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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：
pRS425
- 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：pRS425酿酒酵母质粒
别称: pRS425
质粒属性
质粒简介
This is a YEtype(episomal) yeast shuttle vector. It encodes the beta galactosidase alpha peptide for bluewhite color detection of inserts and also has the T7 and T3 promoters for in vitro RNA synthesis and priming sites for sequencing. It also contains the REP3 and FRT sequences necessary for high copy propagation in yeast. In S. cerevisiae, the copy number is about 20 per haploid cell. In nonselective growth, plasmids are lost through mitotic segregation at rates in the range of 4.6 +/ 0.6% of progeny per doubling. The order of the major features in this plasmid is: ori(f1) lacZ T7 promoter MCS (KpnISacI) T3 promoter lacI ori(pMB1) ampR ori(2 micron) LEU2
质粒图谱
质粒序列
LOCUS Exported 6849 bp ds-DNA circular SYN 12-SEP-2016
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS Untitled 7
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6849)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Monday, September 12, 2016 from SnapGene Viewer 3.1.4
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..6849
/organism="synthetic DNA construct"
/mol_type="other DNA"
CDS complement(663..1757)
/codon_start=1
/gene="S. cerevisiae LEU2"
/product="3-isopropylmalate dehydrogenase, required for
leucine biosynthesis"
/note="LEU2"
/note="yeast auxotrophic marker"
/translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
IGGAAIDATGVPLPDEALEASKKVDAVLLGAVAGPKWGTGSVRPEQGLLKIRKELQLYA
NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
VPEVQRITRMAAFMALQHEPPLPIWSLDKANLLASSRLWRKTVEETIKNEFPTLKVQHQ
LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
GLYEPCHGSAPDLPKNKVDPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
DLGGSNSTTEVGDAVAEEVKKILA"
promoter complement(1770..2174)
/gene="S. cerevisiae LEU2"
/note="LEU2 promoter"
rep_origin complement(2474..2929)
/direction=LEFT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
CDS complement(2712..3290)
/codon_start=1
/gene="lacZ fragment"
/product="LacZ-alpha fragment of beta-galactosidase"
/note="lacZ-alpha"
/translation="MTMITPSAQLTLTKGNKSWSSTAVAAALELVDPPGCRNSISSLSI
PSTSRGGPVPNSPYSESYYARSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEAR
TDRPSQQLRSLNGEWRDAPCSGALSAAGVVVTRSVTATLASALAPAPFAFFPSFLATFA
GFPRQALNRGLPLGFRFSALRHLDPKKLD"
primer_bind 3074..3090
/note="M13 fwd"
/note="common sequencing primer, one of multiple similar
variants"
promoter 3100..3118
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
misc_feature 3127..3234
/note="MCS"
/note="pBluescript multiple cloning site"
primer_bind 3144..3160
/note="KS primer"
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(3194..3210)
/note="SK primer"
/note="common sequencing primer, one of multiple similar
variants"
promoter complement(3247..3265)
/note="T3 promoter"
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind complement(3286..3302)
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 3310..3326
/bound_moiety="lac repressor encoded by lacI"
/note="lac operator"
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
promoter complement(3334..3364)
/note="lac promoter"
/note="promoter for the E. coli lac operon"
rep_origin complement(3688..4276)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(4447..5307)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(5308..5412)
/gene="bla"
/note="AmpR promoter"
rep_origin 5439..6781
/note="2u ori"
/note="yeast 2u plasmid origin of replication"
protein_bind complement(6402..6449)
/bound_moiety="FLP recombinase from the Saccharomyces
cerevisiae 2u plasmid"
/note="FRT"
/note="FLP-mediated recombination occurs in the 8-bp core
sequence TCTAGAAA (Turan and Bode, 2011)."
