pAcGFP1-C1哺乳动物表达载体说明

pAcGFP1-C1

编号 载体名称

北京华越洋生物VECT6156 pAcGFP1-­‐C1 pAcGFP1-­‐C1载体基本信息

载体名称: pAcGFP1-C1

质粒类型: 哺乳动物细胞表达载体；荧光报告载体高拷贝/低拷贝: 高拷贝

克隆方法: 限制性内切酶，多克隆位点

启动子: CMV IE

载体大小: 4731 bp

5' 测序引物及序列: --

3' 测序引物及序列: --

载体标签: AcGFP1 (N-端）

载体抗性: 卡那霉素

筛选标记: 新霉素（Neomycin）

克隆菌株: DH5α, HB101

宿主细胞（系）: 常规细胞系（293、CV-1、CHO等）

备注: 哺乳动物载体pAcGFP1-C1组成型表达N端AcGFP融合蛋白；AcGFP1与GFP相比，密码子经过了优化，更适合在哺乳动物细胞中高水平表达，同时也增强了亮度。

稳定性: 稳表达或瞬表达

组成型/诱导型: 组成型

病毒/非病毒: 非病毒

pAcGFP1-­‐C1载体质粒图谱和多克隆位点信息

pAcGFP1-C1载体描述

pAcGFP1-C1 encodes a green fluorescent protein (GFP) from Aequorea coerulescens (excitation maximum = 475 nm; emission maximum = 505 nm). Sequences flanking AcGFP1 have been converted to a Kozak consensus translation initiation site (1) to further increase the translation efficiency in eukaryotic cells. The MCS in pAcGFP1-C1 is downstream of the AcGFP1 coding region, allowing the contruction of a C-terminal fusion protein with the AcGFP1 when genes are cloned in the same reading frame as AcGFP1 and there are no intervening stop codons. SV40 polyadenylation signals downstream of the AcGFP1 gene direct proper processing of the 3' end of the AcGFP1 mRNA. The vector backbone also contains an SV40 origin

for replication in mammalian cells expressing the SV40 T-antigen. A neomycin resistance cassette (Neor), consisting 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, allows stably transfected eukaryotic cells to be selected using G418. A bacterial promoter upstream of the gene expresses kanamycin resistance in E. coli. The pAcGFP1-C1 backbone also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.

Fusions to the C terminus of AcGFP1 retain the fluorescent properties of the native protein allowing the localization of the fusion protein in vivo. The target gene should be cloned into pAcGFP1-C1 so that it is in frame with the AcGFP1 coding sequences, with no intervening in-frame stop codons. The recombinant AcGFP1 vector can be transfected into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (2). pAcGFP1-C1 can also be used simply to express AcGFP1 in a cell line of interest (e.g., as a transfection marker). Propagation in E. coli

Suitable host strains: DH5α, HB101, and other general purpose strains. Single-stranded DNA production requires

a host containing an F plasmid such as JM109 or XL1-Blue.

Selectable marker: plasmid confers resistance to kanamycin (30 μg/ml) to E. coli hosts.

E. coli replication origin: pUC

Copy number: ≈500

Plasmid incompatibility group: pMB1/ColE1

pAcGFP1-­‐C1载体序列

ORIGIN

1 TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT AGCCCATATA TGGAGTTCCG 61 CGTTACATAA CTTACGGTAA ATGGCCCGCC TGGCTGACCG CCCAACGACC CCCGCCCATT 121 GACGTCAATA ATGACGTATG TTCCCATAGT AACGCCAATA GGGACTTTCC ATTGACGTCA 181 ATGGGTGGAG TATTTACGGT AAACTGCCCA CTTGGCAGTA CATCAAGTGT ATCATATGCC 241 AAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC GCCTGGCATT ATGCCCAGTA 301 CATGACCTTA TGGGACTTTC CTACTTGGCA GTACATCTAC GTATTAGTCA TCGCTATTAC 361 CATGGTGATG CGGTTTTGGC AGTACATCAA TGGGCGTGGA TAGCGGTTTG ACTCACGGGG 421 ATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG TTTTGGCACC AAAATCAACG 481 GGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG CAAATGGGCG GTAGGCGTGT 541 ACGGTGGGAG GTCTATATAA GCAGAGCTGG TTTAGTGAAC CGTCAGATCC GCTAGCGCTA 601 CCGGTCGCCA CCATGGTGAG CAAGGGCGCC GAGCTGTTCA CCGGCATCGT GCCCATCCTG 661 ATCGAGCTGA ATGGCGATGT GAATGGCCAC AAGTTCAGCG TGAGCGGCGA GGGCGAGGGC 721 GATGCCACCT ACGGCAAGCT GACCCTGAAG TTCATCTGCA CCACCGGCAA GCTGCCTGTG 781 CCCTGGCCCA CCCTGGTGAC CACCCTGAGC TACGGCGTGC AGTGCTTCTC ACGCTACCCC 841 GATCACATGA AGCAGCACGA CTTCTTCAAG AGCGCCATGC CTGAGGGCTA CATCCAGGAG