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保存条件 :-20
保质期 :2年
英文名 :Plasmid#65974
库存 :100
供应商 :上海烜雅生物科技有限公司
CAS号 :/
规格 :干粉/液体
基本信息名称:T7opt in pCAGGS(T7 RNA polymerase)聚合酶基因质粒
别称: Plasmid#65974
启动子:CMV
复制子: pUC
原核抗性: Amp
克隆菌株: DH5a
培养条件: 37度
质粒属性
载体宿主:哺乳细胞
载体用途:蛋白表达
基因种属:
基因类型:ORF
原核抗性:Amp
真核抗性:
荧光蛋白:
质粒简介
T7opt in pCAGGS(T7 RNA polymerase)是一个T7 RNA 聚合酶表达质粒。
质粒图谱
质粒序列
LOCUS Exported 7381 bp ds-DNA circular SYN 26-DEC-2017
DEFINITION synthetic circular DNA
FEATURES Location/Qualifiers
source 1..7381
/organism="synthetic DNA construct"
/mol_type="other DNA"
CDS 1725..4376
/codon_start=1
/label=T7 RNA polymerase
/translation="MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYE
MGEARFRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQ
FLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFK
KNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIE
STGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGI
TGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANV
ITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLE
FMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYY
WLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFE
YAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAK
KVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVM
TLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVE
AMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLG
QFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHD
SFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLN
LRDILESDFAFA"
polyA_site 4450..4505
/label=-beta-globin poly(A) signal
/note="rabbit beta-globin polyadenylation signal"
promoter 5039..5234
/label=SV40 promoter
/note="SV40 early promoter"
rep_origin 5085..5220
/label=SV40 ori
/note="SV40 origin of replication"
polyA_signal 5240..5374
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
rep_origin complement(5613..6201)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(6372..7232)
/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"
promoter complement(7233..7337)
/gene="bla"
/label=AmpR promoter
misc_feature 7368..1664
/label=CAG promoter
ORIGIN
1 actagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
61 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
121 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
181 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
241 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
301 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
361 accatggtcg aggtgagccc cacgttctgc ttcactctcc ccatctcccc cccctcccca
421 cccccaattt tgtatttatt tattttttaa ttattttgtg cagcgatggg ggcggggggg
481 gggggggggc gcgcgccagg cggggcgggg cggggcgagg ggcggggcgg ggcgaggcgg
541 agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa agtttccttt tatggcgagg
601 cggcggcggc ggcggcccta taaaaagcga agcgcgcggc gggcggggag tcgctgcgac
661 gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac
721 tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt
781 agcgcttggt ttaatgacgg cttgtttctt ttctgtggct gcgtgaaagc cttgaggggc
841 tccgggaggg ccctttgtgc ggggggagcg gctcgggggg tgcgtgcgtg tgtgtgtgcg
901 tggggagcgc cgcgtgcggc tccgcgctgc ccggcggctg tgagcgctgc gggcgcggcg
961 cggggctttg tgcgctccgc agtgtgcgcg aggggagcgc ggccgggggc ggtgccccgc
1021 ggtgcggggg gggctgcgag gggaacaaag gctgcgtgcg gggtgtgtgc gtgggggggt
1081 gagcaggggg tgtgggcgcg tcggtcgggc tgcaaccccc cctgcacccc cctccccgag
1141 ttgctgagca cggcccggct tcgggtgcgg ggctccgtac ggggcgtggc gcggggctcg
1201 ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg ccgcctcggg
1261 ccggggaggg ctcgggggag gggcgcggcg gcccccggag cgccggcggc tgtcgaggcg