ORIGIN
1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
181 accatatcga ctacgtcgta aggccgtttc tgacagagta aaattcttga gggaactttc
241 accattatgg gaaatgcttc aagaaggtat tgacttaaac tccatcaaat ggtcaggtca
301 ttgagtgttt tttatttgtt gtattttttt ttttttagag aaaatcctcc aatatcaaat
361 taggaatcgt agtttcatga ttttctgtta cacctaactt tttgtgtggt gccctcctcc
421 ttgtcaatat taatgttaaa gtgcaattct ttttccttat cacgttgagc cattagtatc
481 aatttgctta cctgtattcc tttactatcc tcctttttct ccttcttgat aaatgtatgt
541 agattgcgta tatagtttcg tctaccctat gaacatattc cattttgtaa tttcgtgtcg
601 tttctattat gaatttcatt tataaagttt atgtacaaat atcataaaaa aagagaatct
661 ttttaagcaa ggattttctt aacttcttcg gcgacagcat caccgacttc ggtggtactg
721 ttggaaccac ctaaatcacc agttctgata cctgcatcca aaaccttttt aactgcatct
781 tcaatggcct taccttcttc aggcaagttc aatgacaatt tcaacatcat tgcagcagac
841 aagatagtgg cgatagggtc aaccttattc tttggcaaat ctggagcaga accgtggcat
901 ggttcgtaca aaccaaatgc ggtgttcttg tctggcaaag aggccaagga cgcagatggc
961 aacaaaccca aggaacctgg gataacggag gcttcatcgg agatgatatc accaaacatg
1021 ttgctggtga ttataatacc atttaggtgg gttgggttct taactaggat catggcggca
1081 gaatcaatca attgatgttg aaccttcaat gtagggaatt cgttcttgat ggtttcctcc
1141 acagtttttc tccataatct tgaagaggcc aaaagattag ctttatccaa ggaccaaata
1201 ggcaatggtg gctcatgttg tagggccatg aaagcggcca ttcttgtgat tctttgcact
1261 tctggaacgg tgtattgttc actatcccaa gcgacaccat caccatcgtc ttcctttctc
1321 ttaccaaagt aaatacctcc cactaattct ctgacaacaa cgaagtcagt acctttagca
1381 aattgtggct tgattggaga taagtctaaa agagagtcgg atgcaaagtt acatggtctt
1441 aagttggcgt acaattgaag ttctttacgg atttttagta aaccttgttc aggtctaaca
1501 ctaccggtac cccatttagg accagccaca gcacctaaca aaacggcatc aaccttcttg
1561 gaggcttcca gcgcctcatc tggaagtggg acacctgtag catcgatagc agcaccacca
1621 attaaatgat tttcgaaatc gaacttgaca ttggaacgaa catcagaaat agctttaaga
1681 accttaatgg cttcggctgt gatttcttga ccaacgtggt cacctggcaa aacgacgatc
1741 ttcttagggg cagacatagg ggcagacatt agaatggtat atccttgaaa tatatatata
1801 tattgctgaa atgtaaaagg taagaaaagt tagaaagtaa gacgattgct aaccacctat
1861 tggaaaaaac aataggtcct taaataatat tgtcaacttc aagtattgtg atgcaagcat
1921 ttagtcatga acgcttctct attctatatg aaaagccggt tccggcctct cacctttcct
1981 ttttctccca atttttcagt tgaaaaaggt atatgcgtca ggcgacctct gaaattaaca
2041 aaaaatttcc agtcatcgaa tttgattctg tgcgatagcg cccctgtgtg ttctcgttat
2101 gttgaggaaa aaaataatgg ttgctaagag attcgaactc ttgcatctta cgatacctga
2161 gtattcccac agttaactgc ggtcaagata tttcttgaat caggcgcctt agaccgctcg
2221 gccaaacaac caattacttg ttgagaaata gagtataatt atcctataaa tataacgttt
2281 ttgaacacac atgaacaagg aagtacagga caattgattt tgaagagaat gtggattttg
2341 atgtaattgt tgggattcca tttttaataa ggcaataata ttaggtatgt ggatatacta
2401 gaagttctcc tcgaccgtcg atatgcggtg tgaaataccg cacagatgcg taaggagaaa
2461 ataccgcatc aggaaattgt aaacgttaat attttgttaa aattcgcgtt aaatttttgt
2521 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
2581 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
2641 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
2701 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
2761 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
2821 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
2881 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc gcgccattcg
2941 ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc
3001 cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc
3061 cagtcacgac gttgtaaaac gacggccagt gagcgcgcgt aatacgactc actatagggc
3121 gaattgggta ccgggccccc cctcgaggtc gacggtatcg ataagcttga tatcgaattc
3181 ctgcagcccg ggggatccac tagttctaga gcggccgcca ccgcggtgga gctccagctt
3241 ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc
3301 tgtgtgaaat tgttatccgc tcacaattcc acacaacata ggagccggaa gcataaagtg
3361 taaagcctgg ggtgcctaat gagtgaggta actcacatta attgcgttgc gctcactgcc
3421 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
3481 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
3541 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
3601 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
