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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：
pRS424
- 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：pRS424酿酒酵母质粒
别称: pRS424
质粒属性
质粒简介
Restriction digests of the clone give the following sizes (kb): BamHI--5.6; EcoRI--5.6; PvuII--5.2, 0.5; XbaI--3.3, 1.4, 1.0.Contains the REP3 and FRT sequences necessary for high copy propagation in yeast.In S. cerevisiae, the number is about 20 per haploid cell. In non-selective growth, plasmids are lost through mitotic segregation at rates in the range of 1.5 + or - 1.7% 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.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) - TRP1.
质粒图谱
质粒序列
LOCUS Exported 5616 bp ds-DNA circular SYN 12-SEP-2016
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS Untitled 6
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5616)
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..5616
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 187..467
/gene="S. cerevisiae TRP1"
/note="TRP1 promoter"
CDS 468..1142
/codon_start=1
/gene="S. cerevisiae TRP1"
/product="phosphoribosylanthranilate isomerase, required
for tryptophan biosynthesis"
/note="TRP1"
/note="yeast auxotrophic marker"
/translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
KK"
rep_origin complement(1241..1696)
/direction=LEFT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
CDS complement(1479..2057)
/codon_start=1
/gene="lacZ fragment"
/product="LacZ-alpha fragment of beta-galactosidase"
/note="lacZ-alpha"
/translation="MTMITPSAQLTLTKGNKSWSSTAVAAALELVDPPGCRNSISSLSI
PSTSRGGPVPNSPYSESYYARSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEAR
TDRPSQQLRSLNGEWRDAPCSGALSAAGVVVTRSVTATLASALAPAPFAFFPSFLATFA
GFPRQALNRGLPLGFRFSALRHLDPKKLD"
primer_bind 1841..1857
/note="M13 fwd"
/note="common sequencing primer, one of multiple similar
variants"
promoter 1867..1885
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
misc_feature 1894..2001
/note="MCS"
/note="pBluescript multiple cloning site"
primer_bind 1911..1927
/note="KS primer"
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(1961..1977)
/note="SK primer"
/note="common sequencing primer, one of multiple similar
variants"
promoter complement(2014..2032)
/note="T3 promoter"
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind complement(2053..2069)
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 2077..2093
/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(2101..2131)
/note="lac promoter"
/note="promoter for the E. coli lac operon"
rep_origin complement(2455..3043)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(3214..4074)
/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(4075..4179)
/gene="bla"
/note="AmpR promoter"
rep_origin 4206..5548
/note="2u ori"
/note="yeast 2u plasmid origin of replication"
protein_bind complement(5169..5216)
/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 accataaacg acattactat atatataata taggaagcat ttaatagaca gcatcgtaat
241 atatgtgtac tttgcagtta tgacgccaga tggcagtagt ggaagatatt ctttattgaa
301 aaatagcttg tcaccttacg tacaatcttg atccggagct tttctttttt tgccgattaa
361 gaattaattc ggtcgaaaaa agaaaaggag agggccaaga gggagggcat tggtgactat
421 tgagcacgtg agtatacgtg attaagcaca caaaggcagc ttggagtatg tctgttatta
481 atttcacagg tagttctggt ccattggtga aagtttgcgg cttgcagagc acagaggccg
541 cagaatgtgc tctagattcc gatgctgact tgctgggtat tatatgtgtg cccaatagaa
601 agagaacaat tgacccggtt attgcaagga aaatttcaag tcttgtaaaa gcatataaaa
661 atagttcagg cactccgaaa tacttggttg gcgtgtttcg taatcaacct aaggaggatg
721 ttttggctct ggtcaatgat tacggcattg atatcgtcca actgcatgga gatgagtcgt
781 ggcaagaata ccaagagttc ctcggtttgc cagttattaa aagactcgta tttccaaaag
841 actgcaacat actactcagt gcagcttcac agaaacctca ttcgtttatt cccttgtttg
901 attcagaagc aggtgggaca ggtgaacttt tggattggaa ctcgatttct gactgggttg
961 gaaggcaaga gagccccgaa agcttacatt ttatgttagc tggtggactg acgccagaaa
1021 atgttggtga tgcgcttaga ttaaatggcg ttattggtgt tgatgtaagc ggaggtgtgg
1081 agacaaatgg tgtaaaagac tctaacaaaa tagcaaattt cgtcaaaaat gctaagaaat
1141 aggttattac tgagtagtat ttatttaagt attgtttgtg cacttgccta tgcggtgtga
1201 aataccgcac agatgcgtaa ggagaaaata ccgcatcagg aaattgtaaa cgttaatatt
1261 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
1321 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
1381 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
1441 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
1501 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
1561 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
1621 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
1681 ccgctacagg gcgcgtcgcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga
1741 tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga
1801 ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgag
1861 cgcgcgtaat acgactcact atagggcgaa ttgggtaccg ggccccccct cgaggtcgac
