质粒编号
质粒名称
规格
价格
V001133
pHRdSV40-NLS-dCas9-24xGCN4_v4-NLS-P2A-BFP-dWPRE
5 μg
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:
- pHRdSV40-NLS-dCas9-24xGCN4_v4-NLS-P2A-BFP-dWPRE
- 载体抗性:
- Ampicillin
- 载体长度:
- 14914 bp
- 载体类型:
- Mammalian Expression, Lentiviral, CRISPR
- 复制子:
- ori
- 拷贝数:
- High Copy
- 启动子:
- SV40
- 克隆方法:
- Restriction Enzyme
- 5'引物:
- SV40pro-F2
- 3'引物:
- mTagBFP-R
pHRdSV40-NLS-dCas9-24xGCN4_v4-NLS-P2A-BFP-dWPRE 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。
pHRdSV40-NLS-dCas9-24xGCN4_v4-NLS-P2A-BFP-dWPRE 载体序列
LOCUS V001133 14914 bp DNA circular SYN 13-MAY-2021
DEFINITION Exported.
ACCESSION V001133
VERSION V001133
KEYWORDS pHRdSV40-NLS-dCas9-24xGCN4_v4-NLS-P2A-BFP-dWPRE
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 14914)
AUTHORS Tanenbaum ME, Gilbert LA, Qi LS, Weissman JS, Vale RD
TITLE A Protein-Tagging System for Signal Amplification in Gene Expression
and Fluorescence Imaging.
JOURNAL Cell. 2014 Oct 8. pii: S0092-8674(14)01227-6. doi:
10.1016/j.cell.2014.09.039.
PUBMED 25307933
REFERENCE 2 (bases 1 to 14914)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 14914)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Cell. 2014
Oct 8. pii: S0092-8674(14)01227-6. doi: 10.1016/j.cell.2014.09.039."
SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..14914
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 90..419
/label="SV40 promoter"
/note="SV40 enhancer and early promoter"
intron 1553..1618
/label="small t intron"
/note="SV40 (simian virus 40) small t antigen intron"
CDS 1748..1768
/label="SV40 NLS"
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
polyA_signal 2193..2327
/label="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
LTR 2495..3128
/label="3' LTR"
/note="3' long terminal repeat (LTR) from HIV-1"
misc_feature 3175..3300
/label="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 3797..4030
/label="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 4215..4259
/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 4408..4449
/note="Protein Tat from Human immunodeficiency virus type 1
group M subtype B (isolate WMJ22). Accession#: P12509"
/label="Protein Tat"
misc_feature 4544..4661
/label="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 4724..4934
/label="SV40 promoter"
/note="SV40 early promoter"
rep_origin 4793..4928
/label="SV40 ori"
/note="SV40 origin of replication"
CDS 4950..4970
/label="SV40 NLS"
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
CDS 5025..9128
/label="dCas9"
/note="catalytically dead mutant of the Cas9 endonuclease
from the Streptococcus pyogenes Type II CRISPR/Cas system"
CDS 9132..9158
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/label="HA"
/translation="YPYDVPDYA"
CDS 9177..9197
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label="SV40 NLS"
/translation="PKKKRKV"
CDS 9204..9224
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label="SV40 NLS"
/translation="PKKKRKV"
CDS 9303..9359
/codon_start=1
/product="GCN4 peptide optimized to improve solubility
while preserving binding to an scFv-GFP fusion protein
(Tanenbaum et al., 2014)"
/label="GCN4_v4"
/translation="EELLSKNYHLENEVARLKK"
CDS 9375..9431
/codon_start=1
/product="GCN4 peptide optimized to improve solubility
while preserving binding to an scFv-GFP fusion protein
(Tanenbaum et al., 2014)"
/label="GCN4_v4"
/translation="EELLSKNYHLENEVARLKK"
CDS 9447..9503
/codon_start=1
/product="GCN4 peptide optimized to improve solubility
while preserving binding to an scFv-GFP fusion protein
(Tanenbaum et al., 2014)"
/label="GCN4_v4"
/translation="EELLSKNYHLENEVARLKK"
CDS 9519..9575
/codon_start=1
/product="GCN4 peptide optimized to improve solubility
while preserving binding to an scFv-GFP fusion protein
(Tanenbaum et al., 2014)"
/label="GCN4_v4"
/translation="EELLSKNYHLENEVARLKK"
CDS 9591..9647
/codon_start=1
/product="GCN4 peptide optimized to improve solubility
while preserving binding to an scFv-GFP fusion protein
(Tanenbaum et al., 2014)"
/label="GCN4_v4"
/translation="EELLSKNYHLENEVARLKK"
CDS 9663..9719
/codon_start=1
/product="GCN4 peptide optimized to improve solubility
while preserving binding to an scFv-GFP fusion protein
(Tanenbaum et al., 2014)"
/label="GCN4_v4"
/translation="EELLSKNYHLENEVARLKK"
CDS 9735..9791
/codon_start=1
/product="GCN4 peptide optimized to improve solubility
while preserving binding to an scFv-GFP fusion protein
(Tanenbaum et al., 2014)"
/label="GCN4_v4"
/translation="EELLSKNYHLENEVARLKK"
CDS 9807..9863
/codon_start=1
/product="GCN4 peptide optimized to improve solubility
