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名称	规格
pET-24b大肠表达质粒	2μg质粒干粉

 

启动子：T7/lac promoter

复制子：ColE1 ori，F1 ori

终止子：T7 terminator

质粒分类：大肠系列质粒；大肠表达质粒；pET系列质粒

质粒大小：5309bp

质粒标签：N-T7, C-6×His

原核抗性：卡那霉素Kan（50μg/ml）

克隆菌株：大肠杆菌DH5α

培养条件：37℃，有氧，LB

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：T7（TAATACGACTCACTATAGGG）

3'测序引物：T7-ter（TGCTAGTTATTGCTCAGCGG）

备注：需要诱导后才表达
 

        pET-24a质粒是一种原核表达载体，N端含有一个T7标签、C端的6×His affinity标签。该质粒含有多个常用的酶切位点，便于不同基因克隆。表达由宿主细胞提供的T7 RNA聚合酶诱导，目的基因被克隆到质粒载体上，受噬菌体强转录及翻译信号控制。pET系统是有史以来在大肠杆菌中表达重组蛋白的功能**强大的系统，也是现今原核表达方面使用**广泛的系统。该系列质粒能很容易的通过降低诱导物的浓度来削弱蛋白表达。在非诱导条件下，可以使目的基因处于完全沉默状态而不转录。

      The pET-24a-d(+) vectors carry an N-terminal T7•Tag® sequence plus an optional C-terminal His•Tag® sequence. These vectors differ from pET-21a-d(+) only by their selectable marker (kanamycin vs. ampicillin resistance). Unique sites are shown on the circle map. Note that the sequence is numbered by the pBR322 convention, so the T7 expression region is reversed on the circular map. The cloning/expression region of the coding strand transcribed by T7 RNA polymerase is shown below. The f1 origin is oriented so that infection with helper phage will produce virions containing single-stranded DNA that corresponds to the coding strand. Therefore, singlestranded sequencing should be performed using the T7 terminator primer.

