pUSF-3 BAC cloning vector

BAC細(xì)菌人工染色體質(zhì)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/

簡(jiǎn)介：

細(xì)菌人工染色體（Bacterial artificial chromosome,BAC）是指一種以F質(zhì)粒（F-plasmid）為基礎(chǔ)建構(gòu)而成的細(xì)菌染色體克隆載體,長用來克隆150kb左右大小的DNA片段,最多可保存300kb個(gè)堿基對(duì). 　　該質(zhì)粒主要包括oriS,repE(控制F質(zhì)粒復(fù)制）和parA、parB（控制拷貝數(shù)）等成分.以BAC為基礎(chǔ)克隆的載體成嵌合體的頻率較低,轉(zhuǎn)化效率高,而且以環(huán)狀結(jié)構(gòu)存在于細(xì)菌體內(nèi),易于分辨和分離純化,已被科學(xué)界廣泛接受.

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