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- 详细信息
- 文献和实验
- 技术资料
- 保存条件：
-20
- 保质期：
2年
- 英文名：
pCMV-Tet3G
- 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：pCMV-Tet3G哺乳调控质粒
别称: pCMV-Tet3G
质粒属性
质粒简介
The pCMV-Tet3G Vector expresses Tet-On® 3G, a tetracycline-controlled transactivator that exhibits high activity in the presence of the inducer doxycycline (Dox) and exceptionally low activity in its absence. Tet-On 3G results from the fusion of amino acids 1–207 of a mutant Tet repressor (TetR) to 39 amino acids that form three minimal "F"-type transcriptional activation domains from the herpes simplex virus VP16 protein. Tet-On 3G was derived from Tet-On Advanced (1–4); as a result, it’s fully synthetic, lacks cryptic splice sites, and is codon-optimized for stable expression in mammalian cells. Compared to both of its predecessors, this 3rd generation Tet-On transactivator demonstrates increased sensitivity to Dox (1). Constitutive expression of Tet-On 3G is driven by the human cytomegalovirus immediately early promoter (PCMV IE). Note: An EF1α version of this vector is available for cell lines in which the CMV promoter is silenced.
pCMV-Tet3G is used to develop stable Tet-On 3G cell lines, which are hosts for Tet-inducible gene expression systems. To create a Tet-inducible expression system, a vector containing a gene of interest under the control of the Tet-inducible TRE3G promoter (PTRE3G) is transfected into a Tet-On 3G cell line. The addition of Dox to the system causes Tet-On 3G to undergo a conformational change that allows it to bind to PTRE3G, activating transcription of the gene of interest in a highly dose-dependent manner
质粒图谱
质粒序列
LOCUS Exported 7139 bp ds-DNA circular SYN 05-SEP-2016
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS Untitled 6
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7139)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Monday, September 5, 2016 from SnapGene Viewer 3.1.4
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..7139
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 89..468
/note="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 469..672
/note="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
CDS 775..1521
/codon_start=1
/product="modified rtTA protein that binds tightly to
promoters containing the tet operator in the presence of
doxycycline"
/note="Tet-On(R) 3G"
/translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
DALDDFDLDMLPADALDDFDLDMLPG"
polyA_signal 1531..1665
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
rep_origin complement(2385..2973)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(3144..4004)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(4005..4109)
/gene="bla"
/note="AmpR promoter"
polyA_signal 4222..4356
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
CDS complement(4781..4801)
/codon_start=1
/product="nuclear localization signal of SV40 large T
antigen"
/note="SV40 NLS"
/translation="PKKKRKV"
intron 4931..4996
/note="small t intron"
/note="simian virus 40 (SV40) small t antigen intron"
CDS complement(5416..6210)
/codon_start=1
/gene="aph(3')-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/note="NeoR/KanR"
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATC-PFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
promoter complement(6571..6900)
/note="SV40 promoter"
/note="SV40 enhancer and early promoter"
rep_origin 6585..6720
/note="SV40 ori"
/note="SV40 origin of replication"
ORIGIN
1 ctcgaggagc ttggcccatt gcatacgttg tatccatatc ataatatgta catttatatt
61 ggctcatgtc caacattacc gccatgttga cattgattat tgactagtta ttaatagtaa
121 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg
181 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg
241 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta
301 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt
361 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac
421 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt
481 tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac
541 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt
601 cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat
661 ataagcagag ctcgtttagt gaaccgtcag atcgcctgga gacgccatcc acgctgtttt
721 gacctccata gaagacaccg ggaccgatcc agcctccgcg gccccgaatt caccatgtct
781 agactggaca agagcaaagt cataaactct gctctggaat tactcaatgg agtcggtatc
841 gaaggcctga cgacaaggaa actcgctcaa aagctgggag ttgagcagcc taccctgtac
901 tggcacgtga agaacaagcg ggccctgctc gatgccctgc caatcgagat gctggacagg
961 catcataccc actcctgccc cctggaaggc gagtcatggc aagactttct gcggaacaac
1021 gccaagtcat accgctgtgc tctcctctca catcgcgacg gggctaaagt gcatctcggc
1081 acccgcccaa cagagaaaca gtacgaaacc ctggaaaatc agctcgcgtt cctgtgtcag
1141 caaggcttct ccctggagaa cgcactgtac gctctgtccg ccgtgggcca ctttacactg
1201 ggctgcgtat tggaggaaca ggagcatcaa gtagcaaaag aggaaagaga gacacctacc
1261 accgattcta tgcccccact tctgaaacaa gcaattgagc tgttcgaccg gcagggagcc
1321 gaacctgcct tccttttcgg cctggaacta atcatatgtg gcctggagaa acagctaaag
1381 tgcgaaagcg gcgggccgac cgacgccctt gacgattttg acttagacat gctcccagcc
1441 gatgcccttg acgactttga ccttgatatg ctgcctgctg acgctcttga cgattttgac
1501 cttgacatgc tccccgggta actaagtaag gatccagaca tgataagata cattgatgag
1561 tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat
1621 gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc
1681 attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaaag caagtaaaac
1741 ctctacaaat gtggtatggc tgattatgat cctgcaagcc tcgtcgtcct ggccggacca
1801 cgctatctgt gcaaggtccc cggccccgga cgcgcgctcc atgagcagag cgcccgccgc
1861 cgaggcgaag actcgggcgg cgccctgccc gtcccaccag gtcaacaggc ggtaaccggc
1921 ctcttcatcg ggaatgcgcg cgaccttcag catcgccggc atgtccccct ggcggacggg
1981 aagtatccag ctcgaccaag cttggcgaga ttttcaggag ctaaggaagc taaaatggag
2041 aaaaaaatca ctggatatac caccgttgat atatcccaat ggcatcgtaa agaacatttt
2101 gaggcatttc agtcagttgc tcaatgtacc tataaccaga ccgttcagct gcattaatga
2161 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
2221 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
2281 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
2341 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
2401 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
2461 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
2521 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
2581 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
2641 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
2701 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
2761 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
2821 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
2881 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
2941 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
3001 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
3061 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
3121 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
3181 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
3241 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
3301 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
3361 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
3421 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
3481 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
3541 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
3601 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
3661 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
3721 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa cacgggataa taccgcgcca
3781 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
3841 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
3901 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
3961 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
4021 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
4081 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
4141 taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt
4201 cgtcttcact cgaggtcgag ggatccagac atgataagat acattgatga gtttggacaa
4261 accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct
4321 ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt
4381 atgtttcagg ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa
4441 tgtggtatgg ctgattatga tctctagtca aggcactata catcaaatat tccttattaa
4501 cccctttaca aattaaaaag ctaaaggtac acaatttttg agcatagtta ttaatagcag
4561 acactctatg cctgtgtgga gtaagaaaaa acagtatgtt atgattataa ctgttatgcc
4621 tacttataaa ggttacagaa tatttttcca taattttctt gtatagcagt gcagcttttt
