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- 详细信息
- 技术资料
- 保存条件：
-20
- 保质期：
2年
- 英文名：
pYES3-CT
- 库存：
100
- 供应商：
上海烜雅生物科技有限公司
- 规格：
干粉/液体
基本信息
名称：pYES3/CT酿酒酵母质粒
别称: pYES3-CT
质粒属性
质粒简介
质粒图谱
质粒序列
LOCUS Exported 5870 bp ds-DNA circular SYN 29-NOV-2013
DEFINITION High-copy episomal vector with a TRP1 marker for galactose-inducible
expression of C-terminally tagged proteins in S. cerevisiae.
ACCESSION .
VERSION .
KEYWORDS pYES3 CT
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5870)
AUTHORS Invitrogen (Life Technologies)
TITLE Direct Submission
JOURNAL Exported Sunday, September 11, 2016 from SnapGene Viewer 3.1.4
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..5870
/organism="synthetic DNA construct"
/lab_host="Saccharomyces cerevisiae"
/mol_type="other DNA"
promoter 2..443
/gene="S. cerevisiae GAL1"
/note="GAL1 promoter"
/note="inducible promoter, regulated by Gal4"
protein_bind 2..119
/bound_moiety="Gal4"
/note="UAS"
/note="upstream activating sequence mediating
Gal4-dependent induction"
promoter 475..493
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
misc_feature 501..600
/note="MCS"
/note="multiple cloning site"
CDS 607..648
/codon_start=1
/product="epitope tag from simian virus 5"
/note="V5 tag"
/translation="GKPIPNPLLGLDST"
CDS 658..675
/codon_start=1
/product="6xHis affinity tag"
/note="6xHis"
/translation="HHHHHH"
terminator 709..956
/gene="S. cerevisiae CYC1"
/note="CYC1 terminator"
/note="transcription terminator for CYC1"
rep_origin complement(1204..1792)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(1963..2823)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKRSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(2824..2928)
/gene="bla"
/note="AmpR promoter"
promoter 3031..3132
/gene="S. cerevisiae TRP1"
/note="TRP1 promoter"
CDS 3133..3807
/codon_start=1
/gene="S. cerevisiae TRP1"
/product="phosphoribosylanthranilate isomerase, required
for tryptophan biosynthesis"
/note="TRP1"
/note="yeast auxotrophic marker"
/translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
KK"
rep_origin 4449..5329
/note="2u ori"
/note="yeast 2u plasmid origin of replication"
rep_origin complement(5398..5853)
/direction=LEFT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
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 ccattgctac
2281 aggcatcgtg gtgtcacgct 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 cggcgtcaac
2581 acgggataat accgcgccac 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 attattgaag catttatcag ggttattgtc tcatgagcgg
2881 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
2941 aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag
3001 gcgtatcacg aggccctttc gtcttcaaga aattcggtcg aaaaaagaaa aggagagggc
3061 caagagggag ggcattggtg actattgagc acgtgagtat acgtgattaa gcacacaaag
3121 gcagcttgga gtatgtctgt tattaatttc acaggtagtt ctggtccatt ggtgaaagtt
