Puc 19 e 18

Páginas: 2 (363 palavras) Publicado: 8 de outubro de 2011
pUC18, pUC19
pUC18 and pUC19 vectors are small, high copy number, E.coli plasmids, 2686 bp in length. They are identical except that they contain multiple cloning sites (MCS) arranged in oppositeorientations. pUC18/19 plasmids contain: (1) the pMB1 replicon rep responsible for the replication of plasmid (source – plasmid pBR322). The high copy number of pUC plasmids is a result of the lack ofthe rop gene and a single point mutation in rep of pMB1; (2) bla gene, coding for beta-lactamase that confers resistance to ampicillin (source – plasmid pBR322). It differs from that of pBR322 by twopoint mutations; (3) region of E.coli operon lac containing CAP protein binding site, promoter Plac, lac repressor binding site and 5’-terminal part of the lacZ gene encoding the N-terminal fragment ofbeta-galactosidase (source – M13mp19/19). This fragment, whose synthesis can be induced by IPTG, is capable of intra-allelic (alfa) complementation with a defective form of beta-galactosidase encodedby host (mutation lacZΔM15). In the presence of IPTG, bacteria synthesise both fragments of the enzyme and form blue colonies on media with X-gal. Insertion of DNA into the MCS located within thelacZ gene (codons 6-7 of lacZ are replaced by MCS) inactivates the N-terminal fragment of beta-galactosidase and abolishes alfa-complementation. Bacteria carrying recombinant plasmids therefore give riseto white colonies.

The map shows enzymes that cut pUC18/19 DNA once. The coordinates refer to the position of first nucleotide in each recognition sequence.

The exact position of geneticelements is shown on the map (termination codons included). The bla gene nucleotides 2486-2418 (complementary strand) code for a signal peptide. The LacZ polypeptide corresponding to wt beta-galactosidaseand essential for blue/white screening ends at nt position 236 (compl. strand); another 30 codons in the same reading frame are derived from pBR322. The indicated rep region is sufficient to promote...
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