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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：
pRS426
- 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：pRS426酿酒酵母质粒
别称: pRS426
质粒属性
质粒简介
The order of the major features in this plasmid is:ori(f1) - lacZ - T7 promoter - MCS (KpnI-SacI) - T3 promoter - lacI - ori(pMB1) - ampR - ori (2 micron) - URA3. 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 non-selective growth, plasmids are lost through mitotic segregation at rates in the range of 4.4 + or - 1.4% of progeny per doubling. YE-type shuttle vector encoding the beta-galactosidase alpha peptide, permitting blue-white visual detection. Can be used to produce ssDNA. Contains promoters for in vitro RNA synthesis and priming sites useful for sequencing. Restriction digests of the clone give the following sizes (kb): BamHI--5.8; EcoRI--5.8; PstI--4.0, 1.7; PvuII--5.2, 0.54. (ATCC staff) Medium is 1227 LB plus ampicillin. NCBI gi: 416322 Hosts: E.coli DH5alpha, Saccharomyces cerevisiae, E.coli.
质粒图谱
质粒序列
LOCUS Exported 5726 bp ds-DNA circular SYN 12-SEP-2016
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS Untitled 8
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5726)
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..5726
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 201..416
/gene="S. cerevisiae URA3"
/note="URA3 promoter"
CDS 417..1220
/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"
rep_origin complement(1351..1806)
/direction=LEFT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
CDS complement(1589..2167)
/codon_start=1
/gene="lacZ fragment"
/product="LacZ-alpha fragment of beta-galactosidase"
/note="lacZ-alpha"
/translation="MTMITPSAQLTLTKGNKSWSSTAVAAALELVDPPGCRNSISSLSI
PSTSRGGPVPNSPYSESYYARSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEAR
TDRPSQQLRSLNGEWRDAPCSGALSAAGVVVTRSVTATLASALAPAPFAFFPSFLATFA
GFPRQALNRGLPLGFRFSALRHLDPKKLD"
primer_bind 1951..1967
/note="M13 fwd"
/note="common sequencing primer, one of multiple similar
variants"
promoter 1977..1995
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
misc_feature 2004..2111
/note="MCS"
/note="pBluescript multiple cloning site"
primer_bind 2021..2037
/note="KS primer"
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(2071..2087)
/note="SK primer"
/note="common sequencing primer, one of multiple similar
variants"
promoter complement(2124..2142)
/note="T3 promoter"
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind complement(2163..2179)
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 2187..2203
/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(2211..2241)
/note="lac promoter"
/note="promoter for the E. coli lac operon"
rep_origin complement(2565..3153)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(3324..4184)
/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(4185..4289)
/gene="bla"
/note="AmpR promoter"
rep_origin 4316..5658
/note="2u ori"
/note="yeast 2u plasmid origin of replication"
protein_bind complement(5279..5326)
/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 accataccac agcttttcaa ttcaattcat catttttttt ttattctttt ttttgatttc
241 ggtttctttg aaattttttt gattcggtaa tctccgaaca gaaggaagaa cgaaggaagg
301 agcacagact tagattggta tatatacgca tatgtagtgt tgaagaaaca tgaaattgcc
361 cagtattctt aacccaactg cacagaacaa aaacctgcag gaaacgaaga taaatcatgt
421 cgaaagctac atataaggaa cgtgctgcta ctcatcctag tcctgttgct gccaagctat
481 ttaatatcat gcacgaaaag caaacaaact tgtgtgcttc attggatgtt cgtaccacca
541 aggaattact ggagttagtt gaagcattag gtcccaaaat ttgtttacta aaaacacatg
601 tggatatctt gactgatttt tccatggagg gcacagttaa gccgctaaag gcattatccg
661 ccaagtacaa ttttttactc ttcgaagaca gaaaatttgc tgacattggt aatacagtca
721 aattgcagta ctctgcgggt gtatacagaa tagcagaatg ggcagacatt acgaatgcac
781 acggtgtggt gggcccaggt attgttagcg gtttgaagca ggcggcagaa gaagtaacaa
841 aggaacctag aggccttttg atgttagcag aattgtcatg caagggctcc ctatctactg
901 gagaatatac taagggtact gttgacattg cgaagagcga caaagatttt gttatcggct
961 ttattgctca aagagacatg ggtggaagag atgaaggtta cgattggttg attatgacac
1021 ccggtgtggg tttagatgac aagggagacg cattgggtca acagtataga accgtggatg
1081 atgtggtctc tacaggatct gacattatta ttgttggaag aggactattt gcaaagggaa
1141 gggatgctaa ggtagagggt gaacgttaca gaaaagcagg ctgggaagca tatttgagaa
1201 gatgcggcca gcaaaactaa aaaactgtat tataagtaaa tgcatgtata ctaaactcac
1261 aaattagagc ttcaatttaa ttatatcagt tattacccta tgcggtgtga aataccgcac
1321 agatgcgtaa ggagaaaata ccgcatcagg aaattgtaaa cgttaatatt ttgttaaaat
1381 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa
1441 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca
1501 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg
1561 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta
1621 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg
1681 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa
1741 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg
1801 gcgcgtcgcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg
1861 cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg
1921 taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgag cgcgcgtaat