3181 tgcggcttgc agagcacaga ggccgcagaa tgtgctctag attccgatgc tgacttgctg
3241 ggtattatat gtgtgcccaa tagaaagaga acaattgacc cggttattgc aaggaaaatt
3301 tcaagtcttg taaaagcata taaaaatagt tcaggcactc cgaaatactt ggttggcgtg
3361 tttcgtaatc aacctaagga ggatgttttg gctctggtca atgattacgg cattgatatc
3421 gtccaactgc acggagatga gtcgtggcaa gaataccaag agttcctcgg tttgccagtt
3481 attaaaagac tcgtatttcc aaaagactgc aacatactac tcagtgcagc ttcacagaaa
3541 cctcattcgt ttattccctt gtttgattca gaagcaggtg ggacaggtga acttttggat
3601 tggaactcga tttctgactg ggttggaagg caagagagcc ccgagagctt acattttatg
3661 ttagctggtg gactgacgcc agaaaatgtt ggtgatgcgc ttagattaaa tggcgttatt
3721 ggtgttgatg taagcggagg tgtggagaca aatggtgtaa aagactctaa caaaatagca
3781 aatttcgtca aaaatgctaa gaaataggtt attactgagt agtatttatt taagtattgt
3841 ttgtgcactt gccctagctt atcgatgata agctgtcaaa gatgagaatt aattccacgg
3901 actatagact atactagata ctccgtctac tgtacgatac acttccgctc aggtccttgt
3961 cctttaacga ggccttacca ctcttttgtt actctattga tccagctcag caaaggcagt
4021 gtgatctaag attctatctt cgcgatgtag taaaactagc tagaccgaga aagagactag
4081 aaatgcaaaa ggcacttcta caatggctgc catcattatt atccgatgtg acgctgcagc
4141 ttctcaatga tattcgaata cgctttgagg agatacagcc taatatccga caaactgttt
4201 tacagattta cgatcgtact tgttacccat cattgaattt tgaacatccg aacctgggag
4261 ttttccctga aacagatagt atatttgaac ctgtataata atatatagtc tagcgcttta
4321 cggaagacaa tgtatgtatt tcggttcctg gagaaactat tgcatctatt gcataggtaa
4381 tcttgcacgt cgcatccccg gttcattttc tgcgtttcca tcttgcactt caatagcata
4441 tctttgttaa cgaagcatct gtgcttcatt ttgtagaaca aaaatgcaac gcgagagcgc
4501 taatttttca aacaaagaat ctgagctgca tttttacaga acagaaatgc aacgcgaaag
4561 cgctatttta ccaacgaaga atctgtgctt catttttgta aaacaaaaat gcaacgcgac
4621 gagagcgcta atttttcaaa caaagaatct gagctgcatt tttacagaac agaaatgcaa
4681 cgcgagagcg ctattttacc aacaaagaat ctatacttct tttttgttct acaaaaatgc
4741 atcccgagag cgctattttt ctaacaaagc atcttagatt actttttttc tcctttgtgc
4801 gctctataat gcagtctctt gataactttt tgcactgtag gtccgttaag gttagaagaa
4861 ggctactttg gtgtctattt tctcttccat aaaaaaagcc tgactccact tcccgcgttt
4921 actgattact agcgaagctg cgggtgcatt ttttcaagat aaaggcatcc ccgattatat
4981 tctataccga tgtggattgc gcatactttg tgaacagaaa gtgatagcgt tgatgattct
5041 tcattggtca gaaaattatg aacggtttct tctattttgt ctctatatac tacgtatagg
5101 aaatgtttac attttcgtat tgttttcgat tcactctatg aatagttctt actacaattt
5161 ttttgtctaa agagtaatac tagagataaa cataaaaaat gtagaggtcg agtttagatg
5221 caagttcaag gagcgaaagg tggatgggta ggttatatag ggatatagca cagagatata
5281 tagcaaagag atacttttga gcaatgtttg tggaagcggt attcgcaatg ggaagctcca
5341 ccccggttga taatcagaaa agccccaaaa acaggaagat tgtataagca aatatttaaa
5401 ttgtaaacgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
5461 ttaacgaata gcccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag
5521 ggttgagtgt tgttccagtt tccaacaaga gtccactatt aaagaacgtg gactccaacg
5581 tcaaagggcg aaaaagggtc tatcagggcg atggcccact acgtgaacca tcaccctaat
