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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：
pRS316
- 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：pRS316酿酒酵母质粒
别称: pRS316
质粒属性
质粒简介
This is one of a series of pBluescriptbased YCtype (centromeric) yeast shuttle vectors (ATCC 7714277145) differing in the yeast selectable marker gene. It encodes the beta galactosidase alpha peptide for blue white color detection of inserts and also has the T7 and T3 promoters for in vitro RNA synthesis and priming sites for sequencing. It is useful in plasmid shuffling experiments. It was constructed by inserting a 1.112 kb fragment containing the URA3 gene into the NdeI site of the pRSS56 vector and also inserting a cassette containing CEN6 and the ARS associated with histone 4 (ARSH4) into the AatII site of the same vector. All ends were blunted. The pRSS56 vector was constructed by ligating a PvuI fragment (bp 4982412) of pBluescript KS+ and the fragment from the unique NdeI and AatII sites between bla and f1 origin of pBS(+). The order of the major features in this plasmid is: URA3 f1 ori (NaeI) T7 promoter lacZ/MCS T3 promoter pMB1 ori bla CEN6 ARSH4.
质粒图谱
质粒序列
LOCUS Exported 4887 bp ds-DNA circular SYN 12-SEP-2016
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS Untitled 2
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4887)
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..4887
/organism="synthetic DNA construct"
/mol_type="other DNA"
misc_feature 70..573
/note="CEN/ARS"
/note="S. cerevisiae CEN6 centromere fused to an
autonomously replicating sequence"
promoter 610..714
/gene="bla"
/note="AmpR promoter"
CDS 715..1575
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFFHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 1746..2334
/direction=RIGHT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
promoter 2658..2688
/note="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 2696..2712
/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)."
primer_bind 2720..2736
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
CDS 2732..3307
/codon_start=1
/gene="lacZ fragment"
/product="LacZ-alpha fragment of beta-galactosidase"
/note="lacZ-alpha"
/translation="MTMITPSSELTLTKGNKSWVPGPPSRSTVSISLISNSCSPGDPLV
LERPPPRWSSNSPYSESYYNSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
DRPSQQLRSLNGEWRDAPCSGALSAAGVVVTRSVTATLASALAPAPFAFFPSFLATFAG
FPRQALNRGLPLGFRFSALRHLDPKKLD"
promoter 2757..2775
/note="T3 promoter"
/note="promoter for bacteriophage T3 RNA polymerase"
misc_feature 2788..2895
/note="MCS"
/note="pBluescript multiple cloning site"
primer_bind 2805..2821
/note="KS primer"
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(2855..2871)
/note="SK primer"
/note="common sequencing primer, one of multiple similar
variants"
promoter complement(2904..2922)
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(2929..2945)
/note="M13 fwd"
/note="common sequencing primer, one of multiple similar
variants"
rep_origin 3090..3545
/direction=RIGHT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
CDS complement(3674..4477)
/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(4478..4693)
/gene="S. cerevisiae URA3"
/note="URA3 promoter"
ORIGIN
1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
61 cttaggacgg atcgcttgcc tgtaacttac acgcgcctcg tatcttttaa tgatggaata
121 atttgggaat ttactctgtg tttatttatt tttatgtttt gtatttggat tttagaaagt
181 aaataaagaa ggtagaagag ttacggaatg aagaaaaaaa aataaacaaa ggtttaaaaa
241 atttcaacaa aaagcgtact ttacatatat atttattaga caagaaaagc agattaaata
301 gatatacatt cgattaacga taagtaaaat gtaaaatcac aggattttcg tgtgtggtct
361 tctacacaga caagatgaaa caattcggca ttaatacctg agagcaggaa gagcaagata
421 aaaggtagta tttgttggcg atccccctag agtcttttac atcttcggaa aacaaaaact
481 attttttctt taatttcttt ttttactttc tatttttaat ttatatattt atattaaaaa
541 atttaaatta taattatttt tatagcacgt gatgaaaagg acccaggtgg cacttttcgg
601 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
661 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
721 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
781 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
841 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
901 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
961 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
1021 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
1081 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
1141 ccgaaggagc taaccgcttt ttttcacaac atgggggatc atgtaactcg ccttgatcgt
1201 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
1261 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
1321 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
1381 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
1441 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
1501 ggcagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
1561 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
1621 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
