Related Documents:
pTZ19R
GenBank/EMBL accession number Y14835.pTZ19U
GenBank/EMBL accession number Y14836.Not available from Fermentas
Additional Information:
CAP protein binding site - 837-800 (compl. strand);
- mRNA (LacZ) starts at nt position 753 (compl. strand);
- lac repressor (LacI) binding site - 753-733 (compl. strand);
- f1 packaging signal - 3-94 (pTZ19R) and 364-455 (pTZ19U).
pTZ19R/U are small phagemids 2862 bp in length. They were constructed by inserting the DNA of phage f1 intergenic region (IG) in pUC19 as well as introduction of T7 promoter sequence near to the MCS of pUC19. The phagemids differ in the orientation of cloned f1 IG region. They are designed for DNA cloning, dideoxy DNA sequencing, in vitro mutagenesis and in vitro transcription in one system. A host strain harboring these phagemids will produce single-stranded DNA if superinfected with the helper phage M13K07. pTZ19R/U phagemids contain: (1) the pMB1 replicon rep responsible for the replication of phagemid (source - plasmid pUC19); (2) bla gene, coding for beta-lactamase that confers resistance to ampicillin (source - plasmid pUC19); (3) 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 (source - pUC19). This fragment allows blue/white screening of recombinant phagemids in the same way as described in pUC18/19 section; (4) T7 promoter inserted near to the MCS of pUC19 allowing in vitro synthesis of large amounts of specific RNA; (5) phage f1 intergenic region carrying the sequences required in cis for initiation and termination of phage f1 DNA synthesis (+ and - strands) and for packaging of DNA into bacteriophage particles. Synthesis of single-stranded (plus) DNA requires the phage-encoded gene II, X and V proteins. It is initiated at ori (+) and proceeds in indicated direction. 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 synthesized by host's RNA polymerase and starting at ori (-).
The map show enzymes that cut pTZ19R 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). In the case of the bla gene nucleotides 2732-2664 (complementary strand) code for a signal peptide. LacZ polypeptide corresponding to wt beta-galactosidase and essential for blue/white screening ends at nt position 458 (complementary strand). Remaining amino acids in LacZ reading frame are encoded by f1 DNA. The indicated rep region is sufficient to promote replication. DNA replication initiates in the complementary DNA strand at position 1112 (+/- 1) and proceeds in the direction indicated. Phagemids carrying the pMB1 and ColE1 replicons are incompatible with one another, but are fully compatible with those carrying p15A replicon (pACYC177, pACYC184). pMB1-derived plasmids can be amplified using chloramphenicol.
Reference
Mead, D.A., Szczesna-Skorupa, E. and Kemper, B., Single-stranded DNA áblue' T7 promoter plasmids: a versatile tandem promoter system for cloning and protein engineering, Protein Eng., 1, 67-74, 1986.
Enzymes which cut pTZ19R DNA once:
Acc65I 627, AdeI 230, AflIII 1052, AloI 279, BamHI 636, BcgI 2461, BsaAI 230, BseYI 1356, BveI 652, CaiI 1463, Cfr9I 631, Eam1105I 1940, Ecl136II 621, Eco31I 2012, Eco88I 631, EcoRI 615, GsuI 2030, Hin1I 2481, HincII 648, HindIII 666, KpnI 627, PaeI 660, PdiI 127, PdmI 2540, PscI 1052, PsiI 358, PstI 654, SacI 621, SalI 648, SapI 929, ScaI 2423, SdaI 653, SmaI 631, TatI 2423, XbaI 642, XmiI 648.
Multiple Cloning Sites
pTZ19R/U
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Updated lapkričio 08, 2006 14:19