4681 cctttgtggt gtaaatagca aagcaagcaa gagttctatt actaaacaca gcatgactca
4741 aaaaacttag caattctgaa ggaaagtcct tggggtcttc tacctttctc ttcttttttg
4801 gaggagtaga atgttgagag tcagcagtag cctcatcatc actagatggc atttcttctg
4861 agcaaaacag gttttcctca ttaaaggcat tccaccactg ctcccattca tcagttccat
4921 aggttggaat ctaaaataca caaacaatta gaatcagtag tttaacacat tatacactta
4981 aaaattttat atttacctta gagctttaaa tctctgtagg tagtttgtcc aattatgtca
5041 caccacagaa gtaaggttcc ttcacaaaga tccggaccaa agcggccatc gtgcctcccc
5101 actcctgcag ttcgggggca tggatgcgcg gatagccgct gctggtttcc tggatgccga
5161 cggatttgca ctgccggtag aactccgcga ggtcgtccag cctcaggcag cagctgaacc
5221 aactcgcgag gggatcgagc ccggggtggg cgaagaactc cagcatgaga tccccgcgct
5281 ggaggatcat ccagccggcg tcccggaaaa cgattccgaa gcccaacctt tcatagaagg
5341 cggcggtgga atcgaaatct cgtgatggca ggttgggcgt cgcttggtcg gtcatttcga
5401 accccagagt cccgctcaga agaactcgtc aagaaggcga tagaaggcga tgcgctgcga
5461 atcgggagcg gcgataccgt aaagcacgag gaagcggtca gcccattcgc cgccaagctc
5521 ttcagcaata tcacgggtag ccaacgctat gtcctgatag cggtccgcca cacccagccg
5581 gccacagtcg atgaatccag aaaagcggcc attttccacc atgatattcg gcaagcaggc
5641 atcgccatgg gtcacgacga gatcctcgcc gtcgggcatg cgcgccttga gcctggcgaa
5701 cagttcggct ggcgcgagcc cctgatgctc ttcgtccaga tcatcctgat cgacaagacc
5761 ggcttccatc cgagtacgtg ctcgctcgat gcgatgtttc gcttggtggt cgaatgggca
5821 ggtagccgga tcaagcgtat gcagccgccg cattgcatca gccatgatgg atactttctc
5881 ggcaggagca aggtgagatg acaggagatc ctgccccggc acttcgccca atagcagcca
5941 gtcccttccc gcttcagtga caacgtcgag cacagctgcg caaggaacgc ccgtcgtggc
6001 cagccacgat agccgcgctg cctcgtcctg cagttcattc agggcaccgg acaggtcggt
6061 cttgacaaaa agaaccgggc gcccctgcgc tgacagccgg aacacggcgg catcagagca
6121 gccgattgtc tgttgtgccc agtcatagcc gaatagcctc tccacccaag cggccggaga
6181 acctgcgtgc aatccatctt gttcaatcat gcgaaacgat cctcatcctg tctcttgatc
6241 agatcttgat cccctgcgcc atcagatcct tggcggcaag aaagccatcc agtttacttt
6301 gcagggcttc ccaaccttac cagagggcgc cccagctggc aattccggtt cgcttgctgt
6361 ccataaaacc gcccagtcta gctatcgcca tgtaagccca ctgcaagcta cctgctttct
6421 ctttgcgctt gcgttttccc ttgtccagat agcccagtag ctgacattca tccggggtca
6481 gcaccgtttc tgcggactgg ctttctacgt gttccgcttc ctttagcagc ccttgcgccc
6541 tgagtgcttg cggcagcgtg aagctagctt tttgcaaaag cctaggcctc caaaaaagcc
6601 tcctcactac ttctggaata gctcagaggc cgaggcggcc tcggcctctg cataaataaa
6661 aaaaattagt cagccatggg gcggagaatg ggcggaactg ggcggagtta ggggcgggat
6721 gggcggagtt aggggcggga ctatggttgc tgactaattg agatgcatgc tttgcatact
6781 tctgcctgct ggggagcctg gggactttcc acacctggtt gctgactaat tgagatgcat
6841 gctttgcata cttctgcctg ctggggagcc tggggacttt ccacacccta actgacacac
6901 attccacagc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct
6961 cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg
7021 cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag
7081 cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcacct
//
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：pCMV-Tet3G哺乳调控质粒
别称: pCMV-Tet3G
| 启动子: | CMV |
|---|---|
| 复制子: | pUC |
| 终止子: | SV40 poly(A) signal |
| 质粒分类: | 哺乳细胞，四环素调控系统载体 |
| 质粒大小: | 7139bp |
| 原核抗性: | Amp |
| 克隆菌株: | DH5a |
| 培养条件: | 37度 |
| 表达宿主: | 哺乳细胞 |
| 诱导方式: | 四环素诱导 |
| 5'测序引物: | CMV-F：CGCAAATGGGCGGTAGGCGTG |
| 3'测序引物: | 根据序列设计引物 |
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
The pCMV-Tet3G Vector expresses Tet-On® 3G, a tetracycline-controlled transactivator that exhibits high activity in the presence of the inducer doxycycline (Dox) and exceptionally low activity in its absence. Tet-On 3G results from the fusion of amino acids 1–207 of a mutant Tet repressor (TetR) to 39 amino acids that form three minimal "F"-type transcriptional activation domains from the herpes simplex virus VP16 protein. Tet-On 3G was derived from Tet-On Advanced (1–4); as a result, it’s fully synthetic, lacks cryptic splice sites, and is codon-optimized for stable expression in mammalian cells. Compared to both of its predecessors, this 3rd generation Tet-On transactivator demonstrates increased sensitivity to Dox (1). Constitutive expression of Tet-On 3G is driven by the human cytomegalovirus immediately early promoter (PCMV IE). Note: An EF1α version of this vector is available for cell lines in which the CMV promoter is silenced.
