The nucleotide sequence of pACYC184

TLDR: It appears that the precursor was also digested with Haell which generated the Haell Cm resistance segment and the Hael fragments found between the Tet and Cm genes.

Abstract: pACYC184 ii a commonly used multicopy cloning vector which was constructed by ligating restriction fragments from pSClOl, Tn9, and pl5A each of which have been previously sequence*! (1,23,4,5). Despite its wide use, the complete nucleotide sequence of pACYC 184 has never been repented. The sequence was completed by using oligonudeotide primers designed to span the junctions between each of the previously sequenced regions. pACYC184is 4244 bp in length with nucleotide 1 corresponding to the EcoRI site in the original map (1). The chtorampbenicol resistance (Cm) segment from Tn9 extends from the Haell site at base 3505 to the Haell site at base 585 with bases 219 (ATG) to 3804 encoding the Cm gene. Part of an IS1 (5) from Tn9 extends from bases 443 to 583. Bases 1494 to 3275 are derived from pSClOl with the tetracycline (Tet) resistance gene encoded by bases 1580 (ATG) to 2770. The pl5A origin of replication extends from bases 581 to 1492. Three fragments; an Alul (3276) to Haell (3368), a Haen (3368) to Haell (3422), and a Haell (3422) to Haell (3505) are located between Cm and Tet gene and are all derived from different regions of the Tet gene. During the construction of pACYCl84 a precursor plasmid, pACYC175, was digested with Haem, Alul, and Hindi, to remove extraneous DNA and to reduce the size of the plasmid. It appears that the precursor was also digested with Haell which generated the Haell Cm resistance segment and the Haell fragments found between the Tet and Cm genes. The underlined sequence was determined while the rest of the sequence was taken from the published sequences of pSClOl (2), Tn9 (3,4), and pl5A (5).