1681 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
1741 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
1801 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
1861 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
1921 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
1981 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
2041 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
2101 acgacctaca ccgaactgag atacctacag cgtgagcatt gagaaagcgc cacgcttccc
2161 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
2221 agggagcttc caggggggaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
2281 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc cgagcctatg gaaaaacgcc
2341 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
2401 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
2461 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
2521 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
2581 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttacctcact
2641 cattaggcac cccaggcttt acactttatg cttccggctc ctatgttgtg tggaattgtg
2701 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa gctcggaatt
2761 aaccctcact aaagggaaca aaagctgggt accgggcccc ccctcgaggt cgacggtatc
2821 gataagcttg atatcgaatt cctgcagccc gggggatcca ctagttctag agcggccgcc
2881 accgcggtgg agctccaatt cgccctatag tgagtcgtat tacaattcac tggccgtcgt
2941 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca
3001 tccccccttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
3061 gttgcgcagc ctgaatggcg aatggcgcga cgcgccctgt agcggcgcat taagcgcggc
3121 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
3181 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
3241 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
3301 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
3361 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
3421 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
3481 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac
3541 aatttcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcagggta
3601 ataactgata taattaaatt gaagctctaa tttgtgagtt tagtatacat gcatttactt
3661 ataatacagt tttttagttt tgctggccgc atcttctcaa atatgcttcc cagcctgctt
3721 ttctgtaacg ttcaccctct accttagcat cccttccctt tgcaaatagt cctcttccaa
3781 caataataat gtcagatcct gtagagacca catcatccac ggttctatac tgttgaccca
3841 atgcgtctcc cttgtcatct aaacccacac cgggtgtcat aatcaaccaa tcgtaacctt
3901 catctcttcc acccatgtct ctttgagcaa taaagccgat aacaaaatct ttgtcgctct
3961 tcgcaatgtc aacagtaccc ttagtatatt ctccagtaga tagggagccc ttgcatgaca
4021 attctgctaa catcaaaagg cctctaggtt cctttgttac ttcttctgcc gcctgcttca
4081 aaccgctaac aatacctggg cccaccacac cgtgtgcatt cgtaatgtct gcccattctg
4141 ctattctgta tacacccgca gagtactgca atttgactgt attaccaatg tcagcaaatt
4201 ttctgtcttc gaagagtaaa aaattgtact tggcggataa tgcctttagc ggcttaactg
4261 tgccctccat ggaaaaatca gtcaagatat ccacatgtgt ttttagtaaa caaattttgg
4321 gacctaatgc ttcaactaac tccagtaatt ccttggtggt acgaacatcc aatgaagcac
4381 acaagtttgt ttgcttttcg tgcatgatat taaatagctt ggcagcaaca ggactaggat
4441 gagtagcagc acgttcctta tatgtagctt tcgacatgat ttatcttcgt ttcctgcagg
4501 tttttgttct gtgcagttgg gttaagaata ctgggcaatt tcatgtttct tcaacactac
4561 atatgcgtat atataccaat ctaagtctgt gctccttcct tcgttcttcc ttctgttcgg
4621 agattaccga atcaaaaaaa tttcaaagaa accgaaatca aaaaaaagaa taaaaaaaaa
4681 atgatgaatt gaattgaaaa gcgtggtgca ctctcagtac aatctgctct gatgccgcat
4741 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
4801 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
4861 tttcaccgtc atcaccgaaa cgcgcga
//
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：pRS316酿酒酵母质粒
别称: pRS316
| 启动子: | URA3，T7,T3 |
|---|---|
| 复制子: | pUC |
| 质粒分类: | 酵母系列，酿酒酵母表达载体 |
| 质粒大小: | 4887bp |
| 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 |
| 诱导方式: | 半乳糖诱导 |
| 5'测序引物: | T7:TAATACGACTCACTATAGGG |
| 3'测序引物: | M13R:CAGGAAACAGCTATGACC |
质粒属性
| 载体宿主： | 酵母菌 |
|---|---|
| 载体用途： | 蛋白表达 |
| 基因种属： | |
| 基因类型： | ORF |
| 原核抗性： | Amp |
| 筛选标记： | URA3 |
| 荧光蛋白： |
质粒简介
This is one of a series of pBluescriptbased YCtype (centromeric) yeast shuttle vectors (ATCC 7714277145) differing in the yeast selectable marker gene. It encodes the beta galactosidase alpha peptide for blue white color detection of inserts and also has the T7 and T3 promoters for in vitro RNA synthesis and priming sites for sequencing. It is useful in plasmid shuffling experiments. It was constructed by inserting a 1.112 kb fragment containing the URA3 gene into the NdeI site of the pRSS56 vector and also inserting a cassette containing CEN6 and the ARS associated with histone 4 (ARSH4) into the AatII site of the same vector. All ends were blunted. The pRSS56 vector was constructed by ligating a PvuI fragment (bp 4982412) of pBluescript KS+ and the fragment from the unique NdeI and AatII sites between bla and f1 origin of pBS(+). The order of the major features in this plasmid is: URA3 f1 ori (NaeI) T7 promoter lacZ/MCS T3 promoter pMB1 ori bla CEN6 ARSH4.