pCMV-Tet3G is used to develop stable Tet-On 3G cell lines, which are hosts for Tet-inducible gene expression systems. To create a Tet-inducible expression system, a vector containing a gene of interest under the control of the Tet-inducible TRE3G promoter (PTRE3G) is transfected into a Tet-On 3G cell line. The addition of Dox to the system causes Tet-On 3G to undergo a conformational change that allows it to bind to PTRE3G, activating transcription of the gene of interest in a highly dose-dependent manner
质粒图谱
质粒序列
LOCUS Exported 7139 bp ds-DNA circular SYN 05-SEP-2016
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS Untitled 6
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7139)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Monday, September 5, 2016 from SnapGene Viewer 3.1.4
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..7139
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 89..468
/note="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 469..672
/note="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
CDS 775..1521
/codon_start=1
/product="modified rtTA protein that binds tightly to
promoters containing the tet operator in the presence of
doxycycline"
/note="Tet-On(R) 3G"
/translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
DALDDFDLDMLPADALDDFDLDMLPG"
polyA_signal 1531..1665
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
rep_origin complement(2385..2973)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(3144..4004)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(4005..4109)
/gene="bla"
/note="AmpR promoter"
polyA_signal 4222..4356
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
CDS complement(4781..4801)
/codon_start=1
/product="nuclear localization signal of SV40 large T
antigen"
/note="SV40 NLS"
/translation="PKKKRKV"
intron 4931..4996
/note="small t intron"
/note="simian virus 40 (SV40) small t antigen intron"
CDS complement(5416..6210)
/codon_start=1
/gene="aph(3')-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/note="NeoR/KanR"
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATC-PFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
promoter complement(6571..6900)
/note="SV40 promoter"
/note="SV40 enhancer and early promoter"
rep_origin 6585..6720
/note="SV40 ori"
/note="SV40 origin of replication"
ORIGIN
1 ctcgaggagc ttggcccatt gcatacgttg tatccatatc ataatatgta catttatatt
61 ggctcatgtc caacattacc gccatgttga cattgattat tgactagtta ttaatagtaa
121 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg
181 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg
241 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta
301 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt
361 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac
421 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt
481 tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac
541 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt
601 cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat
661 ataagcagag ctcgtttagt gaaccgtcag atcgcctgga gacgccatcc acgctgtttt
721 gacctccata gaagacaccg ggaccgatcc agcctccgcg gccccgaatt caccatgtct
781 agactggaca agagcaaagt cataaactct gctctggaat tactcaatgg agtcggtatc
841 gaaggcctga cgacaaggaa actcgctcaa aagctgggag ttgagcagcc taccctgtac
901 tggcacgtga agaacaagcg ggccctgctc gatgccctgc caatcgagat gctggacagg
961 catcataccc actcctgccc cctggaaggc gagtcatggc aagactttct gcggaacaac
1021 gccaagtcat accgctgtgc tctcctctca catcgcgacg gggctaaagt gcatctcggc
1081 acccgcccaa cagagaaaca gtacgaaacc ctggaaaatc agctcgcgtt cctgtgtcag
1141 caaggcttct ccctggagaa cgcactgtac gctctgtccg ccgtgggcca ctttacactg
1201 ggctgcgtat tggaggaaca ggagcatcaa gtagcaaaag aggaaagaga gacacctacc
1261 accgattcta tgcccccact tctgaaacaa gcaattgagc tgttcgaccg gcagggagcc
1321 gaacctgcct tccttttcgg cctggaacta atcatatgtg gcctggagaa acagctaaag
