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- 技术资料
- 保存条件：
-20
- 保质期：
2年
- 英文名：
pY26TEF-GPD
- 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：pY26TEF-GPD酵母双框质粒
别称: pY26TEF-GPD
质粒属性
质粒简介
pY26TEF-GPD是一个酿酒酵母高拷贝双向表达质粒，TEF1 promoter，GAP promoter分别驱动两个基因的表达。
质粒图谱
质粒序列
LOCUS Exported 7430 bp ds-DNA circular SYN 05-NOV-2017
DEFINITION synthetic circular DNA
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7430)
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..7430
/organism="synthetic DNA construct"
/mol_type="other DNA"
rep_origin 69..1411
/label=2u ori
/note="yeast 2u plasmid origin of replication"
protein_bind 401..448
/label=FRT
/bound_moiety="FLP recombinase from the Saccharomyces
cerevisiae 2u plasmid"
/note="FLP-mediated recombination occurs in the 8-bp core
sequence TCTAGAAA (Turan and Bode, 2011)."
promoter 1438..1542
/gene="bla"
/label=AmpR promoter
CDS 1543..2403
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 2574..3162
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
terminator 3643..3807
/gene="S. cerevisiae ADH1"
/label=ADH1 terminator
/note="transcription terminator for the S. cerevisiae
alcohol dehydrogenase 1 (ADH1) gene"
misc_feature 3915..3982
/label=MCS1
promoter complement(3998..4396)
/gene="S. cerevisiae TEF1"
/label=TEF1 promoter
/note="promoter for EF-1-alpha"
promoter 4452..5095
/gene="S. cerevisiae TDH3"
/label=GAP promoter
/note="promoter for glyceraldehyde-3-phosphate
dehydrogenase"
misc_feature 5107..5169
/label=MCS2
terminator 5170..5417
/gene="S. cerevisiae CYC1"
/label=CYC1 terminator
/note="transcription terminator for CYC1"
rep_origin 5625..6080
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
CDS complement(6211..7014)
/codon_start=1
/gene="S. cerevisiae URA3"
/product="orotidine-5'-phosphate decarboxylase, required
for uracil biosynthesis"
/label=URA3
/note="yeast auxotrophic marker, counterselectable with
5-fluoroorotic acid (5-FOA)"
/translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
promoter complement(7015..7230)
/gene="S. cerevisiae URA3"
/label=URA3 promoter
ORIGIN
1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
61 cttagtatga tccaatatca aaggaaatga tagcattgaa ggatgagact aatccaattg
121 aggagtggca gcatatagaa cagctaaagg gtagtgctga aggaagcata cgataccccg
181 catggaatgg gataatatca caggaggtac tagactacct ttcatcctac ataaatagac
241 gcatataagt acgcatttaa gcataaacac gcactatgcc gttcttctca tgtatatata
301 tatacaggca acacgcagat ataggtgcga cgtgaacagt gagctgtatg tgcgcagctc
361 gcgttgcatt ttcggaagcg ctcgttttcg gaaacgcttt gaagttccta ttccgaagtt
421 cctattctct agaaagtata ggaacttcag agcgcttttg aaaaccaaaa gcgctctgaa
481 gacgcacttt caaaaaacca aaaacgcacc ggactgtaac gagctactaa aatattgcga
541 ataccgcttc cacaaacatt gctcaaaagt atctctttgc tatatatctc tgtgctatat
601 ccctatataa cctacccatc cacctttcgc tccttgaact tgcatctaaa ctcgacctct
661 acatttttta tgtttatctc tagtattact ctttagacaa aaaaattgta gtaagaacta
721 ttcatagagt gaatcgaaaa caatacgaaa atgtaaacat ttcctatacg tagtatatag
781 agacaaaata gaagaaaccg ttcataattt tctgaccaat gaagaatcat caacgctatc
841 actttctgtt cacaaagtat gcgcaatcca catcggtata gaatataatc ggggatgcct
901 ttatcttgaa aaaatgcacc cgcagcttcg ctagtaatca gtaaacgcgg gaagtggagt
961 caggcttttt ttatggaaga gaaaatagac accaaagtag ccttcttcta accttaacgg
1021 acctacagtg caaaaagtta tcaagagact gcattataga gcgcacaaag gagaaaaaaa
1081 gtaatctaag atgctttgtt agaaaaatag cgctctcggg atgcattttt gtagaacaaa
1141 aaagaagtat agattctttg ttggtaaaat agcgctctcg cgttgcattt ctgttctgta
1201 aaaatgcagc tcagattctt tgtttgaaaa attagcgctc tcgcgttgca tttttgtttt
1261 acaaaaatga agcacagatt cttcgttggt aaaatagcgc tttcgcgttg catttctgtt
1321 ctgtaaaaat gcagctcaga ttctttgttt gaaaaattag cgctctcgcg ttgcattttt
1381 gttctacaaa atgaagcaca gatgcttcgt tcaggtggca cttttcgggg aaatgtgcgc