质粒图谱
质粒序列
LOCUS Exported 4887 bp ds-DNA circular SYN 12-SEP-2016
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS Untitled 2
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4887)
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..4887
/organism="synthetic DNA construct"
/mol_type="other DNA"
misc_feature 70..573
/note="CEN/ARS"
/note="S. cerevisiae CEN6 centromere fused to an
autonomously replicating sequence"
promoter 610..714
/gene="bla"
/note="AmpR promoter"
CDS 715..1575
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFFHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 1746..2334
/direction=RIGHT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
promoter 2658..2688
/note="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 2696..2712
/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)."
primer_bind 2720..2736
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
CDS 2732..3307
/codon_start=1
/gene="lacZ fragment"
/product="LacZ-alpha fragment of beta-galactosidase"
/note="lacZ-alpha"
/translation="MTMITPSSELTLTKGNKSWVPGPPSRSTVSISLISNSCSPGDPLV
LERPPPRWSSNSPYSESYYNSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
DRPSQQLRSLNGEWRDAPCSGALSAAGVVVTRSVTATLASALAPAPFAFFPSFLATFAG
FPRQALNRGLPLGFRFSALRHLDPKKLD"
promoter 2757..2775
/note="T3 promoter"
/note="promoter for bacteriophage T3 RNA polymerase"
misc_feature 2788..2895
/note="MCS"
/note="pBluescript multiple cloning site"
primer_bind 2805..2821
/note="KS primer"
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(2855..2871)
/note="SK primer"
/note="common sequencing primer, one of multiple similar
variants"
promoter complement(2904..2922)
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(2929..2945)
/note="M13 fwd"
/note="common sequencing primer, one of multiple similar
variants"
rep_origin 3090..3545
/direction=RIGHT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
CDS complement(3674..4477)
/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(4478..4693)
/gene="S. cerevisiae URA3"
/note="URA3 promoter"
ORIGIN
1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
61 cttaggacgg atcgcttgcc tgtaacttac acgcgcctcg tatcttttaa tgatggaata
121 atttgggaat ttactctgtg tttatttatt tttatgtttt gtatttggat tttagaaagt
181 aaataaagaa ggtagaagag ttacggaatg aagaaaaaaa aataaacaaa ggtttaaaaa
241 atttcaacaa aaagcgtact ttacatatat atttattaga caagaaaagc agattaaata
301 gatatacatt cgattaacga taagtaaaat gtaaaatcac aggattttcg tgtgtggtct
361 tctacacaga caagatgaaa caattcggca ttaatacctg agagcaggaa gagcaagata
421 aaaggtagta tttgttggcg atccccctag agtcttttac atcttcggaa aacaaaaact
481 attttttctt taatttcttt ttttactttc tatttttaat ttatatattt atattaaaaa
541 atttaaatta taattatttt tatagcacgt gatgaaaagg acccaggtgg cacttttcgg
601 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
661 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
721 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
781 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
841 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
901 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
961 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
1021 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
1081 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
1141 ccgaaggagc taaccgcttt ttttcacaac atgggggatc atgtaactcg ccttgatcgt
1201 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
1261 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
1321 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
1381 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
1441 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
1501 ggcagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
1561 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
1621 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
1681 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
1741 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
1801 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
1861 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
