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:
- dCAS9-VP64_GFP
- 载体抗性:
- Ampicillin
- 载体长度:
- 14382 bp
- 载体类型:
- CRISPR Plasmids
- 复制子:
- ori
- 载体来源:
- Konermann S, Brigham MD, Trevino AE, Joung J, Abudayyeh OO, Barcena
- 拷贝数:
- High copy number
- 启动子:
- EF-1α
dCAS9-VP64_GFP 载体载体图谱
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。
dCAS9-VP64_GFP 载体载体序列
LOCUS V012347 14382 bp DNA circular SYN 01-JAN-1980
DEFINITION Exported.
ACCESSION V012347
VERSION V012347
KEYWORDS dCAS9-VP64_GFP
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 14382)
AUTHORS Konermann S, Brigham MD, Trevino AE, Joung J, Abudayyeh OO, Barcena
C, Hsu PD, Habib N, Gootenberg JS, Nishimasu H, Nureki O, Zhang F.
TITLE Genome-scale transcriptional activation by an engineered CRISPR-Cas9
complex.
JOURNAL Nature 2015;517:583-8.
PUBMED 25494202
REFERENCE 2 (bases 1 to 14382)
AUTHORS Zhang Lab / Addgene #61422
TITLE Direct Submission
REFERENCE 3 (bases 1 to 14382)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nature";
date: "2015"; volume: "517"; pages: "583-8"
SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..14382
/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 2682..3859
/label="EF-1-alpha promoter"
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
CDS 3872..3874
/codon_start=1
/product="start codon"
/label="start codon"
/note="ATG"
/translation="M"
CDS 3878..3898
/codon_start=1
/product="nuclear localization signal of SV40 large T
antigen"
/note="SV40 NLS"
/translation="PKKKRKV"
CDS 3908..8008
/label="dCas9"
/note="catalytically dead mutant of the Cas9 endonuclease
from the Streptococcus pyogenes Type II CRISPR/Cas system"
CDS 8063..8083
/label="SV40 NLS"
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
CDS 8108..8257
/codon_start=1
/product="tetrameric repeat of the minimal activation
domain of herpes simplex virus VP16 (Beerli et al., 1998)"
/label="tetrameric repeat of the minimal activation dom"
/note="VP64"
/translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
DLDML"
CDS 8279..8332
/codon_start=1
/product="2A peptide from Thosea asigna virus capsid
protein"
/label="2A peptide from Thosea asigna virus capsid protein"
/note="T2A"
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="EGRGSLLTCGDVEENPGP"
CDS 8333..9046
/label="EGFP"
/note="enhanced GFP"
misc_feature 9074..9662
/label="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 10187..10367
/label="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
polyA_signal 10399..10623
/label="bGH poly(A) signal"
/note="bovine growth hormone polyadenylation signal"
rep_origin 10669..11097
/label="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 11111..11440
/label="SV40 promoter"
/note="SV40 enhancer and early promoter"
promoter 11488..11535
/label="EM7 promoter"
/note="synthetic bacterial promoter"
CDS 11554..11925
/label="BleoR"
/note="antibiotic-binding protein"
polyA_signal 12058..12191
/label="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
promoter complement(12276..12306)
/label="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind complement(12321..12342)
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(12630..13218)
/direction=LEFT
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(13392..14249)
/label="AmpR"
/note="beta-lactamase"
promoter complement(14250..14354)
/label="AmpR promoter"