1441 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
1501 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
1561 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
1621 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
1681 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
1741 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
1801 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
1861 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
1921 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
1981 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
2041 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
2101 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
2161 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
2221 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
2281 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
2341 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
2401 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
2461 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
2521 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
2581 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
2641 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
2701 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
2761 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
2821 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
2881 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
2941 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
3001 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
3061 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
3121 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
3181 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
3241 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
3301 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa
3361 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
3421 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt acctcactca ttaggcaccc
3481 caggctttac actttatgct tccggctcct atgttgtgtg gaattgtgag cggataacaa
3541 tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa ccctcactaa
3601 agggaacaaa agctggagct cgctattacg ccagctgaat tggagcgacc tcatgctata
3661 ctgagaaagc aacctgacct acaggaaaga gttactcaag aataagaatt ttcgttttaa
3721 aacctaagag tcactttaaa atttgtatac acttattttt tttataactt atttaataat
3781 aaaaatcata aatcataaga aattcgctta tttagaagtg tcaacaacgt atctaccaac
3841 gatttgaccc ttttccatct tttcgtaaat ttctggcaag gtagacaagc cgacaacctt
3901 gattggagac ttgaccaaac ctctggcgaa gaattgttaa ttaaagatct ccgcggtccg
3961 gagttaactg atcagcggcc gcgctagttc tagaaaactt agattagatt gctatgcttt
4021 ctttctaatg agcaagaagt aaaaaaagtt gtaatagaac aagaaaaatg aaactgaaac
4081 ttgagaaatt gaagaccgtt tattaactta aatatcaatg ggaggtcatc gaaagagaaa
4141 aaaatcaaaa aaaaaatttt caagaaaaag aaacgtgata aaaattttta ttgccttttt
4201 cgacgaagaa aaagaaacga ggcggtctct tttttctttt ccaaaccttt agtacgggta
4261 attaacgaca ccctagagga agaaagaggg gaaatttagt atgctgtgct tgggtgtttt
4321 gaagtggtac ggcgatgcgc ggagtccgag aaaatctgga agagtaaaaa aggagtagaa
4381 acattttgaa gctatgagct ccagcttttg ttccctttag tgagggttaa ttgcgcgccg
4441 agctcagttt atcattatca atactcgcca tttcaaagaa tacgtaaata attaatagta
4501 gtgattttcc taactttatt tagtcaaaaa attagccttt taattctgct gtaacccgta
4561 catgcccaaa atagggggcg ggttacacag aatatataac atcgtaggtg tctgggtgaa
4621 cagtttattc ctggcatcca ctaaatataa tggagcccgc tttttaagct ggcatccaga
4681 aaaaaaaaga atcccagcac caaaatattg ttttcttcac caaccatcag ttcataggtc
4741 cattctctta gcgcaactac agagaacagg ggcacaaaca ggcaaaaaac gggcacaacc
4801 tcaatggagt gatgcaacct gcctggagta aatgatgaca caaggcaatt gacccacgca
4861 tgtatctatc tcattttctt acaccttcta ttaccttctg ctctctctga tttggaaaaa
4921 gctgaaaaaa aaggttgaaa ccagttccct gaaattattc ccctacttga ctaataagta
