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:
- pHAGE EF1α dCas9-VP64
- 载体抗性:
- Ampicillin
- 载体长度:
- 12794 bp
- 载体类型:
- Mammalian Expression, Lentiviral, CRISPR
- 复制子:
- ori
- 筛选标记:
- Puromycin
- 拷贝数:
- High Copy
- 启动子:
- EF-1α
- 克隆方法:
- Gateway Cloning
- 5'引物:
- AGAGCTCGTTTAGTGAACCG
- 3'引物:
- MSCV-rev
pHAGE EF1α dCas9-VP64 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。
pHAGE EF1α dCas9-VP64 载体序列
LOCUS V000725 12794 bp DNA circular SYN 13-MAY-2021
DEFINITION Exported.
ACCESSION V000725
VERSION V000725
KEYWORDS pHAGE EF1-alpha dCas9-VP64
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 12794)
AUTHORS Kearns NA, Genga RM, Enuameh MS, Garber M, Wolfe SA, Maehr R
TITLE Cas9 effector-mediated regulation of transcription and
differentiation in human pluripotent stem cells.
JOURNAL Development. 2014 Jan;141(1):219-23. doi: 10.1242/dev.103341.
PUBMED 24346702
REFERENCE 2 (bases 1 to 12794)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 12794)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; doi: "10.1242/dev";
journalName: "Development"; date: "2014-01"; volume: "141"; issue:
"1"; pages: "219-23"
SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..12794
/mol_type="other DNA"
/organism="synthetic DNA construct"
CDS 67..93
/label="HA"
/note="HA (human influenza hemagglutinin) epitope tag"
promoter 126..625
/label="PGK promoter"
/note="mouse phosphoglycerate kinase 1 promoter"
CDS 646..1242
/label="PuroR"
/note="puromycin N-acetyltransferase"
misc_feature 1262..1850
/label="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 1925..2158
/label="3' LTR (Delta-U3)"
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
primer_bind complement(2277..2295)
/label="pBRforEco"
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 2363..2467
/label="AmpR promoter"
CDS 2468..3325
/label="AmpR"
/note="beta-lactamase"
rep_origin 3499..4087
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 4241..4258
/label="L4440"
/note="L4440 vector, forward primer"
primer_bind 4350..4369
/label="SV40pro-F"
/note="SV40 promoter/origin, forward primer"
protein_bind 4498..4519
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 4534..4564
/label="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 4572..4588
/label="lac operator"
/bound_moiety="lac repressor encoded by lacI"
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
primer_bind 4577..4599
/label="M13/pUC Reverse"
/note="In lacZ gene"
primer_bind 4596..4612
/label="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 4596..4612
/label="M13 Reverse"
/note="In lacZ gene. Also called M13-rev"
primer_bind 4662..4681
/label="EBV-rev"
/note="SV40 polyA terminator, reverse primer"
LTR 4873..5506
/label="3' LTR"
/note="3' long terminal repeat (LTR) from HIV-1"
misc_feature 5553..5678
/label="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 6175..6408
/label="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 6593..6637
/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 6786..6827
/note="Protein Tat from Human immunodeficiency virus type 1
group M subtype B (isolate WMJ22). Accession#: P12509"
/label="Protein Tat"
misc_feature 6930..7047
/label="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 7093..8271
/label="EF-1-alpha promoter"
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
protein_bind 8306..8330
/gene="mutant version of attB"
/label="attB1"
/bound_moiety="BP Clonase(TM)"
/note="recombination site for the Gateway(R) BP reaction"
CDS 8340..12443
/label="dCas9"
/note="catalytically dead mutant of the Cas9 endonuclease
from the Streptococcus pyogenes Type II CRISPR/Cas system"
CDS 12456..12476
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label="SV40 NLS"
/translation="PKKKRKV"
CDS 12477..12566
/label="3xHA"
/note="three tandem HA epitope tags"
CDS 12588..12737
/label="VP64"
/note="tetrameric repeat of the minimal activation domain
of herpes simplex virus VP16 (Beerli et al., 1998)"