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- 详细信息
- 文献和实验
- 技术资料
- 保存条件:
-20℃
- 保质期:
6个月
- 英文名:
pYES2-NTA
- 库存:
大量
- 供应商:
钰博生物
- 规格:
1ug/5ug
载体基本信息
| 出品公司: | Ybscience |
|---|---|
| 载体名称: | pYES2-NTA, pYES2/NT A |
| 质粒类型: | 酿酒酵母蛋白表达载体 |
| 表达水平: | 高拷贝 |
| 诱导方法: | 半乳糖 |
| 启动子: | GAL1 |
| 克隆方法: | 多克隆位点,限制性内切酶 |
| 载体大小: | 6038 bp |
| 5' 测序引物及序列: | T7: TAATACGACTCACTATAGGG |
| 3' 测序引物及序列: | CYC1 Terminator: GTGACATAACTAATTACATGATG |
| 载体标签: | N-His, N-Xpress, C-His, C-V5 |
| 载体抗性: | 氨苄 |
| 筛选标记: | URA3 |
| 备注: | 利用半乳糖诱导蛋白在酿酒酵母中表达。 |
| 稳定性: | 稳定 Stable |
| 组成型/诱导型: | 诱导型 |
| 病毒/非病毒: | 非病毒 |
载体质粒图谱和多克隆位点信息


载体简介
pYES2 NT A, B, C的是一个6.0 kb的载体,设计用来在酿酒酵母(Saccharomyces cerevisiae)中诱导表达重组蛋白。
该载体包含以下元素:
1. 酵母GAL1启动子,能够在酿酒酵母中被半乳糖高水平的诱导蛋白表达目的蛋白,同时能够被葡萄糖抑制表达
2.多克隆位点可以使用的很多限制酶切位点,便于基因插入。
3.CYC1终止子能够有效终止mRNA的转录。
4.能够利用URA3基因筛选带有ura3基因型的酵母宿主菌株转化子。
5.氨苄抗性基因能够方便在大肠杆菌中的进行载体筛选。
6. 载体本身带有N端His和Xpress标签,同时载体C端也带有V5抗原表位和His纯化标签。
8. 在酵母中,利用载体上的2μ复制子维持质粒的高拷贝。
载体序列
LOCUS pYES2/NTA 6038 bp DNA circular SYN
DEFINITION pYES2/NTA
ACCESSION
KEYWORDS
SOURCE
ORGANISM other sequences; artificial sequences; vectors.
COMMENT This file is created by Vector NTI
http://www.biofeng.com/
COMMENT VNTAUTHORNAME|biofeng.com|
FEATURES Location/Qualifiers
source 1..6038
/organism="pYES2/NT A"
/mol_type="other DNA"
promoter 1..451
/label="GAL1_promoter"
misc_feature 414..437
/label="GAL1_primer"
promoter 475..493
/label="T7_promoter"
misc_feature 542..560
/label="Xpress_fwd_primer"
misc_feature 543..575
/label="T7_leader"
misc_feature 579..602
/label="Xpress_EK"
terminator 791..1036
/label="CYC1_terminator"
misc_feature complement(800..818)
/label="CYC1_primer"
misc_feature complement(800..818)
/label="pYESTrp_rev_primer"
rep_origin complement(1264..1883)
/label="pBR322_origin"
CDS complement(2038..2898)
/label="ORF frame 3"
CDS complement(2994..3797)
/label="ORF frame 1"
gene complement(2997..3797)
/label="URA3"
/gene="URA3"
CDS 3286..3762
/label="ORF frame 1"
promoter complement(3799..4024)
/label="URA3_promoter"
rep_origin 4027..5498
