pGEX-4T-1大肠表达质粒

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