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。
所有的产品都严格仅供科学研究使用,不能应用于临床试验,包括人体摄入、注射或外用的药理学用途。
确保质粒的关键元件正确,但是我们并不能保证实验结果。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。
- 载体名称:
- pEBDuet23A
- 载体抗性:
- Kanamycin
- 载体长度:
- 5207 bp
- 载体类型:
- Expression vector
- 复制子:
- RSF ori
- 载体来源:
- Buchinger E, Aachmann FL, Aranko AS, Valla S, Skjak-Braek G, Iwai H, Wimmer R.
pEBDuet23A 载体载体图谱
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。
pEBDuet23A 载体载体序列
LOCUS 40924_16560 5207 bp DNA circular SYN 17-DEC-2018 DEFINITION Expression vector pEBDuet23A, complete sequence. ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5207) AUTHORS Buchinger E, Aachmann FL, Aranko AS, Valla S, Skjak-Braek G, Iwai H, Wimmer R. TITLE Use of protein trans-splicing to produce active and segmentally (2)H, (15)N labeled mannuronan C5-epimerase AlgE4 JOURNAL Protein Sci. 19 (8), 1534-1543 (2010) PUBMED 20552686 REFERENCE 2 (bases 1 to 5207) AUTHORS Buchinger E, Aachmann FL, Aranko S, Valla S, Sjaak-Braek G, Iwai H, Wimmer R. TITLE Direct Submission JOURNAL Submitted (23-MAR-2010) Aalborg University, Sohngaardsholmsvej 49, Aalborg 9000, Denmark REFERENCE 3 (bases 1 to 5207) TITLE Direct Submission REFERENCE 4 (bases 1 to 5207) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Protein Sci."; date: "2010"; volume: "19"; issue: "8"; pages: "1534-1543" COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted (23-MAR-2010) Aalborg University, Sohngaardsholmsvej 49, Aalborg 9000, Denmark" COMMENT SGRef: number: 3; type: "Journal Article" FEATURES Location/Qualifiers source 1..5207 /mol_type="other DNA" /organism="synthetic DNA construct" promoter 30..48 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 49..73 /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 88..110 /label=RBS /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 117..1568 /codon_start=1 /product="A-IntN" /label=A-IntN /note="fusion protein of Azotobacter vinelandii mannuronan C5-epimerase AlgE4 A-module (A) and Nostoc punctiforme DnaE intein (IntN) N-terminal fragment" /protein_id="ADG44968.1" /translation="MDYNVKDFGALGDGVSDDRASIQAAIDAAYAAGGGTVYLPAGEYR VSAAGEPGDGCLMLKDGVYLAGAGMGETVIKLIDGSDQKITGMVRSAYGEETSNFGMRD LTLDGNRDNTSGKVDGWFNGYIPGGDGADRDVTIERVEVREMSGYGFDPHEQTINLTIR DSVAHDNGLDGFVADYLVDSVFENNVAYANDRHGFNVVTSTHDFVMTNNVAYGNGSSGL VVQRGLEDLALPSNILIDGGAYYDNAREGVLLKMTSDITLQNADIHGNGSSGVRVYGAQ DVQILDNQIHDNAQAAAVPEVLLQSFDDTAGASGTYYTTLNTRIEGNTISGSANSTYGI QERNDGTDYSSLIDNDIAGVQQPIQLYGPHSTVSGEPGATKCLSYETEILTVEYGLLPI GKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTV DGQMLPIDEIFERELDLMRVDNLPN" promoter 1638..1656 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 1657..1681 /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)." CDS 1790..1834 /codon_start=1 /label=S-Tag /note="affinity and epitope tag derived from pancreatic ribonuclease A" /translation="KETAAAKFERQHMDS" terminator 1886..1933 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" CDS complement(2166..2978) /codon_start=1 /label=KanR /note="aminoglycoside phosphotransferase" /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" promoter complement(2979..3070) /label=AmpR promoter rep_origin complement(3086..3835) /direction=LEFT /label=RSF ori /note="Plasmids containing the RSF 1030 origin of replication can be propagated in E. coli cells that contain additional plasmids with compatible origins." protein_bind complement(3997..4018) /label=CAP binding site /note="CAP binding activates transcription in the presence of cAMP." CDS complement(4034..5113) /codon_start=1 /label=lacI /note="lac repressor" /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR ALADSLMQLARQVSRLESGQ" promoter complement(5114..5191) /label=lacI promoter