| 质粒名称: | pEF1a-Usp9x_CD-mCherry |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 170380 |
| 存储实验室: | Miguel Ramalho-Santos |
|
载体骨架基本信息 |
|
| 载体名称: | pEF1a-IRES-Neo (Plasmid #28019) |
| 载体出品公司: | -- |
| 空载体大小: | -- |
| 完整质粒大小: | 7613 bp |
| 载体修饰: | -- |
| 载体类型: | 哺乳动物表达 |
| 筛选标记: | 新霉素（G418筛选） |
| 载体抗性: | 氨苄青霉素，100μg/mL |
| 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: | 泛素特异性蛋白酶9x催化结构域 |
| 物种来源: | M.musculus（小鼠） |
| 插入大小: | 2007 bp |
| 基因突变: | Catalytic domain |
| GenBank编号 | NM_009481.2 |
| 启动子: | -- |
| 标签/融合蛋白: | mCherry-3xFlag（插入件上的C端子） |
| 克隆方法: | Gibson Cloning |
| 5’克隆位点: | -- |
| 3’克隆位点: | -- |
| 5’测序引物 | taatacgactcactatagg |
| 3’测序引物 | -- |
|
文献引用方法 |
|
| 材料和方法部分: | pEF1a-Usp9x_CD-mCherry was a gift from Miguel Ramalho-Santos (Addgene plasmid # 170380 ; http://n2t.net/addgene:170380 ; RRID:Addgene_170380) |
| 参考文献部分: | The deubiquitinase Usp9x regulates PRC2-mediated chromatin reprogramming during mouse development. Macrae TA, Ramalho-Santos M. Nat Commun. 2021 Mar 25;12(1):1865. doi: 10.1038/s41467-021-21910-0. 10.1038/s41467-021-21910-0 PubMed 33767158 |
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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 pEF1a-Usp9x_CD-mCherry 7613 bp ds-DNA circular SYN 17-JUN-2021
DEFINITION Vector for mammalian expression of the Usp9x catalytic domain tagged
with mCherry, in pEF1a-IRES-Neo backbone..
ACCESSION .
VERSION .
KEYWORDS pEF1a-Usp9x_CD-mCherry
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7613)
AUTHORS Macrae TA, Ramalho-Santos M
TITLE The deubiquitinase Usp9x regulates PRC2-mediated chromatin
reprogramming during mouse development.
JOURNAL Nat Commun. 2021 Mar 25;12(1):1865. doi: 10.1038/s41467-021-21910-0.
PUBMED 33767158
REFERENCE 2 (bases 1 to 7613)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Jun 17, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..7613
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 1..1163
/label=EF-1-alpha promoter
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
intron 232..1154
/label=EF-1-alpha intron A
/note="intron upstream of the start codon of human
EF-1-alpha"
primer_bind 1111..1131
/label=EF1a-F
/note="Human elongation factor-1a promoter, forward primer"
intron 1202..1334
/label=chimeric intron
/note="chimera between introns from human beta-globin and
immunoglobulin heavy chain genes"
primer_bind 1317..1336
/label=pMT2-F
/note="Synthetic intron, forward primer"
promoter 1379..1397
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 2639..3346
/codon_start=1
/product="monomeric derivative of DsRed fluorescent protein
(Shaner et al., 2004)"
/label=mCherry
/note="mammalian codon-optimized"
/translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
EGRHSTGGMDELYK"
primer_bind complement(2784..2802)
/label=mCherry-R
/note="mCherry, reverse primer"
primer_bind 3052..3071
/label=mCherry-F
/note="mCherry, forward primer"
CDS 3347..3412
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=3xFLAG
/translation="DYKDHDGDYKDHDIDYKDDDDK"
misc_feature 3454..4006
/label=IRES
/note="internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)"
primer_bind complement(3598..3615)
/label=IRES reverse
/note="IRES internal ribosome entry site, reverse primer.
