pET-41a大肠表达质粒

基本信息
名称：pET-41a大肠表达质粒
别称: pET41a

启动子:	T7
复制子:	pBR322
终止子:	T7 terminator
质粒分类:	大肠杆菌载体；PET系列表达质粒
质粒大小:	5933bp
质粒标签:	N-GST, N-6×His, N-Thrombin
原核抗性:	Kan
克隆菌株:	DH5a
培养条件:	37度
表达宿主:	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:	T7:TAATACGACTCACTATAGGG
3'测序引物:	T7-ter:TGCTAGTTATTGCTCAGCGG

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：	Kan
真核抗性：
荧光蛋白：

质粒简介
   pET41系列载体是设计用来克隆和高水平表达蛋白质的载体，载体融合表达一个含有220个氨基酸的GST标签蛋白纯化片段。pET41a载体的单一的多克隆位点见上面的环状质粒图谱。注意：载体序列是以pBR322质粒的编码规矩进行编码的，所以T7蛋白表达区在质粒图谱上面是反向的。
       T7 RNA聚合酶启动的克隆和表达区域在质粒图谱中也被标注了出来。质粒的F1复制子是被定向的，所以在T7噬菌体聚合酶的作用下，包含有蛋白编码序列的病毒 粒子能够产生，并启动蛋白表达，同时蛋白表达将被T7终止子序列的作用下终止蛋白翻译。pET-41a载体上面含有一个EK蛋白酶切位点，当将目的基因使用载体上面的PshAI限制性内切酶位点插入进去时，可以通过EK蛋白酶将载体上的融合的所有氨基酸序列包括GST标签序列，全部切除掉。

