质粒编号
质粒名称
规格
价格
V004199
pECFP-MEM
5 μg (冻干粉)
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。
pECFP-Mem encodes a fusion protein consisting of the N-terminal 20 amino acids of neuromodulin, also called GAP-43 (1), and a cyan fluorescent variant of the enhanced green fluorescent protein (EGFP). The neuromodulin fragment contains a signal for posttranslational palmitoylation of cysteines 3 and 4 that targets ECFP to cellular membranes. The ECFP gene contains amino acid substitutions that give ECFP fluorescence excitation (major peak at 433 nm and a minor peak at 453 nm) and emission (major peak at 475 nm and a minor peak at 501 nm) spectra similar to other cyan emission variants (2, 3). These substitutions also enhance the brightness and solubility of the protein (2, 4, 5). In addition to the chromophore mutations, ECFP contains over 190 silent mutations that create an open reading frame comprised almost entirely of preferred human codons (6, 7). Furthermore, upstream sequences flanking the ECFP-Mem fusion protein have been converted to a Kozak consensus translation initiation site (8). These changes increase the translational efficiency of the fusion protein and consequently its expression in mammalian cells.Expression of ECFP-Mem is driven by the immediate early promoter of CMV ( P CMV IE ). The vector contains an SV40 origin of replication and a neomycin resistance (Neo r ) gene for selection in mammalian cells. A bacterial promoter upstream of this cassette ( P ) expresses kanamycin resistance in E. coli . The vector backbone also provides a pUC19 origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.pECFP-Mem can be transfected into mammalian cells using any standard method. If required, stable transformants can be selected using G418 (9). Expression of ECFP-Mem in mammalian cells results in strong labeling of the plasma membrane and allows easy tracking of individual cells in a population. This membrane labeling also permits study of fine cellular processes such as neuronal axons (10), leading edges of migrating cells, filopodia, or microvilli on cell surfaces. pECFP-Mem cannot be used as an exclusive plasma membrane marker because it also partially labels intracellular membranes.
- 载体名称:
- pECFP-MEM
- 载体抗性:
- Kanamycin
- 载体长度:
- 4793 bp
- 载体类型:
- Fluorescent Protein Reporter Vectors
- 复制子:
- ori
- 筛选标记:
- Neomycin
- 启动子:
- CMV
- 载体标签:
- ECFP
pECFP-MEM 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。
pECFP-MEM 载体序列
LOCUS 40924_16730 4793 bp DNA circular SYN 13-JAN-2022
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4793)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 4793)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..4793
/mol_type="other DNA"
/organism="synthetic DNA construct"
enhancer 61..364
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 365..568
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
misc_feature 591..671
/label=MCS
/note="multiple cloning site"
CDS 739..1455
/codon_start=1
/label=ECFP
/note="enhanced CFP"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
polyA_signal 1581..1702
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
rep_origin complement(1709..2164)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 2191..2295
/label=AmpR promoter
promoter 2297..2654
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
CDS 2689..3480
/codon_start=1
/label=NeoR/KanR
/note="aminoglycoside phosphotransferase"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
polyA_signal 3715..3762
/label=HSV TK poly(A) signal
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
rep_origin 4091..4679
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"