/label="2micron_origin"
ORIGIN
1 ACGGATTAGA AGCCGCCGAG CGGGTGACAG CCCTCCGAAG GAAGACTCTC CTCCGTGCGT
61 CCTCGTCTTC ACCGGTCGCG TTCCTGAAAC GCAGATGTGC CTCGCGCCGC ACTGCTCCGA
121 ACAATAAAGA TTCTACAATA CTAGCTTTTA TGGTTATGAA GAGGAAAAAT TGGCAGTAAC
181 CTGGCCCCAC AAACCTTCAA ATGAACGAAT CAAATTAACA ACCATAGGAT GATAATGCGA
241 TTAGTTTTTT AGCCTTATTT CTGGGGTAAT TAATCAGCGA AGCGATGATT TTTGATCTAT
301 TAACAGATAT ATAAATGCAA AAACTGCATA ACCACTTTAA CTAATACTTT CAACATTTTC
361 GGTTTGTATT ACTTCTTATT CAAATGTAAT AAAAGTATCA ACAAAAAATT GTTAATATAC
421 CTCTATACTT TAACGTCAAG GAGAAAAAAC CCCGGATCGG ACTACTAGCA GCTGTAATAC
481 GACTCACTAT AGGGAATATT AAGCTTACCA TGGGGGGTTC TCATCATCAT CATCATCATG
541 GTATGGCTAG CATGACTGGT GGACAGCAAA TGGGTCGGGA TCTGTACGAC GATGACGATA
601 AGGTACCTAG GATCCAGTGT GGTGGAATTC TGCAGATATC CAGCACAGTG GCGGCCGCTC
661 GAGTCTAGAG GGCCCTTCGA AGGTAAGCCT ATCCCTAACC CTCTCCTCGG TCTCGATTCT
721 ACGCGTACCG GTCATCATCA CCATCACCAT TGAGTTTAAA CCCGCTGATC CTAGAGGGCC
781 GCATCATGTA ATTAGTTATG TCACGCTTAC ATTCACGCCC TCCCCCCACA TCCGCTCTAA
841 CCGAAAAGGA AGGAGTTAGA CAACCTGAAG TCTAGGTCCC TATTTATTTT TTTATAGTTA
901 TGTTAGTATT AAGAACGTTA TTTATATTTC AAATTTTTCT TTTTTTTCTG TACAGACGCG
961 TGTACGCATG TAACATTATA CTGAAAACCT TGCTTGAGAA GGTTTTGGGA CGCTCGAAGG
1021 CTTTAATTTG CAAGCTGCGG CCCTGCATTA ATGAATCGGC CAACGCGCGG GGAGAGGCGG
1081 TTTGCGTATT GGGCGCTCTT CCGCTTCCTC GCTCACTGAC TCGCTGCGCT CGGTCGTTCG
1141 GCTGCGGCGA GCGGTATCAG CTCACTCAAA GGCGGTAATA CGGTTATCCA CAGAATCAGG
1201 GGATAACGCA GGAAAGAACA TGTGAGCAAA AGGCCAGCAA AAGCCCAGGA ACCGTAAAAA
1261 GGCCGCGTTG CTGGCGTTTT TCCATAGGCT CCGCCCCCCT GACGAGCATC ACAAAAATCG
1321 ACGCTCAAGT CAGAGGTGGC GAAACCCGAC AGGACTATAA AGATACCAGG CGTTTCCCCC
1381 TGGAAGCTCC CTCGTGCGCT CTCCTGTTCC GACCCTGCCG CTTACCGGAT ACCTGTCCGC
1441 CTTTCTCCCT TCGGGAAGCG TGGCGCTTTC TCATAGCTCA CGCTGTAGGT ATCTCAGTTC
1501 GGTGTAGGTC GTTCGCTCCA AGCTGGGCTG TGTGCACGAA CCCCCCGTTC AGCCCGACCG
1561 CTGCGCCTTA TCCGGTAACT ATCGTCTTGA GTCCAACCCG GTAAGACACG ACTTATCGCC
1621 ACTGGCAGCA GCCACTGGTA ACAGGATTAG CAGAGCGAGG TATGTAGGCG GTGCTACAGA
1681 GTTCTTGAAG TGGTGGCCTA ACTACGGCTA CACTAGAAGG ACAGTATTTG GTATCTGCGC
