pGEX-4T-3大肠表达质粒

基本信息
名称：pGEX-4T-3大肠表达质粒
别称: pGEX4T3;pGEX4T-3

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:	4968bp
质粒标签:	N-GST, N-thrombin
原核抗性:	Amp
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

载体宿主：	大肠杆菌
载体用途：	蛋白表达
基因种属：	空载体
基因类型：	ORF
原核抗性：	Amp
真核抗性：
荧光蛋白：

质粒简介
  pGEX-4T-3是一个大肠杆菌表达载体质粒，可通过MCS处的酶切位点连入目的基因，Tac启动子启动GST促溶标签和目的基因融合表达。
         其余类似载体：

质粒图谱


质粒序列
LOCUS       Exported File           4968 bp ds-DNA    circular SYN 28-1-2015
KEYWORDS    pGEX4T-3
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4968)
  TITLE     Direct Submission
  JOURNAL   Exported 2015-1-28 from SnapGene 2.0.1
            http://www.miaolingbio.com
FEATURES             Location/Qualifiers
     source          1..4968
                     /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
                     /gene="
                     "
                     /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        1271..1375
                     /gene="bla"
                     /note="AmpR promoter"
     CDS             1376..2236
                     /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      2407..2995
                     /direction=RIGHT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     promoter        3239..3316
                     /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             3317..4399
                     /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    4412..4433
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding site"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        4448..4478
                     /note="lac promoter"
                     /note="promoter for the E. coli lac operon"
     protein_bind    4486..4502
                     /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     4510..4526
                     /note="M13 rev"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     primer_bind     complement(4542..4558)
                     /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 gatccccgaa ttcccgggtc gactcgagcg
      961 gccgcatcgt gactgactga cgatctgcct cgcgcgtttc ggtgatgacg gtgaaaacct
     1021 ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag
     1081 acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca
     1141 gtcacgtagc gatagcggag tgtataattc ttgaagacga aagggcctcg tgatacgcct
     1201 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     1261 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     1321 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     1381 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     1441 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     1501 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     1561 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt
     1621 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     1681 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     1741 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     1801 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     1861 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgc
     1921 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     1981 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     2041 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     2101 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     2161 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     2221 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     2281 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     2341 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     2401 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     2461 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     2521 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
     2581 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     2641 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     2701 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     2761 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     2821 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     2881 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     2941 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     3001 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     3061 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     3121 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     3181 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca taaattccga
     3241 caccatcgaa tggtgcaaaa cctttcgcgg tatggcatga tagcgcccgg aagagagtca
     3301 attcagggtg gtgaatgtga aaccagtaac gttatacgat gtcgcagagt atgccggtgt
     3361 ctcttatcag accgtttccc gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg
     3421 ggaaaaagtg gaagcggcga tggcggagct gaattacatt cccaaccgcg tggcacaaca
     3481 actggcgggc aaacagtcgt tgctgattgg cgttgccacc tccagtctgg ccctgcacgc
     3541 gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat caactgggtg ccagcgtggt
     3601 ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct
     3661 cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg gatgaccagg atgccattgc
     3721 tgtggaagct gcctgcacta atgttccggc gttatttctt gatgtctctg accagacacc
     3781 catcaacagt attattttct cccatgaaga cggtacgcga ctgggcgtgg agcatctggt
     3841 cgcattgggt caccagcaaa tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg
     3901 tctgcgtctg gctggctggc ataaatatct cactcgcaat caaattcagc cgatagcgga
     3961 acgggaaggc gactggagtg ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga
     4021 gggcatcgtt cccactgcga tgctggttgc caacgatcag atggcgctgg gcgcaatgcg
     4081 cgccattacc gagtccgggc tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga
     4141 taccgaagac agctcatgtt atatcccgcc gttaaccacc atcaaacagg attttcgcct
     4201 gctggggcaa accagcgtgg accgcttgct gcaactctct cagggccagg cggtgaaggg
     4261 caatcagctg ttgcccgtct cactggtgaa aagaaaaacc accctggcgc ccaatacgca
     4321 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
     4381 actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac
     4441 cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac
     4501 aatttcacac aggaaacagc tatgaccatg attacggatt cactggccgt cgttttacaa
     4561 cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct
     4621 ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
     4681 agcctgaatg gcgaatggcg ctttgcctgg tttccggcac cagaagcggt gccggaaagc
     4741 tggctggagt gcgatcttcc tgaggccgat actgtcgtcg tcccctcaaa ctggcagatg
     4801 cacggttacg atgcgcccat ctacaccaac gtaacctatc ccattacggt caatccgccg
     4861 tttgttccca cggagaatcc gacgggttgt tactcgctca catttaatgt tgatgaaagc
     4921 tggctacagg aaggccagac gcgaattatt tttgatggcg ttggaatt
//

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

P2087/pENTR223-CDC34人源基因质粒

P2088/pENTR223-ABCD3人源基因质粒

P2089/pENTR223-C6orf142-A596C人源基因质粒

P2091/pENTR223-CLDN12人源基因质粒

P2092/pENTR223-C1QC人源基因质粒

P2093/pENTR223-FAM108A1人源基因质粒

P2094/pENTR223-ZNF174人源基因质粒

P2095/pENTR223-EXOC7人源基因质粒

P2096/pENTR223-UBE2U人源基因质粒

P2097/pENTR223-SCAMP4-G7A人源基因质粒

P2098/pENTR223-UBTD1人源基因质粒