3661 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
3721 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
3781 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
3841 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
3901 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
3961 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
4021 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
4081 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg
4141 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
4201 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
4261 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
4321 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
4381 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
4441 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
4501 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
4561 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
4621 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
4681 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
4741 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
4801 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
4861 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
4921 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
4981 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
5041 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
5101 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
5161 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
5221 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
5281 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
5341 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
5401 aggggttccg cgcacatttc cccgaaaagt gccacctgaa cgaagcatct gtgcttcatt
5461 ttgtagaaca aaaatgcaac gcgagagcgc taatttttca aacaaagaat ctgagctgca
5521 tttttacaga acagaaatgc aacgcgaaag cgctatttta ccaacgaaga atctgtgctt
5581 catttttgta aaacaaaaat gcaacgcgag agcgctaatt tttcaaacaa agaatctgag
5641 ctgcattttt acagaacaga aatgcaacgc gagagcgcta ttttaccaac aaagaatcta
5701 tacttctttt ttgttctaca aaaatgcatc ccgagagcgc tatttttcta acaaagcatc
5761 ttagattact ttttttctcc tttgtgcgct ctataatgca gtctcttgat aactttttgc
5821 actgtaggtc cgttaaggtt agaagaaggc tactttggtg tctattttct cttccataaa
5881 aaaagcctga ctccacttcc cgcgtttact gattactagc gaagctgcgg gtgcattttt
5941 tcaagataaa ggcatccccg attatattct ataccgatgt ggattgcgca tactttgtga
6001 acagaaagtg atagcgttga tgattcttca ttggtcagaa aattatgaac ggtttcttct
6061 attttgtctc tatatactac gtataggaaa tgtttacatt ttcgtattgt tttcgattca
6121 ctctatgaat agttcttact acaatttttt tgtctaaaga gtaatactag agataaacat
6181 aaaaaatgta gaggtcgagt ttagatgcaa gttcaaggag cgaaaggtgg atgggtaggt
6241 tatataggga tatagcacag agatatatag caaagagata cttttgagca atgtttgtgg
6301 aagcggtatt cgcaatattt tagtagctcg ttacagtccg gtgcgttttt ggttttttga
6361 aagtgcgtct tcagagcgct tttggttttc aaaagcgctc tgaagttcct atactttcta
6421 gagaatagga acttcggaat aggaacttca aagcgtttcc gaaaacgagc gcttccgaaa
6481 atgcaacgcg agctgcgcac atacagctca ctgttcacgt cgcacctata tctgcgtgtt
6541 gcctgtatat atatatacat gagaagaacg gcatagtgcg tgtttatgct taaatgcgta
6601 cttatatgcg tctatttatg taggatgaaa ggtagtctag tacctcctgt gatattatcc
6661 cattccatgc ggggtatcgt atgcttcctt cagcactacc ctttagctgt tctatatgct
6721 gccactcctc aattggatta gtctcatcct tcaatgctat catttccttt gatattggat
6781 catactaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc
6841 cctttcgtc
//
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：pRS425酿酒酵母质粒
别称: pRS425
| 启动子: | LEU2，T7,T3 |
|---|---|
| 复制子: | 2U |
| 质粒分类: | 酵母系列，酿酒酵母表达载体 |
| 质粒大小: | 6849bp |
| 原核抗性: | Amp |
| 筛选标记: | LEU2 |
| 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: | M13R:CAGGAAACAGCTATGACC |
| 3'测序引物: | 根据序列设计引物 |
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： | LEU2 |
| 荧光蛋白： |
质粒简介