1741 TCTGCTGAAG CCAGTTACCT TCGGAAAAAG AGTTGGTAGC TCTTGATCCG GCAAACAAAC
1801 CACCGCTGGT AGCGGTGGTT TTTTTGTTTG CAAGCAGCAG ATTACGCGCA GAAAAAAAGG
1861 ATCTCAAGAA GATCCTTTGA TCTTTTCTAC GGGGTCTGAC GCTCAGTGGA ACGAAAACTC
1921 ACGTTAAGGG ATTTTGGTCA TGAGATTATC AAAAAGGATC TTCACCTAGA TCCTTTTAAA
1981 TTAAAAATGA AGTTTTAAAT CAATCTAAAG TATATATGAG TAAACTTGGT CTGACAGTTA
2041 CCAATGCTTA ATCAGTGAGG CACCTATCTC AGCGATCTGT CTATTTCGTT CATCCATAGT
2101 TGCCTGACTC CCCGTCGTGT AGATAACTAC GATACGGGAG CGCTTACCAT CTGGCCCCAG
2161 TGCTGCAATG ATACCGCGAG ACCCACGCTC ACCGGCTCCA GATTTATCAG CAATAAACCA
2221 GCCAGCCGGA AGGGCCGAGC GCAGAAGTGG TCCTGCAACT TTATCCGCCT CCATCCAGTC
2281 TATTAATTGT TGCCGGGAAG CTAGAGTAAG TAGTTCGCCA GTTAATAGTT TGCGCAACGT
2341 TGTTGGCATT GCTACAGGCA TCGTGGTGTC ACTCTCGTCG TTTGGTATGG CTTCATTCAG
2401 CTCCGGTTCC CAACGATCAA GGCGAGTTAC ATGATCCCCC ATGTTGTGCA AAAAAGCGGT
2461 TAGCTCCTTC GGTCCTCCGA TCGTTGTCAG AAGTAAGTTG GCCGCAGTGT TATCACTCAT
2521 GGTTATGGCA GCACTGCATA ATTCTCTTAC TGTCATGCCA TCCGTAAGAT GCTTTTCTGT
2581 GACTGGTGAG TACTCAACCA AGTCATTCTG AGAATAGTGT ATGCGGCGAC CGAGTTGCTC
2641 TTGCCCGGCG TCAATACGGG ATAATAGTGT ATCACATAGC AGAACTTTAA AAGTGCTCAT
2701 CATTGGAAAA CGTTCTTCGG GGCGAAAACT CTCAAGGATC TTACCGCTGT TGAGATCCAG
2761 TTCGATGTAA CCCACTCGTG CACCCAACTG ATCTTCAGCA TCTTTTACTT TCACCAGCGT
2821 TTCTGGGTGA GCAAAAACAG GAAGGCAAAA TGCCGCAAAA AAGGGAATAA GGGCGACACG
2881 GAAATGTTGA ATACTCATAC TCTTCCTTTT TCAATGGGTA ATAACTGATA TAATTAAATT
2941 GAAGCTCTAA TTTGTGAGTT TAGTATACAT GCATTTACTT ATAATACAGT TTTTTAGTTT
3001 TGCTGGCCGC ATCTTCTCAA ATATGCTTCC CAGCCTGCTT TTCTGTAACG TTCACCCTCT
3061 ACCTTAGCAT CCCTTCCCTT TGCAAATAGT CCTCTTCCAA CAATAATAAT GTCAGATCCT
3121 GTAGAGACCA CATCATCCAC GGTTCTATAC TGTTGACCCA ATGCGTCTCC CTTGTCATCT
3181 AAACCCACAC CGGGTGTCAT AATCAACCAA TCGTAACCTT CATCTCTTCC ACCCATGTCT
3241 CTTTGAGCAA TAAAGCCGAT AACAAAATCT TTGTCGCTCT TCGCAATGTC AACAGTACCC
3301 TTAGTATATT CTCCAGTAGA TAGGGAGCCC TTGCATGACA ATTCTGCTAA CATCAAAAGG
3361 CCTCTAGGTT CCTTTGTTAC TTCTTCTGCC GCCTGCTTCA AACCGCTAAC AATACCTGGG