1921 ggtatcgata agcttgatat cgaattcctg cagcccgggg gatccactag ttctagagcg
1981 gccgccaccg cggtggagct ccagcttttg ttccctttag tgagggttaa ttgcgcgctt
2041 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
2101 caacatagga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgaggtaact
2161 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
2221 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
2281 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
2341 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
2401 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
2461 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
2521 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
2581 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
2641 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
2701 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
2761 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
2821 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
2881 cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
2941 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
3001 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
3061 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
3121 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
3181 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
3241 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
3301 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
3361 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
3421 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
3481 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
3541 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
3601 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
3661 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
3721 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
3781 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
3841 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
3901 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
3961 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
4021 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
4081 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
4141 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
4201 acctgaacga agcatctgtg cttcattttg tagaacaaaa atgcaacgcg agagcgctaa
4261 tttttcaaac aaagaatctg agctgcattt ttacagaaca gaaatgcaac gcgaaagcgc
4321 tattttacca acgaagaatc tgtgcttcat ttttgtaaaa caaaaatgca acgcgagagc
4381 gctaattttt caaacaaaga atctgagctg catttttaca gaacagaaat gcaacgcgag
4441 agcgctattt taccaacaaa gaatctatac ttcttttttg ttctacaaaa atgcatcccg
4501 agagcgctat ttttctaaca aagcatctta gattactttt tttctccttt gtgcgctcta
4561 taatgcagtc tcttgataac tttttgcact gtaggtccgt taaggttaga agaaggctac
4621 tttggtgtct attttctctt ccataaaaaa agcctgactc cacttcccgc gtttactgat
4681 tactagcgaa gctgcgggtg cattttttca agataaaggc atccccgatt atattctata
4741 ccgatgtgga ttgcgcatac tttgtgaaca gaaagtgata gcgttgatga ttcttcattg
4801 gtcagaaaat tatgaacggt ttcttctatt ttgtctctat atactacgta taggaaatgt
4861 ttacattttc gtattgtttt cgattcactc tatgaatagt tcttactaca atttttttgt
4921 ctaaagagta atactagaga taaacataaa aaatgtagag gtcgagttta gatgcaagtt
4981 caaggagcga aaggtggatg ggtaggttat atagggatat agcacagaga tatatagcaa
5041 agagatactt ttgagcaatg tttgtggaag cggtattcgc aatattttag tagctcgtta
5101 cagtccggtg cgtttttggt tttttgaaag tgcgtcttca gagcgctttt ggttttcaaa
5161 agcgctctga agttcctata ctttctagag aataggaact tcggaatagg aacttcaaag
5221 cgtttccgaa aacgagcgct tccgaaaatg caacgcgagc tgcgcacata cagctcactg
5281 ttcacgtcgc acctatatct gcgtgttgcc tgtatatata tatacatgag aagaacggca
5341 tagtgcgtgt ttatgcttaa atgcgtactt atatgcgtct atttatgtag gatgaaaggt
5401 agtctagtac ctcctgtgat attatcccat tccatgcggg gtatcgtatg cttccttcag
5461 cactaccctt tagctgttct atatgctgcc actcctcaat tggattagtc tcatccttca
5521 atgctatcat ttcctttgat attggatcat attaagaaac cattattatc atgacattaa
5581 cctataaaaa 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：pRS424酿酒酵母质粒
别称: pRS424
| 启动子: | TRP1，T3,T7 |
|---|---|
| 复制子: | 2U |
| 质粒分类: | 酵母系列，酿酒酵母表达载体 |
| 质粒大小: | 5616bp |
| 原核抗性: | Amp |
| 筛选标记: | TRP1 |
| 克隆菌株: | DH5a |
| 培养条件: | 37度 |
| 表达宿主: | 酵母细胞 |
| 诱导方式: | 半乳糖诱导 |
| 5'测序引物: | T7:TAATACGACTCACTATAGGG |
| 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： | TRP1 |
| 荧光蛋白： |
质粒简介
Restriction digests of the clone give the following sizes (kb): BamHI--5.6; EcoRI--5.6; PvuII--5.2, 0.5; XbaI--3.3, 1.4, 1.0.Contains the REP3 and FRT sequences necessary for high copy propagation in yeast.In S. cerevisiae, the number is about 20 per haploid cell. In non-selective growth, plasmids are lost through mitotic segregation at rates in the range of 1.5 + or - 1.7% 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.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) - TRP1.
