pYES2/CT
pYES2/CT
pYES2/CT
编号 | 载体名称 |
北京华越洋生物VECT2850 | pYES2/CT |
pYES2/CT载体基本信息
| 出品公司: | -- |
|---|---|
| 载体名称: | pYES2-CT, pYES2/CT |
| 质粒类型: | 酿酒酵母表达载体 |
| 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: | 5963 bp |
| 5' 测序引物及序列: | GAL1-F: AATATACCTCTATACTTTAACGTC |
| 3' 测序引物及序列: | CYC1-R: GCGTGAATGTAAGCGTGAC |
| 载体标签: | C-His, C-V5 |
| 载体抗性: | 氨苄青霉素 |
| 筛选标记: | URA3报告基因 |
| 克隆菌株: | TOP10, E.coli |
| 表达菌株: | INVSc1, Saccharomyces cerevisiae |
| 备注: | 酿酒酵母表达载体pYES2/CT含有URA3报告基因； GAL1启动子驱动目的基因高水平表达； 半乳糖诱导目基因表达，葡萄糖阻遏目的基因表达，二者起到分子开关作用。 |
| 产品目录号: | -- |
| 稳定性: | 稳表达 |
| 组成型/诱导型: | 诱导型 |
| 病毒/非病毒: | 非病毒 |
pYES2/CT载体质粒图谱和多克隆位点信息
载体简介
pYES2/CT酿酒酵母表达载体pYES2/CT载体大小为6.0 kb左右，用于重组蛋白在酿酒酵母细胞中的诱导型表达。诱导剂为半乳糖。重组蛋白的C-端融合了6XHis标签，用于重组蛋白的纯化和检测。 pYES2/CT载体含有以下元件： Yeast GAL1 promoter for high level inducible protein expression in yeast by galactose and repression by glucose (Giniger et al., 1985; West et al., 1984) Multiple cloning site (MCS) with 8 or 9 unique sites (plus two BstX I sites) to facilitate in-frame cloning with the C-terminal peptide C-terminal peptide encoding the V5 epitope and a polyhistidine (6xHis) tag for detection and purification of your recombinant fusion protein 2μ origin for episomal maintenance and high copy replication or CEN6/ARSH4 sequence for non-integrative centromeric maintenance and low copy replication (pYC2/CT) URA3 auxotrophic marker for selection of yeast transformants Ampicillin resistance gene for selection in E. coli使用pYES2/CT载体表达目的基因的实验流程如下：1 Consult the multiple cloning site described on page 7 to determine a strategy to clone your gene in frame with the C-terminal peptide. 2 Ligate your insert into the appropriate vector and transform into E. coli. Select transformants on LB plates containing 50 to 100 μg/mL ampicillin. 3 Analyze your transformants for the presence of insert by restriction digestion. 4 Select a transformant with the correct restriction pattern and sequence to confirm that your gene is cloned in frame with the C-terminal peptide. 5 Transform your construct into competent INVSc1 cells and select for the appropriate amino acid prototrophy. 6 Test for expression of your recombinant protein by western blot analysis or functional assay. 7 Use metal-chelating resin such as ProBond to purify your recombinant protein.