3421 CCCACCACAC CGTGTGCATT CGTAATGTCT GCCCATTCTG CTATTCTGTA TACACCCGCA
3481 GAGTACTGCA ATTTGACTGT ATTACCAATG TCAGCAAATT TTCTGTCTTC GAAGAGTAAA
3541 AAATTGTACT TGGCGGATAA TGCCTTTAGC GGCTTAACTG TGCCCTCCAT GGAAAAATCA
3601 GTCAAGATAT CCACATGTGT TTTTAGTAAA CAAATTTTGG GACCTAATGC TTCAACTAAC
3661 TCCAGTAATT CCTTGGTGGT ACGAACATCC AATGAAGCAC ACAAGTTTGT TTGCTTTTCG
3721 TGCATGATAT TAAATAGCTT GGCAGCAACA GGACTAGGAT GAGTAGCAGC ACGTTCCTTA
3781 TATGTAGCTT TCGACATGAT TTATCTTCGT TTCCTGCAGG TTTTTGTTCT GTGCAGTTGG
3841 GTTAAGAATA CTGGGCAATT TCATGTTTCT TCAACACTAC ATATGCGTAT ATATACCAAT
3901 CTAAGTCTGT GCTCCTTCCT TCGTTCTTCC TTCTGTTCGG AGATTACCGA ATCAAAAAAA
3961 TTTCAAAGAA ACCGAAATCA AAAAAAAGAA TAAAAAAAAA ATGATGAATT GAATTGAAAA
4021 GCTAGCTTAT CGATGATAAG CTGTCAAAGA TGAGAATTAA TTCCACGGAC TATAGACTAT
4081 ACTAGATACT CCGTCTACTG TACGATACAC TTCCGCTCAG GTCCTTGTCC TTTAACGAGG
4141 CCTTACCACT CTTTTGTTAC TCTATTGATC CAGCTCAGCA AAGGCAGTGT GATCTAAGAT
4201 TCTATCTTCG CGATGTAGTA AAACTAGCTA GACCGAGAAA GAGACTAGAA ATGCAAAAGG
4261 CACTTCTACA ATGGCTGCCA TCATTATTAT CCGATGTGAC GCTGCAGCTT CTCAATGATA
4321 TTCGAATACG CTTTGAGGAG ATACAGCCTA ATATCCGACA AACTGTTTTA CAGATTTACG
4381 ATCGTACTTG TTACCCATCA TTGAATTTTG AACATCCGAA CCTGGGAGTT TTCCCTGAAA
4441 CAGATAGTAT ATTTGAACCT GTATAATAAT ATATAGTCTA GCGCTTTACG GAAGACAATG
4501 TATGTATTTC GGTTCCTGGA GAAACTATTG CATCTATTGC ATAGGTAATC TTGCACGTCG
4561 CATCCCCGGT TCATTTTCTG CGTTTCCATC TTGCACTTCA ATAGCATATC TTTGTTAACG
4621 AAGCATCTGT GCTTCATTTT GTAGAACAAA AATGCAACGC GAGAGCGCTA ATTTTTCAAA
4681 CAAAGAATCT GAGCTGCATT TTTACAGAAC AGAAATGCAA CGCGAAAGCG CTATTTTACC
4741 AACGAAGAAT CTGTGCTTCA TTTTTGTAAA ACAAAAATGC AACGCGACGA GAGCGCTAAT
4801 TTTTCAAACA AAGAATCTGA GCTGCATTTT TACAGAACAG AAATGCAACG CGAGAGCGCT
4861 ATTTTACCAA CAAAGAATCT ATACTTCTTT TTTGTTCTAC AAAAATGCAT CCCGAGAGCG
4921 CTATTTTTCT AACAAAGCAT CTTAGATTAC TTTTTTTCTC CTTTGTGCGC TCTATAATGC
4981 AGTCTCTTGA TAACTTTTTG CACTGTAGGT CCGTTAAGGT TAGAAGAAGG CTACTTTGGT
5041 GTCTATTTTC TCTTCCATAA AAAAAGCCTG ACTCCACTTC CCGCGTTTAC TGATTACTAG
5101 CGAAGCTGCG GGTGCATTTT TTCAAGATAA AGGCATCCCC GATTATATTC TATACCGATG
