Related Documents:
M13mp18
GenBank/EMBL accession number M77815.Not available from Fermentas
M13mp19
GenBank/EMBL accession number L08821.Not available from Fermentas
Additional Information:
DNA from E.coli lac operon extends from nt 5869 to nt 6711;
- CAP protein binding site – 6095-6132;
- mRNA (LacZ) starts at nt position 6179;
- lac repressor binding site – 6179-6199.
The M13mp18 and M13mp19 vectors are derivatives of the single-stranded, male-specific filamentous DNA bacteriophage M13. Both vectors are 7250 bp in length and have the same MCS inserted in opposing orientations. The vector DNAs contain a region of E.coli operon lac containing CAP protein binding site, promoter Plac, lac repressor binding site and 5'-terminal part of lacZ gene encoding the N-terminal fragment of beta-galactosidase (codons 6-7 of lacZ are replaced by MCS). This fragment, whose synthesis can be induced by IPTG, is capable of intra-allelic (a) complementation with a defective form of beta-galactosidase encoded by host (mutation lacZDM15). This results in appearance of blue plaques on media containing IPTG and X-gal. Recombinant phages containing inserts that destroy the reading frame of lacZ are revealed as colorless plaques. Synthesis of viral (plus) single-stranded DNA requires the phage-encoded gene II, X and V proteins. It is initiated at ori (+) and proceeds in the direction indicated. The conversion of plus DNA strands to double strands does not require any of the phage genes. DNA synthesis is initiated by a 30-nucleotide RNA primer synthesised by host's RNA polymerase and starting at ori (-). Double-stranded circular DNA (replicative form, or RF) can be isolated from cells by standard plasmid preparation techniques and used for cloning experiments, while the single-stranded viral DNA (+ strand) can be isolated from phage particles collected from culture medium.
The M13mp18/19 genes are shown on the map (M13 genes are transcribed clockwise). The map shows enzymes that cut M13mp18/19 DNA once. Enzymes produced by Fermentas are shown in green. The coordinates refer to the position of first nucleotide in each recognition sequence.
References
Norrander, J., Kempe, T. and Messing, J., Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis, Gene, 26, 101-106, 1983.
- 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 M13mp18 DNA once:
AasI 5759, Acc65I 6243, AdeI 5716, AloI 5765, Alw44I* 4743, BamHI 6252, BfiI 6317, BglI 6431, BglII 6935, BseRI 2008, Bsp1407I 1021, CaiI 2187, Cfr9I 6247, Cfr10I 5613, Ecl136II 6237, Eco47I 5914, Eco81I 6508, Eco105I 1268, EcoRI 6231, EheI 6001, Esp3I 5971, Hin1I 6001, HincII 6264, HindIII 6282, KpnI 6243, MlsI 5080, Mva1269I 1746, NsbI 6425, OliI* 6573, PacI 4132, PaeI 6276, PagI 1299, PdiI 5613, PpiI 5766, PstI 6270, PvuI 6405, SacI 6237, SalI 6264, SdaI 6269, SmaI 6247, SmiI 6783, TstI 6618, XbaI 6258, XmiI 6264.* According to our experimental data:
Alw44I does not cut M13mp18/19;
- Eco47III has only one recognition site at a position 3039;
- BseSI has only one recognition site at a position 2088.
- OliI has one recognition site at a position 6573.
Coordinates of M13mp18/19 genes (termination codons included):
I 3196-4242
II 6849-831
III 1579-2853
IV 4220-5500
V 843-1106
VI 2856-3194
VII 1108-1209
VIII 1301-1522
IX 1206-1304
X 496-831Multiple Cloning Sites
M13mp18
M13mp19
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Updated lapkričio 08, 2006 14:13
