Enger-Valk B E, Heyneker H L, Oosterbaan R A, Pouwels P H
Gene. 1980 Apr;9(1-2):69-85. doi: 10.1016/0378-1119(80)90167-5.
In vitro recombination techniques were used to construct a series of new cloning vehicles with genes of the tryptophan (trp) operon of Escherichia coli as selective marker. To construct these plasmids we have made a restriction cleavage map of the trp operon for the enzymes AosI, AvaI, BglI, BglII, HindIII, HpaI, PvuII, SalI, SstI and XhoI. The constructed plasmids pHP39, pEP392, pEP3921 and pEP3923 are derived from the amplifiable plasmid pBR345 and carry two or more genes of the trp operon, which are controlled by the trp regulatory elements. Plasmid pEP3921 (7.0 kb) carries intact trpE and trpA genes and contains single BglII and SstI sites in trpE, a single HindIII site located between trpE and trpA, and single EcoRI, SalI and XhoI sites located outside the trp genes. Plasmid pEP121 (12 kb) is similar to pEP3921, but has an extra selective marker conferring bacterial resistance to ampicillin. Plasmid pEP3923 (7.4 kb) comprises intact trpB and trpA genes and single BglII, HindIII, EcoRI, SalI and XhoI sites. Plasmids pHP39 (9.8 kb) and pEP392 (9.8 kb) carry an intact trp operon and have two and one EcoRI site, respectively. Plasmid pHP3 (18 kb) carries an intact trp operon and markers for tetracycline and ampicillin resistance.
利用体外重组技术构建了一系列新的克隆载体，以大肠杆菌色氨酸（trp）操纵子的基因作为选择标记。为构建这些质粒，我们绘制了trp操纵子对于AosI、AvaI、BglI、BglII、HindIII、HpaI、PvuII、SalI、SstI和XhoI等酶的限制性酶切图谱。构建的质粒pHP39、pEP392、pEP3921和pEP3923源自可扩增质粒pBR345，并携带两个或更多trp操纵子的基因，这些基因受trp调控元件控制。质粒pEP3921（7.0 kb）携带完整的trpE和trpA基因，在trpE中有单个BglII和SstI位点，在trpE和trpA之间有单个HindIII位点，在trp基因外部有单个EcoRI、SalI和XhoI位点。质粒pEP121（12 kb）与pEP3921相似，但有一个额外的赋予细菌对氨苄青霉素抗性的选择标记。质粒pEP3923（7.4 kb）包含完整的trpB和trpA基因以及单个BglII、HindIII、EcoRI、SalI和XhoI位点。质粒pHP39（9.8 kb）和pEP392（9.8 kb）分别携带完整的trp操纵子，且分别有两个和一个EcoRI位点。质粒pHP3（18 kb）携带完整的trp操纵子以及四环素和氨苄青霉素抗性标记。
