| 质粒名称: | pRK5-HA-Ubiquitin-K29R |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 22900 |
| 存储实验室: | Sandra Weller |
|
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: | 4993 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: | K29R; V5 deleted |
| 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-K29R was a gift from Ted Dawson (Addgene plasmid # 17602 ; http://n2t.net/addgene:17602 ; RRID:Addgene_17602)
|
| 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-K29R 4993 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Mammalian expression of HA tagged ubiquitin K29R.
ACCESSION .
VERSION .
KEYWORDS pRK5-HA-Ubiquitin-K29R
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4993)
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 4993)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..4993
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 14..393
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 394..597
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 548..568
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
primer_bind 594..618
/label=LNCX
/note="Human CMV promoter, forward primer"
promoter 828..846
/label=SP6 promoter
/note="promoter for bacteriophage SP6 RNA polymerase"
primer_bind 828..845
/label=SP6
/note="SP6 promoter, forward primer"
primer_bind 859..878
/label=pMT2-F
/note="Synthetic intron, forward primer"
regulatory 963..972
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 975..1001
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/label=HA
/translation="YPYDVPDYA"
regulatory 1293..1302
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
polyA_signal 1306..1440
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(1343..1362)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 1397..1416
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
promoter 1509..1866
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
primer_bind complement(1532..1552)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
rep_origin 1717..1852
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 1779..1798
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
primer_bind complement(1886..1903)
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(1886..1902)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(1895..1917)
/label=M13/pUC Forward
/note="In lacZ gene"
rep_origin 2115..2570
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(2202..2221)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 2412..2433
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(2587..2606)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 2706..2728
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(2766..2784)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 2852..2956
/gene="bla"
/label=AmpR promoter
CDS 2957..3817
/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(3175..3194)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 3988..4576
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 4477..4496
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 4730..4747
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 4864..4885
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 4900..4930
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 4938..4954
/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 4943..4965
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 4962..4978
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 4962..4978
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
ORIGIN
1 ttcgagctcg cccgacattg attattgact agttattaat agtaatcaat tacggggtca
61 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
121 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
181 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
241 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
301 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
361 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
421 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
481 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
541 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt
601 ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga
661 caccgggacc gatccagcct ccgcggccgg gaacggtgca ttggaacgcg gattccccgt
721 gccaagagtg acgtaagtac cgcctataga gtctataggc ccaccccctt ggcttcgtta
781 gaacgcggct acaattaata cataacctta tgtatcatac acatacgatt taggtgacac
841 tatagaataa catccacttt gcctttctct ccacaggtgt ccactcccag gtccaactgc
901 acctcggttc tatcgattga attcccgaac cgacagtcgg tctcttcacc aaggccattc
961 gcgccaccat gggctacccc tatgatgtgc ctgactacgc agatctcaat ggtggtggtg
1021 gtgggtcgac catgcagatc ttcaagacgt taaccggtaa aaccataact ctagaagttg
1081 aaccatccga taccatcgaa aacgttaagg ctaggattca agacaaggaa ggcattccac
1141 ctgatcaaca aagattgatc tttgccggta agcagctcga ggacggtaga acgctgtctg
1201 attacaacat tcagaaggag tccaccctgc acctggtcct ccgtctcaga ggtggttgag
1261 cggccgctaa gtaagtaagg atccccagct tggccgccat ggcccaactt gtttattgca
1321 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
1381 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc
1441 gggaattaat tcggcgcagc accatggcct gaaataacct ctgaaagagg aacttggtta
1501 ggtaccttct gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag
1561 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
1621 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
1681 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
1741 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
1801 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
1861 tgcaaaaagc tgttaacagc ttggcactgg ccgtcgtttt acaacgtcgt gactgggaaa
1921 accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta
1981 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat
2041 ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc
2101 aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
2161 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
2221 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
2281 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg
2341 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
2401 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcgggcta
2461 ttcttttaat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
2521 ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac
2581 tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc
2641 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
2701 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg
2761 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta
2821 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
2881 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
2941 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
3001 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
3061 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
3121 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
3181 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
3241 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
3301 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
3361 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
3421 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
3481 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
3541 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
3601 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
3661 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
3721 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
3781 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
3841 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
3901 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
3961 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
4021 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
4081 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
4141 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
4201 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
4261 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
4321 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
4381 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
4441 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
4501 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
4561 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
4621 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
4681 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
4741 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
4801 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
4861 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc
4921 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacat
4981 gattacgaat taa
//
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