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保存条件 :-20
保质期 :2年
英文名 :pGEX4T1;pGEX4T-1
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:pGEX-4T-1大肠表达质粒
别称: pGEX4T1;pGEX4T-1
启动子: | Tac |
---|---|
复制子: | pBR322 |
质粒分类: | 大肠杆菌载体;pGEX系列表达质粒 |
质粒大小: | 4969bp |
质粒标签: | N-GST, N-thrombin |
克隆菌株: | DH5a |
培养条件: | 37度 |
表达宿主: | 大肠杆菌BL21 DE3 |
培养条件: | 37℃,有氧,LB |
诱导方式: | IPTG或乳糖及其类似物 |
5'测序引物: | pGEX5: GGGCTGGCAAGCCACGTTTGGTG |
3'测序引物: | pGEX3: CCGGGAGCTGCATGTGTCAGAGG |
备注: | 可用GST亲和柱纯化重组蛋白 |
质粒属性
质粒宿主: | 大肠杆菌 |
---|---|
质粒用途: | 蛋白表达 |
片段类型: | ORF |
片段物种: | 空载体 |
原核抗性: | Amp |
真核抗性: | |
荧光标记: |
质粒简介
pGEX-4T-1质粒是一个大肠杆菌表达载体,Tac强启动子可以驱动GST促溶标签和目的基因融合表达,LacI阻碍蛋白和Laco操作子使本质粒在没有加入IPTG之前禁止表达,防止其影响细菌生长。表达后的融合蛋白可用凝血酶蛋白酶切割,再过一次GST柱子即可清除掉GST标签。
质粒图谱
质粒序列
LOCUS Exported 4969 bp ds-DNA circular SYN 03-8-2015
DEFINITION synthetic circular DNA
KEYWORDS pGEX-4T-1
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4969)
TITLE Direct Submission
JOURNAL Exported 2015-8-3 from MLCC
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..4969
/organism="synthetic DNA construct"
/mol_type="other DNA"
misc_feature 183..211
/note="tac promoter"
/note="strong E. coli promoter; hybrid between the trp and
lac UV5 promoters"
protein_bind 219..235
/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)."
CDS 258..911
/codon_start=1
/product="glutathione S-transferase from Schistosoma
japonicum"
/note="GST"
/translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
CDS 918..935
/codon_start=1
/product="thrombin recognition and cleavage site"
/note="thrombin site"
/translation="LVPRGS"
promoter 1272..1376
/gene="bla"
/note="AmpR promoter"
CDS 1377..2237
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 2408..2996
/direction=RIGHT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
promoter 3240..3317
/gene="lacI (mutant)"
/note="lacIq promoter"
/note="In the lacIq allele, a single base change in the
promoter boosts expression of the lacI gene about 10-fold."
CDS 3318..4400
/codon_start=1
/gene="lacI"
/product="lac repressor"
/note="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"
protein_bind 4413..4434
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 4449..4479
/note="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 4487..4503
/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)."
primer_bind 4511..4527
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
CDS 4523..81
/codon_start=1
/gene="lacZ fragment"
/product="LacZ-alpha fragment of beta-galactosidase"
/note="lacZ-alpha"
/translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGITLSTARCTNASGVR
QPSEAVVWLCRS"
primer_bind complement(4543..4559)
/note="M13 fwd"
/note="common sequencing primer, one of multiple similar
variants"
ORIGIN
1 acgttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg
61 gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt
121 tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc
181 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca
241 cacaggaaac agtattcatg tcccctatac taggttattg gaaaattaag ggccttgtgc
301 aacccactcg acttcttttg gaatatcttg aagaaaaata tgaagagcat ttgtatgagc
361 gcgatgaagg tgataaatgg cgaaacaaaa agtttgaatt gggtttggag tttcccaatc
421 ttccttatta tattgatggt gatgttaaat taacacagtc tatggccatc atacgttata
481 tagctgacaa gcacaacatg ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc
541 ttgaaggagc ggttttggat attagatacg gtgtttcgag aattgcatat agtaaagact
601 ttgaaactct caaagttgat tttcttagca agctacctga aatgctgaaa atgttcgaag
661 atcgtttatg tcataaaaca tatttaaatg gtgatcatgt aacccatcct gacttcatgt
721 tgtatgacgc tcttgatgtt gttttataca tggacccaat gtgcctggat gcgttcccaa
781 aattagtttg ttttaaaaaa cgtattgaag ctatcccaca aattgataag tacttgaaat
841 ccagcaagta tatagcatgg cctttgcagg gctggcaagc cacgtttggt ggtggcgacc
901 atcctccaaa atcggatctg gttccgcgtg gatccccgga attcccgggt cgactcgagc
961 ggccgcatcg tgactgactg acgatctgcc tcgcgcgttt cggtgatgac ggtgaaaacc
1021 tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca
1081 gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc
1141 agtcacgtag cgatagcgga gtgtataatt cttgaagacg aaagggcctc gtgatacgcc
1201 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc
1261 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
1321 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