载体序列
LOCUS pYES2/CT 5963 bp DNA circular SYN DEFINITION pYES2/CT ACCESSION KEYWORDS SOURCE ORGANISM other sequences; artificial sequences; vectors. COMMENT This file is created by Vector NTI FEATURES Location/Qualifiers source 1..5963 /organism="pYES2/CT" /mol_type="other DNA" promoter 1..451 /label="GAL1_promoter" misc_feature 414..437 /label="GAL1_primer" promoter 475..493 /label="T7_promoter" terminator 716..961 /label="CYC1_terminator" misc_feature complement(725..743) /label="pYESTrp_rev_primer" misc_feature complement(725..743) /label="CYC1_primer" rep_origin complement(1189..1808) /label="pBR322_origin" CDS complement(1963..2823) /label="ORF frame 3" CDS complement(2919..3722) /label="ORF frame 1" gene complement(2922..3722) /label="URA3" /gene="URA3" CDS 3211..3687 /label="ORF frame 1" promoter complement(3724..3949) /label="URA3_promoter" rep_origin 3952..5423 /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 AAGCTTGGTA CCGAGCTCGG ATCCACTAGT AACGGCCGCC 541 AGTGTGCTGG AATTCTGCAG ATATCCAGCA CAGTGGCGGC CGCTCGAGTC TAGAGGGCCC 601 TTCGAAGGTA AGCCTATCCC TAACCCTCTC CTCGGTCTCG ATTCTACGCG TACCGGTCAT 661 CATCACCATC ACCATTGAGT TTAAACCCGC TGATCCTAGA GGGCCGCATC ATGTAATTAG 721 TTATGTCACG CTTACATTCA CGCCCTCCCC CCACATCCGC TCTAACCGAA AAGGAAGGAG 781 TTAGACAACC TGAAGTCTAG GTCCCTATTT ATTTTTTTAT AGTTATGTTA GTATTAAGAA 841 CGTTATTTAT ATTTCAAATT TTTCTTTTTT TTCTGTACAG ACGCGTGTAC GCATGTAACA 901 TTATACTGAA AACCTTGCTT GAGAAGGTTT TGGGACGCTC GAAGGCTTTA ATTTGCAAGC 961 TGCGGCCCTG CATTAATGAA TCGGCCAACG CGCGGGGAGA GGCGGTTTGC GTATTGGGCG 1021 CTCTTCCGCT TCCTCGCTCA CTGACTCGCT GCGCTCGGTC GTTCGGCTGC GGCGAGCGGT 1081 ATCAGCTCAC TCAAAGGCGG TAATACGGTT ATCCACAGAA TCAGGGGATA ACGCAGGAAA 1141 GAACATGTGA GCAAAAGGCC AGCAAAAGCC CAGGAACCGT AAAAAGGCCG CGTTGCTGGC 1201 GTTTTTCCAT AGGCTCCGCC CCCCTGACGA GCATCACAAA AATCGACGCT CAAGTCAGAG 1261 GTGGCGAAAC CCGACAGGAC TATAAAGATA CCAGGCGTTT CCCCCTGGAA GCTCCCTCGT 1321 GCGCTCTCCT GTTCCGACCC TGCCGCTTAC CGGATACCTG TCCGCCTTTC TCCCTTCGGG 1381 AAGCGTGGCG CTTTCTCATA GCTCACGCTG TAGGTATCTC AGTTCGGTGT AGGTCGTTCG 1441 CTCCAAGCTG GGCTGTGTGC ACGAACCCCC CGTTCAGCCC GACCGCTGCG CCTTATCCGG 1501 TAACTATCGT CTTGAGTCCA ACCCGGTAAG ACACGACTTA TCGCCACTGG CAGCAGCCAC 1561 TGGTAACAGG ATTAGCAGAG CGAGGTATGT AGGCGGTGCT ACAGAGTTCT TGAAGTGGTG 1621 GCCTAACTAC GGCTACACTA GAAGGACAGT ATTTGGTATC TGCGCTCTGC TGAAGCCAGT 1681 TACCTTCGGA AAAAGAGTTG GTAGCTCTTG ATCCGGCAAA CAAACCACCG CTGGTAGCGG 1741 TGGTTTTTTT GTTTGCAAGC AGCAGATTAC GCGCAGAAAA AAAGGATCTC AAGAAGATCC 