质粒图谱
质粒序列
LOCUS Exported 5616 bp ds-DNA circular SYN 12-SEP-2016
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS Untitled 6
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5616)
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..5616
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 187..467
/gene="S. cerevisiae TRP1"
/note="TRP1 promoter"
CDS 468..1142
/codon_start=1
/gene="S. cerevisiae TRP1"
/product="phosphoribosylanthranilate isomerase, required
for tryptophan biosynthesis"
/note="TRP1"
/note="yeast auxotrophic marker"
/translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
KK"
rep_origin complement(1241..1696)
/direction=LEFT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
CDS complement(1479..2057)
/codon_start=1
/gene="lacZ fragment"
/product="LacZ-alpha fragment of beta-galactosidase"
/note="lacZ-alpha"
/translation="MTMITPSAQLTLTKGNKSWSSTAVAAALELVDPPGCRNSISSLSI
PSTSRGGPVPNSPYSESYYARSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEAR
TDRPSQQLRSLNGEWRDAPCSGALSAAGVVVTRSVTATLASALAPAPFAFFPSFLATFA
GFPRQALNRGLPLGFRFSALRHLDPKKLD"
primer_bind 1841..1857
/note="M13 fwd"
/note="common sequencing primer, one of multiple similar
variants"
promoter 1867..1885
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
misc_feature 1894..2001
/note="MCS"
/note="pBluescript multiple cloning site"
primer_bind 1911..1927
/note="KS primer"
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(1961..1977)
/note="SK primer"
/note="common sequencing primer, one of multiple similar
variants"
promoter complement(2014..2032)
/note="T3 promoter"
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind complement(2053..2069)
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 2077..2093
/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(2101..2131)
/note="lac promoter"
/note="promoter for the E. coli lac operon"
rep_origin complement(2455..3043)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(3214..4074)
/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(4075..4179)
/gene="bla"
/note="AmpR promoter"
rep_origin 4206..5548
/note="2u ori"
/note="yeast 2u plasmid origin of replication"
protein_bind complement(5169..5216)
/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 accataaacg acattactat atatataata taggaagcat ttaatagaca gcatcgtaat
241 atatgtgtac tttgcagtta tgacgccaga tggcagtagt ggaagatatt ctttattgaa
301 aaatagcttg tcaccttacg tacaatcttg atccggagct tttctttttt tgccgattaa
361 gaattaattc ggtcgaaaaa agaaaaggag agggccaaga gggagggcat tggtgactat
421 tgagcacgtg agtatacgtg attaagcaca caaaggcagc ttggagtatg tctgttatta
481 atttcacagg tagttctggt ccattggtga aagtttgcgg cttgcagagc acagaggccg
541 cagaatgtgc tctagattcc gatgctgact tgctgggtat tatatgtgtg cccaatagaa
601 agagaacaat tgacccggtt attgcaagga aaatttcaag tcttgtaaaa gcatataaaa
661 atagttcagg cactccgaaa tacttggttg gcgtgtttcg taatcaacct aaggaggatg
721 ttttggctct ggtcaatgat tacggcattg atatcgtcca actgcatgga gatgagtcgt
781 ggcaagaata ccaagagttc ctcggtttgc cagttattaa aagactcgta tttccaaaag
841 actgcaacat actactcagt gcagcttcac agaaacctca ttcgtttatt cccttgtttg
901 attcagaagc aggtgggaca ggtgaacttt tggattggaa ctcgatttct gactgggttg
961 gaaggcaaga gagccccgaa agcttacatt ttatgttagc tggtggactg acgccagaaa
1021 atgttggtga tgcgcttaga ttaaatggcg ttattggtgt tgatgtaagc ggaggtgtgg
1081 agacaaatgg tgtaaaagac tctaacaaaa tagcaaattt cgtcaaaaat gctaagaaat
1141 aggttattac tgagtagtat ttatttaagt attgtttgtg cacttgccta tgcggtgtga
1201 aataccgcac agatgcgtaa ggagaaaata ccgcatcagg aaattgtaaa cgttaatatt
1261 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
1321 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
1381 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
1441 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
1501 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
1561 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
1621 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
1681 ccgctacagg gcgcgtcgcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga
1741 tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga
1801 ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgag
1861 cgcgcgtaat acgactcact atagggcgaa ttgggtaccg ggccccccct cgaggtcgac
1921 ggtatcgata agcttgatat cgaattcctg cagcccgggg gatccactag ttctagagcg
1981 gccgccaccg cggtggagct ccagcttttg ttccctttag tgagggttaa ttgcgcgctt