4981 tataaagacg gtaggtattg attgtaattc tgtaaatcta tttcttaaac ttcttaaatt
5041 ctacttttat agttagtctt ttttttagtt ttaaaacacc agaacttagt ttcgacggat
5101 tctagaacta gtggatcccc cgggctgcag gaattcgata tcaagcttat cgataccgtc
5161 gacctcgagt catgtaatta gttatgtcac gcttacattc acgccctccc cccacatccg
5221 ctctaaccga aaaggaagga gttagacaac ctgaagtcta ggtccctatt tattttttta
5281 tagttatgtt agtattaaga acgttattta tatttcaaat ttttcttttt tttctgtaca
5341 gacgcgtgta cgcatgtaac attatactga aaaccttgct tgagaaggtt ttgggacgct
5401 cgaaggcttt aatttgcggc cggtacccaa ttcgccctat agtgagtcgt attacgcgcg
5461 ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
5521 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga
5581 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgcgacgcgc cctgtagcgg
5641 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
5701 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
5761 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
5821 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
5881 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
5941 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
6001 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
6061 aatattaacg tttacaattt cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
6121 tcacaccgca tagggtaata actgatataa ttaaattgaa gctctaattt gtgagtttag
6181 tatacatgca tttacttata atacagtttt ttagttttgc tggccgcatc ttctcaaata
6241 tgcttcccag cctgcttttc tgtaacgttc accctctacc ttagcatccc ttccctttgc
6301 aaatagtcct cttccaacaa taataatgtc agatcctgta gagaccacat catccacggt
6361 tctatactgt tgacccaatg cgtctccctt gtcatctaaa cccacaccgg gtgtcataat
6421 caaccaatcg taaccttcat ctcttccacc catgtctctt tgagcaataa agccgataac
6481 aaaatctttg tcgctcttcg caatgtcaac agtaccctta gtatattctc cagtagatag
6541 ggagcccttg catgacaatt ctgctaacat caaaaggcct ctaggttcct ttgttacttc
6601 ttctgccgcc tgcttcaaac cgctaacaat acctgggccc accacaccgt gtgcattcgt
6661 aatgtctgcc cattctgcta ttctgtatac acccgcagag tactgcaatt tgactgtatt
6721 accaatgtca gcaaattttc tgtcttcgaa gagtaaaaaa ttgtacttgg cggataatgc
6781 ctttagcggc ttaactgtgc cctccatgga aaaatcagtc aagatatcca catgtgtttt
6841 tagtaaacaa attttgggac ctaatgcttc aactaactcc agtaattcct tggtggtacg
6901 aacatccaat gaagcacaca agtttgtttg cttttcgtgc atgatattaa atagcttggc
6961 agcaacagga ctaggatgag tagcagcacg ttccttatat gtagctttcg acatgattta
7021 tcttcgtttc ctgcaggttt ttgttctgtg cagttgggtt aagaatactg ggcaatttca
7081 tgtttcttca acactacata tgcgtatata taccaatcta agtctgtgct ccttccttcg
7141 ttcttccttc tgttcggaga ttaccgaatc aaaaaaattt caaagaaacc gaaatcaaaa
7201 aaaagaataa aaaaaaaatg atgaattgaa ttgaaaagct gtggtatggt gcactctcag
7261 tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga
7321 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
7381 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
//
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：pY26TEF-GPD酵母双框质粒
别称: pY26TEF-GPD
| 启动子: | TEF1+GAP |
|---|---|
| 复制子: | 2μ ori，pBR322 ori，F1 ori |
| 终止子: | ADH1 terminator，CYC1 terminator |
| 质粒大小: | 7430bp |
| 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: | 酿酒酵母 |
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
pY26TEF-GPD是一个酿酒酵母高拷贝双向表达质粒，TEF1 promoter，GAP promoter分别驱动两个基因的表达。
质粒图谱
质粒序列
LOCUS Exported 7430 bp ds-DNA circular SYN 05-NOV-2017
DEFINITION synthetic circular DNA
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7430)
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..7430
/organism="synthetic DNA construct"
/mol_type="other DNA"
rep_origin 69..1411
/label=2u ori
/note="yeast 2u plasmid origin of replication"
protein_bind 401..448
/label=FRT
/bound_moiety="FLP recombinase from the Saccharomyces
cerevisiae 2u plasmid"
/note="FLP-mediated recombination occurs in the 8-bp core
sequence TCTAGAAA (Turan and Bode, 2011)."
