| Comments |
Restriction digests of the clone give the following sizes (kb): PvuII--3.75, 2.9, 2.0, 0.7, 0.5; HindIII--6.3, 3.6; BglII/HindIII--3.6, 3.2, 2.3, 0.7; EcoRI--8.8, 1.1; BamHI--5.0, 2.8, 1.1, 0.6, 0.3. The plasmid contains the following restriction sites (nt from an arbitrary origin): PvuII--613, 1150, 1854, 3818, 7564; HindIII--1000, 7220; AflII--1023, 4245, 6916; AatII--9786. Can be linearized by a AatII digestion before introduction to host cells. The vector sequences permit generation of high copy number by methotrexate treatment, and high levels of expression. The 3.2 kb AflII fragment (1023-4245) contains the MIS cDNA, polyadenylation signal, and 3' flanking sequence. The 0.657 kb BglII/HindIII fragment (6563-7220) contains the Dhfr gene. Constructed from pSV2-dhfr, a construct including the Mullerian inhibiting substance gene, and a synthetic oligonucleotide homologous to the human gastrin transcriptional terminator. The gene sequences include both genomic and cDNA. The order of the major features in this plasmid is: SV40 enhancer - adenovirus-2 major late promoter - MIS cDNA - MIS polyadenylation signal - MIS genomic 3' flanking sequence - SV40 polyadenylation signal - gastrin terminator. SV40 polyadenylation signal - SV40 splice signal - mouse Dhfr - SV40 promoter - pMB1 ori - bla. |
| References |
Barsoum JG. Method for producing cells containing stably integrated foreign DNA at a high copy number, the cells produced by this method, and the use of these cells to produce the polypeptides coded by the foreign DNA. US Patent 4,956,288 dated Sep 11 1990
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