1321 cggcgagccg cagccattgc cttttatggt aatcgtgcga gagggcgcag ggacttcctt
1381 tgtcccaaat ctgtgcggag ccgaaatctg ggaggcgccg ccgcaccccc tctagcgggc
1441 gcggggcgaa gcggtgcggc gccggcagga aggaaatggg cggggagggc cttcgtgcgt
1501 cgccgcgccg ccgtcccctt ctccctctcc agcctcgggg ctgtccgcgg ggggacggct
1561 gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc ggcggctcta
1621 gagcctctgc taaccatgtt catgccttct tctttttcct acagctcctg ggcaacgtgc
1681 tggttattgt gctgtctcat cattttggca aagaattcgc caccatgaac accatcaata
1741 ttgccaagaa cgacttttct gatatcgagc tggccgctat tccattcaat acactggctg
1801 accactacgg agagcggctg gcccgcgaac agctggctct ggagcatgaa agctatgaga
1861 tgggagaagc ccgattcagg aagatgtttg agaggcagct gaaagctggg gaagtggcag
1921 acaacgcagc cgctaagcca ctgattacca cactgctgcc caaaatgatc gccagaatta
1981 atgattggtt cgaggaagtg aaggcaaaac gaggaaagag gcctaccgcc ttccagtttc
2041 tgcaggagat caagccagaa gctgtggcat acatcaccat caagactacc ctggcatgcc
2101 tgacaagcgc cgacaacaca actgtgcagg cagtcgcttc cgcaatcgga cgagctattg
2161 aggacgaagc acgctttggg agaatccggg atctggaggc caagcacttc aagaaaaacg
2221 tggaggaaca gctgaacaag agggtggggc atgtctataa gaaggccttc atgcaggtgg
2281 tcgaggccga catgctgtca aagggactgc tgggaggaga ggcctggagc tcctggcaca
2341 aagaagatag catccatgtg ggagtccgct gcatcgagat gctgattgaa tctactggga
2401 tggtgagtct gcaccgacag aacgccggcg tggtcggaca ggactctgag acaatcgaac
2461 tggctcccga gtatgccgaa gctattgcaa ctagagccgg agctctggca gggatcagtc
2521 caatgttcca gccctgcgtg gtccccccta agccttggac tggcatcacc gggggcggat
2581 actgggctaa tggaaggaga ccactggcac tggtgcgaac acactctaag aaagccctga
2641 tgagatacga ggatgtctat atgcccgaag tgtataaggc catcaacatt gctcagaata
2701 cagcatggaa aattaacaag aaagtgctgg ccgtcgctaa tgtgatcact aagtggaaac
2761 attgtcccgt ggaggacatc cctgccattg aacgggagga actgcctatg aagccagagg
2821 acatcgatat gaaccctgaa gctctgaccg catggaagcg cgcagccgct gcagtgtaca
2881 gaaaggataa agcccggaag tcccggcgca tttctctgga gttcatgctg gaacaggcca
2941 acaagtttgc taatcacaaa gcaatctggt tcccctacaa catggactgg cgcggacgag
3001 tctatgccgt gagcatgttc aaccctcagg ggaatgatat gacaaagggc ctgctgactc
3061 tggctaaggg gaaaccaatt gggaaggagg gctactattg gctgaaaatc cacggggcca
3121 attgcgctgg cgtcgacaag gtgccattcc ccgagaggat caagttcatc gaggaaaacc
3181 atgaaaatat tatggcatgt gccaagtctc ccctggagaa cacatggtgg gccgaacagg
3241 atagtccttt ctgctttctg gccttctgtt ttgagtacgc tggagtgcag caccatgggc
3301 tgagttataa ttgctccctg ccactggcct ttgacggctc ttgtagtgga atccagcact
3361 tctccgcaat gctgagggat gaggtcggag gaagagcagt gaacctgctg ccatctgaga
3421 cagtgcagga catctacggc attgtcgcca agaaagtgaa tgagatcctg caggctgacg
3481 caattaacgg gactgataat gaggtggtca ccgtcacaga tgaaaacact ggcgagatca
3541 gcgaaaaggt gaaactggga accaaggccc tggctggaca gtggctggca tacggagtca
3601 cccgctcagt gacaaagcga agcgtgatga ccctggctta tggcagcaaa gagttcggct
3661 tcaggcagca ggtgctggaa gacaccatcc agccagccat tgattccgga aaggggctga
3721 tgtttacaca gcccaaccag gccgctggct acatggccaa gctgatctgg gagtcagtga
3781 gcgtcacagt ggtcgcagcc gtggaagcta tgaattggct gaagtccgct gcaaaactgc
3841 tggccgctga ggtgaaggac aagaaaactg gcgaaattct gaggaaaaga tgcgccgtcc
3901 actgggtgac ccctgatgga ttcccagtgt ggcaggagta taagaaaccc atccagacca
3961 gactgaacct gatgttcctg ggccagtttc ggctgcagcc tacaatcaac actaataagg
4021 acagtgagat tgatgctcat aaacaggaat cagggattgc acctaatttt gtgcacagcc
4081 aggacggctc ccatctgcgg aagactgtgg tctgggctca cgagaaatac ggcatcgaat
4141 ccttcgcact gattcatgac tcttttggaa ccatcccagc cgatgcagcc aacctgttca
4201 aggctgtccg cgagactatg gtggacacct acgaaagttg tgatgtgctg gccgacttct
4261 atgatcagtt tgctgaccag ctgcacgagt cacagctgga taagatgccc gcactgcctg
4321 ccaaaggcaa cctgaatctg agagacatcc tggagtccga tttcgcattt gcctgactcg