1381 tgcgaaagcg gcgggccgac cgacgccctt gacgattttg acttagacat gctcccagcc
1441 gatgcccttg acgactttga ccttgatatg ctgcctgctg acgctcttga cgattttgac
1501 cttgacatgc tccccgggta actaagtaag gatccagaca tgataagata cattgatgag
1561 tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat
1621 gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc
1681 attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaaag caagtaaaac
1741 ctctacaaat gtggtatggc tgattatgat cctgcaagcc tcgtcgtcct ggccggacca
1801 cgctatctgt gcaaggtccc cggccccgga cgcgcgctcc atgagcagag cgcccgccgc
1861 cgaggcgaag actcgggcgg cgccctgccc gtcccaccag gtcaacaggc ggtaaccggc
1921 ctcttcatcg ggaatgcgcg cgaccttcag catcgccggc atgtccccct ggcggacggg
1981 aagtatccag ctcgaccaag cttggcgaga ttttcaggag ctaaggaagc taaaatggag
2041 aaaaaaatca ctggatatac caccgttgat atatcccaat ggcatcgtaa agaacatttt
2101 gaggcatttc agtcagttgc tcaatgtacc tataaccaga ccgttcagct gcattaatga
2161 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
2221 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
2281 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
2341 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
2401 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
2461 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
2521 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
2581 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
2641 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
2701 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
2761 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
2821 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
2881 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
2941 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
3001 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
3061 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
3121 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
3181 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
3241 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
3301 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
3361 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
3421 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
3481 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
3541 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
3601 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
3661 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
3721 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa cacgggataa taccgcgcca
3781 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
3841 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
3901 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
3961 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
4021 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
4081 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
4141 taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt
4201 cgtcttcact cgaggtcgag ggatccagac atgataagat acattgatga gtttggacaa
4261 accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct
4321 ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt
4381 atgtttcagg ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa
4441 tgtggtatgg ctgattatga tctctagtca aggcactata catcaaatat tccttattaa
4501 cccctttaca aattaaaaag ctaaaggtac acaatttttg agcatagtta ttaatagcag
4561 acactctatg cctgtgtgga gtaagaaaaa acagtatgtt atgattataa ctgttatgcc
4621 tacttataaa ggttacagaa tatttttcca taattttctt gtatagcagt gcagcttttt
4681 cctttgtggt gtaaatagca aagcaagcaa gagttctatt actaaacaca gcatgactca
4741 aaaaacttag caattctgaa ggaaagtcct tggggtcttc tacctttctc ttcttttttg
4801 gaggagtaga atgttgagag tcagcagtag cctcatcatc actagatggc atttcttctg
4861 agcaaaacag gttttcctca ttaaaggcat tccaccactg ctcccattca tcagttccat
4921 aggttggaat ctaaaataca caaacaatta gaatcagtag tttaacacat tatacactta
