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:
- pTKIP-hygro
- 载体抗性:
- Ampicillin
- 载体长度:
- 4491 bp
- 载体类型:
- Donor vector
- 复制子:
- ori
- 载体来源:
- Kuhlman TE, Cox EC.
- 启动子:
- mPGK
pTKIP-hygro 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。
pTKIP-hygro 载体载体序列
LOCUS 40924_43548 4491 bp DNA circular SYN 18-DEC-2018 DEFINITION Donor vector pTKIP-hygro, complete sequence. ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4491) AUTHORS Kuhlman TE, Cox EC. TITLE Site-specific chromosomal integration of large synthetic constructs JOURNAL Nucleic Acids Res. 38 (6), E92 (2010) PUBMED 20047970 REFERENCE 2 (bases 1 to 4491) AUTHORS Kuhlman TE, Cox EC. TITLE Direct Submission JOURNAL Submitted (17-DEC-2009) Molecular Biology, Princeton University, Washington Rd., Princeton, NJ 08544, USA REFERENCE 3 (bases 1 to 4491) TITLE Direct Submission REFERENCE 4 (bases 1 to 4491) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nucleic Acids Res."; date: "2010"; volume: "38"; issue: "6"; pages: "E92" COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted (17-DEC-2009) Molecular Biology, Princeton University, Washington Rd., Princeton, NJ 08544, USA" COMMENT SGRef: number: 3; type: "Journal Article" FEATURES Location/Qualifiers source 1..4491 /mol_type="other DNA" /organism="synthetic DNA construct" CDS 238..426 /codon_start=1 /label=rop /note="Rop protein, which maintains plasmids at low copy number" /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" misc_feature 531..671 /label=bom /note="basis of mobility region from pBR322" rep_origin complement(857..1445) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(1619..2476) /codon_start=1 /label=AmpR /note="beta-lactamase" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" promoter complement(2477..2581) /label=AmpR promoter misc_feature 2607..2624 /label=I-SceI recognition site /note="I-SceI recognition site" misc_feature 2625..2649 /label=Landing Pad Region 1 /note="Landing Pad Region 1" primer_bind 2668..2684 /label=KS primer /note="common sequencing primer, one of multiple similar variants" protein_bind 2706..2739 /label=FRT (minimal) /note="supports FLP-mediated excision but not integration (Turan and Bode, 2011)" promoter 2803..3302 /label=PGK promoter /note="mouse phosphoglycerate kinase 1 promoter" promoter 3314..3361 /label=EM7 promoter /note="synthetic bacterial promoter" CDS 3380..4378 /codon_start=1 /label=HygR /note="aminoglycoside phosphotransferase from E. coli" /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRP" misc_feature 4415..4448 /label=FRT /note="FRT" protein_bind 4415..4448 /label=FRT (minimal) /bound_moiety="FLP recombinase from the Saccharomyces cerevisiae 2u plasmid" /note="supports FLP-mediated excision but not integration (Turan and Bode, 2011)" misc_feature 4449..4473 /label=Landing Pad Region 2 /note="Landing Pad Region 2" misc_feature 4474..4491 /label=I-SceI recognition site /note="I-SceI recognition site"