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。
pDsRed1-N1 encodes a novel red fluorescent protein (RFP; 1) that has been optimized for high expression in mammalian cells (excitation maximum = 558 nm; emission maximum = 583 nm). RFP was isolated from an IndoPacific sea anemone-relative, Discosoma sp; DsRed1’s coding sequence contains 144 silent base pair changes, which correspond to human codon-usage preferences for high expression in mammalian cells (2). Sequences upstream of DsRed1 have been converted to a Kozak consensus translation initiation site (3) to increase translation efficiency in eukaryotic cells. The MCS is between the immediate early promoter of CMV (PCMV IE) and the DsRed1 coding sequence. Genes cloned into the MCS as described below are expressed as fusions to the N-terminus of DsRed1. SV40 polyadenylation signals downstream of the DsRed1 gene direct proper processing of the 3' end of the DsRed1 mRNA. The vector backbone contains an SV40 origin for replication in mammalian cells expressing the SV40 T antigen. A neomycin-resistance cassette (Neor ) allows stably transfected eukaryotic cells to be selected using G418. This cassette consists of the SV40 early promoter, the neomycin/kanamycin resistance gene of Tn5, and polyadenylation signals from the Herpes simplex virus thymidine kinase (HSV TK) gene. A bacterial promoter upstream of the cassette confers kanamycin resistance to E. coli. The pDsRed1-N1 backbone also has a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.Fusions to the N terminus of DsRed1 typically do not alter the fluorescence properties of native DsRed1, allowing in vivo localization of the fusion protein.The target gene should be cloned into pDsRed1-N1 in frame with the DsRed1 coding sequence, with no intervening in-frame stop codons. The inserted gene should include an initiating ATG codon. Recombinant pDsRed1-N1 can be transfected into mammalian cells using any standard transfection method. If required, stable transfectants can be selected using G418 (4). Unmodified pDsRed1-N1 can also be used to express DsRed1 in a cell line of interest (e.g., for use as a transfection marker).
- 载体名称:
- pDsRed1-N1
- 载体抗性:
- Kanamycin
- 载体长度:
- 4692 bp
- 载体类型:
- Fluorescent Protein Reporter Vectors
- 复制子:
- ori
- 筛选标记:
- Neomycin
- 启动子:
- CMV
pDsRed1-N1 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。
pDsRed1-N1 载体序列
LOCUS 40924_15685 4692 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 4692)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 4692)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..4692
/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 679..1356
/codon_start=1
/label=DsRed1
/note="wild-type DsRed"
/translation="MVRSSKNVIKEFMRFKVRMEGTVNGHEFEIEGEGEGRPYEGHNTV
KLKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
VVTVTQDSSLQDGCFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
KALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERTEGRH
HLFL"
polyA_signal 1480..1601
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
rep_origin complement(1608..2063)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 2090..2194
/label=AmpR promoter
promoter 2196..2553
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
CDS 2588..3379
/codon_start=1
/label=NeoR/KanR
/note="aminoglycoside phosphotransferase"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
polyA_signal 3614..3661
/label=HSV TK poly(A) signal
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
rep_origin 3990..4578
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"