4981 aaaattttat atttacctta gagctttaaa tctctgtagg tagtttgtcc aattatgtca
5041 caccacagaa gtaaggttcc ttcacaaaga tccggaccaa agcggccatc gtgcctcccc
5101 actcctgcag ttcgggggca tggatgcgcg gatagccgct gctggtttcc tggatgccga
5161 cggatttgca ctgccggtag aactccgcga ggtcgtccag cctcaggcag cagctgaacc
5221 aactcgcgag gggatcgagc ccggggtggg cgaagaactc cagcatgaga tccccgcgct
5281 ggaggatcat ccagccggcg tcccggaaaa cgattccgaa gcccaacctt tcatagaagg
5341 cggcggtgga atcgaaatct cgtgatggca ggttgggcgt cgcttggtcg gtcatttcga
5401 accccagagt cccgctcaga agaactcgtc aagaaggcga tagaaggcga tgcgctgcga
5461 atcgggagcg gcgataccgt aaagcacgag gaagcggtca gcccattcgc cgccaagctc
5521 ttcagcaata tcacgggtag ccaacgctat gtcctgatag cggtccgcca cacccagccg
5581 gccacagtcg atgaatccag aaaagcggcc attttccacc atgatattcg gcaagcaggc
5641 atcgccatgg gtcacgacga gatcctcgcc gtcgggcatg cgcgccttga gcctggcgaa
5701 cagttcggct ggcgcgagcc cctgatgctc ttcgtccaga tcatcctgat cgacaagacc
5761 ggcttccatc cgagtacgtg ctcgctcgat gcgatgtttc gcttggtggt cgaatgggca
5821 ggtagccgga tcaagcgtat gcagccgccg cattgcatca gccatgatgg atactttctc
5881 ggcaggagca aggtgagatg acaggagatc ctgccccggc acttcgccca atagcagcca
5941 gtcccttccc gcttcagtga caacgtcgag cacagctgcg caaggaacgc ccgtcgtggc
6001 cagccacgat agccgcgctg cctcgtcctg cagttcattc agggcaccgg acaggtcggt
6061 cttgacaaaa agaaccgggc gcccctgcgc tgacagccgg aacacggcgg catcagagca
6121 gccgattgtc tgttgtgccc agtcatagcc gaatagcctc tccacccaag cggccggaga
6181 acctgcgtgc aatccatctt gttcaatcat gcgaaacgat cctcatcctg tctcttgatc
6241 agatcttgat cccctgcgcc atcagatcct tggcggcaag aaagccatcc agtttacttt
6301 gcagggcttc ccaaccttac cagagggcgc cccagctggc aattccggtt cgcttgctgt
6361 ccataaaacc gcccagtcta gctatcgcca tgtaagccca ctgcaagcta cctgctttct
6421 ctttgcgctt gcgttttccc ttgtccagat agcccagtag ctgacattca tccggggtca
6481 gcaccgtttc tgcggactgg ctttctacgt gttccgcttc ctttagcagc ccttgcgccc
6541 tgagtgcttg cggcagcgtg aagctagctt tttgcaaaag cctaggcctc caaaaaagcc
6601 tcctcactac ttctggaata gctcagaggc cgaggcggcc tcggcctctg cataaataaa
6661 aaaaattagt cagccatggg gcggagaatg ggcggaactg ggcggagtta ggggcgggat
6721 gggcggagtt aggggcggga ctatggttgc tgactaattg agatgcatgc tttgcatact
6781 tctgcctgct ggggagcctg gggactttcc acacctggtt gctgactaat tgagatgcat
6841 gctttgcata cttctgcctg ctggggagcc tggggacttt ccacacccta actgacacac
6901 attccacagc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct
6961 cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg
7021 cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag
7081 cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcacct
//
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
| P2817/pENTR223-MLC1-C74A人源基因质粒 |
| P2818/pENTR223-GNAZ-C404A人源基因质粒 |
| P2819/pENTR223-FGD6人源基因质粒 |
| P2820/pENTR223-IQUB（1-1119bp）人源基因质粒 |
| P2821/pENTR223-PTPN2-C15G人源基因质粒 |
| P2822/pENTR223-NFKBIE（418-1503bp）人源基因质粒 |
| P2823/pENTR223-DNAJB11人源基因质粒 |
| P2824/pENTR223-B4GALT3（2同义突变）人源基因质粒 |
| P2825/pENTR223-KCNJ13（3点突变）人源基因质粒 |
| P2848/pENTR223-NGEF人源基因质粒 |
| P1600/pENTR-GFP-MDC1 (770-7)人源基因质粒 |
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文献和实验相关实验
1、质粒的概念 质粒（Plasmid）是一种存在于细菌、酵母等微生物细胞中的小型环状双链 DNA 分子，能够独立于宿主染色体进行自我复制，并通常携带对宿主生存非必需但具有特定功能的基因（如抗生素抗性基因）。质粒是分子生物学和基因工程中最重要的工具之一。 质粒根据其用途可以分为多种不同的类型，如用于保存序列信息的克隆质粒（如：pUC 系列质粒），用于表达蛋白的质粒（如哺乳动物细胞表达所用的 pcDNA3.1 系列质粒），用于基因编辑的质粒（如哺乳动物细胞基因编辑所用的 espCas9-2A
将克隆化基因插入合适载体后导入大肠杆菌用于表达大量蛋白质的方法一般称为原核表达。这种方法在蛋白纯化、定位及功能分析等方面都有应用。大肠杆菌用于表达重组蛋白有以下特点： 易于生长和控制；用于细菌培养的材料不及哺乳动物细胞系统的材料昂贵；有各种各样的大肠杆菌菌株及与之匹配的具各种特性的质粒可供选择。但是，在大肠杆菌中表达的蛋白由于缺少修饰和糖基化、磷酸化等翻译后加工，常形成包涵体而影响表达蛋白的生物学活性及构象。 表达载体在基因工程中具有十分重要的作用，原核表达载体通常为质粒，典型的表达
1、优越的灵敏度，比Westernbloting灵敏度高1000倍以上；2、内源性低，哺乳动物无内源性表达；3、荧光素酶检测不受细胞内其他物质影响；4、发光检测，检测方便；5、灵敏度高，10‐20摩尔荧光素酶分子；6、检测范围广，大于7个数量级。四、主要应用领域1、潜在启动子/启动子核心区域检测；2、潜在增强子/抑制子等调控子核心元件检测；3、启动子区可能的转录因子结合位点检测；4、启动子/增强子与转录因子的相互作用；5、病毒/细胞相互作用；6、药物等化学诱导因素对启动子活性的调节（抑制或增强
pCMV-Tet3G哺乳调控质粒
￥100 - 1000
