pCLIP-All-EFS-ZsGreen 载体 (V012270)

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

pCLIP-All-EFS-ZsGreen13009 bp6001200180024003000360042004800540060006600720078008400900096001020010800114001200012600CMV enhancer5' LTR (truncated)HIV-1 PsiRREgp41 peptideProtein TatcPPT/CTSU6 promoterStuffergRNA scaffoldpolIII terminatorEF-1-alpha core promoterCas9nucleoplasmin NLSFLAGP2AZsGreen1Factor Xa site3' LTR (Delta-U3)SV40 promoteroriAmpRAmpR 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。

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"