This is a YEtype(episomal) yeast shuttle vector. It encodes the beta galactosidase alpha peptide for bluewhite color detection of inserts and also has the T7 and T3 promoters for in vitro RNA synthesis and priming sites for sequencing. It also contains the REP3 and FRT sequences necessary for high copy propagation in yeast. In S. cerevisiae, the copy number is about 20 per haploid cell. In nonselective growth, plasmids are lost through mitotic segregation at rates in the range of 4.6 +/ 0.6% of progeny per doubling. The order of the major features in this plasmid is: ori(f1) lacZ T7 promoter MCS (KpnISacI) T3 promoter lacI ori(pMB1) ampR ori(2 micron) LEU2
质粒图谱
质粒序列
LOCUS Exported 6849 bp ds-DNA circular SYN 12-SEP-2016
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS Untitled 7
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6849)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Monday, September 12, 2016 from SnapGene Viewer 3.1.4
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..6849
/organism="synthetic DNA construct"
/mol_type="other DNA"
CDS complement(663..1757)
/codon_start=1
/gene="S. cerevisiae LEU2"
/product="3-isopropylmalate dehydrogenase, required for
leucine biosynthesis"
/note="LEU2"
/note="yeast auxotrophic marker"
/translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
IGGAAIDATGVPLPDEALEASKKVDAVLLGAVAGPKWGTGSVRPEQGLLKIRKELQLYA
NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
VPEVQRITRMAAFMALQHEPPLPIWSLDKANLLASSRLWRKTVEETIKNEFPTLKVQHQ
LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
GLYEPCHGSAPDLPKNKVDPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
DLGGSNSTTEVGDAVAEEVKKILA"
promoter complement(1770..2174)
/gene="S. cerevisiae LEU2"
/note="LEU2 promoter"
rep_origin complement(2474..2929)
/direction=LEFT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
CDS complement(2712..3290)
/codon_start=1
/gene="lacZ fragment"
/product="LacZ-alpha fragment of beta-galactosidase"
/note="lacZ-alpha"
/translation="MTMITPSAQLTLTKGNKSWSSTAVAAALELVDPPGCRNSISSLSI
PSTSRGGPVPNSPYSESYYARSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEAR
TDRPSQQLRSLNGEWRDAPCSGALSAAGVVVTRSVTATLASALAPAPFAFFPSFLATFA
GFPRQALNRGLPLGFRFSALRHLDPKKLD"
primer_bind 3074..3090
/note="M13 fwd"
/note="common sequencing primer, one of multiple similar
variants"
promoter 3100..3118
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
misc_feature 3127..3234
/note="MCS"
/note="pBluescript multiple cloning site"
primer_bind 3144..3160
/note="KS primer"
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(3194..3210)
/note="SK primer"
/note="common sequencing primer, one of multiple similar
variants"
promoter complement(3247..3265)
/note="T3 promoter"
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind complement(3286..3302)
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 3310..3326
/bound_moiety="lac repressor encoded by lacI"
/note="lac operator"
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
promoter complement(3334..3364)
/note="lac promoter"
/note="promoter for the E. coli lac operon"
rep_origin complement(3688..4276)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(4447..5307)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(5308..5412)
/gene="bla"
/note="AmpR promoter"
rep_origin 5439..6781
/note="2u ori"
/note="yeast 2u plasmid origin of replication"
protein_bind complement(6402..6449)
/bound_moiety="FLP recombinase from the Saccharomyces
cerevisiae 2u plasmid"
/note="FRT"
/note="FLP-mediated recombination occurs in the 8-bp core
sequence TCTAGAAA (Turan and Bode, 2011)."
ORIGIN
1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
181 accatatcga ctacgtcgta aggccgtttc tgacagagta aaattcttga gggaactttc
241 accattatgg gaaatgcttc aagaaggtat tgacttaaac tccatcaaat ggtcaggtca
301 ttgagtgttt tttatttgtt gtattttttt ttttttagag aaaatcctcc aatatcaaat
361 taggaatcgt agtttcatga ttttctgtta cacctaactt tttgtgtggt gccctcctcc
421 ttgtcaatat taatgttaaa gtgcaattct ttttccttat cacgttgagc cattagtatc
481 aatttgctta cctgtattcc tttactatcc tcctttttct ccttcttgat aaatgtatgt
541 agattgcgta tatagtttcg tctaccctat gaacatattc cattttgtaa tttcgtgtcg
601 tttctattat gaatttcatt tataaagttt atgtacaaat atcataaaaa aagagaatct
