| 出品公司: | ZYbscience |
|---|---|
| 载体名称: | pMAL-c5x |
| 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: | 5677bp |
| 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: | -- |
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- 详细信息
- 文献和实验
- 技术资料
- 保存条件:
-20℃低温保存
- 保质期:
三年
- 英文名:
pMAL-c5x
- 库存:
20
- 供应商:
泽叶生物
载体基本信息
载体质粒图谱和多克隆位点信息
载体简介
The vector pMAL-c5X is designed to produce maltose-binding protein (MBP) fusions, where the protein of interest can be cleaved from MBP with the specific protease Factor Xa (NEB #P8010).
MBP fusions made with this vector are expressed cytoplasmically. The MBP has been engineered for tighter 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 maltose-binding protein). The fusion protein thus produced can be purified by amylose affinity chromatography. The sequence coding for the four amino acids Ile-Glu-Gly-Arg is present just upstream of the XmnI site. This allows the protein of interest to be cleaved from maltose-binding protein with the specific protease Factor Xa. Fragments inserted in the XmnI site (cleaves GAAGG↓ATTTC) will produce a fusion protein that, after Factor Xa cleavage, contains no vector-derived residues on the protein of interest.
载体序列
LOCUS pMAL-c5X 5677 bp DNA circular 13-NOV-2008
DEFINITION Cloning vector pMAL-c5X, complete sequence.
ACCESSION
VERSION
KEYWORDS .
SOURCE Cloning vector pMAL-c5X
ORGANISM Cloning vector pMAL-c5X
other sequences; artificial sequences; vectors.
REFERENCE 1 (bases 1 to 5677)
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 5677)
AUTHORS Riggs,P.D.
TITLE Direct Submission
JOURNAL Submitted (13-NOV-2008) Research Department, New England Biolabs,
240 County Road, Ipswich, MA 01938, USA
FEATURES Location/Qualifiers
source 1..5677
/organism="Cloning vector pMAL-c5X"
/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..2968
/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..2754
/gene="malE expression cassette"
/note="malE sequence 1528..2628; spacer and factor Xa site
2629..2687; multiple cloning site region 2688..2754"
/codon_start=1
/product="maltose binding protein (MBP) fusion"
/translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKL
EEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNG
KLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWP
LIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAF
NKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKEL
AKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNI
PQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGIEGRISHMS
MGGRDIVDGSEFPAGN"
primer_bind 2587..2610
/note="malE primer sequence (NEB #S1237S)"
misc_feature 2695..2747
/gene="malE expression cassette"
/note="multiple cloning site (NdeI-SbfI)"
terminator 2766..2809
/gene="malE expression cassette"
/note="rrnB T1 transcription terminator"
primer_bind complement(2823..2846)
/note="MCS reverse primer sequence (NEB #SxxxxS)"
terminator 2941..2968
/gene="malE expression cassette"
/note="rrnB T2 transcription terminator"
gene 3017..3947
/gene="bla"
-35_signal 3017..3022
/gene="bla"
/note="bla promoter (clockwise); TTCAAA"
-10_signal 3040..3045
/gene="bla"
/note="bla promoter (clockwise); GACAAT"
CDS 3087..3947
/gene="bla"
/note="ampR (confers resistance to ampicillin)"
/codon_start=1
/product="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVE
YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
EIGASLIKHW"
rep_origin 4035..4623
/note="pMB1 origin of replication (clockwise) (RNAII -35
to RNA/DNA switch point)"
gene complement(4993..5184)
/gene="rop"
CDS complement(4993..5184)
/gene="rop"
/codon_start=1
/transl_table=11
/product="ROP protein"
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDH
ADELYRSCLARFGDDGENL"
BASE COUNT 1418 a 1430 c 1523 g 1306 t
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文献和实验的工业化前景. IMPACT-TWIN系统包括两个大肠杆菌表达载体――pTWIN1,pTWIN2.之所以叫做Twin,原因在于pTWIN的设计在多克隆位点两端都各有一段intein和CBD,排列如下:几丁质结合蛋白域―intein1―MCS―Intein2―几丁质结合蛋白.这个巧妙的设计有3重便利:N-端,C-端或者N-端和C-端同时融合短的内含肽序列.想做N端融合,你可以将目的基因替换MCS―Intein2―几丁质结合蛋白,如果想做C 端融合表达,就可以将目的基因替换MCS前面的几丁质
如下:几丁质结合蛋白域―intein1―MCS―Intein2―几丁质结合蛋白。这个巧妙的设计有3重便利:N-端,C-端或者N-端和C-端同时融合短的内含肽序列。想做N端融合,你可以将目的基因替换MCS―Intein2―几丁质结合蛋白,如果想做C端融合表达,就可以将目的基因替换MCS前面的几丁质结合蛋白域―intein1―MCS,不用分2个载体;而且做过克隆的人会很有体会:很多时候双酶切载体,由于2个酶切位点都多克隆位点上,没有小片断切下来,所以是否成功双酶切就不好判断,如果用Twin载体就会切下一个片断
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