Note: pETDuet-1 is a commonly used E. coli protein dual expression vector plasmid
基本信息
- 载体名称:
- pETDuet-1
- 载体抗性:
- Ampicillin
- 载体长度:
- 5417 bp
- 载体类型:
- pET & Duet Vectors (Novagen)
- 复制子:
- ori
- 载体来源:
- Novagen (EMD Millipore)
- 拷贝数:
- High copy number
- 感受态:
- DH10B
- 培养温度:
- 37℃
产品信息
质粒编号:V010930
质粒名称:pETDuet-1
规格:5 μg (冻干粉)
下载资源
我们所展示的质粒图谱主要是从文献和开放数据库中收集而来，主要是为了方便研究工作，其中一小部分质粒进行了质量控制，供学术研究使用。
确保质粒的关键元件正确，但是我们并不能保证实验效果。页面展示的图谱序列为理论序列，可能与测序结果不一致，请自行比对后确定是否满足要求。(如果测过序，本页面一般会提供下载)
开放数据库中的大多数载体序列都没有被完全测序。如果实际序列与参考序列的相似度超过99%，则将其视为正确。
由于科学研究是在探索未知，具有很大的不确定性，在任何情况下，我们都不承担超出质粒本身的额外经济损失或责任。
质粒操作方法
1. 发货形式：质粒干粉(常温运输，存于-20度，请务必先转化提质粒后使用)
2. 收到质粒干粉后请先5000rpm离心1min，再加入20μl ddH2O溶解质粒;（质粒复测的浓度有时候与标称值差距较大，这可能是因为冻干质粒在管中的位置、复溶效率、测量偏差以及管壁的吸附导致，因此建议先转化提质粒后再使用）
3. 取1支100μl 感受态于冰上解冻10min，加入2μl质粒，再冰浴30min后，42℃热激60s，不要搅动，再冰浴2min；
4. 加入900μl无抗的LB液体培养基，180rpm震荡37℃培养45min (30℃培养1-1.5小时);
5. 6000rpm离心5min，仅留100μl上清液重悬细菌沉淀，并涂布至目标质粒抗性的LB平板上;
6. 将平板倒置37℃培养14h，如果要求是30℃则培养20h; (菌落过多则将质粒稀释后再转化；没有菌落则加入10μl质粒转化；建议不要直接转表达感受态，要先转克隆感受态，重提质粒后再导入表达感受态);
7. 挑取单菌落至LB液体培养基中，加入对应抗生素，220rpm震荡培养14h，根据实验需要和质粒提取试剂盒说明书提取质粒。
参考文献
- Chang Z, Liu D, Yang Z, Wu J, Zhuang W, Niu H, Ying H. Efficient Xylitol Production from Cornstalk Hydrolysate Using Engineered Escherichia coli Whole Cells. J Agric Food Chem. 2018 Dec 19;66(50):13209-13216. doi: 10.1021/acs.jafc.8b04666. Epub 2018 Dec 5. PMID: 30465421.
- Lan J, Ji S, Yang C, Cai G, Lu J, Li X. Extracellular Expression of Feruloyl Esterase and Xylanase in Escherichia coli for Ferulic Acid Production from Agricultural Residues. Microorganisms. 2023 Jul 25;11(8):1869. doi: 10.3390/microorganisms11081869. PMID: 37630429; PMCID: PMC10456899.
- Özcan D, SİpahİoĞlu HM. Simultaneous production of alpha and beta amylase enzymes using separate gene bearing recombinant vectors in the same Escherichia coli cells. Turk J Biol. 2020 Aug 19;44(4):201-207. doi: 10.3906/biy-2001-71. PMID: 32922127; PMCID: PMC7478135.
pETDuet-1 质粒 (编号: V010930)序列
LOCUS Exported 5417 bp DNA circular SYN 10-SEP-2025
DEFINITION Bacterial vector for the co-expression of two genes.
ACCESSION .
VERSION .
KEYWORDS pETDuet-1
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5417)
AUTHORS Novagen (EMD Millipore)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 5417)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 5417)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 5417)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
COMMENT SGRef: number: 2; type: "Journal Article"
COMMENT SGRef: number: 3; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..5417
/mol_type="other DNA"
/organism="synthetic DNA construct"
source join(303..5417,1..302)
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 286..304
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 305..329
/label=lac operator
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
RBS 344..366
/label=RBS
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 373..375
/codon_start=1
/product="start codon"
/label=start codon
/note="ATG"
/translation="M"
CDS 385..402
/label=6xHis
/note="6xHis affinity tag"
promoter 516..534
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 535..559
/label=lac repressor encoded by lacI binding site
/bound_moiety="lac repressor encoded by lacI"
/note="lac operator"
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
RBS 588..593
/note="ribosome binding site"
CDS 602..604
/codon_start=1
/product="start codon"
/label=start codon
/note="ATG"
/translation="M"
CDS 668..712
/label=S-Tag
/note="affinity and epitope tag derived from pancreatic
ribonuclease A"
terminator 764..811
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
rep_origin 848..1303
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
CDS complement(1418..2278)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(2279..2371)
/label=AmpR promoter
rep_origin 2452..3040
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
misc_feature complement(3226..3365)
/label=bom
/note="basis of mobility region from pBR322"
CDS complement(3467..3658)
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/label=rop
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
protein_bind complement(4196..4217)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
CDS complement(4230..5312)
/codon_start=1
/gene="lacI"
/product="lac repressor"
/label=lacI
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
/translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
ALADSLMQLARQVSRLESGQ"
promoter complement(5313..5390)
/label=lacI promoter