pJET1: description & restriction map
Additional Information:
CAP protein binding site - 1010-973 (compl. strand);
- lac repressor binding site -926-906 (compl. strand).
The positive selection vector pJET1 is a small, high copy number E.coli plasmid, 3128 bp in length. It was derived from pUC19 by replacing the 5’-terminal part of the lacZ gene encoding the N-terminal fragment of beta-galactosidase with the gene coding for the Eco47I restriction enzyme. In the absence of cognate methylation, the endonuclease is lethal to E.coli host cells. Insertional inactivation of the eco47IR gene is the mechanism to obtain a positive selection using the plasmid pJET1. For convenience, the multiple cloning site (MCS) used for positive selection was incorporated into eco47IR by silent mutagenesis. In addition, polylinkers 1 and 2 that flank the positive selection cassette were introduced for gene mapping/subcloning purposes. The pJET1 plasmid contains: (1) the pMB1 replicon rep responsible for the replication of plasmid (source - plasmid pUC19). The high copy number of pJET1 is a result of the lack of the rop gene and a single point mutation in the replicon rep of pMB1; (2) the bla gene, coding for beta-lactamase, that confers resistance to ampicillin (source - plasmid pUC19); (3) the region of E.coli lac operon containing a CAP protein binding site, promoter PlacUV5 which differs from the wild-type promoter Plac by two point mutations in -10 region, and lac repressor binding site (source - plasmid pUC19; mutations were introduced by site-specific mutagenesis); (4) the modified eco47IR gene, coding for Eco47I restriction enzyme. The gene is toxic for all E.coli strains that are not protected by cognate methylation. The background transcription from PlacUV5 ensures the efficient killing of recipient cells even in case if they encode the lacIq mutation which ensures elevated intracellular concentration of LacI repressor. Therefore, the induction of Eco47I synthesis by IPTG is not required for positive selection purposes. After the cloning of DNA into eco47IR the integrity of this gene is disrupted. Therefore, only those cells survive after the transformation and are able to form a colony in presence of ampicillin which have the recombinant plasmid. This feature ensures positive selection of recombinant clones.
The map (see below) shows enzymes which have unique targets within pJET1 (enzymes produced by Fermentas are shown in orange). The coordinates refer to the position of the first nucleotide in each recognition sequence.
The exact positions of the genetic elements are shown on the map (termination codons included). The bla gene nucleotides 2928-2860 (complementary strand) code for a signal peptide. The indicated rep region is sufficient to promote replication. DNA replication initiates at position 1308 (+/-1) and proceeds in the direction indicated. 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.Enzymes which cut pJET1 DNA once:
AdeI 291, AloI* 455, ApaI 1030, BamHI 1023, BbvCI 843, BceAI 1734, BcgI 2657, BfiI 2186, BglI 2255, BglII* 509, BpiI 229, Bpu10I 843, BsaXI 1101, BseYI 1552, Bsp119I 181, Bsp120I 1030, BspTI 141, BstXI* 466, Bsu15I* 543, BtgI* 534, BveI 390, CaiI 1659, Cfr10I 2221, Cfr9I 1027, Csp6I 2620, Eam1105I 2136, Ecl136II 162, Eco130I* 534, Eco31I 2208, Eco32I* 495, Eco47III 1040, Eco52I 155, Eco72I 173, EcoRI 178, FaqI 274, GsuI 2226, HincII 168, HindIII 750, Kpn2I* 469, LguI 1125, MssI 887, MunI 1018, Mva1269I 848, NcoI* 534, NheI 146, NotI 154, NsbI 2361, PaeI 150, PdmI 2736, PfoI 46, Ppu21I 173, PspXI* 477, PstI 1036, PvuI 2508, RsaI 2620, SacI 162, SalI 168, ScaI 2619, SmaI 1027, TatI 2619, XbaI* 503, XhoI* 478, XmiI 168
* - MCS
pJET1 Multiple Cloning Site (MCS)
Note
Note that only fragments inserted into Kpn2I, XhoI, Eco32I, XbaI and BglII targets might be sequenced with the indicated primers.
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Updated lapkričio 08, 2006 14:16
