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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：
2年
- 英文名：
pYES2/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：pYES2/CT酿酒酵母质粒
别称: pYES2/CT
质粒属性
质粒简介
The pYC2/CT vector contains the CEN6/ARSH4 sequence (Sikorski and Hieter, 1989) for maintenance and replication in yeast. The CEN6/ARSH4 sequence is a 518 bp hybrid DNA fragment that contains a yeast centromere sequence (CEN) and an autonomously replicating sequence (ARS) (Sikorski and Hieter, 1989). The CEN6 sequence is derived from the CEN6 locus of yeast chromosome 6 (Panzeri and Philippsen, 1982) while the ARSH4 sequence is derived from the yeast histone H4-associated ARS (Bouton and Smith, 1986). When placed in a heterologous expression plasmid (i.e. pYC2/CT), the presence of the CEN6/ARSH4 sequence allows non-integrative centromeric maintenance and low copy number replication of the plasmid (generally 1–2 copies per cell).
质粒图谱
质粒序列
LOCUS Exported 5963 bp ds-DNA circular SYN 11-SEP-2016
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS Untitled 4
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5963)
AUTHORS .
TITLE Direct Submission
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..5963
/organism="synthetic DNA construct"
/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"
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
ELDLNSGKILESFRPEERFPMMSTFKVLLCDTLLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDESDTTMPVAMPTTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKRSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
CDS complement(2919..3722)
/codon_start=1
/gene="S. cerevisiae URA3"
/product="orotidine-5'-phosphate decarboxylase, required
for uracil biosynthesis"
/note="URA3"
/note="yeast auxotrophic marker, counterselectable with
5-fluoroorotic acid (5-FOA)"
/translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
promoter complement(3723..3938)
/gene="S. cerevisiae URA3"
/note="URA3 promoter"
rep_origin 4542..5422
/note="2u ori"
/note="yeast 2u plasmid origin of replication"
rep_origin complement(5491..5946)
/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 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
//
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：pYES2/CT酿酒酵母质粒
别称: pYES2/CT
| 启动子: | GAL1 |
|---|---|
| 复制子: | pUC,2U |
| 终止子: | CYC1 |
| 质粒大小: | 5963bp |
| 原核抗性: | Amp |
| 筛选标记: | URA3 |
| 克隆菌株: | DH5a |
| 培养条件: | 37度 |
| 表达宿主: | 酵母细胞 |
| 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： | URA3 |
| 荧光蛋白： |
质粒简介
The pYC2/CT vector contains the CEN6/ARSH4 sequence (Sikorski and Hieter, 1989) for maintenance and replication in yeast. The CEN6/ARSH4 sequence is a 518 bp hybrid DNA fragment that contains a yeast centromere sequence (CEN) and an autonomously replicating sequence (ARS) (Sikorski and Hieter, 1989). The CEN6 sequence is derived from the CEN6 locus of yeast chromosome 6 (Panzeri and Philippsen, 1982) while the ARSH4 sequence is derived from the yeast histone H4-associated ARS (Bouton and Smith, 1986). When placed in a heterologous expression plasmid (i.e. pYC2/CT), the presence of the CEN6/ARSH4 sequence allows non-integrative centromeric maintenance and low copy number replication of the plasmid (generally 1–2 copies per cell).
质粒图谱
质粒序列
LOCUS Exported 5963 bp ds-DNA circular SYN 11-SEP-2016
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS Untitled 4
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5963)
AUTHORS .
TITLE Direct Submission
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..5963
/organism="synthetic DNA construct"
/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"
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
ELDLNSGKILESFRPEERFPMMSTFKVLLCDTLLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDESDTTMPVAMPTTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKRSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
CDS complement(2919..3722)
/codon_start=1
/gene="S. cerevisiae URA3"
/product="orotidine-5'-phosphate decarboxylase, required
for uracil biosynthesis"
/note="URA3"
/note="yeast auxotrophic marker, counterselectable with
5-fluoroorotic acid (5-FOA)"
/translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
promoter complement(3723..3938)
/gene="S. cerevisiae URA3"
/note="URA3 promoter"
rep_origin 4542..5422
/note="2u ori"
/note="yeast 2u plasmid origin of replication"
rep_origin complement(5491..5946)
/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 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
//
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
| P1269/pESC-HIS酿酒酵母质粒 |
| P0224/pYES2酿酒酵母质粒 |
| P0240/pYES2/CT酿酒酵母质粒 |
| P1258/pYES2/CT-α-Factor酿酒酵母质粒 |
| P1680/pYES2/NTA酿酒酵母质粒 |
| P0410/pYES3/CT酿酒酵母质粒 |
| P1259/pYES6/CT酿酒酵母质粒 |
| P2169/pYES260酿酒酵母质粒 |
| P0434/pYEp352酿酒酵母质粒 |
| P1696/YEplac181酿酒酵母质粒 |
| P1807/YEplac195酿酒酵母质粒 |
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文献和实验相关实验
, we may find much more references regarding this issue. http://books.google.com/books?id=3mgPzNFjcWAC&pg=PA51&lpg=PA51&dq=gsk+tyrosine+phosphorylation+and+activity&source=bl&ots=x5ueTldpez&sig=CqRFJleXtEBn9OfelhODPAc28xs&hl=en&ei=KerNSu-XGcem8Abm-qmABA&sa=X&oi=book_result&ct
相关专题 酶联免疫斑点法（ELISpot） 酵母细胞的细胞壁比较厚，不容易破壁，不如大肠杆菌的质粒 容易提取，最近做了些酿酒酵母的实验，从酿酒酵母中提取质粒，现在就总结下实验的方法和步骤。 酵母细胞质粒 提取步骤 1. 接种单菌落(待检测酵母细胞)于25mLYNB(补加氨基酸营养物)培养基中，30℃振荡培养过夜。 2.第二天取一滴菌液于进行显微镜下观察,目镜用16,物镜用40倍观察细胞壁破碎前
pYES2.0 酵母质粒 Amp 酿酒酵母载体 pRS403 酵母质粒 pRS423 酵母质粒 pRC426gal 酵母质粒
pYES2/CT酿酒酵母质粒
￥100 - 1000
