| 质粒名称: | pRK5-HA-Ubiquitin-KO |
|---|---|
| 出品公司： | addgene |
| 目录编号: | 17603 |
| 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: | 4800bp |
| 完整质粒大小: | 49996bp |
| 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: | KO: all lysines mutated to arginines |
| 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-KO was a gift from Ted Dawson (Addgene plasmid # 17603 ; http://n2t.net/addgene:17603 ; RRID:Addgene_17603) |
| 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-KO 4996 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Mammalian expression of HA tagged ubiquitin carrying zero lysines
(K0).
ACCESSION .
VERSION .
KEYWORDS pRK5-HA-Ubiquitin-KO
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 May 12, 2021 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 74..91
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 208..229
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 244..274
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 282..298
/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 287..309
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 306..322
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 306..322
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
enhancer 351..730
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 731..934
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 885..905
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
primer_bind 931..955
/label=LNCX
/note="Human CMV promoter, forward primer"
promoter 1165..1183
/label=SP6 promoter
/note="promoter for bacteriophage SP6 RNA polymerase"
primer_bind 1165..1182
/label=SP6
/note="SP6 promoter, forward primer"
primer_bind 1196..1215
/label=pMT2-F
/note="Synthetic intron, forward primer"
regulatory 1300..1309
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 1312..1338
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/label=HA
/translation="YPYDVPDYA"
CDS 1369..1596
/codon_start=1
/product="human ubiquitin variant in which all of the
lysines have been mutated to arginine"
/label=ubiquitin-K0
/note="also known as ubiquitin-KO"
/translation="MQIFVRTLTGRTITLEVEPSDTIENVRARIQDREGIPPDQQRLIF
AGRQLEDGRTLSDYNIQRESTLHLVLRLRGG"
regulatory 1633..1642
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
polyA_signal 1646..1780
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(1683..1702)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 1737..1756
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
promoter 1849..2206
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
primer_bind complement(1872..1892)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
rep_origin 2057..2192
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 2119..2138
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
primer_bind complement(2226..2243)
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(2226..2242)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(2235..2257)
/label=M13/pUC Forward
/note="In lacZ gene"
rep_origin 2455..2910
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(2542..2561)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 2752..2773
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(2927..2946)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 3046..3068
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(3106..3124)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 3192..3296
/gene="bla"
/label=AmpR promoter
CDS 3297..4157
/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(3515..3534)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 4328..4916
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 4817..4836
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
ORIGIN
1 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
61 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc
121 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
181 gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca
241 ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt
301 tcacacagga aacagctatg acatgattac gaattaattc gagctcgccc gacattgatt
361 attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga
421 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
481 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg
541 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
601 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
661 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
721 tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc
781 acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa
841 tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag
901 gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc agatcgcctg
961 gagacgccat ccacgctgtt ttgacctcca tagaagacac cgggaccgat ccagcctccg
1021 cggccgggaa cggtgcattg gaacgcggat tccccgtgcc aagagtgacg taagtaccgc
1081 ctatagagtc tataggccca cccccttggc ttcgttagaa cgcggctaca attaatacat
1141 aaccttatgt atcatacaca tacgatttag gtgacactat agaataacat ccactttgcc
1201 tttctctcca caggtgtcca ctcccaggtc caactgcacc tcggttctat cgattgaatt
1261 cccgaaccga cagtcggtct cttcaccaag gccattcgcg ccaccatggg ctacccctat
1321 gatgtgcctg actacgcaga tctcaatggt ggtggtggtg ggtcgaccat gcagatcttc
1381 gtcagaacgt taaccggtag aaccataact ctagaagttg aaccatccga taccatcgaa
1441 aacgttagag ctagaattca agacagagaa ggcattccac ctgatcaaca aagattgatc
1501 tttgccggta gacagctcga ggacggtaga acgctgtctg attacaacat tcagagagag
1561 tcgaccttac atcttgtctt aagactaaga ggtggttgag cggccgctaa gtaagtaagg
1621 atccccagct tggccgccat ggcccaactt gtttattgca gcttataatg gttacaaata
1681 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
1741 tttgtccaaa ctcatcaatg tatcttatca tgtctggatc gggaattaat tcggcgcagc
1801 accatggcct gaaataacct ctgaaagagg aacttggtta ggtaccttct gaggcggaaa
1861 gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg
1921 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg
1981 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc
2041 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
2101 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt
2161 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tgttaacagc
2221 ttggcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt
2281 aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc
2341 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt
2401 ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc
2461 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
2521 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
2581 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
2641 tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc
2701 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
2761 tgttccaaac tggaacaaca ctcaacccta tctcgggcta ttcttttaat ttataaggga
2821 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
2881 attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg
2941 atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
3001 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
3061 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc
3121 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc
3181 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
3241 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
3301 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
3361 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
3421 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
3481 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
3541 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
3601 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
3661 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
3721 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
3781 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
3841 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
3901 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
3961 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
4021 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
4081 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
4141 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
4201 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
4261 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
4321 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
4381 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
4441 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
4501 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
4561 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
4621 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
4681 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
4741 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
4801 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
4861 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
4921 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
4981 ttcctgcgtt atcccc
//
