| 出品公司: | |
|---|---|
| 别名: | pET33b, pET 33b |
| 质粒类型: | 大肠杆菌蛋白表达 |
| 表达水平: | 高 |
| 克隆方法: | 多克隆位点,限制性内切酶 |
| 载体大小: | 5383 bp |
| 5' 测序引物: | T7 |
| 5' 测序引物序列: | T7: 5'-TAATACGACTCACTATAGGG-3' |
| 载体标签: | His (中间和C端), N-Thrombin |
| 载体抗性: | Kanamycin |
| 备注: |
Production of target proteins suitable for site-specific 32P-labeling; Nterm thrombin cleavage site; Nterm enterokinase cleavage site
|
| 产品目录号: | ZY69054-3 |
| 稳定性: | 瞬时表达 Transient |
| 组成型: | 组成型 Constitutive |
| 病毒/非病毒: | 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:
pET-33b(+)
- 库存:
20
- 供应商:
泽叶生物
pET33b载体基本信息
pET33b载体质粒图谱和多克隆位点信息
| Feature Name | Start | End | |
|---|---|---|---|
| T7_terminator | 129 | 1 | |
| T7_Terminal_primer | 69 | 87 | |
| T7_leader | 238 | 206 | |
| Xpress_fwd_primer | 239 | 221 | |
| T7_transl_en_RBS | 337 | 321 | |
| lacO | 382 | 355 | |
| T7_promoter | 400 | 382 | |
| tet (300 - 563) | 432 | 695 | |
| pBRrevBam_primer | 503 | 484 | |
| lacI | 778 | 1869 | |
| tet (576 - 851) | 1928 | 2203 | |
| ROP | 2678 | 2869 | |
| pGEX_3_primer | 2885 | 2863 | |
| pBR322_origin | 3903 | 3284 | |
| KanR2 | 4009 | 4824 |
| ORF | Start | End | |
|---|---|---|---|
| ORF frame 3 | 537 | 1037 | |
| ORF frame 1 | 910 | 1869 | |
| ORF frame 1 | 4009 | 4824 |
| Enzyme Name | Cut |
|---|---|
| XhoI | 158 |
| NotI | 166 |
| EagI | 166 |
| HindIII | 173 |
| SalI | 179 |
| SacI | 190 |
| EcoRI | 192 |
| BamHI | 198 |
| NheI | 230 |
| NdeI | 237 |
| NcoI | 310 |
| XbaI | 349 |
| BglII | 415 |
| BclI | 1151 |
| ApaI | 1348 |
| EcoRV | 1587 |
| HpaI | 1643 |
| FspI | 2219 |
| NruI | 4097 |
| ClaI | 4131 |
| XmaI | 4312 |
| SmaI | 4314 |
pET33b载体简介
The pET-33b(+) vector (Cat. No. 69054-3) is derived from pET-28b(+) and carries a 15bp sequence encoding the protein kinase A (PKA) site RRASV, located between the thrombin cleavage and Nde I sites (1). Proteins expressed in pET-33b(+) can be easily purified by metal chelation chromatography (via either N- or C-terminal His•Tag® sequences) and efficiently labeled with 32P- or 33P-gATP and the catalytic subunit of cAMP-dependent protein kinase from heart muscle. Labeled proteins can be used as direct probes in protein-protein interaction studies. 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 circle 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, single-stranded sequencing should be performed using the T7 terminator primer (Cat. No. 69337-3).
pET33b载体序列
DEFINITION pET-33b(+)
ACCESSION
KEYWORDS
SOURCE
ORGANISM other sequences; artificial sequences; vectors.
