pJH290
(Plasmid #13330)

• Depositing Lab: Martin Gellert
• Publication: Lewis et al Cell. 1988 Dec 23. 55(6):1099-107.

Backbone

• Vector backbone: pUC13
• Backbone size w/o insert (bp): 2680

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: Unknown

Gene/Insert

• Gene/Insert name: recombination substrate

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ sequencing primer: M13 reverse
• 3′ sequencing primer: M13 forward 20

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor Comments

pJH290 was derived from pJH288. pJH288 (approximately 8000bp) was constructed by stepwise insertion of fragments into the multipurpose cloning sites of pUC13. A fragment containing the early region of polyoma virus was generated by double digestion of PY-pBR322 with BamHI (base 4632) and HincII (base 2962) followed by end-filling with the Klenow fragment of DNA polymerase I. This 3600bp fragment was inserted into the EcoRI site (first blunt-ended using klenow) of pUC13. A 500bp fragment containing the bacteriophage f1 intergenic region (isolated by EcoRI-HindIIIdouble digestion of pUC-f1) was inserted using BglII linkers into the AvrII site (base 4707) of polyoma, which had first been converted to a BglII site, also by using linker DNA. A fragment containing the cat structural gene was generated by double digestion of pRSVcat with HindIII (base 5027) and DpnI (base 4241), subcloning into a suitable vector, and re-isolation using flanking SmaI sites. This 800bp fragment was inserted into the SmaI site of pUC13. A 200bp fragment containing the oop transcription terminator of bacteriophage lambda was isolated from a V(D)J recombinant of pJH195 by digestion at the HincII sites flanking a coding junction consisting of tandem terminators generated by an inversion event. This fragment, which contains a single intact copy of the oop terminator, includes lambda phage sequences from 38,478-38,666, but in a permuted order. Using ClaI linkers, it was inserted into pUC13 at the XbaI site, which had first been converted to a ClaI site using linker DNA.

The 39bp fragment containing the 12-spacer signal, created by annealing of synthesized complementary oligonucleotides with protruding ends compatible with SalI, was inserted into the SalI site of pUC13. The synthesized 49bp fragment containing the 23-spacer signal was inserted into the BamHI site of pUC13.

The difference between pJH290 and pJH288 is the orientation of the 23-spacer signal at the BamHI site.