5161 TGGATTGCGC ATACTTTGTG AACAGAAAGT GATAGCGTTG ATGATTCTTC ATTGGTCAGA
5221 AAATTATGAA CGGTTTCTTC TATTTTGTCT CTATATACTA CGTATAGGAA ATGTTTACAT
5281 TTTCGTATTG TTTTCGATTC ACTCTATGAA TAGTTCTTAC TACAATTTTT TTGTCTAAAG
5341 AGTAATACTA GAGATAAACA TAAAAAATGT AGAGGTCGAG TTTAGATGCA AGTTCAAGGA
5401 GCGAAAGGTG GATGGGTAGG TTATATAGGG ATATAGCACA GAGATATATA GCAAAGAGAT
5461 ACTTTTGAGC AATGTTTGTG GAAGCGGTAT TCGCAATGGG AAGCTCCACC CCGGTTGATA
5521 ATCAGAAAAG CCCCAAAAAC AGGAAGATTG TATAAGCAAA TATTTAAATT GTAAACGTTA
5581 ATATTTTGTT AAAATTCGCG TTAAATTTTT GTTAAATCAG CTCATTTTTT AACGAATAGC
5641 CCGAAATCGG CAAAATCCCT TATAAATCAA AAGAATAGAC CGAGATAGGG TTGAGTGTTG
5701 TTCCAGTTTC CAACAAGAGT CCACTATTAA AGAACGTGGA CTCCAACGTC AAAGGGCGAA
5761 AAAGGGTCTA TCAGGGCGAT GGCCCACTAC GTGAACCATC ACCCTAATCA AGTTTTTTGG
5821 GGTCGAGGTG CCGTAAAGCA GTAAATCGGA AGGGTAAACG GATGCCCCCA TTTAGAGCTT
5881 GACGGGGAAA GCCGGCGAAC GTGGCGAGAA AGGAAGGGAA GAAAGCGAAA GGAGCGGGGG
5941 CTAGGGCGGT GGGAAGTGTA GGGGTCACGC TGGGCGTAAC CACCACACCC GCCGCGCTTA
6001 ATGGGGCGCT ACAGGGCGCG TGGGGATGAT CCACTAGT
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文献和实验首先,我们先看原理: 纳米颗粒示踪分析仪(NTA)利用光散射和布朗运动的属性,以获得液体悬浮液中样品的粒径分布。 颗粒的运动速度和其粒径有着直接的关联;此技术观测的是颗粒的散射光斑,而不是颗粒本身; 「眼见为实」,确保分析的是目标颗粒而不是背景颗粒或噪音。 然后,我们看下设备介绍:(声明:本次操作基于最近正在使用的 NS300。) 然后,我们看下具体是如何操作的: 开机→清洗→上样→设置参数→调整视野→设置保存路径→检测 →仪器自动分析结果→出报告→保存后下样关机 说明:散射光模式
6xHis-tagged protein purification using Qiagen Ni-NTA Column
)Centrifuge lysate at 10,000xg for 20’ at 40℃to pellet the cellular debris and save supernatant.Add 5μl 2xSB to 5μl Supernatant and store at 200℃for SDS-PAGE analysisBatch purification under Native ConditionsAdd 2.5 ml of the 50% Ni-NTA slurry to 10 ml cleared
One-Step Purification of Recombinant Proteins with the 6xHis Tag and Ni-NTA Resin
The 6xHis/Ni-NTA system is a fast and versatile tool for the affinity purification of recombinant proteins and antigenic peptides. It is based on the high-affinity binding of six consecutive histidine residues (the 6xHis tag) to immobilized
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