661 ttttaagcaa ggattttctt aacttcttcg gcgacagcat caccgacttc ggtggtactg
721 ttggaaccac ctaaatcacc agttctgata cctgcatcca aaaccttttt aactgcatct
781 tcaatggcct taccttcttc aggcaagttc aatgacaatt tcaacatcat tgcagcagac
841 aagatagtgg cgatagggtc aaccttattc tttggcaaat ctggagcaga accgtggcat
901 ggttcgtaca aaccaaatgc ggtgttcttg tctggcaaag aggccaagga cgcagatggc
961 aacaaaccca aggaacctgg gataacggag gcttcatcgg agatgatatc accaaacatg
1021 ttgctggtga ttataatacc atttaggtgg gttgggttct taactaggat catggcggca
1081 gaatcaatca attgatgttg aaccttcaat gtagggaatt cgttcttgat ggtttcctcc
1141 acagtttttc tccataatct tgaagaggcc aaaagattag ctttatccaa ggaccaaata
1201 ggcaatggtg gctcatgttg tagggccatg aaagcggcca ttcttgtgat tctttgcact
1261 tctggaacgg tgtattgttc actatcccaa gcgacaccat caccatcgtc ttcctttctc
1321 ttaccaaagt aaatacctcc cactaattct ctgacaacaa cgaagtcagt acctttagca
1381 aattgtggct tgattggaga taagtctaaa agagagtcgg atgcaaagtt acatggtctt
1441 aagttggcgt acaattgaag ttctttacgg atttttagta aaccttgttc aggtctaaca
1501 ctaccggtac cccatttagg accagccaca gcacctaaca aaacggcatc aaccttcttg
1561 gaggcttcca gcgcctcatc tggaagtggg acacctgtag catcgatagc agcaccacca
1621 attaaatgat tttcgaaatc gaacttgaca ttggaacgaa catcagaaat agctttaaga
1681 accttaatgg cttcggctgt gatttcttga ccaacgtggt cacctggcaa aacgacgatc
1741 ttcttagggg cagacatagg ggcagacatt agaatggtat atccttgaaa tatatatata
1801 tattgctgaa atgtaaaagg taagaaaagt tagaaagtaa gacgattgct aaccacctat
1861 tggaaaaaac aataggtcct taaataatat tgtcaacttc aagtattgtg atgcaagcat
1921 ttagtcatga acgcttctct attctatatg aaaagccggt tccggcctct cacctttcct
1981 ttttctccca atttttcagt tgaaaaaggt atatgcgtca ggcgacctct gaaattaaca
2041 aaaaatttcc agtcatcgaa tttgattctg tgcgatagcg cccctgtgtg ttctcgttat
2101 gttgaggaaa aaaataatgg ttgctaagag attcgaactc ttgcatctta cgatacctga
2161 gtattcccac agttaactgc ggtcaagata tttcttgaat caggcgcctt agaccgctcg
2221 gccaaacaac caattacttg ttgagaaata gagtataatt atcctataaa tataacgttt
2281 ttgaacacac atgaacaagg aagtacagga caattgattt tgaagagaat gtggattttg
2341 atgtaattgt tgggattcca tttttaataa ggcaataata ttaggtatgt ggatatacta
2401 gaagttctcc tcgaccgtcg atatgcggtg tgaaataccg cacagatgcg taaggagaaa
2461 ataccgcatc aggaaattgt aaacgttaat attttgttaa aattcgcgtt aaatttttgt
2521 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
2581 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
2641 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
2701 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
2761 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
2821 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
2881 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc gcgccattcg
2941 ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc
3001 cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc
3061 cagtcacgac gttgtaaaac gacggccagt gagcgcgcgt aatacgactc actatagggc
3121 gaattgggta ccgggccccc cctcgaggtc gacggtatcg ataagcttga tatcgaattc
3181 ctgcagcccg ggggatccac tagttctaga gcggccgcca ccgcggtgga gctccagctt
3241 ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc
3301 tgtgtgaaat tgttatccgc tcacaattcc acacaacata ggagccggaa gcataaagtg
3361 taaagcctgg ggtgcctaat gagtgaggta actcacatta attgcgttgc gctcactgcc
3421 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
3481 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
3541 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
3601 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
3661 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
3721 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
3781 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
3841 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
3901 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
3961 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
4021 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
4081 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg
4141 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
4201 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
4261 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