2041 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
2101 caacatagga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgaggtaact
2161 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
2221 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
2281 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
2341 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
2401 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
2461 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
2521 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
2581 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
2641 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
2701 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
2761 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
2821 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
2881 cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
2941 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
3001 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
3061 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
3121 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
3181 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
3241 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
3301 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
3361 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
3421 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
3481 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
3541 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
3601 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
3661 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
3721 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
3781 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
3841 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
3901 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
3961 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
4021 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
4081 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
4141 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
4201 acctgaacga agcatctgtg cttcattttg tagaacaaaa atgcaacgcg agagcgctaa
4261 tttttcaaac aaagaatctg agctgcattt ttacagaaca gaaatgcaac gcgaaagcgc
4321 tattttacca acgaagaatc tgtgcttcat ttttgtaaaa caaaaatgca acgcgagagc
4381 gctaattttt caaacaaaga atctgagctg catttttaca gaacagaaat gcaacgcgag
4441 agcgctattt taccaacaaa gaatctatac ttcttttttg ttctacaaaa atgcatcccg
4501 agagcgctat ttttctaaca aagcatctta gattactttt tttctccttt gtgcgctcta
4561 taatgcagtc tcttgataac tttttgcact gtaggtccgt taaggttaga agaaggctac
4621 tttggtgtct attttctctt ccataaaaaa agcctgactc cacttcccgc gtttactgat
4681 tactagcgaa gctgcgggtg cattttttca agataaaggc atccccgatt atattctata
4741 ccgatgtgga ttgcgcatac tttgtgaaca gaaagtgata gcgttgatga ttcttcattg
4801 gtcagaaaat tatgaacggt ttcttctatt ttgtctctat atactacgta taggaaatgt
4861 ttacattttc gtattgtttt cgattcactc tatgaatagt tcttactaca atttttttgt
4921 ctaaagagta atactagaga taaacataaa aaatgtagag gtcgagttta gatgcaagtt
4981 caaggagcga aaggtggatg ggtaggttat atagggatat agcacagaga tatatagcaa
5041 agagatactt ttgagcaatg tttgtggaag cggtattcgc aatattttag tagctcgtta
5101 cagtccggtg cgtttttggt tttttgaaag tgcgtcttca gagcgctttt ggttttcaaa
5161 agcgctctga agttcctata ctttctagag aataggaact tcggaatagg aacttcaaag
5221 cgtttccgaa aacgagcgct tccgaaaatg caacgcgagc tgcgcacata cagctcactg
5281 ttcacgtcgc acctatatct gcgtgttgcc tgtatatata tatacatgag aagaacggca
5341 tagtgcgtgt ttatgcttaa atgcgtactt atatgcgtct atttatgtag gatgaaaggt
5401 agtctagtac ctcctgtgat attatcccat tccatgcggg gtatcgtatg cttccttcag
5461 cactaccctt tagctgttct atatgctgcc actcctcaat tggattagtc tcatccttca
5521 atgctatcat ttcctttgat attggatcat attaagaaac cattattatc atgacattaa
5581 cctataaaaa 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
| P2065/pENTR223-GSTM4人源基因质粒 |
| P2066/pENTR223-FAM119A人源基因质粒 |
| P2067/pENTR223-PRELID1人源基因质粒 |
| P2068/pENTR223-TMED7人源基因质粒 |
| P2069/pENTR223-RNF114人源基因质粒 |
| P2070/pENTR223-NIPAL3-T113C人源基因质粒 |
| P2071/pENTR223-RBCK1人源基因质粒 |
| P2072/pENTR223-C2orf49人源基因质粒 |
| P2073/pENTR223-IMAGE4149333人源基因质粒 |
| P2074/pENTR223-EEF1AKMT3人源基因质粒 |
| P2075/pENTR223-RCAN2-C11人源基因质粒 |
| P2076/pENTR223-C16orf79-T16A人源基因质粒 |
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pRS424酿酒酵母质粒
￥100 - 1000
