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pET-21a(+) 质粒 (编号: V011023)

Note:

pET-21a(+)5439 bp60012001800240030003600420048005400T7 promoterlac operatorRBST7 tag (gene 10 leader)6xHisT7 terminatorf1 oriAmpR promoterAmpRoribomroplacIlacI promoter
复制序列 NovoBuilder中查看图谱

基本信息

载体名称:
pET-21a(+)
载体抗性:
Ampicillin
载体长度:
5439 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:V011023
质粒名称:pET-21a(+)
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，可供科学家使用。

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

  • Kim YJ, Shin JS, Lee KW, Eom HJ, Jo BG, Lee JW, Kim JH, Kim SY, Kang JH, Choi JW. Expression, Purification, and Characterization of Plasmodium vivax Lactate Dehydrogenase from Bacteria without Codon Optimization. Int J Mol Sci. 2023 Jul 4;24(13):11083. doi: 10.3390/ijms241311083. PMID: 37446261; PMCID: PMC10342390.

  • Zafar A, Aftab MN, Asif A, Karadag A, Peng L, Celebioglu HU, Afzal MS, Hamid A, Iqbal I. Efficient biomass saccharification using a novel cellobiohydrolase from Clostridium clariflavum for utilization in biofuel industry. RSC Adv. 2021 Mar 1;11(16):9246-9261. doi: 10.1039/d1ra00545f. Erratum in: RSC Adv. 2021 Mar 18;11(19):11387. doi: 10.1039/d1ra90090k. PMID: 35423428; PMCID: PMC8695235.

  • Wang Y, Xuan G, Lin H, Fei Z, Wang J. Phage resistance of Salmonella enterica obtained by transposon Tn5-mediated SefR gene silent mutation. J Basic Microbiol. 2023 May;63(5):530-541. doi: 10.1002/jobm.202200532. Epub 2023 Apr 9. PMID: 37032321.

pET-21a(+) 质粒 (编号: V011023)序列

LOCUS       Exported                5439 bp DNA     circular SYN 03-SEP-2024
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pET-21a(+)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5439)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..5439
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        124..142
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    143..167
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by 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)."
     RBS             182..204
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             212..244
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             294..311
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      378..425
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      462..917
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        943..1047
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1048..1908
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      2079..2667
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    2853..2992
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(3094..3285)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4094..5176)
                     /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(5177..5254)
                     /gene="lacI"
                     /label=lacI promoter