1981 acgactcact atagggcgaa ttgggtaccg ggccccccct cgaggtcgac ggtatcgata
2041 agcttgatat cgaattcctg cagcccgggg gatccactag ttctagagcg gccgccaccg
2101 cggtggagct ccagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca
2161 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatagga
2221 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgaggtaact cacattaatt
2281 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
2341 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
2401 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
2461 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
2521 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
2581 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
2641 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
2701 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
2761 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
2821 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
2881 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
2941 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
3001 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
3061 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
3121 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
3181 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
3241 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
3301 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
3361 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
3421 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
3481 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
3541 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
3601 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
3661 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
3721 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
3781 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
3841 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
3901 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
3961 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
4021 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
4081 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
4141 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
4201 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
4261 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgaacga
4321 agcatctgtg cttcattttg tagaacaaaa atgcaacgcg agagcgctaa tttttcaaac
4381 aaagaatctg agctgcattt ttacagaaca gaaatgcaac gcgaaagcgc tattttacca
4441 acgaagaatc tgtgcttcat ttttgtaaaa caaaaatgca acgcgagagc gctaattttt
4501 caaacaaaga atctgagctg catttttaca gaacagaaat gcaacgcgag agcgctattt
4561 taccaacaaa gaatctatac ttcttttttg ttctacaaaa atgcatcccg agagcgctat
4621 ttttctaaca aagcatctta gattactttt tttctccttt gtgcgctcta taatgcagtc
4681 tcttgataac tttttgcact gtaggtccgt taaggttaga agaaggctac tttggtgtct
4741 attttctctt ccataaaaaa agcctgactc cacttcccgc gtttactgat tactagcgaa
4801 gctgcgggtg cattttttca agataaaggc atccccgatt atattctata ccgatgtgga
4861 ttgcgcatac tttgtgaaca gaaagtgata gcgttgatga ttcttcattg gtcagaaaat
4921 tatgaacggt ttcttctatt ttgtctctat atactacgta taggaaatgt ttacattttc
4981 gtattgtttt cgattcactc tatgaatagt tcttactaca atttttttgt ctaaagagta
5041 atactagaga taaacataaa aaatgtagag gtcgagttta gatgcaagtt caaggagcga
5101 aaggtggatg ggtaggttat atagggatat agcacagaga tatatagcaa agagatactt
5161 ttgagcaatg tttgtggaag cggtattcgc aatattttag tagctcgtta cagtccggtg
5221 cgtttttggt tttttgaaag tgcgtcttca gagcgctttt ggttttcaaa agcgctctga
5281 agttcctata ctttctagag aataggaact tcggaatagg aacttcaaag cgtttccgaa
5341 aacgagcgct tccgaaaatg caacgcgagc tgcgcacata cagctcactg ttcacgtcgc
5401 acctatatct gcgtgttgcc tgtatatata tatacatgag aagaacggca tagtgcgtgt
5461 ttatgcttaa atgcgtactt atatgcgtct atttatgtag gatgaaaggt agtctagtac
5521 ctcctgtgat attatcccat tccatgcggg gtatcgtatg cttccttcag cactaccctt
5581 tagctgttct atatgctgcc actcctcaat tggattagtc tcatccttca atgctatcat
5641 ttcctttgat attggatcat actaagaaac cattattatc atgacattaa cctataaaaa
5701 taggcgtatc acgaggccct ttcgtc
//
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：pRS426酿酒酵母质粒
别称: pRS426
| 启动子: | URA3，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: | 5726bp |
| 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: | 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： | URA3 |
| 荧光蛋白： |
质粒简介
The order of the major features in this plasmid is:ori(f1) - lacZ - T7 promoter - MCS (KpnI-SacI) - T3 promoter - lacI - ori(pMB1) - ampR - ori (2 micron) - URA3. 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 non-selective growth, plasmids are lost through mitotic segregation at rates in the range of 4.4 + or - 1.4% of progeny per doubling. YE-type shuttle vector encoding the beta-galactosidase alpha peptide, permitting blue-white visual detection. Can be used to produce ssDNA. Contains promoters for in vitro RNA synthesis and priming sites useful for sequencing. Restriction digests of the clone give the following sizes (kb): BamHI--5.8; EcoRI--5.8; PstI--4.0, 1.7; PvuII--5.2, 0.54. (ATCC staff) Medium is 1227 LB plus ampicillin. NCBI gi: 416322 Hosts: E.coli DH5alpha, Saccharomyces cerevisiae, E.coli.