while preserving binding to an scFv-GFP fusion protein
(Tanenbaum et al., 2014)"
/label="GCN4_v4"
/translation="EELLSKNYHLENEVARLKK"
CDS 9879..9935
/codon_start=1
/product="GCN4 peptide optimized to improve solubility
while preserving binding to an scFv-GFP fusion protein
(Tanenbaum et al., 2014)"
/label="GCN4_v4"
/translation="EELLSKNYHLENEVARLKK"
CDS 9951..10007
/codon_start=1
/product="GCN4 peptide optimized to improve solubility
while preserving binding to an scFv-GFP fusion protein
(Tanenbaum et al., 2014)"
/label="GCN4_v4"
/translation="EELLSKNYHLENEVARLKK"
CDS 10023..10079
/codon_start=1
/product="GCN4 peptide optimized to improve solubility
while preserving binding to an scFv-GFP fusion protein
(Tanenbaum et al., 2014)"
/label="GCN4_v4"
/translation="EELLSKNYHLENEVARLKK"
CDS 10095..10151
/codon_start=1
/product="GCN4 peptide optimized to improve solubility
while preserving binding to an scFv-GFP fusion protein
(Tanenbaum et al., 2014)"
/label="GCN4_v4"
/translation="EELLSKNYHLENEVARLKK"
CDS 10167..10223
/codon_start=1
/product="GCN4 peptide optimized to improve solubility
while preserving binding to an scFv-GFP fusion protein
(Tanenbaum et al., 2014)"
/label="GCN4_v4"
/translation="EELLSKNYHLENEVARLKK"
CDS 10239..10295
/codon_start=1
/product="GCN4 peptide optimized to improve solubility
while preserving binding to an scFv-GFP fusion protein
(Tanenbaum et al., 2014)"
/label="GCN4_v4"
/translation="EELLSKNYHLENEVARLKK"
CDS 10311..10367
/codon_start=1
/product="GCN4 peptide optimized to improve solubility
while preserving binding to an scFv-GFP fusion protein
(Tanenbaum et al., 2014)"
/label="GCN4_v4"
/translation="EELLSKNYHLENEVARLKK"
CDS 10383..10439
/codon_start=1
/product="GCN4 peptide optimized to improve solubility
while preserving binding to an scFv-GFP fusion protein
(Tanenbaum et al., 2014)"
/label="GCN4_v4"
/translation="EELLSKNYHLENEVARLKK"
CDS 10455..10511
/codon_start=1
/product="GCN4 peptide optimized to improve solubility
while preserving binding to an scFv-GFP fusion protein
(Tanenbaum et al., 2014)"
/label="GCN4_v4"
/translation="EELLSKNYHLENEVARLKK"
CDS 10527..10583
/codon_start=1
/product="GCN4 peptide optimized to improve solubility
while preserving binding to an scFv-GFP fusion protein
(Tanenbaum et al., 2014)"
/label="GCN4_v4"
/translation="EELLSKNYHLENEVARLKK"
CDS 10599..10655
/codon_start=1
/product="GCN4 peptide optimized to improve solubility
while preserving binding to an scFv-GFP fusion protein
(Tanenbaum et al., 2014)"
/label="GCN4_v4"
/translation="EELLSKNYHLENEVARLKK"
CDS 10671..10727
/codon_start=1
/product="GCN4 peptide optimized to improve solubility
while preserving binding to an scFv-GFP fusion protein
(Tanenbaum et al., 2014)"
/label="GCN4_v4"
/translation="EELLSKNYHLENEVARLKK"
CDS 10743..10799
/codon_start=1
/product="GCN4 peptide optimized to improve solubility
while preserving binding to an scFv-GFP fusion protein
(Tanenbaum et al., 2014)"
/label="GCN4_v4"
/translation="EELLSKNYHLENEVARLKK"
CDS 10815..10871
/codon_start=1
/product="GCN4 peptide optimized to improve solubility
while preserving binding to an scFv-GFP fusion protein
(Tanenbaum et al., 2014)"
/label="GCN4_v4"
/translation="EELLSKNYHLENEVARLKK"
CDS 10959..11015
/codon_start=1
/product="GCN4 peptide optimized to improve solubility
while preserving binding to an scFv-GFP fusion protein
(Tanenbaum et al., 2014)"
/label="GCN4_v4"
/translation="EELLSKNYHLENEVARLKK"
CDS 11055..11075
/label="SV40 NLS"
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
CDS 11085..11141
/codon_start=1
/product="2A peptide from porcine teschovirus-1
polyprotein"
/label="P2A"
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="ATNFSLLKQAGDVEENPGP"
CDS 11154..11852
/label="TagBFP"
/note="monomeric blue fluorescent protein"
LTR 11970..12203
/label="3' LTR (Delta-U3)"
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
promoter complement(12228..12246)
/label="SP6 promoter"
/note="promoter for bacteriophage SP6 RNA polymerase"
primer_bind complement(12536..12555)
/label="pBRrevBam"
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
primer_bind complement(12769..12788)
/label="pRS-marker"
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 12888..12910
/label="pGEX 3'"
/note="pGEX vectors, reverse primer"
primer_bind complement(12948..12966)
/label="pBRforEco"
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 13034..13138
/label="AmpR promoter"
CDS 13139..13996
/label="AmpR"
/note="beta-lactamase"
rep_origin 14170..14758
/direction=RIGHT
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"