1801 TTTGATCTTT TCTACGGGGT CTGACGCTCA GTGGAACGAA AACTCACGTT AAGGGATTTT 1861 GGTCATGAGA TTATCAAAAA GGATCTTCAC CTAGATCCTT TTAAATTAAA AATGAAGTTT 1921 TAAATCAATC TAAAGTATAT ATGAGTAAAC TTGGTCTGAC AGTTACCAAT GCTTAATCAG 1981 TGAGGCACCT ATCTCAGCGA TCTGTCTATT TCGTTCATCC ATAGTTGCCT GACTCCCCGT 2041 CGTGTAGATA ACTACGATAC GGGAGCGCTT ACCATCTGGC CCCAGTGCTG CAATGATACC 2101 GCGAGACCCA CGCTCACCGG CTCCAGATTT ATCAGCAATA AACCAGCCAG CCGGAAGGGC 2161 CGAGCGCAGA AGTGGTCCTG CAACTTTATC CGCCTCCATC CAGTCTATTA ATTGTTGCCG 2221 GGAAGCTAGA GTAAGTAGTT CGCCAGTTAA TAGTTTGCGC AACGTTGTTG GCATTGCTAC 2281 AGGCATCGTG GTGTCACTCT CGTCGTTTGG TATGGCTTCA TTCAGCTCCG GTTCCCAACG 2341 ATCAAGGCGA GTTACATGAT CCCCCATGTT GTGCAAAAAA GCGGTTAGCT CCTTCGGTCC 2401 TCCGATCGTT GTCAGAAGTA AGTTGGCCGC AGTGTTATCA CTCATGGTTA TGGCAGCACT 2461 GCATAATTCT CTTACTGTCA TGCCATCCGT AAGATGCTTT TCTGTGACTG GTGAGTACTC 2521 AACCAAGTCA TTCTGAGAAT AGTGTATGCG GCGACCGAGT TGCTCTTGCC CGGCGTCAAT 2581 ACGGGATAAT AGTGTATCAC ATAGCAGAAC TTTAAAAGTG CTCATCATTG GAAAACGTTC 2641 TTCGGGGCGA AAACTCTCAA GGATCTTACC GCTGTTGAGA TCCAGTTCGA TGTAACCCAC 2701 TCGTGCACCC AACTGATCTT CAGCATCTTT TACTTTCACC AGCGTTTCTG GGTGAGCAAA 2761 AACAGGAAGG CAAAATGCCG CAAAAAAGGG AATAAGGGCG ACACGGAAAT GTTGAATACT 2821 CATACTCTTC CTTTTTCAAT GGGTAATAAC TGATATAATT AAATTGAAGC TCTAATTTGT 2881 GAGTTTAGTA TACATGCATT TACTTATAAT ACAGTTTTTT AGTTTTGCTG GCCGCATCTT 2941 CTCAAATATG CTTCCCAGCC TGCTTTTCTG TAACGTTCAC CCTCTACCTT AGCATCCCTT 3001 CCCTTTGCAA ATAGTCCTCT TCCAACAATA ATAATGTCAG ATCCTGTAGA GACCACATCA 3061 TCCACGGTTC TATACTGTTG ACCCAATGCG TCTCCCTTGT CATCTAAACC CACACCGGGT 3121 GTCATAATCA ACCAATCGTA ACCTTCATCT CTTCCACCCA TGTCTCTTTG AGCAATAAAG 3181 CCGATAACAA AATCTTTGTC GCTCTTCGCA ATGTCAACAG TACCCTTAGT ATATTCTCCA 3241 GTAGATAGGG AGCCCTTGCA TGACAATTCT GCTAACATCA AAAGGCCTCT AGGTTCCTTT 3301 GTTACTTCTT CTGCCGCCTG CTTCAAACCG CTAACAATAC CTGGGCCCAC CACACCGTGT 3361 GCATTCGTAA TGTCTGCCCA TTCTGCTATT CTGTATACAC CCGCAGAGTA CTGCAATTTG 3421 ACTGTATTAC CAATGTCAGC AAATTTTCTG TCTTCGAAGA GTAAAAAATT GTACTTGGCG 3481 GATAATGCCT TTAGCGGCTT AACTGTGCCC TCCATGGAAA AATCAGTCAA GATATCCACA 3541 TGTGTTTTTA GTAAACAAAT TTTGGGACCT AATGCTTCAA CTAACTCCAG TAATTCCTTG 3601 GTGGTACGAA CATCCAATGA AGCACACAAG TTTGTTTGCT TTTCGTGCAT GATATTAAAT 3661 AGCTTGGCAG CAACAGGACT AGGATGAGTA GCAGCACGTT CCTTATATGT AGCTTTCGAC 3721 ATGATTTATC TTCGTTTCCT GCAGGTTTTT GTTCTGTGCA GTTGGGTTAA GAATACTGGG 3781 CAATTTCATG TTTCTTCAAC ACTACATATG CGTATATATA CCAATCTAAG TCTGTGCTCC 3841 TTCCTTCGTT CTTCCTTCTG TTCGGAGATT ACCGAATCAA