lentiCas9-EGFP 载体 (V012330)

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:
lentiCas9-EGFP
载体抗性:
Ampicillin
载体长度:
13177 bp
载体类型:
CRISPR Plasmids
复制子:
ori
载体来源:
Chen S, Sanjana NE, Zheng K, Shalem O, Lee K, Shi X, Scott DA, Song
拷贝数:
High copy number
启动子:
EM7

lentiCas9-EGFP 载体载体图谱

lentiCas9-EGFP13177 bp6001200180024003000360042004800540060006600720078008400900096001020010800114001200012600CMV enhancerCMV promoter5' LTR (truncated)HIV-1 PsiRREgp41 peptideProtein TatcPPT/CTSEF-1-alpha core promoterCas9nucleoplasmin NLSFLAGP2AEGFPWPRE5' LTR (truncated)bGH poly(A) signalf1 oriSV40 promoterEM7 promoterBleoRSV40 poly(A) signallac promoterCAP binding siteoriAmpRAmpR promoter

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。

lentiCas9-EGFP 载体载体序列

LOCUS       V012330                13177 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  Exported.
ACCESSION   V012330
VERSION     V012330
KEYWORDS    lentiCas9-EGFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 13177)
  AUTHORS   Chen S, Sanjana NE, Zheng K, Shalem O, Lee K, Shi X, Scott DA, Song
            J, Pan JQ, Weissleder R, Lee H, Zhang F, Sharp PA.
  TITLE     Genome-wide CRISPR screen in a mouse model of tumor growth and
            metastasis.
  JOURNAL   Cell 2015;160:1246-60.
   PUBMED   25748654
REFERENCE   2  (bases 1 to 13177)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 13177)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell";
            date: "2015"; volume: "160"; pages: "1246-60"
            SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..13177
                     /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        2650..2861
                     /label="EF-1-alpha core promoter"
                     /note="core promoter for human elongation factor
                     EF-1-alpha"
     CDS             2886..6989
                     /label="Cas9"
                     /note="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
     CDS             6990..7037
                     /codon_start=1
                     /product="bipartite nuclear localization signal from
                     nucleoplasmin"
                     /label="bipartite nuclear localization signal from nucl"
                     /note="nucleoplasmin NLS"
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7038..7061
                     /label="FLAG"
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
     CDS             7071..7127
                     /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             7128..7841
                     /label="EGFP"
                     /note="enhanced GFP"
     misc_feature    7869..8457
                     /label="WPRE"
                     /note="woodchuck hepatitis virus posttranscriptional
                     regulatory element"
     LTR             8982..9162
                     /label="5' LTR (truncated)"
                     /note="5' LTR (truncated)"
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     polyA_signal    9194..9418
                     /label="bGH poly(A) signal"
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      9464..9892
                     /label="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        10017..10227
                     /label="SV40 promoter"
                     /note="SV40 early promoter"
     promoter        10283..10330
                     /label="EM7 promoter"
                     /note="synthetic bacterial promoter"
     CDS             10349..10720
                     /label="BleoR"
                     /note="antibiotic-binding protein"
     polyA_signal    10853..10986
                     /label="SV40 poly(A) signal"
                     /note="SV40 polyadenylation signal"
     promoter        complement(11071..11101)
                     /label="lac promoter"
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(11116..11137)
                     /label="CAP binding site"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(11425..12013)
                     /direction=LEFT
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     CDS             complement(12187..13044)
                     /label="AmpR"
                     /note="beta-lactamase"
     promoter        complement(13045..13149)
                     /label="AmpR promoter"