promoter 1438..1542
/gene="bla"
/label=AmpR promoter
CDS 1543..2403
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 2574..3162
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
terminator 3643..3807
/gene="S. cerevisiae ADH1"
/label=ADH1 terminator
/note="transcription terminator for the S. cerevisiae
alcohol dehydrogenase 1 (ADH1) gene"
misc_feature 3915..3982
/label=MCS1
promoter complement(3998..4396)
/gene="S. cerevisiae TEF1"
/label=TEF1 promoter
/note="promoter for EF-1-alpha"
promoter 4452..5095
/gene="S. cerevisiae TDH3"
/label=GAP promoter
/note="promoter for glyceraldehyde-3-phosphate
dehydrogenase"
misc_feature 5107..5169
/label=MCS2
terminator 5170..5417
/gene="S. cerevisiae CYC1"
/label=CYC1 terminator
/note="transcription terminator for CYC1"
rep_origin 5625..6080
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
CDS complement(6211..7014)
/codon_start=1
/gene="S. cerevisiae URA3"
/product="orotidine-5'-phosphate decarboxylase, required
for uracil biosynthesis"
/label=URA3
/note="yeast auxotrophic marker, counterselectable with
5-fluoroorotic acid (5-FOA)"
/translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
promoter complement(7015..7230)
/gene="S. cerevisiae URA3"
/label=URA3 promoter
ORIGIN
1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
61 cttagtatga tccaatatca aaggaaatga tagcattgaa ggatgagact aatccaattg
121 aggagtggca gcatatagaa cagctaaagg gtagtgctga aggaagcata cgataccccg
181 catggaatgg gataatatca caggaggtac tagactacct ttcatcctac ataaatagac
241 gcatataagt acgcatttaa gcataaacac gcactatgcc gttcttctca tgtatatata
301 tatacaggca acacgcagat ataggtgcga cgtgaacagt gagctgtatg tgcgcagctc
361 gcgttgcatt ttcggaagcg ctcgttttcg gaaacgcttt gaagttccta ttccgaagtt
421 cctattctct agaaagtata ggaacttcag agcgcttttg aaaaccaaaa gcgctctgaa
481 gacgcacttt caaaaaacca aaaacgcacc ggactgtaac gagctactaa aatattgcga
541 ataccgcttc cacaaacatt gctcaaaagt atctctttgc tatatatctc tgtgctatat
601 ccctatataa cctacccatc cacctttcgc tccttgaact tgcatctaaa ctcgacctct
661 acatttttta tgtttatctc tagtattact ctttagacaa aaaaattgta gtaagaacta
721 ttcatagagt gaatcgaaaa caatacgaaa atgtaaacat ttcctatacg tagtatatag
781 agacaaaata gaagaaaccg ttcataattt tctgaccaat gaagaatcat caacgctatc
841 actttctgtt cacaaagtat gcgcaatcca catcggtata gaatataatc ggggatgcct
901 ttatcttgaa aaaatgcacc cgcagcttcg ctagtaatca gtaaacgcgg gaagtggagt
961 caggcttttt ttatggaaga gaaaatagac accaaagtag ccttcttcta accttaacgg
1021 acctacagtg caaaaagtta tcaagagact gcattataga gcgcacaaag gagaaaaaaa
1081 gtaatctaag atgctttgtt agaaaaatag cgctctcggg atgcattttt gtagaacaaa
1141 aaagaagtat agattctttg ttggtaaaat agcgctctcg cgttgcattt ctgttctgta
1201 aaaatgcagc tcagattctt tgtttgaaaa attagcgctc tcgcgttgca tttttgtttt
1261 acaaaaatga agcacagatt cttcgttggt aaaatagcgc tttcgcgttg catttctgtt
1321 ctgtaaaaat gcagctcaga ttctttgttt gaaaaattag cgctctcgcg ttgcattttt
1381 gttctacaaa atgaagcaca gatgcttcgt tcaggtggca cttttcgggg aaatgtgcgc
1441 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
1501 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
1561 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
1621 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
1681 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
1741 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
1801 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