质粒图谱
质粒序列
LOCUS Exported 5726 bp ds-DNA circular SYN 12-SEP-2016
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS Untitled 8
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5726)
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..5726
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 201..416
/gene="S. cerevisiae URA3"
/note="URA3 promoter"
CDS 417..1220
/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"
rep_origin complement(1351..1806)
/direction=LEFT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
CDS complement(1589..2167)
/codon_start=1
/gene="lacZ fragment"
/product="LacZ-alpha fragment of beta-galactosidase"
/note="lacZ-alpha"
/translation="MTMITPSAQLTLTKGNKSWSSTAVAAALELVDPPGCRNSISSLSI
PSTSRGGPVPNSPYSESYYARSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEAR
TDRPSQQLRSLNGEWRDAPCSGALSAAGVVVTRSVTATLASALAPAPFAFFPSFLATFA
GFPRQALNRGLPLGFRFSALRHLDPKKLD"
primer_bind 1951..1967
/note="M13 fwd"
/note="common sequencing primer, one of multiple similar
variants"
promoter 1977..1995
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
misc_feature 2004..2111
/note="MCS"
/note="pBluescript multiple cloning site"
primer_bind 2021..2037
/note="KS primer"
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(2071..2087)
/note="SK primer"
/note="common sequencing primer, one of multiple similar
variants"
promoter complement(2124..2142)
/note="T3 promoter"
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind complement(2163..2179)
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 2187..2203
/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(2211..2241)
/note="lac promoter"
/note="promoter for the E. coli lac operon"
rep_origin complement(2565..3153)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(3324..4184)
/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(4185..4289)
/gene="bla"
/note="AmpR promoter"
rep_origin 4316..5658
/note="2u ori"
/note="yeast 2u plasmid origin of replication"
protein_bind complement(5279..5326)
/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 accataccac agcttttcaa ttcaattcat catttttttt ttattctttt ttttgatttc
241 ggtttctttg aaattttttt gattcggtaa tctccgaaca gaaggaagaa cgaaggaagg
301 agcacagact tagattggta tatatacgca tatgtagtgt tgaagaaaca tgaaattgcc
361 cagtattctt aacccaactg cacagaacaa aaacctgcag gaaacgaaga taaatcatgt
421 cgaaagctac atataaggaa cgtgctgcta ctcatcctag tcctgttgct gccaagctat
481 ttaatatcat gcacgaaaag caaacaaact tgtgtgcttc attggatgtt cgtaccacca
541 aggaattact ggagttagtt gaagcattag gtcccaaaat ttgtttacta aaaacacatg
601 tggatatctt gactgatttt tccatggagg gcacagttaa gccgctaaag gcattatccg
661 ccaagtacaa ttttttactc ttcgaagaca gaaaatttgc tgacattggt aatacagtca
721 aattgcagta ctctgcgggt gtatacagaa tagcagaatg ggcagacatt acgaatgcac
781 acggtgtggt gggcccaggt attgttagcg gtttgaagca ggcggcagaa gaagtaacaa
841 aggaacctag aggccttttg atgttagcag aattgtcatg caagggctcc ctatctactg
901 gagaatatac taagggtact gttgacattg cgaagagcga caaagatttt gttatcggct
961 ttattgctca aagagacatg ggtggaagag atgaaggtta cgattggttg attatgacac
1021 ccggtgtggg tttagatgac aagggagacg cattgggtca acagtataga accgtggatg
1081 atgtggtctc tacaggatct gacattatta ttgttggaag aggactattt gcaaagggaa
1141 gggatgctaa ggtagagggt gaacgttaca gaaaagcagg ctgggaagca tatttgagaa
1201 gatgcggcca gcaaaactaa aaaactgtat tataagtaaa tgcatgtata ctaaactcac
1261 aaattagagc ttcaatttaa ttatatcagt tattacccta tgcggtgtga aataccgcac
1321 agatgcgtaa ggagaaaata ccgcatcagg aaattgtaaa cgttaatatt ttgttaaaat
1381 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa
1441 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca
1501 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg
1561 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta
1621 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg
1681 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa
1741 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg
1801 gcgcgtcgcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg
1861 cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg
1921 taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgag cgcgcgtaat
1981 acgactcact atagggcgaa ttgggtaccg ggccccccct cgaggtcgac ggtatcgata
2041 agcttgatat cgaattcctg cagcccgggg gatccactag ttctagagcg gccgccaccg
2101 cggtggagct ccagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca
2161 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatagga
2221 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgaggtaact cacattaatt
2281 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
2341 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
2401 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
2461 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
2521 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
2581 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
2641 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
2701 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
2761 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
2821 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
2881 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
2941 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
3001 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
3061 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
3121 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
3181 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
3241 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
3301 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
3361 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
3421 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
3481 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
3541 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
3601 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
3661 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
3721 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
3781 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
3841 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
3901 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
3961 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
4021 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
4081 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
4141 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
4201 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
4261 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgaacga
4321 agcatctgtg cttcattttg tagaacaaaa atgcaacgcg agagcgctaa tttttcaaac
4381 aaagaatctg agctgcattt ttacagaaca gaaatgcaac gcgaaagcgc tattttacca
4441 acgaagaatc tgtgcttcat ttttgtaaaa caaaaatgca acgcgagagc gctaattttt
4501 caaacaaaga atctgagctg catttttaca gaacagaaat gcaacgcgag agcgctattt
4561 taccaacaaa gaatctatac ttcttttttg ttctacaaaa atgcatcccg agagcgctat
4621 ttttctaaca aagcatctta gattactttt tttctccttt gtgcgctcta taatgcagtc
4681 tcttgataac tttttgcact gtaggtccgt taaggttaga agaaggctac tttggtgtct
4741 attttctctt ccataaaaaa agcctgactc cacttcccgc gtttactgat tactagcgaa
4801 gctgcgggtg cattttttca agataaaggc atccccgatt atattctata ccgatgtgga
4861 ttgcgcatac tttgtgaaca gaaagtgata gcgttgatga ttcttcattg gtcagaaaat
4921 tatgaacggt ttcttctatt ttgtctctat atactacgta taggaaatgt ttacattttc
4981 gtattgtttt cgattcactc tatgaatagt tcttactaca atttttttgt ctaaagagta
5041 atactagaga taaacataaa aaatgtagag gtcgagttta gatgcaagtt caaggagcga
5101 aaggtggatg ggtaggttat atagggatat agcacagaga tatatagcaa agagatactt
5161 ttgagcaatg tttgtggaag cggtattcgc aatattttag tagctcgtta cagtccggtg
5221 cgtttttggt tttttgaaag tgcgtcttca gagcgctttt ggttttcaaa agcgctctga
5281 agttcctata ctttctagag aataggaact tcggaatagg aacttcaaag cgtttccgaa
5341 aacgagcgct tccgaaaatg caacgcgagc tgcgcacata cagctcactg ttcacgtcgc
5401 acctatatct gcgtgttgcc tgtatatata tatacatgag aagaacggca tagtgcgtgt
5461 ttatgcttaa atgcgtactt atatgcgtct atttatgtag gatgaaaggt agtctagtac
5521 ctcctgtgat attatcccat tccatgcggg gtatcgtatg cttccttcag cactaccctt
5581 tagctgttct atatgctgcc actcctcaat tggattagtc tcatccttca atgctatcat
5641 ttcctttgat attggatcat actaagaaac cattattatc atgacattaa cctataaaaa
5701 taggcgtatc acgaggccct ttcgtc
//
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
| P2098/pENTR223-UBTD1人源基因质粒 |
| P2099/pENTR223-ING5人源基因质粒 |
| P2100/pENTR223-CHMP4B人源基因质粒 |
| P2101/pENTR223-RNF125-A694G人源基因质粒 |
| P2103/pENTR223-PPP2R3B-A8C人源基因质粒 |
| P2104/pENTR223-RAB7A-T8G人源基因质粒 |
| P2105/pENTR223-FAM76B人源基因质粒 |
| P2106/pENTR223-DDIT4人源基因质粒 |
| P2107/pENTR223-GSTT1-C7T人源基因质粒 |
| P2129/pENTR223-SCAMP5人源基因质粒 |
| P2130/pENTR223-CBR4人源基因质粒 |
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pRS426酿酒酵母质粒
￥100 - 1000