COMMENT This file is created by Vector NTI
http://www.biofeng.com/
COMMENT ORIGDB|GenBank
COMMENT VNTAUTHORNAME|biofeng.com|
COMMENT VNTOAUTHORAD1|Novagen Inc.|
FEATURES Location/Qualifiers
source 1..5383
/organism="pET-33b(+)"
/mol_type="other DNA"
terminator complement(1..129)
/label="T7_terminator"
misc_feature 69..87
/label="T7_Terminal_primer"
misc_feature complement(206..238)
/label="T7_leader"
misc_feature complement(221..239)
/label="Xpress_fwd_primer"
misc_feature complement(321..337)
/label="T7_transl_en_RBS"
misc_feature complement(355..382)
/label="lacO"
promoter complement(382..400)
/label="T7_promoter"
gene 432..695
/label="tet (300 - 563)"
/gene="tet (300 - 563)"
misc_feature complement(484..503)
/label="pBRrevBam_primer"
CDS 537..1037
/label="ORF frame 3"
/translation="MSACFGVGMVAGPVAGGLLGAISLHAPFLAAAVLNGLNLLLGCF
LMQESHKGERRDPGHHRMAQNLSRYGMIAPGRESIQGGECETSNVIRCRRVCRCLLSD
RFPRGEPGQPRFCENAGKSGSGDGGAELHSQPRGTTTGGQTVVADWRCHLQSGPARAV
ANCRGD*"
misc_feature 778..1869
/label="lacI"
/translation="MSACFGVGMVAGPVAGGLLGAISLHAPFLAAAVLNGLNLLLGCF
LMQESHKGERRDPGHHRMAQNLSRYGMIAPGRESIQGGECETSNVIRCRRVCRCLLSD
RFPRGEPGQPRFCENAGKSGSGDGGAELHSQPRGTTTGGQTVVADWRCHLQSGPARAV
ANCRGD*"
CDS 910..1869
/label="ORF frame 1"
/translation="MAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKS
RADQLGASVVVSMVERSGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNV
PALFLDVSDQTPINSIIFSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGW
HKYLTRNQIQPIAEREGDWSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITE
SGLRVGADISVVGYDDTEDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQ
LLPVSLVKRKTTLAPNTQTASPRALADSLMQLARQVSRLESGQ*"
gene 1928..2203
/label="tet (576 - 851)"
/gene="tet (576 - 851)"
/translation="MAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKS
RADQLGASVVVSMVERSGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNV
PALFLDVSDQTPINSIIFSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGW
HKYLTRNQIQPIAEREGDWSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITE
SGLRVGADISVVGYDDTEDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQ
LLPVSLVKRKTTLAPNTQTASPRALADSLMQLARQVSRLESGQ*"
misc_feature 2678..2869
/label="ROP"
/translation="MAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKS
RADQLGASVVVSMVERSGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNV
PALFLDVSDQTPINSIIFSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGW
HKYLTRNQIQPIAEREGDWSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITE
SGLRVGADISVVGYDDTEDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQ
LLPVSLVKRKTTLAPNTQTASPRALADSLMQLARQVSRLESGQ*"
misc_feature complement(2863..2885)
/label="pGEX_3_primer"
/translation="MAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKS
RADQLGASVVVSMVERSGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNV
PALFLDVSDQTPINSIIFSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGW
HKYLTRNQIQPIAEREGDWSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITE
SGLRVGADISVVGYDDTEDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQ
LLPVSLVKRKTTLAPNTQTASPRALADSLMQLARQVSRLESGQ*"
rep_origin complement(3284..3903)
/label="pBR322_origin"
/translation="MAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKS
RADQLGASVVVSMVERSGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNV
PALFLDVSDQTPINSIIFSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGW
HKYLTRNQIQPIAEREGDWSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITE
SGLRVGADISVVGYDDTEDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQ
LLPVSLVKRKTTLAPNTQTASPRALADSLMQLARQVSRLESGQ*"
gene 4009..4824
/label="KanR2"
/gene="KanR2"
/translation="MAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKS
RADQLGASVVVSMVERSGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNV
PALFLDVSDQTPINSIIFSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGW
HKYLTRNQIQPIAEREGDWSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITE
SGLRVGADISVVGYDDTEDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQ
LLPVSLVKRKTTLAPNTQTASPRALADSLMQLARQVSRLESGQ*"
CDS 4009..4824
/label="ORF frame 1"
/translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLY
GKPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIP
GKTAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGL
VDASDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVG
RVGIADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF*"
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 gaacagatgc acgacggctg ccgcgcggca ccaggccgct gctgtgatga tgatgatgat
301 ggctgctgcc catggtatat ctccttctta aagttaaaca aaattatttc tagaggggaa
361 ttgttatccg ctcacaattc ccctatagtg agtcgtatta atttcgcggg atcgagatct
421 cgatcctcta cgccggacgc atcgtggccg gcatcaccgg cgccacaggt gcggttgctg
481 gcgcctatat cgccgacatc accgatgggg aagatcgggc tcgccacttc gggctcatga
541 gcgcttgttt cggcgtgggt atggtggcag gccccgtggc cgggggactg ttgggcgcca
601 tctccttgca tgcaccattc cttgcggcgg cggtgctcaa cggcctcaac ctactactgg
661 gctgcttcct aatgcaggag tcgcataagg gagagcgtcg agatcccgga caccatcgaa
721 tggcgcaaaa cctttcgcgg tatggcatga tagcgcccgg aagagagtca attcagggtg
781 gtgaatgtga aaccagtaac gttatacgat gtcgcagagt atgccggtgt ctcttatcag
841 accgtttccc gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg
901 gaagcggcga tggcggagct gaattacatt cccaaccgcg tggcacaaca actggcgggc
961 aaacagtcgt tgctgattgg cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa
1021 attgtcgcgg cgattaaatc tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg
1081 gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc
1141 gtcagtgggc tgatcattaa ctatccgctg gatgaccagg atgccattgc tgtggaagct