5641 caagtttttt ggggtcgagg tgccgtaaag cagtaaatcg gaagggtaaa cggatgcccc
5701 catttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
5761 aaggagcggg ggctagggcg gtgggaagtg taggggtcac gctgggcgta accaccacac
5821 ccgccgcgct taatggggcg ctacagggcg cgtggggatg atccactagt
//
质粒菌株产品操作说明书
一、扩增流程
收到产品后，请先根据产品管壁标签来判断产品形式，并在扩增前准确查找该质粒菌株的抗性、感受态和培养温度。
1、质粒干粉（常温运输，存于-20度，90天保质期，请务必转化挑单克隆培养，不要直接使用和测序）
①收到质粒干粉后请先5000rpm离心1min，再加入20μl ddH2O去离子水溶解质粒；
②取1支100μl 感受态于冰上解冻10min，加入2μl质粒，再冰浴30min后，42℃热激60s，不要搅动，再冰浴2min；（从第二步开始均要在超净工作台中无菌操作）
③加入900μl无抗的LB液体培养基，180rpm震荡37℃培养45min；
④6000rpm离心5min，仅留100μl上清液重悬细菌沉淀，并涂布至目标质粒抗性的LB平板上；（可使用本平台的平板涂布专用玻璃珠进行涂布，可以比传统涂布方法获得更多转化子）
⑤将平板正向培养1h，再倒置37℃培养14h。如果要求是30度则培养20h；
（菌落过多则将质粒稀释后再转化。没有菌落则加入10μl质粒转化。另不要直接转表达感受态，要先转克隆感受态，重提质粒后再导入表达感受态）
⑥挑取单菌落至LB液体培养基中，加入对应抗生素，220rpm震荡培养14h，根据实验需要和质粒提取试剂盒说明书提取质粒。
2、甘油菌种（冰袋运输，存于-80℃，保质期90天，请务必划线挑单克隆培养）
四区划线后挑单菌落培养，酵母菌需要先液体复苏再四区划线，再挑单菌落液体培养。
3、穿刺菌种（冰袋运输，存于4℃，保质期7天）
穿刺接种，液体培养后四区划线，再挑单菌落液体培养。
4、菌落平板（冰袋运输，存于4℃，保质期7天）
直接挑取单菌落至液体培养基中。
5、液体质粒（冰袋运输，存于-20℃，保质期90天）
单独提取的液体质粒收到后可直接使用。
6、滤纸质粒（常温运输，存于-20度，90天保质期，请务必转化挑单克隆培养，不要直接使用和测序）
收到货后将滤纸画圈部分剪下放入EP管中，加100ul无菌水将滤纸浸湿并浸泡5min，吸取5ul质粒转化，离心全涂。
二、转化图片
名称：pYES3/CT酿酒酵母质粒
别称: pYES3-CT
| 启动子: | GAL1 |
|---|---|
| 复制子: | 2μ |
| 终止子: | CYC1 |
| 质粒分类: | 酵母系列，酿酒酵母表达载体 |
| 质粒大小: | 5870bp |
| 原核抗性: | Amp |
| 筛选标记: | TRP1 |
| 克隆菌株: | DH5a |
| 培养条件: | 37度 |
| 表达宿主: | 酵母细胞 |
| 诱导方式: | 半乳糖诱导 |
| 5'测序引物: | GAL1-F: ATTTTCGGTTTGTATTACTTC |
| 3'测序引物: | GALI-R: GTTCTTAATACTAACATAACT |
质粒属性
| 载体宿主： | 酵母菌 |
|---|---|
| 载体用途： | 蛋白表达 |
| 基因种属： | 空载体 |
| 基因类型： | ORF |
| 原核抗性： | Amp |
| 筛选标记： | TRP1 |
质粒简介
质粒图谱
质粒序列
LOCUS Exported 5870 bp ds-DNA circular SYN 29-NOV-2013
DEFINITION High-copy episomal vector with a TRP1 marker for galactose-inducible
expression of C-terminally tagged proteins in S. cerevisiae.
ACCESSION .
VERSION .
KEYWORDS pYES3 CT
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5870)
AUTHORS Invitrogen (Life Technologies)
TITLE Direct Submission
JOURNAL Exported Sunday, September 11, 2016 from SnapGene Viewer 3.1.4
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..5870
/organism="synthetic DNA construct"
/lab_host="Saccharomyces cerevisiae"
/mol_type="other DNA"
promoter 2..443
/gene="S. cerevisiae GAL1"
/note="GAL1 promoter"
/note="inducible promoter, regulated by Gal4"
protein_bind 2..119
/bound_moiety="Gal4"
/note="UAS"
/note="upstream activating sequence mediating
Gal4-dependent induction"
promoter 475..493
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
misc_feature 501..600
/note="MCS"
/note="multiple cloning site"
CDS 607..648
/codon_start=1
/product="epitope tag from simian virus 5"
/note="V5 tag"
/translation="GKPIPNPLLGLDST"
CDS 658..675
/codon_start=1
/product="6xHis affinity tag"
/note="6xHis"
/translation="HHHHHH"
terminator 709..956
/gene="S. cerevisiae CYC1"
/note="CYC1 terminator"
/note="transcription terminator for CYC1"