1921 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
1981 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
2041 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
2101 acgacctaca ccgaactgag atacctacag cgtgagcatt gagaaagcgc cacgcttccc
2161 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
2221 agggagcttc caggggggaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
2281 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc cgagcctatg gaaaaacgcc
2341 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
2401 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
2461 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
2521 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
2581 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttacctcact
2641 cattaggcac cccaggcttt acactttatg cttccggctc ctatgttgtg tggaattgtg
2701 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa gctcggaatt
2761 aaccctcact aaagggaaca aaagctgggt accgggcccc ccctcgaggt cgacggtatc
2821 gataagcttg atatcgaatt cctgcagccc gggggatcca ctagttctag agcggccgcc
2881 accgcggtgg agctccaatt cgccctatag tgagtcgtat tacaattcac tggccgtcgt
2941 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca
3001 tccccccttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
3061 gttgcgcagc ctgaatggcg aatggcgcga cgcgccctgt agcggcgcat taagcgcggc
3121 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
3181 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
3241 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
3301 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
3361 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
3421 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
3481 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac
3541 aatttcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcagggta
3601 ataactgata taattaaatt gaagctctaa tttgtgagtt tagtatacat gcatttactt
3661 ataatacagt tttttagttt tgctggccgc atcttctcaa atatgcttcc cagcctgctt
3721 ttctgtaacg ttcaccctct accttagcat cccttccctt tgcaaatagt cctcttccaa
3781 caataataat gtcagatcct gtagagacca catcatccac ggttctatac tgttgaccca
3841 atgcgtctcc cttgtcatct aaacccacac cgggtgtcat aatcaaccaa tcgtaacctt
3901 catctcttcc acccatgtct ctttgagcaa taaagccgat aacaaaatct ttgtcgctct
3961 tcgcaatgtc aacagtaccc ttagtatatt ctccagtaga tagggagccc ttgcatgaca
4021 attctgctaa catcaaaagg cctctaggtt cctttgttac ttcttctgcc gcctgcttca
4081 aaccgctaac aatacctggg cccaccacac cgtgtgcatt cgtaatgtct gcccattctg
4141 ctattctgta tacacccgca gagtactgca atttgactgt attaccaatg tcagcaaatt
4201 ttctgtcttc gaagagtaaa aaattgtact tggcggataa tgcctttagc ggcttaactg
4261 tgccctccat ggaaaaatca gtcaagatat ccacatgtgt ttttagtaaa caaattttgg
4321 gacctaatgc ttcaactaac tccagtaatt ccttggtggt acgaacatcc aatgaagcac
4381 acaagtttgt ttgcttttcg tgcatgatat taaatagctt ggcagcaaca ggactaggat
4441 gagtagcagc acgttcctta tatgtagctt tcgacatgat ttatcttcgt ttcctgcagg
4501 tttttgttct gtgcagttgg gttaagaata ctgggcaatt tcatgtttct tcaacactac
4561 atatgcgtat atataccaat ctaagtctgt gctccttcct tcgttcttcc ttctgttcgg
4621 agattaccga atcaaaaaaa tttcaaagaa accgaaatca aaaaaaagaa taaaaaaaaa
4681 atgatgaatt gaattgaaaa gcgtggtgca ctctcagtac aatctgctct gatgccgcat
4741 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
4801 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
4861 tttcaccgtc atcaccgaaa cgcgcga
//
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
| P1995/pENTR223-RAB5A人源基因质粒 |
| P1996/pENTR223-NMNAT3人源基因质粒 |
| P1997/pENTR223-ZC3HC1人源基因质粒 |
| P1998/pENTR223-F8人源基因质粒 |
| P1999/pENTR223-MOB1B人源基因质粒 |
| P2000/pENTR223-TIMP3-T9G-A620人源基因质粒 |
| P2001/pENTR223-PITHD1人源基因质粒 |
| P2025/pENTR223-TRIM11人源基因质粒 |
| P2026/pENTR223-TM2D1人源基因质粒 |
| P2027/pENTR223-SCRN3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
(like the pRS316 vector) and transforming the MATalpha strain with, for example, any TRP1 plasmid (like the pRS314 plasmid) and then mating the two strains together--only the diploid will grow on the minimal plates. Materials MATa and MATalpha
pRS316酿酒酵母质粒
￥100 - 1000
