| 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: | -- |
买家导航
载体基本信息
载体图谱质粒图谱和多克隆位点信息
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
//
