pUSF-3 BAC细菌人工染色体质粒-BioVector NTCC质粒载体菌种细胞基因保藏中心

pUSF-3 BAC cloning vector

BAC细菌人工染色体质粒

A BAC vector, pUSF-3, was described in reference. pUSF-4 was constructed by removing a ClaI site from the green fluorescent protein (GFP) expression cassette of pUSF-3. pUSF-5 was formed by digesting pUSF-4 with HindIII to remove two human cytomegalovirus (HCMV) fragments and religation of the plasmid. pUSF-6 was constructed from pUSF-5 by inserting two fragments of VZV DNA with added sequences for BamHI, HindIII, and LoxP sites. PCR primers were designed to amplify two fragments of ∼500 bp between ORF60 and ORF61 of the VZV pOka strain at genomic sites 102080 to 102560 and 102564 to 103039 (NCBI nucleotide accession no. AB097933). The four primers are 5′-GGA TCC AAC GTA CCC CAA ACT TAA AAC GCT C and 5′-AAG CTT ATA ACT TCG TAT AGC ATA CAT TAT ACG AAG TTA TAT TAA AAT GGC CGG AAG GTG GCG G; and 5′-AAG CTT ATA ACT TCG TAT AAT GTA TGC TAT ACG AAG TTA TAA CTT TAA TTG CTA TAA GAC ATA CCC AAA C and 5′-GGA TCC TGT TGG GAG GGG GAA GGA AAT CG. The fragments were then cloned separately into pCR4-TOPO (Invitrogen), sequenced to verify the products, digested with BamHI and HindIII, and finally combined in a triple ligation into pUSF-5 at the HindIII site, creating pUSF-6. pGEM-T TA cloning vector was purchased from Promega (Madison, WI). pGEM-lox-zeo was used for rescue virus generation as described in reference20. pGL3-hyg was derived from plasmid pGL3-control (Promega) by replacing its simian virus 40 (SV40) enhancer sequence with a hygromycin resistance gene expression cassette from pIJ963 (a gift from Issar Smith). pGEM-oriV/kan1 was described in reference/

简介：

细菌人工染色体（Bacterial artificial chromosome,BAC）是指一种以F质粒（F-plasmid）为基础建构而成的细菌染色体克隆载体,长用来克隆150kb左右大小的DNA片段,最多可保存300kb个碱基对. 　　该质粒主要包括oriS,repE(控制F质粒复制）和parA、parB（控制拷贝数）等成分.以BAC为基础克隆的载体成嵌合体的频率较低,转化效率高,而且以环状结构存在于细菌体内,易于分辨和分离纯化,已被科学界广泛接受.

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