Also called pCDH-rev"
primer_bind 3825..3844
/label=IRES-F
/note="IRES internal ribosome entry site, forward primer"
CDS 4006..4803
/codon_start=1
/gene="aph(3')-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/label=NeoR/KanR
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MGIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR
PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLL
SSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEH
QGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDI
ALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
primer_bind complement(4063..4082)
/label=Neo-R
/note="Neomycin resistance gene, reverse primer"
primer_bind 4673..4692
/label=Neo-F
/note="Neomycin resistance gene, forward primer"
polyA_signal 4867..4915
/note="synthetic polyadenylation signal"
rep_origin 5010..5465
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(5097..5116)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 5307..5328
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(5532..5551)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 5651..5673
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(5711..5729)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 5797..5901
/gene="bla"
/label=AmpR promoter
CDS 5902..6762
/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(6120..6139)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 6933..7521
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 7422..7441
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
ORIGIN
1 ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg
61 ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt
121 gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca
181 gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc
241 gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt
301 acttccacct ggctccagta cgtgattctt gatcccgagc tggagccagg ggcgggcctt
361 gcgctttagg agccccttcg cctcgtgctt gagttgaggc ctggcctggg cgctggggcc
421 gccgcgtgcg aatctggtgg caccttcgcg cctgtctcgc tgctttcgat aagtctctag
481 ccatttaaaa tttttgatga cctgctgcga cgcttttttt ctggcaagat agtcttgtaa
541 atgcgggcca ggatctgcac actggtattt cggtttttgg ggccgcgggc ggcgacgggg
601 cccgtgcgtc ccagcgcaca tgttcggcga ggcggggcct gcgagcgcgg ccaccgagaa
661 tcggacgggg gtagtctcaa gctggccggc ctgctctggt gcctggcctc gcgccgccgt
721 gtatcgcccc gccctgggcg gcaaggctgg cccggtcggc accagttgcg tgagcggaaa
781 gatggccgct tcccggccct gctccagggg gctcaaaatg gaggacgcgg cgctcgggag
841 agcgggcggg tgagtcaccc acacaaagga aaggggcctt tccgtcctca gccgtcgctt
901 catgtgactc cacggagtac cgggcgccgt ccaggcacct cgattagttc tggagctttt
961 ggagtacgtc gtctttaggt tggggggagg ggttttatgc gatggagttt ccccacactg
1021 agtgggtgga gactgaagtt aggccagctt ggcacttgat gtaattctcc ttggaatttg
1081 ccctttttga gtttggatct tggttcattc tcaagcctca gacagtggtt caaagttttt
1141 ttcttccatt tcaggtgtcg tgaacacggg ctgcagaagt tggtcgtgag gcactgggca
1201 ggtaagtatc aaggttacaa gacaggttta aggagaccaa tagaaactgg gcttgtcgag
1261 acagagaaga ctcttgcgtt tctgataggc acctattggt cttactgaca tccactttgc
1321 ctttctctcc acaggtgtcc actcccagtt caattacagc tcttaaggct agagtactta
1381 atacgactca ctataggcta gcctcgagat gaaaggattt gtggggctga aaaatgctgg
1441 tgctacttgt tacatgaact ctgtgatcca gcaactctat atgatcccct ctatcaggaa
1501 tggtattctt gccatagaag gcacaggtag tgatgtagat gatgatatgt ctggggatga
1561 gaagcaggac aacgagagca atgttgatcc cagggatgat gtgtttggat atcctcaaca
1621 atttgaagac aaaccaccac tgagtaaaac agaagatagg aaagagtaca atattggtgt
1681 tctaagacac cttcaggtta tctttggcca tttagctgct tctcgactac agtactatgt
1741 gcctagagga ttttggaaac agttcaggct ttggggtgag cctgtcaatc ttcgtgaaca
1801 acatgatgct ttagagttct ttaattcttt ggtggatagt ttagatgaag ctttaaaagc
1861 cttagggcat ccagctatgc taagtaaagt cttgggaggt tcctttgctg atcagaaaat
1921 ttgccaaggc tgcccacata ggtacgaatg tgaagaatct tttacgactc tgaatgtaga
1981 cattagaaac caccaaaatc ttctggattc tttggaacag tatgtcaaag gagacttact
2041 agaaggagca aatgcttatc attgtgaaaa atgcaataaa aaggttgata ctgtaaagcg
2101 cttgctaatt aaaaaattgc ctcctgttct tgctatccaa ctaaaacgat tcgattatga
2161 ctgggaaaga gaatgtgcaa tcaagttcaa tgattacttt gaatttcctc gagagttaga
2221 catggaacct tacacagtgg caggtgttgc taaactggag ggagataatg tcaacccaga
2281 aagtcagctg atacaacaga atgagcagtc tgaaagcgag aaagcaggaa gcacaaaata
2341 cagacttgtg ggtgtgcttg tgcacagtgg tcaagcaagt ggcggacatt actattctta
2401 catcattcag aggaatggag gggatggtga aaaaaatcgt tggtataaat ttgatgatgg
2461 agatgtaacg gaatgtaaaa tggatgacga tgaagaaatg aaaaaccagt gttttggggg
2521 agagtacatg ggagaagtat ttgatcacat gatgaagcgc atgtcgtaca ggcgtcagaa
2581 aagatggtgg aatgcatata tactttttta tgaacgaatg gacggtggat caggcggtat
2641 ggtgagcaag ggcgaggagg ataacatggc catcatcaag gagttcatgc gcttcaaggt
2701 gcacatggag ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg
2761 cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggtggcc ccctgccctt
2821 cgcctgggac atcctgtccc ctcagttcat gtacggctcc aaggcctacg tgaagcaccc
2881 cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt
2941 gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg
3001 cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg gccccgtaat
3061 gcagaagaag accatgggct gggaggcctc ctccgagcgg atgtaccccg aggacggcgc
3121 cctgaagggc gagatcaagc agaggctgaa gctgaaggac ggcggccact acgacgctga
3181 ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa
3241 catcaagttg gacatcacct cccacaacga ggactacacc atcgtggaac agtacgaacg
3301 cgccgagggc cgccactcca ccggcggcat ggacgagctg tacaaggact acaaagacca
3361 tgacggtgat tataaagatc atgacatcga ctacaaggat gacgatgaca agtgaggcgg
3421 ccgctctagc ccaattccgc cccccccccc ctaacgttac tggccgaagc cgcttggaat
3481 aaggccggtg tgcgtttgtc tatatgttat tttccaccat attgccgtct tttggcaatg
3541 tgagggcccg gaaacctggc cctgtcttct tgacgagcat tcctaggggt ctttcccctc
3601 tcgccaaagg aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct ctggaagctt
3661 cttgaagaca aacaacgtct gtagcgaccc tttgcaggca gcggaacccc ccacctggcg
3721 acaggtgcct ctgcggccaa aagccacgtg tataagatac acctgcaaag gcggcacaac
3781 cccagtgcca cgttgtgagt tggatagttg tggaaagagt caaatggctc tcctcaagcg
3841 tattcaacaa ggggctgaag gatgcccaga aggtacccca ttgtatggga tctgatctgg
3901 ggcctcggtg cacatgcttt acatgtgttt agtcgaggtt aaaaaaacgt ctaggccccc
3961 cgaaccacgg ggacgtggtt ttcctttgaa aaacacgatg ataatatggg cattgaacaa
4021 gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg
4081 gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc
4141 ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca
4201 gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc
4261 actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca
4321 tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat
4381 acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca
4441 cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg
4501 ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc
4561 gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct
4621 ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct
4681 acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac
4741 ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc
4801 tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgat
4861 ggccgcaata aaatatcttt attttcatta catctgtgtg ttggtttttt gtgtgaatcg
4921 atagcgataa ggatccgggc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
4981 aacagttgcg cagcctgaat ggcgaatgga cgcgccctgt agcggcgcat taagcgcggc
5041 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
5101 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
5161 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
5221 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
5281 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
5341 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
5401 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac
5461 aatttcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatatgg
5521 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca
5581 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
5641 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
5701 agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt
5761 tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
5821 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
5881 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
5941 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
6001 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
6061 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
6121 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
6181 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
6241 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
6301 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
6361 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
6421 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
6481 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
6541 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
6601 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
6661 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
6721 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
6781 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
6841 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
6901 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
6961 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
7021 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
7081 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
7141 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
7201 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
7261 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
7321 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
7381 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
7441 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
7501 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
7561 tgctggcctt ttgctcacat ggctcgacag atcccccaat tcaagcttcg tga
//
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