4381 aggatctttt tccctctgcc aaaaattatg gggacatcat gaagcccctt gagcatctga
4441 cttctggcta ataaaggaaa tttattttca ttgcaatagt gtgttggaat tttttgtgtc
4501 tctcactcgg aaggacatat gggagggcaa atcatttaaa acatcagaat gagtatttgg
4561 tttagagttt ggcaacatat gcccatatgc tggctgccat gaacaaaggt tggctataaa
4621 gaggtcatca gtatatgaaa cagccccctg ctgtccattc cttattccat agaaaagcct
4681 tgacttgagg ttagattttt tttatatttt gttttgtgtt atttttttct ttaacatccc
4741 taaaattttc cttacatgtt ttactagcca gatttttcct cctctcctga ctactcccag
4801 tcatagctgt ccctcttctc ttatggagat ccctcgacct gcagcccaag cttggcgtaa
4861 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
4921 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
4981 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gcggatccgc
5041 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
5101 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
5161 ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
5221 taggcttttg caaaaagcta acttgtttat tgcagcttat aatggttaca aataaagcaa
5281 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
5341 caaactcatc aatgtatctt atcatgtctg gatccgctgc attaatgaat cggccaacgc
5401 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
5461 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
5521 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
5581 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
5641 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
5701 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
5761 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
5821 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
5881 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
5941 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
6001 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
6061 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
6121 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
6181 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
6241 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
6301 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
6361 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
6421 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
6481 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
6541 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
6601 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
6661 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
6721 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
6781 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
6841 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
6901 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
6961 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
7021 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
7081 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
7141 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
7201 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
7261 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
7321 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgggtcgac attgattatt
7381 g
//
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
P3929/pCMV-SPORT6-MYD88人源基因质粒 |
P3930/pCMV-SPORT6-LMF1人源基因质粒 |
P3931/pCMV-SPORT6-DLX4人源基因质粒 |
P3932/pCMV-SPORT6-RAB35人源基因质粒 |
P3933/pCMV-SPORT6-ELK1人源基因质粒 |
P3934/pCMV-SPORT6-MEF2A(1同义突变)人源基因质粒 |
P3935/pCMV-SPORT6-CANT1人源基因质粒 |
P3936/pCMV-SPORT6-ZFP36人源基因质粒 |
P3937/pCMV-SPORT6-PPM1M人源基因质粒 |
P3938/pCMV-SPORT6-SARAF人源基因质粒 |
P3939/pCMV-SPORT6-CHRAC1人源基因质粒 |
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