| 质粒名称: | pRK5-HA-Ubiquitin-K48 |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 17605 |
| 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K48，其他赖氨酸突变为精氨酸。增强蛋白质的G76-K48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-K48 was a gift from Ted Dawson (Addgene plasmid # 17605 ; http://n2t.net/addgene:17605 ; RRID:Addgene_17605) |
| 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-K48 4996 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION Mammalian expression of HA tagged ubiquitin with only K48, other
lysines mutated to arginines.
ACCESSION .
VERSION .
KEYWORDS pRK5-HA-Ubiquitin-K48
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"
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"
CDS 1032..1259
/codon_start=1
/product="human ubiquitin variant containing Lys-48, with
all other lysines mutated to arginine"
/label=ubiquitin-K48
/translation="MQIFVRTLTGRTITLEVEPSDTIENVRARIQDREGIPPDQQRLIF
AGKQLEDGRTLSDYNIQRESTLHLVLRLRGG"
regulatory 1296..1305
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
polyA_signal 1309..1443
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(1346..1365)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 1400..1419
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
promoter 1512..1869
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
primer_bind complement(1535..1555)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
rep_origin 1720..1855
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 1782..1801
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
primer_bind complement(1889..1906)
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(1889..1905)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(1898..1920)
/label=M13/pUC Forward
/note="In lacZ gene"
rep_origin 2118..2573
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(2205..2224)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 2415..2436
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(2590..2609)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 2709..2731
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(2769..2787)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 2855..2959
/gene="bla"
/label=AmpR promoter
CDS 2960..3820
/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(3178..3197)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 3991..4579
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 4480..4499
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 4733..4750
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 4867..4888
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 4903..4933
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 4941..4957
/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 4946..4968
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 4965..4981
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 4965..4981
/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 ttcgtcagaa cgttaaccgg tagaaccata actctagaag
1081 ttgaaccatc cgataccatc gaaaacgtta gagctagaat tcaagacaga gaaggcattc
1141 cacctgatca acaaagattg atctttgccg gtaagcagct cgaggacggt agaacgctgt
1201 ctgattacaa cattcagaga gagtccaccc tgcacctggt cctccgtctc agaggtggtt
1261 gagcggccgc taagtaagta aggatcccca gcttggccgc catggcccaa cttgtttatt
1321 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
1381 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg
1441 atcgggaatt aattcggcgc agcaccatgg cctgaaataa cctctgaaag aggaacttgg
1501 ttaggtacct tctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga
1561 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
1621 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
1681 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
1741 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
1801 gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
1861 ttttgcaaaa agctgttaac agcttggcac tggccgtcgt tttacaacgt cgtgactggg
1921 aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc
1981 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg
2041 aatggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac
2101 gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
2161 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
2221 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
2281 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga
2341 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
2401 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggg
2461 ctattctttt aatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
2521 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg
2581 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac
2641 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
2701 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
2761 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
2821 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
2881 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
2941 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
3001 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
3061 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
3121 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
3181 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
3241 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
3301 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
3361 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
3421 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
3481 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
3541 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
3601 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
3661 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
3721 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
3781 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
3841 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
3901 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
3961 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
4021 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
4081 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
4141 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
4201 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
4261 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
4321 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
4381 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
4441 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
4501 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
4561 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
4621 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
4681 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
4741 agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc
4801 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa
4861 cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc
4921 ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga
4981 catgattacg aattaa
//
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