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保存条件 :-20
保质期 :2年
英文名 :Plasmid#29581
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:pMCSG10-Map3k1人源基因质粒
别称: Plasmid#29581
启动子: | T7 |
---|
质粒分类: | 基因库质粒 |
---|
克隆菌株: DH5a
培养条件: 37度
质粒属性
载体宿主:大肠杆菌
载体用途:蛋白表达
基因种属:人
基因类型:ORF
原核抗性:Amp
真核抗性:
荧光蛋白:
质粒简介
pMCSG10-Map3k1是一个人源基因大肠表达质粒。
质粒图谱
质粒序列
LOCUS Exported 7735 bp ds-DNA circular SYN 02-NOV-2017
FEATURES Location/Qualifiers
source 1..7735
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 285..388
/gene="bla"
/label=AmpR promoter
CDS 389..1249
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 1420..2008
/direction=RIGHT
/label=ori
/note="high-copy-number colE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(2438..2629)
/codon_start=1
/gene="rop"
/product="Rop protein"
/label=rop
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
CDS complement(3438..4520)
/codon_start=1
/gene="lacI"
/product="lac repressor"
/label=lacI
/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)."
/translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
ALADSLMQLARQVSRLESGQ"
promoter complement(4521..4598)
/gene="lacI"
/label=lacI promoter
promoter 4911..4929
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 4930..4954
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/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)."
CDS 5002..5019
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
CDS 5038..5691
/codon_start=1
/product="glutathione S-transferase from Schistosoma
japonicum"
/label=GST
/translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
CDS 5719..5739
/codon_start=1
/product="tobacco etch virus (TEV) recognition and cleavage
site"
/label=TEV site
/translation="ENLYFQS"
misc_feature 5740..7515
/label=MAP3K13
terminator 7663..7710
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
ORIGIN
1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
61 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
241 acattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
301 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
361 tgcttcaata atttgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
421 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
481 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
541 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
601 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
661 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
721 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
781 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
841 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
901 accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact
961 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
1021 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
1081 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
1141 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
1201 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
1261 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
1321 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
1381 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
1441 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
1501 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
1561 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
1621 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
1681 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
1741 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
1801 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
1861 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
1921 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
1981 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
2041 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
2101 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
2161 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
2221 tctgtgcggt atttcacacc gcatatatgg tgcactctca gtacaatctg ctctgatgcc
2281 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc
2341 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
2401 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
2461 cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga
2521 tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc
2581 ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg
2641 tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca
2701 cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac
2761 tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg
2821 ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga
2881 acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga
2941 agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc
3001 gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg
3061 tcctcaacga caggagcacg atcatgcgca cccgtggggc cgccatgccg gcgataatgg
3121 cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt
3181 gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt
3241 cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga
3301 agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg
3361 ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta
3421 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
3481 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt
