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:
- pCRISPRia-v2
- 载体抗性:
- Ampicillin
- 载体长度:
- 8893 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:
- Restriction Enzyme
- 5'引物:
- gcgccaattctgcagacaaa
- 3'引物:
- CCTTCTCTAGGCACCGGTTC
pCRISPRia-v2 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。
pCRISPRia-v2 载体序列
LOCUS V000732 8893 bp DNA circular SYN 20-DEC-2021
DEFINITION Exported.
ACCESSION V000732
VERSION V000732
KEYWORDS pCRISPRia-v2
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 8893)
AUTHORS Horlbeck MA, Gilbert LA, Villalta JE, Adamson B, Pak RA, Chen Y,
Fields AP, Park CY, Corn JE, Kampmann M, Weissman JS
TITLE Compact and highly active next-generation libraries for
CRISPR-mediated gene repression and activation.
JOURNAL Elife. 2016 Sep 23;5. pii: e19760. doi: 10.7554/eLife.19760.
PUBMED 27661255
REFERENCE 2 (bases 1 to 8893)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 8893)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Elife.";
date: "2016-09-23"; pages: "
10.7554/eLife.19760"
SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..8893
/mol_type="other DNA"
/organism="synthetic DNA construct"
misc_feature 293..526
/label="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 711..755
/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 904..945
/note="Protein Tat from Human immunodeficiency virus type 1
group M subtype B (isolate WMJ22). Accession#: P12509"
/label="Protein Tat"
misc_feature 1053..1170
/label="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
protein_bind 1431..1464
/label="loxP"
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
primer_bind 1468..1488
/label="mU6-F"
/note="Mouse U6 promoter, forward primer"
promoter 1728..2900
/label="EF-1-alpha promoter"
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
CDS 2912..3508
/label="PuroR"
/note="puromycin N-acetyltransferase"
CDS 3518..3571
/codon_start=1
/product="2A peptide from Thosea asigna virus capsid
protein"
/label="T2A"
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="EGRGSLLTCGDVEENPGP"
CDS 3584..4279
/label="TagBFP"
/note="monomeric blue fluorescent protein"
protein_bind complement(4302..4335)
/label="loxP"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (ATGTATGC) (Shaw et al., 2021)."
misc_feature 4391..4979
/label="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(4982..4998)
/label="KS primer"
/note="common sequencing primer, one of multiple similar
variants"
LTR 5508..5688
/label="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
rep_origin complement(5750..6338)
/direction=LEFT
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(6512..7369)
/label="AmpR"
/note="beta-lactamase"
promoter complement(7370..7474)
/label="AmpR promoter"
primer_bind complement(7549..7568)
/label="pRS-marker"
/note="pRS vectors, use to sequence yeast selectable
marker"
enhancer 7740..8119
/label="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 8120..8322
/label="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
LTR 8337..8517
/label="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 8564..8689
/label="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"