1201 gcctgcacta atgttccggc gttatttctt gatgtctctg accagacacc catcaacagt
1261 attattttct cccatgaaga cggtacgcga ctgggcgtgg agcatctggt cgcattgggt
1321 caccagcaaa tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg tctgcgtctg
1381 gctggctggc ataaatatct cactcgcaat caaattcagc cgatagcgga acgggaaggc
1441 gactggagtg ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt
1501 cccactgcga tgctggttgc caacgatcag atggcgctgg gcgcaatgcg cgccattacc
1561 gagtccgggc tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga taccgaagac
1621 agctcatgtt atatcccgcc gttaaccacc atcaaacagg attttcgcct gctggggcaa
1681 accagcgtgg accgcttgct gcaactctct cagggccagg cggtgaaggg caatcagctg
1741 ttgcccgtct cactggtgaa aagaaaaacc accctggcgc ccaatacgca aaccgcctct
1801 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc
1861 gggcagtgag cgcaacgcaa ttaatgtaag ttagctcact cattaggcac cgggatctcg
1921 accgatgccc ttgagagcct tcaacccagt cagctccttc cggtgggcgc ggggcatgac
1981 tatcgtcgcc gcacttatga ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc
2041 agcgctctgg gtcattttcg gcgaggaccg ctttcgctgg agcgcgacga tgatcggcct
2101 gtcgcttgcg gtattcggaa tcttgcacgc cctcgctcaa gccttcgtca ctggtcccgc
2161 caccaaacgt ttcggcgaga agcaggccat tatcgccggc atggcggccc cacgggtgcg
2221 catgatcgtg ctcctgtcgt tgaggacccg gctaggctgg cggggttgcc ttactggtta
2281 gcagaatgaa tcaccgatac gcgagcgaac gtgaagcgac tgctgctgca aaacgtctgc
2341 gacctgagca acaacatgaa tggtcttcgg tttccgtgtt tcgtaaagtc tggaaacgcg
2401 gaagtcagcg ccctgcacca ttatgttccg gatctgcatc gcaggatgct gctggctacc
2461 ctgtggaaca cctacatctg tattaacgaa gcgctggcat tgaccctgag tgatttttct
2521 ctggtcccgc cgcatccata ccgccagttg tttaccctca caacgttcca gtaaccgggc
2581 atgttcatca tcagtaaccc gtatcgtgag catcctctct cgtttcatcg gtatcattac
2641 ccccatgaac agaaatcccc cttacacgga ggcatcagtg accaaacagg aaaaaaccgc
2701 ccttaacatg gcccgcttta tcagaagcca gacattaacg cttctggaga aactcaacga
2761 gctggacgcg gatgaacagg cagacatctg tgaatcgctt cacgaccacg ctgatgagct
2821 ttaccgcagc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct
2881 cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg
2941 cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag
3001 cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat
3061 atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgctctt
3121 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
3181 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
3241 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
3301 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
3361 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
3421 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
3481 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
3541 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
3601 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
3661 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
3721 actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct
3781 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
3841 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
3901 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
3961 tgaacaataa aactgtctgc ttacataaac agtaatacaa ggggtgttat gagccatatt
4021 caacgggaaa cgtcttgctc taggccgcga ttaaattcca acatggatgc tgatttatat
4081 gggtataaat gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgattgtat
4141 gggaagcccg atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat
4201 gttacagatg agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc
4261 aagcatttta tccgtactcc tgatgatgca tggttactca ccactgcgat ccccgggaaa
4321 acagcattcc aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg
4381 gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat
4441 cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt
4501 gattttgatg acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataaa
4561 cttttgccat tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt
4621 atttttgacg aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac
4681 cgataccagg atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag
4741 aaacggcttt ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat
4801 ttgatgctcg atgagttttt ctaagaatta attcatgagc ggatacatat ttgaatgtat
4861 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgaaat
4921 tgtaaacgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt
4981 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg
5041 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
5101 caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc
5161 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg
5221 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa
5281 aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
5341 cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg cca
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文献和实验表达载体的构建是生物技术和所需蛋白质产生的基本工具,本文总结了pET-32α(+)载体技术的构建,该技术通常用于研究实验室。该方法包括获得用于构建载体的外源DNA片段,将DNA片段亚克隆到pET-32α(+)表达载体中,在大肠杆菌 BL21(DE3)中进行蛋白质表达以及在大肠杆菌中在天然条件下进行蛋白质纯化。裂解液。介绍作为分子生物学实验室中非常有用的实践,重组DNA技术(或基因克隆)是指将DNA片段从一种生物体转移到自我复制的遗传元件如表达载体。然后插入的DNA可以在外源宿主细胞中繁殖
相关专题 1)噬菌体DE3溶源化的菌株如BL21(DE3)是最常用的表达菌株,噬菌体DE3是λ噬菌体的衍生株,构建好的表达载体 可以直接转入表达菌株中,诱导蛋白的表达方式与lac启动子一样都是IPTG诱导。 2)表达菌株BL21(DE3)上带有lacI基因,T7 RNA 聚合酶编码基因以及lacUV5启动子,lacUV5启动子可以启动T7 RNA 聚合酶的表达; 3)表达质粒 如pET-28质粒带有T7启动子和编码阻遏
Expression System、HisPatch ThioFusion System、pBAD Inducible Bacterial System、pL System和T7-based Systems。这四个系列的载体可谓把原核表达的发展史中出现过的几种启动子都囊括进去了,包括T7、pL、pBAD和trc启动子,同时几种经典的调控方式也都出现了,包括有乳糖操纵子、色氨酸操纵子和阿拉伯糖操纵子。 冠军系列 Champion™ pET Expression System之所以取
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