rep_origin complement(1204..1792)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(1963..2823)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKRSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(2824..2928)
/gene="bla"
/note="AmpR promoter"
promoter 3031..3132
/gene="S. cerevisiae TRP1"
/note="TRP1 promoter"
CDS 3133..3807
/codon_start=1
/gene="S. cerevisiae TRP1"
/product="phosphoribosylanthranilate isomerase, required
for tryptophan biosynthesis"
/note="TRP1"
/note="yeast auxotrophic marker"
/translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
KK"
rep_origin 4449..5329
/note="2u ori"
/note="yeast 2u plasmid origin of replication"
rep_origin complement(5398..5853)
/direction=LEFT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
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 ccattgctac
2281 aggcatcgtg gtgtcacgct 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 cggcgtcaac
2581 acgggataat accgcgccac 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 attattgaag catttatcag ggttattgtc tcatgagcgg
2881 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
2941 aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag
3001 gcgtatcacg aggccctttc gtcttcaaga aattcggtcg aaaaaagaaa aggagagggc
3061 caagagggag ggcattggtg actattgagc acgtgagtat acgtgattaa gcacacaaag
3121 gcagcttgga gtatgtctgt tattaatttc acaggtagtt ctggtccatt ggtgaaagtt
3181 tgcggcttgc agagcacaga ggccgcagaa tgtgctctag attccgatgc tgacttgctg
3241 ggtattatat gtgtgcccaa tagaaagaga acaattgacc cggttattgc aaggaaaatt
3301 tcaagtcttg taaaagcata taaaaatagt tcaggcactc cgaaatactt ggttggcgtg
3361 tttcgtaatc aacctaagga ggatgttttg gctctggtca atgattacgg cattgatatc
3421 gtccaactgc acggagatga gtcgtggcaa gaataccaag agttcctcgg tttgccagtt
3481 attaaaagac tcgtatttcc aaaagactgc aacatactac tcagtgcagc ttcacagaaa
3541 cctcattcgt ttattccctt gtttgattca gaagcaggtg ggacaggtga acttttggat
3601 tggaactcga tttctgactg ggttggaagg caagagagcc ccgagagctt acattttatg
3661 ttagctggtg gactgacgcc agaaaatgtt ggtgatgcgc ttagattaaa tggcgttatt
3721 ggtgttgatg taagcggagg tgtggagaca aatggtgtaa aagactctaa caaaatagca
3781 aatttcgtca aaaatgctaa gaaataggtt attactgagt agtatttatt taagtattgt
3841 ttgtgcactt gccctagctt atcgatgata agctgtcaaa gatgagaatt aattccacgg
3901 actatagact atactagata ctccgtctac tgtacgatac acttccgctc aggtccttgt
3961 cctttaacga ggccttacca ctcttttgtt actctattga tccagctcag caaaggcagt
4021 gtgatctaag attctatctt cgcgatgtag taaaactagc tagaccgaga aagagactag
4081 aaatgcaaaa ggcacttcta caatggctgc catcattatt atccgatgtg acgctgcagc
4141 ttctcaatga tattcgaata cgctttgagg agatacagcc taatatccga caaactgttt
4201 tacagattta cgatcgtact tgttacccat cattgaattt tgaacatccg aacctgggag
4261 ttttccctga aacagatagt atatttgaac ctgtataata atatatagtc tagcgcttta
4321 cggaagacaa tgtatgtatt tcggttcctg gagaaactat tgcatctatt gcataggtaa
4381 tcttgcacgt cgcatccccg gttcattttc tgcgtttcca tcttgcactt caatagcata
4441 tctttgttaa cgaagcatct gtgcttcatt ttgtagaaca aaaatgcaac gcgagagcgc