质粒图谱


质粒序列
LOCUS       Exported                5933 bp ds-DNA     circular SYN 10-8-2015
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    Untitled
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5933)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported 2015-8-10   from MLCC
            http://www.miaolinhbio.com
FEATURES             Location/Qualifiers
     source          1..5933
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     source          1103..1125
                     /organism="Enterobacteria phage T7"
                     /mol_type="genomic DNA"
                     /db_xref="taxon:10760"
     terminator      26..73
                     /note="T7 terminator"
                     /note="transcription terminator for bacteriophage T7 RNA
                     polymerase"
     CDS             complement(150..173)
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /note="8xHis"
                     /translation="HHHHHHHH"
     CDS             complement(265..279)
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /note="enterokinase site"
                     /translation="DDDDK"
     CDS             complement(310..354)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic
                     ribonuclease A"
                     /note="S-Tag"
                     /translation="KETAAAKFERQHMDS"
     CDS             complement(370..387)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /note="thrombin site"
                     /translation="LVPRGS"
     CDS             complement(397..414)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /note="6xHis"
                     /translation="HHHHHH"
     CDS             complement(442..1095)
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma
                     japonicum"
                     /note="GST"
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     RBS             1103..1125
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1140..1164
                     /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)."
     promoter        complement(1165..1183)
                     /note="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        1496..1573
                     /gene="lacI"
                     /note="lacI promoter"
     CDS             1574..2656
                     /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"
     CDS             3228..3419
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /note="rop"
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     misc_feature    3521..3663
                     /note="bom"
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(3849..4437)
                     /direction=LEFT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     CDS             4559..5374
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /note="KanR"
                     /note="confers resistance to kanamycin in bacteria or G418
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      complement(5467..5922)
                     /direction=LEFT
                     /note="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgtttagcag cctaggtatt aatcaattag tggtggtggt ggtggtggtg gtgctcgagt
      181 gcggccgcaa gcttgtcgac ggagctcgcc tgcaggcgcg ccaaggcctg tacagaattc
      241 ggatccccga tatcccatgg gactcttgtc gtcgtcatca ccggagccac caccggtacc
      301 cagatctggg ctgtccatgt gctggcgttc gaatttagca gcagcggttt ctttcatacc
      361 aattgcagta ctaccgcgtg gcaccagacc cgcggagtga tggtgatggt gatgaccaga
      421 accactagtt gaaccatccg attttggagg atggtcgcca ccaccaaacg tggcttgcca
      481 gccctgcaaa ggccatgcta tatacttgct ggatttcaag tacttatcaa tttgtgggat
      541 agcttcaata cgttttttaa aacaaactaa ttttgggaac gcatccaggc acattgggtc
      601 catgtataaa acaacatcaa gagcgtcata caacatgaag tcaggatggg ttacatgatc
      661 accatttaaa tatgttttat gacataaacg atcttcgaac attttcagca tttcaggtag
      721 cttgctaaga aaatcaactt tgagagtttc aaagtcttta ctatatgcaa ttctcgaaac
      781 accgtatcta atatccaaaa ccgctccttc aagcattgaa atctctgcac gctcttttgg
      841 acaaccaccc aacatgttgt gcttgtcagc tatataacgt atgatggcca tagactgtgt
      901 taatttaaca tcaccatcaa tataataagg aagattggga aactccaaac ccaattcaaa
      961 ctttttgttt cgccatttat caccttcatc gcgctcatac aaatgctctt catatttttc
     1021 ttcaagatat tccaaaagaa gtcgagtggg ttgcacaagg cccttaattt tccaataacc
     1081 tagtataggg gacatatgta tatctccttc ttaaagttaa acaaaattat ttctagaggg
     1141 gaattgttat ccgctcacaa ttcccctata gtgagtcgta ttaatttcgc gggatcgaga
     1201 tcgatctcga tcctctacgc cggacgcatc gtggccggca tcaccggcgc cacaggtgcg
     1261 gttgctggcg cctatatcgc cgacatcacc gatggggaag atcgggctcg ccacttcggg
     1321 ctcatgagcg cttgtttcgg cgtgggtatg gtggcaggcc ccgtggccgg gggactgttg
     1381 ggcgccatct ccttgcatgc accattcctt gcggcggcgg tgctcaacgg cctcaaccta
     1441 ctactgggct gcttcctaat gcaggagtcg cataagggag agcgtcgaga tcccggacac
     1501 catcgaatgg cgcaaaacct ttcgcggtat ggcatgatag cgcccggaag agagtcaatt
     1561 cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc
     1621 ttatcagacc gtttcccgcg tggtgaacca ggccagccac gtttctgcga aaacgcggga
     1681 aaaagtggaa gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg cacaacaact
     1741 ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc agtctggccc tgcacgcgcc