1381 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
1441 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
1501 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
1561 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgtg
1621 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
1681 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
1741 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
1801 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
1861 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
1921 cagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
1981 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
2041 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
2101 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
2161 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
2221 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
2281 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
2341 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
2401 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
2461 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
2521 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
2581 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
2641 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
2701 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
2761 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
2821 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
2881 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
2941 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
3001 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
3061 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
3121 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
3181 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc ataaattccg
3241 acaccatcga atggtgcaaa acctttcgcg gtatggcatg atagcgcccg gaagagagtc
3301 aattcagggt ggtgaatgtg aaaccagtaa cgttatacga tgtcgcagag tatgccggtg
3361 tctcttatca gaccgtttcc cgcgtggtga accaggccag ccacgtttct gcgaaaacgc
3421 gggaaaaagt ggaagcggcg atggcggagc tgaattacat tcccaaccgc gtggcacaac
3481 aactggcggg caaacagtcg ttgctgattg gcgttgccac ctccagtctg gccctgcacg
3541 cgccgtcgca aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg
3601 tggtgtcgat ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg cacaatcttc
3661 tcgcgcaacg cgtcagtggg ctgatcatta actatccgct ggatgaccag gatgccattg
3721 ctgtggaagc tgcctgcact aatgttccgg cgttatttct tgatgtctct gaccagacac
3781 ccatcaacag tattattttc tcccatgaag acggtacgcg actgggcgtg gagcatctgg
3841 tcgcattggg tcaccagcaa atcgcgctgt tagcgggccc attaagttct gtctcggcgc
3901 gtctgcgtct ggctggctgg cataaatatc tcactcgcaa tcaaattcag ccgatagcgg
3961 aacgggaagg cgactggagt gccatgtccg gttttcaaca aaccatgcaa atgctgaatg
4021 agggcatcgt tcccactgcg atgctggttg ccaacgatca gatggcgctg ggcgcaatgc
4081 gcgccattac cgagtccggg ctgcgcgttg gtgcggatat ctcggtagtg ggatacgacg
4141 ataccgaaga cagctcatgt tatatcccgc cgttaaccac catcaaacag gattttcgcc
4201 tgctggggca aaccagcgtg gaccgcttgc tgcaactctc tcagggccag gcggtgaagg
4261 gcaatcagct gttgcccgtc tcactggtga aaagaaaaac caccctggcg cccaatacgc
4321 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
4381 gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca
4441 ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa
4501 caatttcaca caggaaacag ctatgaccat gattacggat tcactggccg tcgttttaca
4561 acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc
4621 tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg
4681 cagcctgaat ggcgaatggc gctttgcctg gtttccggca ccagaagcgg tgccggaaag
4741 ctggctggag tgcgatcttc ctgaggccga tactgtcgtc gtcccctcaa actggcagat
4801 gcacggttac gatgcgccca tctacaccaa cgtaacctat cccattacgg tcaatccgcc
4861 gtttgttccc acggagaatc cgacgggttg ttactcgctc acatttaatg ttgatgaaag
4921 ctggctacag gaaggccaga cgcgaattat ttttgatggc gttggaatt
//
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
P2040/pENTR223-MOBKL2A人源基因质粒 |
P2041/pENTR223-SAMD3人源基因质粒 |
P2042/pENTR223-MAGEH1人源基因质粒 |
P2043/pENTR223-CLTA人源基因质粒 |
P2057/pENTR223-C22orf23人源基因质粒 |
P2058/pENTR223-TMED3人源基因质粒 |
P2059/pENTR223-HEATR2-C13G人源基因质粒 |
P2060/pENTR223-TWSG1人源基因质粒 |
P2061/pENTR223-TMEM86B人源基因质粒 |
P2062/pENTR223-NUDCD3人源基因质粒 |
P2063/pENTR223-TRAPPC4人源基因质粒 |
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