AAAAATTTCA AAGAAACCGA 3901 AATCAAAAAA AAGAATAAAA AAAAAATGAT GAATTGAATT GAAAAGCTAG CTTATCGATG 3961 ATAAGCTGTC AAAGATGAGA ATTAATTCCA CGGACTATAG ACTATACTAG ATACTCCGTC 4021 TACTGTACGA TACACTTCCG CTCAGGTCCT TGTCCTTTAA CGAGGCCTTA CCACTCTTTT 4081 GTTACTCTAT TGATCCAGCT CAGCAAAGGC AGTGTGATCT AAGATTCTAT CTTCGCGATG 4141 TAGTAAAACT AGCTAGACCG AGAAAGAGAC TAGAAATGCA AAAGGCACTT CTACAATGGC 4201 TGCCATCATT ATTATCCGAT GTGACGCTGC AGCTTCTCAA TGATATTCGA ATACGCTTTG 4261 AGGAGATACA GCCTAATATC CGACAAACTG TTTTACAGAT TTACGATCGT ACTTGTTACC 4321 CATCATTGAA TTTTGAACAT CCGAACCTGG GAGTTTTCCC TGAAACAGAT AGTATATTTG 4381 AACCTGTATA ATAATATATA GTCTAGCGCT TTACGGAAGA CAATGTATGT ATTTCGGTTC 4441 CTGGAGAAAC TATTGCATCT ATTGCATAGG TAATCTTGCA CGTCGCATCC CCGGTTCATT 4501 TTCTGCGTTT CCATCTTGCA CTTCAATAGC ATATCTTTGT TAACGAAGCA TCTGTGCTTC 4561 ATTTTGTAGA ACAAAAATGC AACGCGAGAG CGCTAATTTT TCAAACAAAG AATCTGAGCT 4621 GCATTTTTAC AGAACAGAAA TGCAACGCGA AAGCGCTATT TTACCAACGA AGAATCTGTG 4681 CTTCATTTTT GTAAAACAAA AATGCAACGC GACGAGAGCG CTAATTTTTC AAACAAAGAA 4741 TCTGAGCTGC ATTTTTACAG AACAGAAATG CAACGCGAGA GCGCTATTTT ACCAACAAAG 4801 AATCTATACT TCTTTTTTGT TCTACAAAAA TGCATCCCGA GAGCGCTATT TTTCTAACAA 4861 AGCATCTTAG ATTACTTTTT TTCTCCTTTG TGCGCTCTAT AATGCAGTCT CTTGATAACT 4921 TTTTGCACTG TAGGTCCGTT AAGGTTAGAA GAAGGCTACT TTGGTGTCTA TTTTCTCTTC 4981 CATAAAAAAA GCCTGACTCC ACTTCCCGCG TTTACTGATT ACTAGCGAAG CTGCGGGTGC 5041 ATTTTTTCAA GATAAAGGCA TCCCCGATTA TATTCTATAC CGATGTGGAT TGCGCATACT 5101 TTGTGAACAG AAAGTGATAG CGTTGATGAT TCTTCATTGG TCAGAAAATT ATGAACGGTT 5161 TCTTCTATTT TGTCTCTATA TACTACGTAT AGGAAATGTT TACATTTTCG TATTGTTTTC 5221 GATTCACTCT ATGAATAGTT CTTACTACAA TTTTTTTGTC TAAAGAGTAA TACTAGAGAT 5281 AAACATAAAA AATGTAGAGG TCGAGTTTAG ATGCAAGTTC AAGGAGCGAA AGGTGGATGG 5341 GTAGGTTATA TAGGGATATA GCACAGAGAT ATATAGCAAA GAGATACTTT TGAGCAATGT 5401 TTGTGGAAGC GGTATTCGCA ATGGGAAGCT CCACCCCGGT TGATAATCAG AAAAGCCCCA 5461 AAAACAGGAA GATTGTATAA GCAAATATTT AAATTGTAAA CGTTAATATT TTGTTAAAAT 5521 TCGCGTTAAA TTTTTGTTAA ATCAGCTCAT TTTTTAACGA ATAGCCCGAA ATCGGCAAAA 5581 TCCCTTATAA ATCAAAAGAA TAGACCGAGA TAGGGTTGAG TGTTGTTCCA GTTTCCAACA 5641 AGAGTCCACT ATTAAAGAAC GTGGACTCCA ACGTCAAAGG GCGAAAAAGG GTCTATCAGG 5701 GCGATGGCCC ACTACGTGAA CCATCACCCT AATCAAGTTT TTTGGGGTCG AGGTGCCGTA 5761 AAGCAGTAAA TCGGAAGGGT AAACGGATGC CCCCATTTAG AGCTTGACGG GGAAAGCCGG 5821 CGAACGTGGC GAGAAAGGAA GGGAAGAAAG CGAAAGGAGC GGGGGCTAGG GCGGTGGGAA 5881 GTGTAGGGGT CACGCTGGGC GTAACCACCA CACCCGCCGC GCTTAATGGG GCGCTACAGG 5941 GCGCGTGGGG ATGATCCACT AGT //
