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:
- pTAG2K
- 载体抗性:
- Kanamycin
- 载体长度:
- 6688 bp
- 载体类型:
- Fusion protein expression vector
- 复制子:
- ori
- 载体来源:
- Keefe AD, Szostak JW.
pTAG2K 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。
pTAG2K 载体序列
LOCUS 40924_42539 6688 bp DNA circular SYN 18-DEC-2018 DEFINITION Fusion protein expression vector pTAG2K, complete sequence. ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6688) AUTHORS Keefe AD, Szostak JW. TITLE Functional proteins from a random-sequence library JOURNAL Nature 410 (6829), 715-718 (2001) PUBMED 11287961 REFERENCE 2 (bases 1 to 6688) AUTHORS Keefe AD, Wilson DS, Seelig B, Szostak JW. TITLE One-step purification of recombinant proteins using a nanomolar-affinity streptavidin-binding peptide, the SBP-Tag JOURNAL Protein Expr. Purif. 23 (3), 440-446 (2001) PUBMED 11722181 REFERENCE 3 (bases 1 to 6688) AUTHORS Keefe AD, Wilson DS, Seelig B, Szostak JW. TITLE Direct Submission JOURNAL Submitted (27-APR-2001) Genetics, Harvard Medical School, Massachusetts General Hospital, Boston, MA 02114, USA REFERENCE 4 (bases 1 to 6688) TITLE Direct Submission REFERENCE 5 (bases 1 to 6688) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nature"; date: "2001"; volume: "410"; issue: "6829"; pages: "715-718" COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Protein Expr. Purif."; date: "2001"; volume: "23"; issue: "3"; pages: "440-446" COMMENT SGRef: number: 3; type: "Journal Article"; journalName: "Submitted (27-APR-2001) Genetics, Harvard Medical School, Massachusetts General Hospital, Boston, MA 02114, USA" COMMENT SGRef: number: 4; type: "Journal Article" FEATURES Location/Qualifiers source 1..6688 /mol_type="other DNA" /organism="synthetic DNA construct" terminator complement(26..73) /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" CDS complement(140..157) /codon_start=1 /label=6xHis /note="6xHis affinity tag" /translation="HHHHHH" CDS complement(323..340) /codon_start=1 /label=thrombin site /note="thrombin recognition and cleavage site" /translation="LVPRGS" CDS complement(347..364) /codon_start=1 /label=6xHis /note="6xHis affinity tag" /translation="HHHHHH" CDS complement(374..487) /codon_start=1 /label=SBP /note="streptavidin-binding peptide" /translation="MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP" CDS complement(518..1618) /codon_start=1 /label=MBP /note="maltose binding protein from E. coli" /translation="MGIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF WYAVRTAVINAASGRQTVDEALKDAQT" RBS complement(1625..1647) /label=RBS /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" protein_bind complement(1662..1686) /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)." promoter complement(1687..1705) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" promoter 2014..2091 /label=lacI promoter CDS 2092..3171 /codon_start=1 /label=lacI /note="lac repressor" /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR ALADSLMQLARQVSRLESGQ" protein_bind 3187..3208 /label=CAP binding site /note="CAP binding activates transcription in the presence of cAMP." CDS 3983..4171 /codon_start=1 /label=rop /note="Rop protein, which maintains plasmids at low copy number" /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" misc_feature 4276..4418 /label=bom /note="basis of mobility region from pBR322" rep_origin complement(4604..5192) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS 5314..6126 /codon_start=1 /label=KanR /note="aminoglycoside phosphotransferase" /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" rep_origin complement(6222..6677) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis"