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- 详细信息
- 文献和实验
- 技术资料
- 保存条件：
-20℃
- 保质期：
6个月
- 英文名：
pMAL-c5x
- 库存：
大量
- 供应商：
钰博生物
- 规格：
1ug/5ug
pMAL-c5x
载体基本信息
| 出品公司: | Ybscience |
|---|---|
| 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: | -- |
| 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
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
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 aaaatcgaag aaggtaaact ggtaatctgg
1561 attaacggcg ataaaggcta taacggtctc gctgaagtcg gtaagaaatt cgagaaagat
1621 accggaatta aagtcaccgt tgagcatccg gataaactgg aagagaaatt cccacaggtt
1681 gcggcaactg gcgatggccc tgacattatc ttctgggcac acgaccgctt tggtggctac
1741 gctcaatctg gcctgttggc tgaaatcacc ccggacaaag cgttccagga caagctgtat
1801 ccgtttacct gggatgccgt acgttacaac ggcaagctga ttgcttaccc gatcgctgtt
1861 gaagcgttat cgctgattta taacaaagat ctgctgccga acccgccaaa aacctgggaa
1921 gagatcccgg cgctggataa agaactgaaa gcgaaaggta agagcgcgct gatgttcaac
1981 ctgcaagaac cgtacttcac ctggccgctg attgctgctg acgggggtta tgcgttcaag
2041 tatgaaaacg gcaagtacga cattaaagac gtgggcgtgg ataacgctgg cgcgaaagcg
2101 ggtctgacct tcctggttga cctgattaaa aacaaacaca tgaatgcaga caccgattac
2161 tccatcgcag aagctgcctt taataaaggc gaaacagcga tgaccatcaa cggcccgtgg
2221 gcatggtcca acatcgacac cagcaaagtg aattatggtg taacggtact gccgaccttc
2281 aagggtcaac catccaaacc gttcgttggc gtgctgagcg caggtattaa cgccgccagt
2341 ccgaacaaag agctggcaaa agagttcctc gaaaactatc tgctgactga tgaaggtctg
2401 gaagcggtta ataaagacaa accgctgggt gccgtagcgc tgaagtctta cgaggaagag
2461 ttggtgaaag atccgcgtat tgccgccact atggaaaacg cccagaaagg tgaaatcatg
2521 ccgaacatcc cgcagatgtc cgctttctgg tatgccgtgc gtactgcggt gatcaacgcc
2581 gccagcggtc gtcagactgt cgatgaagcc ctgaaagacg cgcagactaa ttcgagctcg
2641 aacaacaaca acaataacaa taacaacaac ctcgggatcg agggaaggat ttcacatatg
2701 tccatgggcg gccgcgatat cgtcgacgga tccgaattcc ctgcaggtaa ttaaataagc
2761 ttcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt
2821 cggtgaacgc tctcctgagt aggacaaatc cgccgggagc ggatttgaac gttgcgaagc
2881 aacggcccgg agggtggcgg gcaggacgcc cgccataaac tgccaggcat caaattaagc
2941 agaaggccat cctgacggat ggcctttttg cgtttctaca aactctttcg gtccgttgtt
3001 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
3061 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
3121 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
3181 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
3241 gtaagatcct tgagagtttt cgccccgaag aacgtttccc aatgatgagc acttttaaag
3301 ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc
3361 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
3421 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
3481 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
3541 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
3601 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
3661 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
3721 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
3781 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
3841 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
3901 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
3961 tttactcata tatactttag attgatttcc ttaggactga gcgtcaaccc cgtagaaaag
4021 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
4081 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
4141 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
4201 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
4261 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
4321 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
4381 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
4441 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
4501 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
4561 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
4621 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
4681 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
4741 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
4801 gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
4861 taaggtgcac tgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc
4921 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
4981 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta
5041 aagctcatca gcgtggtcgt gcagcgattc acagatgtct gcctgttcat ccgcgtccag
5101 ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag
5161 ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg
5221 ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca
5281 tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca
5341 gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca
5401 gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt
5461 ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt
5521 cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg
5581 ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat
5641 gcgcacccgt ggccaggacc caacgctgcc cgaaatt
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文献和实验相关实验
The ribonuclease protection assay (RPA)
) and prerun at 40 watts constant power for ~ 45 minutes, with 0.5X TBE as the running buffer. Gel temperature should be 50°C. 24. Flush the wells again with 0.5X TBE and load the samples (from Step 20). Also load a dilution of the probe set
(+)(-)SP6/M13revT7/M13forpGEM-9Zf(+)(-)T7/M13forSP6/M13revpGEM-TT7/M13forSP6/M13revpGEM-T_EasyT7/M13forSP6/M13revpGene/V5-His_A(_B,_C)pGENE-5BGH rev,V5revpGEX-2TpGEX-5pGEX-3pGEX-2TKpGEX-5pGEX-3pGEX-3XpGEX-5pGEX-3pGEX-4T-1(-2,-3)pGEX-5pGEX-3pGEX-5X
°C). Add 2.5 µl of 6X DNA loading buffer. Prepare the solution for the polyacrylamide gel. Mix 15 ml of 40% acrylamide/bisacrylamide stock solution, 12 ml of 5X TBE buffer, and 32.5 ml of H2O. Add 50 µl of TEMED and 500 µl of freshly prepared 10
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pMAL-c5x
¥800 - 1200
