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:
- pCLIP-All-EFS-ZsGreen
- 载体抗性:
- Ampicillin
- 载体长度:
- 13009 bp
- 载体类型:
- CRISPR Plasmids
- 复制子:
- ori
- 载体来源:
- transOMIC
- 拷贝数:
- High copy number
- 启动子:
- U6
pCLIP-All-EFS-ZsGreen 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。
pCLIP-All-EFS-ZsGreen 载体序列
LOCUS V012270 13009 bp DNA circular SYN 01-JAN-1980 DEFINITION Exported. ACCESSION V012270 VERSION V012270 KEYWORDS pCLIP-All-EFS-ZsGreen SOURCE synthetic DNA construct ORGANISM synthetic DNA construct . REFERENCE 1 (bases 1 to 13009) AUTHORS transOMIC TITLE Direct Submission REFERENCE 2 (bases 1 to 13009) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article" FEATURES Location/Qualifiers source 1..13009 /mol_type="other DNA" /organism="synthetic DNA construct" enhancer 27..404 /label="CMV enhancer" /note="human cytomegalovirus immediate early enhancer" LTR 411..668 /label="5' LTR (truncated)" /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 715..840 /label="HIV-1 Psi" /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1333..1566 /label="RRE" /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 1751..1795 /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 1944..1985 /note="Protein Tat from Human immunodeficiency virus type 1 group M subtype B (isolate WMJ22). Accession#: P12509" /label="Protein Tat" misc_feature 2093..2210 /label="cPPT/CTS" /note="central polypurine tract and central termination sequence of HIV-1" promoter 2261..2501 /label="U6 promoter" /note="RNA polymerase III promoter for human U6 snRNA" misc_feature 2506..4390 /label="Stuffer" /note="Stuffer" misc_RNA 4391..4466 /label="gRNA scaffold" /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" terminator 4467..4472 /note="polIII terminator" /note="RNA polymerase III transcription terminator" promoter 4528..4739 /label="EF-1-alpha core promoter" /note="core promoter for human elongation factor EF-1-alpha" CDS 4764..8867 /label="Cas9" /note="Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" CDS 8868..8915 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label="bipartite nuclear localization signal from nucl" /note="nucleoplasmin NLS" /translation="KRPAATKKAGQAKKKK" CDS 8916..8939 /label="FLAG" /note="FLAG(R) epitope tag, followed by an enterokinase cleavage site" CDS 8949..9005 /codon_start=1 /product="2A peptide from porcine teschovirus-1 polyprotein" /label="2A peptide from porcine teschovirus-1 polyprotein" /note="P2A" /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="ATNFSLLKQAGDVEENPGP" CDS 9006..9698 /label="ZsGreen1" /note="Zoanthus green fluorescent protein" CDS complement(10186..10197) /label="Factor Xa site" /note="Factor Xa recognition and cleavage site" LTR 10511..10744 /label="3' LTR (Delta-U3)" /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" promoter 10840..11169 /label="SV40 promoter" /note="SV40 enhancer and early promoter" rep_origin complement(11258..11843) /direction=LEFT /label="ori" /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(12017..12874) /label="AmpR" /note="beta-lactamase" promoter complement(12875..12979) /label="AmpR promoter"