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:
- pLKO.1
- 载体抗性:
- Ampicillin
- 载体长度:
- 8901 bp
- 载体类型:
- Viral Expression & Packaging Vectors
- 复制子:
- ori
- 载体来源:
- The RNAi Consortium
- 拷贝数:
- High copy number
- 启动子:
- hPGK
pLKO.1 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。
pLKO.1 载体序列
LOCUS V010449 8901 bp DNA circular SYN 01-JAN-1980 DEFINITION Exported. ACCESSION V010449 VERSION V010449 KEYWORDS pLKO.1 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct . REFERENCE 1 (bases 1 to 8901) AUTHORS The RNAi Consortium TITLE Direct Submission REFERENCE 2 (bases 1 to 8901) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article" FEATURES Location/Qualifiers source 1..8901 /mol_type="other DNA" /organism="synthetic DNA construct" promoter 7..507 /label="hPGK promoter" /note="human phosphoglycerate kinase 1 promoter" CDS 529..1125 /label="PuroR" /note="puromycin N-acetyltransferase" LTR 1256..1489 /label="3' LTR (Delta-U3)" /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 1561..1695 /label="SV40 poly(A) signal" /note="SV40 polyadenylation signal" rep_origin 1722..1857 /label="SV40 ori" /note="SV40 origin of replication" promoter complement(1878..1896) /label="T7 promoter" /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(1906..1922) /label="M13 fwd" /note="common sequencing primer, one of multiple similar variants" rep_origin 2064..2519 /label="f1 ori" /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" promoter 2545..2649 /label="AmpR promoter" CDS 2650..3507 /label="AmpR" /note="beta-lactamase" rep_origin 3681..4269 /label="ori" /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" protein_bind 4557..4578 /label="CAP binding site" /note="CAP binding activates transcription in the presence of cAMP." promoter 4593..4623 /label="lac promoter" /note="promoter for the E. coli lac operon" protein_bind 4631..4647 /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)." primer_bind 4655..4671 /label="M13 rev" /note="common sequencing primer, one of multiple similar variants" promoter 4692..4710 /label="T3 promoter" /note="promoter for bacteriophage T3 RNA polymerase" promoter 4738..4964 /label="RSV promoter" /note="Rous sarcoma virus enhancer/promoter" LTR 4965..5145 /label="5' LTR (truncated)" /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 5192..5317 /label="HIV-1 Psi" /note="packaging signal of human immunodeficiency virus type 1" misc_feature 5810..6043 /label="RRE" /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 6228..6272 /label="gp41 peptide" /note="antigenic peptide corresponding to amino acids 655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik et al., 2013)" CDS 6421..6462 /note="Protein Tat from Human immunodeficiency virus type 1 group M subtype B (isolate WMJ22). Accession#: P12509" /label="Protein Tat" promoter 6570..6810 /label="U6 promoter" /note="RNA polymerase III promoter for human U6 snRNA" misc_feature 6816..8696 /label="Stuffer" /note="Stuffer" /note="can be removed with AgeI + EcoRI and replaced with an shRNA" misc_feature 8732..8849 /label="cPPT/CTS" /note="central polypurine tract and central termination sequence of HIV-1" promoter 8898..8901 /label="hPGK promoter" /note="human phosphoglycerate kinase 1 promoter"