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The sequence was compiled from the published construction of pJM703.1 (Miller and Mekalanos, 1988) and its parent vectors and the unpublished construction of pGP704, and with help from Jim Slauch. I have tried to give a complete description of how I put the sequences together. Please distribute this sequence, and email me if you find any errors (I know there are some) or have any updates to the sequence, particularly if you find enzyme sites missing or extra, or have any DNA sequence. Also email me if you would like to me to send you updates when I change the sequence.
The sequence starts at oriV and moves clockwise around the map, through oriT and amp to the multiple cloning site, ending with the EcoRI site.
This is an MboI fragment from R6K (accession number M65025), including the HinDIII site. I suspect that this MboI fragment was cloned into the BamHI site in pBR322. I think that there may be a few bp between oriV and the EcoRI site missing. I plan to sequence outwards from oriV in both directions to confirm the junctions with oriT and the amp resistance gene.
The oriT region was cloned into pJM703.1 from pSUP101.1 (Simon et. al., 1983). pSUP101.1 was constructed by partial Sau3A1 digestions of RP4 (to provide oriT) and pBR325 (to provide a vector).
The RP4 sequence (accession number L27758) was used as the starting point. Since the resulting clone contained the oriT region flanked by BamHI sites the Sau3A1 site used must have been GATCC at the 5' end and GGATC at the 3' end. Moreover since the oriT region of RP4 has been independently sequenced (e.g. in accesion number AF012346) the oriT region in pGP704 must contain bp 50,926 to 51,474 of the RP4 sequence. The potential sites are listed below:
GATCC GGATC 49,246 47,691 49,282 48,301 50,134 48,548 52,614 50,812 52,671 51,857 52,887 52,760 53,228
Since the oriT region is between 1.7 and 1.9 kb, and must include region 50,926 to 51,474, the only possible region is 50,134 to 51,857. The orientation was confirmed by restriction digestion. Also, in pGP704, transfer proceeds from oriT to amp to the MCS, with oriV being transferred last. I need to confirm that this is the correct oriT sequence and in the correct orie ntation.
The pBR322 sequence (accession number J01749) was modified as described below:
Multiple cloning site.
The 66bp fragment from bp 6234 to 6300 (BglII-EcoRI) was taken from the M13tg131 sequence (accession number L08833).
Kolter, R. Inuzuka, M. and Helinski, D. (1978). Cell
The sequence was last updated on 11th August 1998
Go to the pGP704 sequence which is in pseudo-Genbank format and save the following page as a text document. You should be able to use your favorite DNA analysis software to look at it. Alternatively save this genbank file to your computer (Mac: click and hold, Windows: right click, then choose "Save Link As")
pGP704 derivatives We have constructed four improved vectors for allelic exchange. The advantage of these vectors is that they contain sacB, the levansucrase from B. subtilis for counter selection, and a wider range of antibiotic resistance markers for selection.
Check this medline reference for full details, or you can download the full text PDF from Elsevier.
The sequence of these vectors is available here
If you would like me to add vector sequences or descriptions to this page then please email me.
A brief version history is available. If you have any problems email me.
Rob Edwards, 11th August 1998