pMAL-p5e

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载体基本信息

出品公司: NEB
载体名称: pMAL-p5e
质粒类型: 大肠杆菌表达载体
高拷贝/低拷贝: --
启动子: --
克隆方法: 多克隆位点,限制性内切酶
载体大小: 5755bp
5' 测序引物及序列: --
3' 测序引物及序列: --
载体标签: --
载体抗性: Ampicillin
筛选标记: --
备注: --
产品目录号: --
稳定性: --
组成型: --
病毒/非病毒: --

载体质粒图谱和多克隆位点信息

pMal-p5e载体图谱
pMAL-p5e多克隆位点

载体简介

The vector pMAL-p5E is designed to produce maltose-binding protein (MBP) fusions, where the protein of interest can be cleaved from MBP with the specific protease Enterokinase (NEB #P8070). 
MBP fusions made with this vector include an N-terminal signal sequence, so the fusion protein is directed to the periplasm. The MBP has been engineered for tigher binding to amylose resin.
A gene or open reading frame is inserted into a restriction site of the vector polylinker, in the same translational reading frame as the malE gene (encoding MBP). The fusion protein produced from the vector can be purified by amylose affinity chromatography. The sequence coding for the five amino acids Asp-Asp-Asp-Asp-Lys is present just upstream of the KpnI site. This allows the protein of interest to be cleaved from maltose-binding protein with enterokinase. 
pMAL-p5E cut with KpnI followed by treatment with the Quick Blunting Kit (NEB #E1201) produces a blunt end at the lysine codon. This allows blunt-end cloning of an insert where the first three nucleotides code for the first amino acid of the protein of interest, and enterokinase cleavage of the fusion produces a protein with no vector-derived amino acids.