     1801 gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt
     1861 gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc
     1921 gcaacgcgtc agtgggctga tcattaacta tccgctggat gaccaggatg ccattgctgt
     1981 ggaagctgcc tgcactaatg ttccggcgtt atttcttgat gtctctgacc agacacccat
     2041 caacagtatt attttctccc atgaagacgg tacgcgactg ggcgtggagc atctggtcgc
     2101 attgggtcac cagcaaatcg cgctgttagc gggcccatta agttctgtct cggcgcgtct
     2161 gcgtctggct ggctggcata aatatctcac tcgcaatcaa attcagccga tagcggaacg
     2221 ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg
     2281 catcgttccc actgcgatgc tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc
     2341 cattaccgag tccgggctgc gcgttggtgc ggacatctcg gtagtgggat acgacgatac
     2401 cgaagacagc tcatgttata tcccgccgtt aaccaccatc aaacaggatt ttcgcctgct
     2461 ggggcaaacc agcgtggacc gcttgctgca actctctcag ggccaggcgg tgaagggcaa
     2521 tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac
     2581 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
     2641 ggaaagcggg cagtgagcgc aacgcaatta atgtaagtta gctcactcat taggcaccgg
     2701 gatctcgacc gatgcccttg agagccttca acccagtcag ctccttccgg tgggcgcggg
     2761 gcatgactag catgatcgtg ctcctgtcgt tgaggacccg gctaggctgg cggggttgcc
     2821 ttactggtta gcagaatgaa tcaccgatac gcgagcgaac gtgaagcgac tgctgctgca
     2881 aaacgtctgc gacctgagca acaacatgaa tggtcttcgg tttccgtgtt tcgtaaagtc
     2941 tggaaacgcg gaagtcagcg ccctgcacca ttatgttccg gatctgcatc gcaggatgct
     3001 gctggctacc ctgtggaaca cctacatctg tattaacgaa gcgctggcat tgaccctgag
     3061 tgatttttct ctggtcccgc cgcatccata ccgccagttg tttaccctca caacgttcca
     3121 gtaaccgggc atgttcatca tcagtaaccc gtatcgtgag catcctctct cgtttcatcg
     3181 gtatcattac ccccatgaac agaaatcccc cttacacgga ggcatcagtg accaaacagg
     3241 aaaaaaccgc ccttaacatg gcccgcttta tcagaagcca gacattaacg cttctggaga
     3301 aactcaacga gctggacgcg gatgaacagg cagacatctg tgaatcgctt cacgaccacg
     3361 ctgatgagct ttaccgcagc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac
     3421 acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag
     3481 cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac
     3541 gtagcgatag cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag
     3601 agtgcaccat atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc
     3661 aggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     3721 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     3781 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     3841 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     3901 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     3961 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     4021 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     4081 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     4141 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     4201 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     4261 tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag
     4321 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     4381 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     4441 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     4501 attttggtca tgaacaataa aactgtctgc ttacataaac agtaatacaa ggggtgttat
     4561 gagccatatt caacgggaaa cgtcttgctc taggccgcga ttaaattcca acatggatgc
     4621 tgatttatat gggtataaat gggctcgcga taatgtcggg caatcaggtg cgacaatcta
     4681 tcgattgtat gggaagcccg atgcgccaga gttgtttctg aaacatggca aaggtagcgt
     4741 tgccaatgat gttacagatg agatggtcag actaaactgg ctgacggaat ttatgcctct
     4801 tccgaccatc aagcatttta tccgtactcc tgatgatgca tggttactca ccactgcgat
     4861 ccccgggaaa acagcattcc aggtattaga agaatatcct gattcaggtg aaaatattgt
     4921 tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta attgtccttt
     4981 taacagcgat cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata acggtttggt
     5041 tgatgcgagt gattttgatg acgagcgtaa tggctggcct gttgaacaag tctggaaaga
     5101 aatgcataaa cttttgccat tctcaccgga ttcagtcgtc actcatggtg atttctcact
     5161 tgataacctt atttttgacg aggggaaatt aataggttgt attgatgttg gacgagtcgg
     5221 aatcgcagac cgataccagg atcttgccat cctatggaac tgcctcggtg agttttctcc
     5281 ttcattacag aaacggcttt ttcaaaaata tggtattgat aatcctgata tgaataaatt
     5341 gcagtttcat ttgatgctcg atgagttttt ctaagaatta attcatgagc ggatacatat
     5401 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     5461 cacctgaaat tgtaaacgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     5521 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
     5581 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
     5641 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
     5701 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     5761 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     5821 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     5881 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg cca
//

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

P1344/pSK+KanaRpsL抗性基因质粒

P1890/pCY 3010-07抗性基因质粒

P2322/pUCGM抗性基因质粒

P1383/GW1-PercevalHR其它基因质粒

P1581/pEVOL-pBpF球菌基因质粒

P3063/pASKt15C+SrtA金黄色葡萄球菌基因质粒

P1683/pEGFP-H37RV杆菌基因质粒

P1990/pEGFP-H37Rv-Outside杆菌基因质粒

P0490/p11-LacY-wtx1其它基因质粒

P1724/Lact-C2-GFP-p416牛源基因质粒