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pUC18
GenBank/EMBL accession number L09136.pUC19
GenBank/EMBL accession number L09137.Additional Information:
CAP protein binding site - 591-554 (compl. strand);
- mRNA (LacZ) starts at nt position 507 (compl. strand);
- lac repressor binding site - 507-487 (compl. strand).
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 opposite orientations. 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 of the 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 two point 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 of beta-galactosidase (source - M13mp18/19). This fragment, whose synthesis can be induced by IPTG, is capable of intra-allelic (alfa) complementation with a defective form of beta-galactosidase encoded by host (mutation lacZDM15). 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 the lacZ 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 rise to white colonies.
The map shows enzymes that cut pUC18/19 DNA once. Enzymes produced by Fermentas are shown in blue. The coordinates refer to the position of first nucleotide in each recognition sequence.
The exact position of genetic elements 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-galactosidase and 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 replication. DNA replication initiates at position 866 (+/-1) and proceeds in indicated direction. Plasmids carrying the pMB1 and ColE1 replicons are incompatible, but they are fully compatible with those carrying the p15A replicon (pACYC177, pACYC184). pMB1-derived plasmids can be amplified using chloramphenicol.
Reference
Yanisch-Perron, C., Vieira, J. and Messing, J., Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors, Gene 33, 103-119, 1985.
Enzymes which cut pUC18 DNA once:
AatII 2617, Acc65I 438, AflIII 806, BamHI 429, BcgI 2215, BsaXI 659, BseYI 1110, BstAPI 179, BveI 413, CaiI 1217, Cfr9I 434, Cfr10I 1779, Eam1105I 1694, Ecl136II 444, Eco24I 444, Eco31I 1766, Eco88I 434, EcoO109I 2674, EcoRI 450, EheI 235, GsuI 1784, HincII 417, HindIII 399, KpnI 438, NdeI 183, PaeI 405, PdmI 2294, PscI 806, PstI 411, SacI 444, SalI 417, SapI 683, ScaI 2177, SdaI 410, SmaI 434, SspI 2501, XapI 450, XbaI 423, XmiI 417.
Multiple Cloning Sites
pUC18
pUC19
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Updated lapkričio 08, 2006 14:17