3541 tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag
3601 agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg
3661 gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata
3721 tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga
3781 tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt
3841 tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg
3901 cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg
3961 cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc
4021 gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga
4081 aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc
4141 ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta
4201 caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg
4261 gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg
4321 gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg
4381 taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg
4441 gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg
4501 tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat
4561 gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt
4621 atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc
4681 cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc
4741 caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc
4801 atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc
4861 cacgatgcgt ccggcgtaga ggatcgagat cgatctcgat cccgcgaaat taatacgact
4921 cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt tgtttaactt
4981 taagaaggag atatacatat gcaccatcat catcatcatt cttctggtgt agatctgatg
5041 tcccctatac taggttattg gaaaattaag ggccttgtgc aacccactcg acttcttttg
5101 gaatatcttg aagaaaaata tgaagagcat ttgtatgagc gcgatgaagg tgataaatgg
5161 cgaaacaaaa agtttgaatt gggtttggag tttcccaatc ttccttatta tattgatggt
5221 gatgttaaat taacacagtc tatggccatc atacgttata tagctgacaa gcacaacatg
5281 ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc ttgaaggagc ggttttggat
5341 attagatacg gtgtttcgag aattgcatat agtaaagact ttgaaactct caaagttgat
5401 tttcttagca agctacctga aatgctgaaa atgttcgaag atcgtttatg tcataaaaca
5461 tatttaaatg gtgatcatgt aacccatcct gacttcatgt tgtatgacgc tcttgatgtt
5521 gttttataca tggacccaat gtgcctggat gcgttcccaa aattagtttg ttttaaaaaa
5581 cgtattgaag ctatcccaca aattgataag tacttgaaat ccagcaagta tatagcatgg
5641 cctttgcagg gctggcaagc cacgtttggt ggtggcgacc atcctccaaa atcggatgat
5701 tacccagatc tgggtaccga gaacctgtac ttccaatcca atgatgggca tcaggacagc
5761 ttacaggcgc tggcccccgc cagctgtcta gaaaacagct cccttgagca cacagtccat
5821 agagagaaaa ctggaaaagg actaagtgct acgagactga gtgccagctc ggaggacatt
5881 tctgacagac tggccggcgt ctctgtagga cttcccagct caacaacaac agaacaacca
5941 aagccagcgg ttcaaacaaa aggcagaccc cacagtcagt gtttgaactc ctcccctttg
6001 tctcatgctc aattaatgtt cccagcacca tcagcccctt gttcctctgc cccgtctgtc
6061 ccagatattt ctaagcacag accccaggca tttgttccct gcaaaatacc ttccgcatct
6121 cctcagacac agcgcaagtt ctctctacaa ttccagagga actgctctga acaccgagac
6181 tcagaccagc tctccccagt cttcactcag tcaagacccc caccctccag taacatacac
6241 aggccaaagc catcccgacc cgttccgggc agtacaagca aactagggga cgccacaaaa
6301 agtagcatga cacttgatct gggcagtgct tccaggtgtg acgacagctt tggcggcggc
6361 ggcaacagtg gcaacgccgt catacccagc gacgagacag tgttcacgcc ggtggaggac
6421 aagtgcaggt tagatgtgaa caccgagctc aactccagca tcgaggacct tcttgaagca
6481 tccatgcctt caagtgacac gacagtcact ttcaagtccg aagtcnatga agntgagaga
6541 agtagcttta ncgatcgcca tgngantcag cgtctcagat gccctcccca tcgtcctcag
6601 ntgcagtgga aaatggagaa gatattatca tcattcagca ggacacacca gaaactntna
6661 ggacatacca aagcgaaaca gccttacaga gaagacgntg agtggctgaa aggccagcag
6721 atagncctcg gagcattttc ttcctgttac caagcacagg atgtggggac tgggacttta
6781 atggctgtga aacaggtgac gtacgtcaga aacacatcnt ccgagcagga ggaggtggtg
6841 gaagcgttga gggaagagat ccggatgatg ggtcacctca accatccaaa catcatccgg
6901 atgctggggg ccacgtgcga gaagagcaac tacaacctct tcattgagtg gatggcggga
6961 ggatctgtgg ctcacctctt gagtaaatac ggagctttca aggagtcagt cgtcattaac
7021 tacactgagc agttactgcg tggcctttcc tatctccacg agaaccagat cattcacaga
7081 gacgtcaaag gtgccaacct gctcattgac agcaccggtc agaggctgag aattgcagac
7141 tttggagctg ctgccaggtt ggcatcaaaa ggaaccggtg caggagagtt ccagggacag
7201 ttactgggga caattgcatt catggcgcct gaggtcctaa gaggtcagca gtatggtagg
7261 agctgtgatg tatggagtgt tggctgcgcc attatagaaa tggcttgtgc aaaaccacct
7321 tggaatgcag aaaaacactc caatcatctc gccttgatat ttaagattgc tagcgcaact
7381 actgcaccgt ccatcccgtc acacctgtcc ccgggtctgc gcgacgtggc gctgcgctgc
7441 ttagaacttc agcctcagga ccggcctccg tccagagagc tgctgaaaca tccggtcttc
7501 cgtaccacgt ggtagcattg gaagtggata acggatccga attcgagctc cgtcgacaag
7561 cttgcggccg cactcgagca ccaccaccac caccactgag atccggctgc taacaaagcc
7621 cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg
7681 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggat
//
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
P3060/pCDNA3.1-SLC28A1-FLAG(2同义突变)人源基因质粒 |
P0566/pECMV-Tet2-Myc-FLAG人源基因质粒 |
P0567/pEGFP-DHX36人源基因质粒 |
P0570/pCDNA3.1-FLAG-UEV1A人源基因质粒 |
P0583/pEGFP-Rab11a-WT人源基因质粒 |
P0505/pEGFP-Rab11a-S25N人源基因质粒 |
P0572/pEGFP-Rab11b-WT人源基因质粒 |
P0571/pEGFP-Rab11b-S25N人源基因质粒 |
P0573/pCDNA3.1-BMP4人源基因质粒 |
P0574/pCDNA3.1-FLAG-Ubc13人源基因质粒 |
P0575/pCS2-EGFP-EB3人源基因质粒 |
P0576/pEGFP-CagA人源基因质粒 |
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