2-F
/note="Synthetic intron, forward primer"
regulatory 1320..1329
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 1332..1358
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/label=HA
/translation="YPYDVPDYA"
CDS 1389..1616
/codon_start=1
/product="human ubiquitin variant containing Lys-63, with
all other lysines mutated to arginine"
/label=ubiquitin-K63
/translation="MQIFVRTLTGRTITLEVEPSDTIENVRARIQDREGIPPDQQRLIF
AGRQLEDGRTLSDYNIQKESTLHLVLRLRGG"
regulatory 1653..1662
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
polyA_signal 1666..1800
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(1703..1722)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 1757..1776
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
promoter 1869..2226
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
primer_bind complement(1892..1912)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
rep_origin 2077..2212
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 2139..2158
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
primer_bind complement(2246..2263)
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(2246..2262)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(2255..2277)
/label=M13/pUC Forward
/note="In lacZ gene"
rep_origin 2475..2930
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(2562..2581)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 2772..2793
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(2947..2966)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 3066..3088
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(3126..3144)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 3212..3316
/gene="bla"
/label=AmpR promoter
CDS 3317..4177
/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(3535..3554)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 4348..4936
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 4837..4856
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
ORIGIN
1 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
61 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
121 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg
181 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag
241 ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga
301 attgtgagcg gataacaatt tcacacagga aacagctatg acatgattac gaattaattc
361 gagctcgccc gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta
421 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
481 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
541 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
601 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
661 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
721 atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg
781 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg
841 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca
901 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta
961 gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagacac
1021 cgggaccgat ccagcctccg cggccgggaa cggtgcattg gaacgcggat tccccgtgcc
1081 aagagtgacg taagtaccgc ctatagagtc tataggccca cccccttggc ttcgttagaa
1141 cgcggctaca attaatacat aaccttatgt atcatacaca tacgatttag gtgacactat
1201 agaataacat ccactttgcc tttctctcca caggtgtcca ctcccaggtc caactgcacc
1261 tcggttctat cgattgaatt cccgaaccga cagtcggtct cttcaccaag gccattcgcg
1321 ccaccatggg ctacccctat gatgtgcctg actacgcaga tctcaatggt ggtggtggtg
1381 ggtcgaccat gcagatcttc gtcagaacgt taaccggtag aaccataact ctagaagttg
1441 aaccatccga taccatcgaa aacgttagag ctagaattca agacagagaa ggcattccac
1501 ctgatcaaca aagattgatc tttgccggta gacagctcga ggacggtaga acgctgtctg
1561 attacaacat tcagaaggag tccaccctgc acctggtcct ccgtctcaga ggtggttgag
1621 cggccgctaa gtaagtaagg atccccagct tggccgccat ggcccaactt gtttattgca
1681 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
1741 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc
1801 gggaattaat tcggcgcagc accatggcct gaaataacct ctgaaagagg aacttggtta
1861 ggtaccttct gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag
1921 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
1981 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
2041 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
2101 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
2161 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
2221 tgcaaaaagc tgttaacagc ttggcactgg ccgtcgtttt acaacgtcgt gactgggaaa
2281 accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta
2341 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat
2401 ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc
2461 aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
2521 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
2581 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
2641 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg
2701 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
2761 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcgggcta
2821 ttcttttaat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
2881 ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac
2941 tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc
3001 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
3061 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg
3121 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta
3181 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
3241 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
3301 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
3361 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
3421 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
3481 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
3541 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
3601 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
3661 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
3721 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
3781 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
3841 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
3901 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
3961 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
4021 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
4081 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
4141 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
4201 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
4261 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
4321 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
4381 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
4441 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
4501 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
4561 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
4621 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
4681 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
4741 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
4801 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
4861 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
4921 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
4981 gccttttgct cacatg
//