质粒序列
LOCUS       Exported                5309 bp ds-DNA     circular SYN 27-AUG-2016
DEFINITION  synthetic circular DNA
KEYWORDS    pET-24b
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5309)
  TITLE     Direct Submission
  JOURNAL   Exported Saturday, August 27, 2016 from MiaoLingPlasmid
FEATURES             Location/Qualifiers
     source          1..5309
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     terminator      26..73
                     /note="T7 terminator"
                     /note="transcription terminator for bacteriophage T7 RNA
                     polymerase"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /note="6xHis"
                     /translation="HHHHHH"
     CDS             complement(206..238)
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /note="T7 tag (gene 10 leader)"
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     protein_bind    283..307
                     /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(308..326)
                     /note="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        635..712
                     /gene="lacI"
                     /note="lacI promoter"
     CDS             713..1795
                     /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             2604..2795
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein"
                     /note="rop"
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     rep_origin      complement(3225..3813)
                     /direction=LEFT
                     /note="ori"
                     /note="high-copy-number colE1/pMB1/pBR322/pUC origin of
                     replication"
     CDS             3935..4750
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /note="KanR"
                     /note="confers resistance to kanamycin"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      complement(4843..5298)
                     /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 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt
      181 cgacggagct cgaattcgga tcccgaccca tttgctgtcc accagtcatg ctagccatat
      241 gtatatctcc ttcttaaagt taaacaaaat tatttctaga ggggaattgt tatccgctca
      301 caattcccct atagtgagtc gtattaattt cgcgggatcg agatctcgat cctctacgcc
      361 ggacgcatcg tggccggcat caccggcgcc acaggtgcgg ttgctggcgc ctatatcgcc
      421 gacatcaccg atggggaaga tcgggctcgc cacttcgggc tcatgagcgc ttgtttcggc
      481 gtgggtatgg tggcaggccc cgtggccggg ggactgttgg gcgccatctc cttgcatgca
      541 ccattccttg cggcggcggt gctcaacggc ctcaacctac tactgggctg cttcctaatg
      601 caggagtcgc ataagggaga gcgtcgagat cccggacacc atcgaatggc gcaaaacctt
      661 tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga atgtgaaacc
      721 agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg tttcccgcgt
      781 ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc
      841 ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct
      901 gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat
      961 taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg
     1021 cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat
     1081 cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct gcactaatgt
     1141 tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta ttttctccca
     1201 tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc
     1261 gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg gctggcataa
     1321 atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat
     1381 gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct
     1441 ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg
     1501 cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagacagct catgttatat
     1561 cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg
     1621 cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact
     1681 ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     1741 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     1801 acgcaattaa tgtaagttag ctcactcatt aggcaccggg atctcgaccg atgcccttga
     1861 gagccttcaa cccagtcagc tccttccggt gggcgcgggg catgactatc gtcgccgcac
     1921 ttatgactgt cttctttatc atgcaactcg taggacaggt gccggcagcg ctctgggtca
     1981 ttttcggcga ggaccgcttt cgctggagcg cgacgatgat cggcctgtcg cttgcggtat
     2041 tcggaatctt gcacgccctc gctcaagcct tcgtcactgg tcccgccacc aaacgtttcg
     2101 gcgagaagca ggccattatc gccggcatgg cggccccacg ggtgcgcatg atcgtgctcc
     2161 tgtcgttgag gacccggcta ggctggcggg gttgccttac tggttagcag aatgaatcac
     2221 cgatacgcga gcgaacgtga agcgactgct gctgcaaaac gtctgcgacc tgagcaacaa
     2281 catgaatggt cttcggtttc cgtgtttcgt aaagtctgga aacgcggaag tcagcgccct
     2341 gcaccattat gttccggatc tgcatcgcag gatgctgctg gctaccctgt ggaacaccta
     2401 catctgtatt aacgaagcgc tggcattgac cctgagtgat ttttctctgg tcccgccgca
     2461 tccataccgc cagttgttta ccctcacaac gttccagtaa ccgggcatgt tcatcatcag
     2521 taacccgtat cgtgagcatc ctctctcgtt tcatcggtat cattaccccc atgaacagaa
     2581 atccccctta cacggaggca tcagtgacca aacaggaaaa aaccgccctt aacatggccc
     2641 gctttatcag aagccagaca ttaacgcttc tggagaaact caacgagctg gacgcggatg
     2701 aacaggcaga catctgtgaa tcgcttcacg accacgctga tgagctttac cgcagctgcc
     2761 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
     2821 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
     2881 ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtatactg
     2941 gcttaactat gcggcatcag agcagattgt actgagagtg caccatatat gcggtgtgaa
     3001 ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc
     3061 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     3121 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     3181 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     3241 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     3301 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     3361 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     3421 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     3481 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     3541 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     3601 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     3661 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     3721 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     3781 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     3841 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgaa caataaaact
     3901 gtctgcttac ataaacagta atacaagggg tgttatgagc catattcaac gggaaacgtc
     3961 ttgctctagg ccgcgattaa attccaacat ggatgctgat ttatatgggt ataaatgggc
     4021 tcgcgataat gtcgggcaat caggtgcgac aatctatcga ttgtatggga agcccgatgc
     4081 gccagagttg tttctgaaac atggcaaagg tagcgttgcc aatgatgtta cagatgagat
     4141 ggtcagacta aactggctga cggaatttat gcctcttccg accatcaagc attttatccg
     4201 tactcctgat gatgcatggt tactcaccac tgcgatcccc gggaaaacag cattccaggt
     4261 attagaagaa tatcctgatt caggtgaaaa tattgttgat gcgctggcag tgttcctgcg
     4321 ccggttgcat tcgattcctg tttgtaattg tccttttaac agcgatcgcg tatttcgtct
     4381 cgctcaggcg caatcacgaa tgaataacgg tttggttgat gcgagtgatt ttgatgacga
     4441 gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg cataaacttt tgccattctc
     4501 accggattca gtcgtcactc atggtgattt ctcacttgat aaccttattt ttgacgaggg
     4561 gaaattaata ggttgtattg atgttggacg agtcggaatc gcagaccgat accaggatct
     4621 tgccatccta tggaactgcc tcggtgagtt ttctccttca ttacagaaac ggctttttca
     4681 aaaatatggt attgataatc ctgatatgaa taaattgcag tttcatttga tgctcgatga
     4741 gtttttctaa gaattaattc atgagcggat acatatttga atgtatttag aaaaataaac
     4801 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgaaattgta aacgttaata
     4861 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg
     4921 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc
     4981 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
     5041 ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt
     5101 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
     5161 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
     5221 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
     5281 cgccgctaca gggcgcgtcc cattcgcca
//