4321 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
4381 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
4441 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
4501 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
4561 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
4621 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
4681 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
4741 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
4801 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
4861 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
4921 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
4981 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
5041 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
5101 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
5161 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
5221 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
5281 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
5341 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
5401 aggggttccg cgcacatttc cccgaaaagt gccacctgaa cgaagcatct gtgcttcatt
5461 ttgtagaaca aaaatgcaac gcgagagcgc taatttttca aacaaagaat ctgagctgca
5521 tttttacaga acagaaatgc aacgcgaaag cgctatttta ccaacgaaga atctgtgctt
5581 catttttgta aaacaaaaat gcaacgcgag agcgctaatt tttcaaacaa agaatctgag
5641 ctgcattttt acagaacaga aatgcaacgc gagagcgcta ttttaccaac aaagaatcta
5701 tacttctttt ttgttctaca aaaatgcatc ccgagagcgc tatttttcta acaaagcatc
5761 ttagattact ttttttctcc tttgtgcgct ctataatgca gtctcttgat aactttttgc
5821 actgtaggtc cgttaaggtt agaagaaggc tactttggtg tctattttct cttccataaa
5881 aaaagcctga ctccacttcc cgcgtttact gattactagc gaagctgcgg gtgcattttt
5941 tcaagataaa ggcatccccg attatattct ataccgatgt ggattgcgca tactttgtga
6001 acagaaagtg atagcgttga tgattcttca ttggtcagaa aattatgaac ggtttcttct
6061 attttgtctc tatatactac gtataggaaa tgtttacatt ttcgtattgt tttcgattca
6121 ctctatgaat agttcttact acaatttttt tgtctaaaga gtaatactag agataaacat
6181 aaaaaatgta gaggtcgagt ttagatgcaa gttcaaggag cgaaaggtgg atgggtaggt
6241 tatataggga tatagcacag agatatatag caaagagata cttttgagca atgtttgtgg
6301 aagcggtatt cgcaatattt tagtagctcg ttacagtccg gtgcgttttt ggttttttga
6361 aagtgcgtct tcagagcgct tttggttttc aaaagcgctc tgaagttcct atactttcta
6421 gagaatagga acttcggaat aggaacttca aagcgtttcc gaaaacgagc gcttccgaaa
6481 atgcaacgcg agctgcgcac atacagctca ctgttcacgt cgcacctata tctgcgtgtt
6541 gcctgtatat atatatacat gagaagaacg gcatagtgcg tgtttatgct taaatgcgta
6601 cttatatgcg tctatttatg taggatgaaa ggtagtctag tacctcctgt gatattatcc
6661 cattccatgc ggggtatcgt atgcttcctt cagcactacc ctttagctgt tctatatgct
6721 gccactcctc aattggatta gtctcatcct tcaatgctat catttccttt gatattggat
6781 catactaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc
6841 cctttcgtc
//
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
| P2084/pENTR223-RNF141人源基因质粒 |
| P2085/pENTR223-ZBED6CL人源基因质粒 |
| P2086/pENTR223-ATP6V1E2人源基因质粒 |
| P2087/pENTR223-CDC34人源基因质粒 |
| P2088/pENTR223-ABCD3人源基因质粒 |
| P2089/pENTR223-C6orf142-A596C人源基因质粒 |
| P2091/pENTR223-CLDN12人源基因质粒 |
| P2092/pENTR223-C1QC人源基因质粒 |
| P2093/pENTR223-FAM108A1人源基因质粒 |
| P2094/pENTR223-ZNF174人源基因质粒 |
| P2095/pENTR223-EXOC7人源基因质粒 |
| P2096/pENTR223-UBE2U人源基因质粒 |
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文献和实验相关实验
了整合，传送http://www.91bio.com/carriers-website-to-find/），比如查找pRS类质粒图谱（注意，是一类质粒图谱，没关系，照样能找到），直接在搜索框输入pRS，可以看到，之类质粒一共有三十多个。 找到自己需要的质粒名称，点击进入，就可以看到质粒图谱了。 拿第一个质粒pRS413举例，如上图，质粒图谱是不是很难看，对，我也觉得很难看，没关系，看见view sequence了吗？点击进入，我们就得到该质粒图谱的序列了。 如何得到
方法一：使用 Vector NT 软件 invitrogen 公司的这款软件绝对是分子生物学虫子们的福音，要想对质粒图谱了解更直观，安装这款软件是非常必要的。这款软件的软件包里面会包括 invitrogen 公司的所有质粒图谱信息和其他比较常见和经典的质粒图谱。 方法二：查找质粒图谱的网站 1.Vector Database（addgene） 这个网站很页面很人性化，直入主题，以前叫做 lablife，现在网站做了整合，比如查找 pRS 类质粒图谱（注意
相关专题 完美酵母质粒提取实验Protocol 问：请教如何从酿酒酵母抽提质粒 ? 用胞壁松解酶处理以后，再用SDS处理，是否就可以破碎酵母了，因为是抽提质粒，不是抽基因组 DNA，可以不用蛋白酶K处理吗?请各位朋友提宝贵建议(不用玻璃珠破细胞)。我要尽可能把样品中的质粒都抽提出来，然后 REAL TIME PCR定量，好计算每个酵母细胞中的质粒拷贝数目。因为样品少，不能做SOUTHERN BOLT计算拷贝。谢谢
pRS425酿酒酵母质粒
￥100 - 1000