4501 taatttttca aacaaagaat ctgagctgca tttttacaga acagaaatgc aacgcgaaag
4561 cgctatttta ccaacgaaga atctgtgctt catttttgta aaacaaaaat gcaacgcgac
4621 gagagcgcta atttttcaaa caaagaatct gagctgcatt tttacagaac agaaatgcaa
4681 cgcgagagcg ctattttacc aacaaagaat ctatacttct tttttgttct acaaaaatgc
4741 atcccgagag cgctattttt ctaacaaagc atcttagatt actttttttc tcctttgtgc
4801 gctctataat gcagtctctt gataactttt tgcactgtag gtccgttaag gttagaagaa
4861 ggctactttg gtgtctattt tctcttccat aaaaaaagcc tgactccact tcccgcgttt
4921 actgattact agcgaagctg cgggtgcatt ttttcaagat aaaggcatcc ccgattatat
4981 tctataccga tgtggattgc gcatactttg tgaacagaaa gtgatagcgt tgatgattct
5041 tcattggtca gaaaattatg aacggtttct tctattttgt ctctatatac tacgtatagg
5101 aaatgtttac attttcgtat tgttttcgat tcactctatg aatagttctt actacaattt
5161 ttttgtctaa agagtaatac tagagataaa cataaaaaat gtagaggtcg agtttagatg
5221 caagttcaag gagcgaaagg tggatgggta ggttatatag ggatatagca cagagatata
5281 tagcaaagag atacttttga gcaatgtttg tggaagcggt attcgcaatg ggaagctcca
5341 ccccggttga taatcagaaa agccccaaaa acaggaagat tgtataagca aatatttaaa
5401 ttgtaaacgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
5461 ttaacgaata gcccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag
5521 ggttgagtgt tgttccagtt tccaacaaga gtccactatt aaagaacgtg gactccaacg
5581 tcaaagggcg aaaaagggtc tatcagggcg atggcccact acgtgaacca tcaccctaat
5641 caagtttttt ggggtcgagg tgccgtaaag cagtaaatcg gaagggtaaa cggatgcccc
5701 catttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
5761 aaggagcggg ggctagggcg gtgggaagtg taggggtcac gctgggcgta accaccacac
5821 ccgccgcgct taatggggcg ctacagggcg cgtggggatg atccactagt
//
质粒菌株产品操作说明书
一、扩增流程
收到产品后，请先根据产品管壁标签来判断产品形式，并在扩增前准确查找该质粒菌株的抗性、感受态和培养温度。
1、质粒干粉（常温运输，存于-20度，90天保质期，请务必转化挑单克隆培养，不要直接使用和测序）
①收到质粒干粉后请先5000rpm离心1min，再加入20μl ddH2O去离子水溶解质粒；
②取1支100μl 感受态于冰上解冻10min，加入2μl质粒，再冰浴30min后，42℃热激60s，不要搅动，再冰浴2min；（从第二步开始均要在超净工作台中无菌操作）
③加入900μl无抗的LB液体培养基，180rpm震荡37℃培养45min；
④6000rpm离心5min，仅留100μl上清液重悬细菌沉淀，并涂布至目标质粒抗性的LB平板上；（可使用本平台的平板涂布专用玻璃珠进行涂布，可以比传统涂布方法获得更多转化子）
⑤将平板正向培养1h，再倒置37℃培养14h。如果要求是30度则培养20h；
（菌落过多则将质粒稀释后再转化。没有菌落则加入10μl质粒转化。另不要直接转表达感受态，要先转克隆感受态，重提质粒后再导入表达感受态）
⑥挑取单菌落至LB液体培养基中，加入对应抗生素，220rpm震荡培养14h，根据实验需要和质粒提取试剂盒说明书提取质粒。
2、甘油菌种（冰袋运输，存于-80℃，保质期90天，请务必划线挑单克隆培养）
四区划线后挑单菌落培养，酵母菌需要先液体复苏再四区划线，再挑单菌落液体培养。
3、穿刺菌种（冰袋运输，存于4℃，保质期7天）
穿刺接种，液体培养后四区划线，再挑单菌落液体培养。
4、菌落平板（冰袋运输，存于4℃，保质期7天）
直接挑取单菌落至液体培养基中。
5、液体质粒（冰袋运输，存于-20℃，保质期90天）
单独提取的液体质粒收到后可直接使用。
6、滤纸质粒（常温运输，存于-20度，90天保质期，请务必转化挑单克隆培养，不要直接使用和测序）
收到货后将滤纸画圈部分剪下放入EP管中，加100ul无菌水将滤纸浸湿并浸泡5min，吸取5ul质粒转化，离心全涂。
二、转化图片
| P4879/pETconNK酵母表达质粒 |
| P0232/pABAI酵母单杂交质粒 |
| P0233/p53-ABAI酵母单杂交质粒 |
| P0234/pGADT7-Rec2酵母单杂交质粒 |
| P0421/pHIS2酵母单杂交质粒 |
| P1136/p53-His2酵母单杂交质粒 |
| P0948/pDEST22酵母单杂交质粒 |
| P1808/pLacZi酿酒酵母单杂交质粒 |
| P0228/pGADT7-AD酵母双杂交质粒 |
| P1217/pGADT7-GW酵母双杂交质粒 |
风险提示：丁香通仅作为第三方平台，为商家信息发布提供平台空间。用户咨询产品时请注意保护个人信息及财产安全，合理判断，谨慎选购商品，商家和用户对交易行为负责。对于医疗器械类产品，请先查证核实企业经营资质和医疗器械产品注册证情况。
pYES3/CT酿酒酵母质粒
￥100 - 1000
