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。
3rd generation lentiviral plasmid for inducible expression of shRNA; puromycin selection.
- 载体名称:
- Tet-pLKO-puro
- 载体抗性:
- Ampicillin
- 载体长度:
- 10633 bp
- 载体类型:
- RNAi
- 复制子:
- ori
- 载体来源:
- Dmitri Wiederschain
- 筛选标记:
- Puromycin
- 拷贝数:
- High copy number
- 启动子:
- hPGK
- 表达方法:
- Constiutive, Stable / Transient
Tet-pLKO-puro 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。
Tet-pLKO-puro 载体序列
LOCUS V012735 10633 bp DNA circular SYN 11-SEP-2021 DEFINITION Exported. ACCESSION V012735 VERSION V012735 KEYWORDS Tet-pLKO-puro SOURCE synthetic DNA construct ORGANISM synthetic DNA construct . REFERENCE 1 (bases 1 to 10633) TITLE Direct Submission REFERENCE 2 (bases 1 to 10633) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article" FEATURES Location/Qualifiers source 1..10633 /mol_type="other DNA" /organism="synthetic DNA construct" promoter 7..507 /label="hPGK promoter" /note="human phosphoglycerate kinase 1 promoter" intron 516..1088 /label="beta-globin intron" /note="intron from rabbit beta-globin gene" promoter 1143..1161 /label="T7 promoter" /note="promoter for bacteriophage T7 RNA polymerase" CDS 1172..1792 /label="TetR" /note="tetracycline repressor TetR" misc_feature 1827..2400 /label="IRES" /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" CDS 2420..3016 /label="PuroR" /note="puromycin N-acetyltransferase" LTR 3147..3380 /label="3' LTR (Delta-U3)" /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 3452..3586 /label="SV40 poly(A) signal" /note="SV40 polyadenylation signal" rep_origin 3613..3748 /label="SV40 ori" /note="SV40 origin of replication" promoter complement(3769..3787) /label="T7 promoter" /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(3797..3813) /label="M13 fwd" /note="M13 fwd" /note="common sequencing primer, one of multiple similar variants" rep_origin 3955..4410 /label="f1 ori" /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" promoter 4436..4540 /label="AmpR promoter" CDS 4541..5398 /label="AmpR" /note="beta-lactamase" rep_origin 5572..6160 /label="ori" /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" protein_bind 6448..6469 /label="CAP binding site" /note="CAP binding activates transcription in the presence of cAMP." promoter 6484..6514 /label="lac promoter" /note="promoter for the E. coli lac operon" protein_bind 6522..6538 /label="lac repressor encoded by lacI binding site" /bound_moiety="lac repressor encoded by lacI" /note="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 6546..6562 /label="M13 rev" /note="M13 rev" /note="common sequencing primer, one of multiple similar variants" promoter 6583..6601 /label="T3 promoter" /note="promoter for bacteriophage T3 RNA polymerase" promoter 6629..6855 /label="RSV promoter" /note="Rous sarcoma virus enhancer/promoter" LTR 6856..7036 /label="5' LTR (truncated)" /note="5' LTR (truncated)" /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 7083..7208 /label="HIV-1 Psi" /note="packaging signal of human immunodeficiency virus type 1" misc_feature 7701..7934 /label="RRE" /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 8119..8163 /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 8312..8353 /note="Protein Tat from Human immunodeficiency virus type 1 group M subtype B (isolate WMJ22). Accession#: P12509" /label="Protein Tat" promoter 8452..8546 /label="H1-2O2 promoter" /note="doxycycline-inducible variant of the human H1 RNA promoter (Henriksen et al., 2007)" promoter 10000..10507 /label="hPGK promoter" /note="human phosphoglycerate kinase 1 promoter"