1861 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
1921 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
1981 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
2041 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
2101 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
2161 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
2221 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
2281 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
2341 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
2401 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
2461 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
2521 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
2581 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
2641 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
2701 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
2761 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
2821 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
2881 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
2941 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
3001 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
3061 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
3121 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
3181 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
3241 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
3301 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa
3361 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
3421 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt acctcactca ttaggcaccc
3481 caggctttac actttatgct tccggctcct atgttgtgtg gaattgtgag cggataacaa
3541 tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa ccctcactaa
3601 agggaacaaa agctggagct cgctattacg ccagctgaat tggagcgacc tcatgctata
3661 ctgagaaagc aacctgacct acaggaaaga gttactcaag aataagaatt ttcgttttaa
3721 aacctaagag tcactttaaa atttgtatac acttattttt tttataactt atttaataat
3781 aaaaatcata aatcataaga aattcgctta tttagaagtg tcaacaacgt atctaccaac
3841 gatttgaccc ttttccatct tttcgtaaat ttctggcaag gtagacaagc cgacaacctt
3901 gattggagac ttgaccaaac ctctggcgaa gaattgttaa ttaaagatct ccgcggtccg
3961 gagttaactg atcagcggcc gcgctagttc tagaaaactt agattagatt gctatgcttt
4021 ctttctaatg agcaagaagt aaaaaaagtt gtaatagaac aagaaaaatg aaactgaaac
4081 ttgagaaatt gaagaccgtt tattaactta aatatcaatg ggaggtcatc gaaagagaaa
4141 aaaatcaaaa aaaaaatttt caagaaaaag aaacgtgata aaaattttta ttgccttttt
4201 cgacgaagaa aaagaaacga ggcggtctct tttttctttt ccaaaccttt agtacgggta
4261 attaacgaca ccctagagga agaaagaggg gaaatttagt atgctgtgct tgggtgtttt
4321 gaagtggtac ggcgatgcgc ggagtccgag aaaatctgga agagtaaaaa aggagtagaa
4381 acattttgaa gctatgagct ccagcttttg ttccctttag tgagggttaa ttgcgcgccg
4441 agctcagttt atcattatca atactcgcca tttcaaagaa tacgtaaata attaatagta
4501 gtgattttcc taactttatt tagtcaaaaa attagccttt taattctgct gtaacccgta
4561 catgcccaaa atagggggcg ggttacacag aatatataac atcgtaggtg tctgggtgaa
4621 cagtttattc ctggcatcca ctaaatataa tggagcccgc tttttaagct ggcatccaga
4681 aaaaaaaaga atcccagcac caaaatattg ttttcttcac caaccatcag ttcataggtc
4741 cattctctta gcgcaactac agagaacagg ggcacaaaca ggcaaaaaac gggcacaacc
4801 tcaatggagt gatgcaacct gcctggagta aatgatgaca caaggcaatt gacccacgca
4861 tgtatctatc tcattttctt acaccttcta ttaccttctg ctctctctga tttggaaaaa
4921 gctgaaaaaa aaggttgaaa ccagttccct gaaattattc ccctacttga ctaataagta
4981 tataaagacg gtaggtattg attgtaattc tgtaaatcta tttcttaaac ttcttaaatt
5041 ctacttttat agttagtctt ttttttagtt ttaaaacacc agaacttagt ttcgacggat
5101 tctagaacta gtggatcccc cgggctgcag gaattcgata tcaagcttat cgataccgtc
5161 gacctcgagt catgtaatta gttatgtcac gcttacattc acgccctccc cccacatccg
5221 ctctaaccga aaaggaagga gttagacaac ctgaagtcta ggtccctatt tattttttta
5281 tagttatgtt agtattaaga acgttattta tatttcaaat ttttcttttt tttctgtaca
5341 gacgcgtgta cgcatgtaac attatactga aaaccttgct tgagaaggtt ttgggacgct
5401 cgaaggcttt aatttgcggc cggtacccaa ttcgccctat agtgagtcgt attacgcgcg
5461 ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
5521 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga
5581 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgcgacgcgc cctgtagcgg
5641 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
5701 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
5761 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
5821 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
5881 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
5941 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
6001 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
6061 aatattaacg tttacaattt cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
6121 tcacaccgca tagggtaata actgatataa ttaaattgaa gctctaattt gtgagtttag
6181 tatacatgca tttacttata atacagtttt ttagttttgc tggccgcatc ttctcaaata
6241 tgcttcccag cctgcttttc tgtaacgttc accctctacc ttagcatccc ttccctttgc
6301 aaatagtcct cttccaacaa taataatgtc agatcctgta gagaccacat catccacggt
6361 tctatactgt tgacccaatg cgtctccctt gtcatctaaa cccacaccgg gtgtcataat
6421 caaccaatcg taaccttcat ctcttccacc catgtctctt tgagcaataa agccgataac
6481 aaaatctttg tcgctcttcg caatgtcaac agtaccctta gtatattctc cagtagatag
6541 ggagcccttg catgacaatt ctgctaacat caaaaggcct ctaggttcct ttgttacttc
6601 ttctgccgcc tgcttcaaac cgctaacaat acctgggccc accacaccgt gtgcattcgt
6661 aatgtctgcc cattctgcta ttctgtatac acccgcagag tactgcaatt tgactgtatt
6721 accaatgtca gcaaattttc tgtcttcgaa gagtaaaaaa ttgtacttgg cggataatgc
6781 ctttagcggc ttaactgtgc cctccatgga aaaatcagtc aagatatcca catgtgtttt
6841 tagtaaacaa attttgggac ctaatgcttc aactaactcc agtaattcct tggtggtacg
6901 aacatccaat gaagcacaca agtttgtttg cttttcgtgc atgatattaa atagcttggc
6961 agcaacagga ctaggatgag tagcagcacg ttccttatat gtagctttcg acatgattta
7021 tcttcgtttc ctgcaggttt ttgttctgtg cagttgggtt aagaatactg ggcaatttca
7081 tgtttcttca acactacata tgcgtatata taccaatcta agtctgtgct ccttccttcg
7141 ttcttccttc tgttcggaga ttaccgaatc aaaaaaattt caaagaaacc gaaatcaaaa
7201 aaaagaataa aaaaaaaatg atgaattgaa ttgaaaagct gtggtatggt gcactctcag
7261 tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga
7321 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
7381 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
//
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
| P2398/pENTR223-HAX1-C16G人源基因质粒 |
| P2399/pENTR223-BUD23-A833人源基因质粒 |
| P2400/pENTR223-EMC2-A24G-13C人源基因质粒 |
| P2401/pENTR223-PEF1人源基因质粒 |
| P2402/pENTR223-INKA2人源基因质粒 |
| P2403/PENTR223-TSGA13（可引物突变）人源基因质粒 |
| P2404/pENTR223-C5AR1人源基因质粒 |
| P2405/pENTR223-DFFA-C871T人源基因质粒 |
| P2406/pENTR223-LRFN4(934-1908bp)人源基因质粒 |
| P2408/pENTR223-DHRS1-T26C人源基因质粒 |
| P2409/pENTR223-JKAMP-T635C人源基因质粒 |
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pY26TEF-GPD酵母双框质粒
￥100 - 1000
