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:
- lentiCRISPR v2
- 载体抗性:
- Ampicillin
- 载体长度:
- 14873 bp
- 载体类型:
- CRISPR Plasmids
- 复制子:
- ori
- 载体来源:
- Sanjana NE, Shalem O, Zhang F.
- 拷贝数:
- High copy number
- 启动子:
- U6
lentiCRISPR v2 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。
lentiCRISPR v2 载体序列
LOCUS V012328 14873 bp DNA circular SYN 01-JAN-1980 DEFINITION Exported. ACCESSION V012328 VERSION V012328 KEYWORDS lentiCRISPR v2 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct . REFERENCE 1 (bases 1 to 14873) AUTHORS Sanjana NE, Shalem O, Zhang F. TITLE Improved vectors and genome-wide libraries for CRISPR screening. JOURNAL Nat. Methods 2014;11:783-4. PUBMED 25075903 REFERENCE 2 (bases 1 to 14873) AUTHORS Zhang Lab / Addgene #52961 TITLE Direct Submission REFERENCE 3 (bases 1 to 14873) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nat. Methods"; date: "2014"; volume: "11"; pages: "783-4" SGRef: number: 2; type: "Journal Article" Full sequence confirmed by Addgene. FEATURES Location/Qualifiers source 1..14873 /mol_type="other DNA" /organism="synthetic DNA construct" enhancer 238..617 /label="CMV enhancer" /note="human cytomegalovirus immediate early enhancer" promoter 618..820 /label="CMV promoter" /note="human cytomegalovirus (CMV) immediate early promoter" LTR 835..1015 /label="5' LTR (truncated)" /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 1062..1187 /label="HIV-1 Psi" /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1680..1913 /label="RRE" /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 2098..2142 /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 2291..2332 /note="Protein Tat from Human immunodeficiency virus type 1 group M subtype B (isolate WMJ22). Accession#: P12509" /label="Protein Tat" misc_feature 2440..2557 /label="cPPT/CTS" /note="central polypurine tract and central termination sequence of HIV-1" promoter 2608..2848 /label="U6 promoter" /note="RNA polymerase III promoter for human U6 snRNA" misc_feature 2853..4737 /label="filler" /note="filler" /note="digest with BsmBI to replace the filler with an sgRNA " misc_RNA 4738..4813 /label="gRNA scaffold" /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" terminator 4814..4819 /note="polIII terminator" /note="RNA polymerase III transcription terminator" promoter 4875..5086 /label="EF-1-alpha core promoter" /note="core promoter for human elongation factor EF-1-alpha" CDS 5111..9214 /label="Cas9" /note="Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" CDS 9215..9262 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label="bipartite nuclear localization signal from nucl" /note="nucleoplasmin NLS" /translation="KRPAATKKAGQAKKKK" CDS 9263..9286 /label="FLAG" /note="FLAG(R) epitope tag, followed by an enterokinase cleavage site" CDS 9296..9352 /label="P2A" /note="2A peptide from porcine teschovirus-1 polyprotein" CDS 9353..9946 /label="PuroR" /note="puromycin N-acetyltransferase" CDS complement(10436..10447) /label="Factor Xa site" /note="Factor Xa recognition and cleavage site" LTR 10625..10858 /label="3' LTR (Delta-U3)" /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 10890..11114 /label="bGH poly(A) signal" /note="bovine growth hormone polyadenylation signal" rep_origin 11160..11588 /label="f1 ori" /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" promoter 11602..11931 /label="SV40 promoter" /note="SV40 enhancer and early promoter" promoter 11979..12026 /label="EM7 promoter" /note="synthetic bacterial promoter" CDS 12045..12416 /label="BleoR" /note="antibiotic-binding protein" polyA_signal 12549..12682 /label="SV40 poly(A) signal" /note="SV40 polyadenylation signal" promoter complement(12767..12797) /label="lac promoter" /note="promoter for the E. coli lac operon" protein_bind complement(12812..12833) /label="CAP binding site" /note="CAP binding activates transcription in the presence of cAMP." rep_origin complement(13121..13709) /direction=LEFT /label="ori" /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(13883..14740) /label="AmpR" /note="beta-lactamase" promoter complement(14741..14845) /label="AmpR promoter"