首页技术支持质粒载体

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。

The lentiGuide-Puro is a lentiviral vector, also known as pLentiGuide-Puro. It is for expressing a single guide RNA (sgRNA) in Cas9-expressing cells.

载体名称:
lentiGuide-Puro
载体抗性:
Ampicillin
载体长度:
10183 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:
EF-1α
感受态:
Stbl3
培养温度:
37℃

lentiGuide-Puro 载体图谱

lentiGuide-Puro10183 bp50010001500200025003000350040004500500055006000650070007500800085009000950010000EF-1-alpha promoterPuroRWPRE3' LTR (Delta-U3)SV40 poly(A) signalSV40 oriT7 promoterf1 oriAmpR promoterAmpRoriCAP binding sitelac promoterT3 promoterRSV promoter5' LTR (truncated)HIV-1 PsiRREgp41 peptideU6 promoterfillergRNA scaffoldpolIII terminatorcPPT/CTS

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。

lentiGuide-Puro 载体序列

LOCUS       lentiGuide-Puro.       10183 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Zhang lab lentiviral vector, also known as pLentiGuide-Puro, for 
            expressing a single guide RNA (sgRNA) in Cas9-expressing cells. Use 
            together with lentiCas9-Blast.
ACCESSION   .
VERSION     .
KEYWORDS    lentiGuide-Puro
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10183)
  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 10183)
  AUTHORS   Zhang Lab / Addgene #52963
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 10183)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat. 
            Methods"; date: "2014"; volume: "11"; pages: "783-4"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..10183
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        87..1265
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation
                     factor EF-1-alpha"
     CDS             1278..1874
                     /label=PuroR
                     /note="puromycin N-acetyltransferase"
     misc_feature    1893..2481
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional
                     regulatory element"
     LTR             2553..2786
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2864..2998
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      3025..3160
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        complement(3181..3199)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      3367..3822
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        3848..3952
                     /label=AmpR promoter
     CDS             3953..4810
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      4984..5572
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    5860..5881
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        5896..5926
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     promoter        5995..6013
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        6041..6267
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             6268..6448
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6495..6620
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus
                     type 1"
     misc_feature    7113..7346
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7531..7575
                     /label=gp41 peptide
                     /note="antigenic peptide corresponding to amino acids 655
                     to 669 of the HIV envelope protein gp41 (Lutje Hulsik et 
                     al., 2013)"
     promoter        7753..7993
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_feature    7998..9882
                     /label=filler
                     /note="filler"
                     /note="digest with BsmBI to replace the filler with an
                     sgRNA"
     misc_RNA        9883..9958
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      9959..9964
                     /note="polIII terminator"
                     /note="RNA polymerase III transcription terminator"
     misc_feature    10014..10131
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination
                     sequence of HIV-1"