pGP704
information
& sequence.

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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was compiled from the published construction of pJM703.1 (Miller and Mekalanos, 1988) and its parent vectors and the unpublished construction of pGP704, and with help from Jim Slauch.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.

oriV.

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.

oriT region.

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.

Amp resistance.

The pBR322 sequence (accession number J01749) was modified as described below:

• The SphI-PvuII and DraI to NdeI regions were blunted and removed.
  
• SphI was blunted from G|CATGC to G
  
• NdeI was blunted from CA|TATG to CA
  
• The AatII site was blunted from G|ACGTC to G
  
• The BglII region has now been sequenced by Jim Slauch. The sequence has been corrected from ³⁶³⁰GGAAG to ³⁶³⁰CAG. This introduces a second PstI site. Previously I used the linker GGAAGATCTTCC (NEB cat#1066). (This was chosen "at random" by me.

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 15: 1199-1208
Miller, V. and Mekalanos, J. (1988). J. Bact. 170:2575-2583
Simon, R. Priefer, U. and Puhler, A. (1983). Bio/Technology 1:784-791 (not available on medline)

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")

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.

Rob Edwards, 11th August 1998