载体序列

LOCUS       pMAL-p5E                5755 bp    DNA     circular     10-NOV-2008
DEFINITION  Cloning vector pMAL-p5E, complete sequence.
ACCESSION
VERSION
KEYWORDS    .
SOURCE      Cloning vector pMAL-p5E
  ORGANISM  Cloning vector pMAL-p5E
            other sequences; artificial sequences; vectors.
REFERENCE   1  (bases 1 to 5755)
  AUTHORS   Riggs,P.
  TITLE     Expression and purification of maltose-binding protein fusions
  JOURNAL   (in) Asubel,F.A., Brent,R., Kingston,R.E., Moore,D.D.,
            Seidman,J.G., Smith,J.A. and Struhl,K. (Eds.);
            CURRENT PROTOCOLS IN MOLECULAR BIOLOGY: 1-14;
            Greene Publishing and Wiley Interscience, New York, NY (1992)
REFERENCE   2  (bases 1 to 5755)
  AUTHORS   Riggs,P.D.
  TITLE     Direct Submission
  JOURNAL   Submitted (10-NOV-2008) Research Department, New England Biolabs,
            240 County Road, Ipswich, MA 01938, USA
FEATURES             Location/Qualifiers
     source          1..5755
                     /organism="Cloning vector pMAL-p5E"
                     /mol_type="other DNA"
     gene            15..1163
                     /gene="lacIq"
     -35_signal      15..19
                     /gene="lacIq"
                     /note="lacIq promoter (clockwise); TGGTG"
     -10_signal      40..46
                     /gene="lacIq"
                     /note="lacIq promoter (clockwise); CATGATA"
     CDS             81..1163
                     /gene="lacIq"
                     /codon_start=1
                     /transl_table=11
                     /product="lac repressor (LacI)"
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAE
                     LNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERS
                     GVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSII
                     FSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREG
                     DWSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDT
                     EDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNT
                     QTASPRALADSLMQLARQVSRLESGQ"
     gene            1406..3046
                     /gene="malE expression cassette"
     -35_signal      1406..1411
                     /gene="malE expression cassette"
                     /note="Ptac promoter (clockwise); TTGACA"
     -10_signal      1428..1433
                     /gene="malE expression cassette"
                     /note="Ptac promoter (clockwise); TATAAT"
     CDS             1528..2832
                     /gene="malE expression cassette"
                     /note="malE sequence 1528..2703; spacer and enterokinase
                     site 2704..2765; multiple cloning site region 2766..2832"
                     /codon_start=1
                     /product="maltose binding protein (MBP) fusion"
                     /translation="MKIKTGARILALSALTTMMFSASALAKIEEGKLVIWINGDKGYN
                     GLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLL
                     AEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPA
                     LDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGL
                     TFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTF
                     KGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYE
                     EELVKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQT
                     NSSSNNNNNNNNNNLGDDDDKVPHMSMGGRDIVDGSEFPAGN"
     primer_bind     2662..2685
                     /note="malE primer sequence (NEB #S1237S)"
     misc_feature    2766..2825
                     /gene="malE expression cassette"
                     /note="multiple cloning site (KpnI-SbfI)"
     terminator      2844..2887
                     /gene="malE expression cassette"
                     /note="rrnB T1 transcription terminator"
     primer_bind     complement(2901..2924)
                     /note="MCS reverse primer sequence (NEB #SxxxxS)"
     terminator      3019..3046
                     /gene="malE expression cassette"
                     /note="rrnB T2 transcription terminator"
     gene            3095..4025
                     /gene="bla"
     -35_signal      3095..3100
                     /gene="bla"
                     /note="bla promoter (clockwise); TTCAAA"
     -10_signal      3118..3123
                     /gene="bla"
                     /note="bla promoter (clockwise); GACAAT"
     CDS             3165..4025
                     /gene="bla"
                     /note="ampR (confers resistance to ampicillin)"
                     /codon_start=1
                     /product="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVE
                     YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
                     DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
                     LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
                     EIGASLIKHW"
     rep_origin      4113..4701
                     /note="pMB1 origin of replication (clockwise) (RNAII -35
                     to RNA/DNA switch point)"
     gene            complement(5071..5262)
                     /gene="rop"
     CDS             complement(5071..5262)
                     /gene="rop"
                     /codon_start=1
                     /transl_table=11
                     /product="ROP protein"
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDH
                     ADELYRSCLARFGDDGENL"
BASE COUNT     1439 a   1455 c   1538 g   1323 t
ORIGIN
        1 ccgacaccat cgaatggtgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga
       61 gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg
      121 gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa
      181 cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac
      241 aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc
      301 acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg
      361 tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc
      421 ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca
      481 ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga
      541 cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc
      601 tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg
      661 cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag
      721 cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga
      781 atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa
      841 tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga tatttcggta gtgggatacg
      901 acgataccga agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc
      961 gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga
     1021 agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata
     1081 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     1141 cccgactgga aagcgggcag tgagcgcaac gcaattaatg taagttagct cactcattag
     1201 gcacaattct catgtttgac agcttatcat cgactgcacg gtgcaccaat gcttctggcg
     1261 tcaggcagcc atcggaagct gtggtatggc tgtgcaggtc gtaaatcact gcataattcg
     1321 tgtcgctcaa ggcgcactcc cgttctggat aatgtttttt gcgccgacat cataacggtt
     1381 ctggcaaata ttctgaaatg agctgttgac aattaatcat cggctcgtat aatgtgtgga
     1441 attgtgagcg gataacaatt tcacacagga aacagccagt ccgtttaggt gttttcacga
     1501 gcaattgacc aacaaggacc atagattatg aaaataaaaa caggtgcacg catcctcgca
     1561 ttatccgcat taacgacgat gatgttttcc gcctcggctc tcgccaaaat cgaagaaggt
     1621 aaactggtaa tctggattaa cggcgataaa ggctataacg gtctcgctga agtcggtaag
     1681 aaattcgaga aagataccgg aattaaagtc accgttgagc atccggataa actggaagag
     1741 aaattcccac aggttgcggc aactggcgat ggccctgaca ttatcttctg ggcacacgac
     1801 cgctttggtg gctacgctca atctggcctg ttggctgaaa tcaccccgga caaagcgttc
     1861 caggacaagc tgtatccgtt tacctgggat gccgtacgtt acaacggcaa gctgattgct
     1921 tacccgatcg ctgttgaagc gttatcgctg atttataaca aagatctgct gccgaacccg
     1981 ccaaaaacct gggaagagat cccggcgctg gataaagaac tgaaagcgaa aggtaagagc
     2041 gcgctgatgt tcaacctgca agaaccgtac ttcacctggc cgctgattgc tgctgacggg
     2101 ggttatgcgt tcaagtatga aaacggcaag tacgacatta aagacgtggg cgtggataac
     2161 gctggcgcga aagcgggtct gaccttcctg gttgacctga ttaaaaacaa acacatgaat
     2221 gcagacaccg attactccat cgcagaagct gcctttaata aaggcgaaac agcgatgacc
     2281 atcaacggcc cgtgggcatg gtccaacatc gacaccagca aagtgaatta tggtgtaacg
     2341 gtactgccga ccttcaaggg tcaaccatcc aaaccgttcg ttggcgtgct gagcgcaggt
     2401 attaacgccg ccagtccgaa caaagagctg gcaaaagagt tcctcgaaaa ctatctgctg
     2461 actgatgaag gtctggaagc ggttaataaa gacaaaccgc tgggtgccgt agcgctgaag
     2521 tcttacgagg aagagttggt gaaagatccg cgtattgccg ccactatgga aaacgcccag
     2581 aaaggtgaaa tcatgccgaa catcccgcag atgtccgctt tctggtatgc cgtgcgtact
     2641 gcggtgatca acgccgccag cggtcgtcag actgtcgatg aagccctgaa agacgcgcag
     2701 actaattcga gctcgaacaa caacaacaat aacaataaca acaacctcgg ggatgacgat
     2761 gacaaggtac cgcatatgtc catgggcggc cgcgatatcg tcgacggatc cgaattccct
     2821 gcaggtaatt aaataagctt caaataaaac gaaaggctca gtcgaaagac tgggcctttc
     2881 gttttatctg ttgtttgtcg gtgaacgctc tcctgagtag gacaaatccg ccgggagcgg
     2941 atttgaacgt tgcgaagcaa cggcccggag ggtggcgggc aggacgcccg ccataaactg
     3001 ccaggcatca aattaagcag aaggccatcc tgacggatgg cctttttgcg tttctacaaa
     3061 ctctttcggt ccgttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     3121 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     3181 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     3241 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     3301 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgtttcccaa
     3361 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc
     3421 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     3481 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     3541 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     3601 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     3661 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     3721 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     3781 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     3841 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     3901 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     3961 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     4021 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttcctt aggactgagc
     4081 gtcaaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     4141 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     4201 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     4261 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     4321 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     4381 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     4441 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     4501 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     4561 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     4621 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     4681 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     4741 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     4801 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     4861 tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc
     4921 ggtatttcac accgcatata aggtgcactg tgactgggtc atggctgcgc cccgacaccc
     4981 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     5041 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     5101 cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtgc agcgattcac agatgtctgc
     5161 ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat
     5221 aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg
     5281 gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg
     5341 ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt
     5401 atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac
     5461 agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat
     5521 ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat
     5581 tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg
     5641 tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa
     5701 cgacaggagc acgatcatgc gcacccgtgg ccaggaccca acgctgcccg aaatt
//

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