pBS2E
(Plasmid #55169)

• Purpose: (Empty Backbone) Empty vector, integration at lacA, ampr, mlsr
• Depositing Lab: Thorsten Mascher
• Publication: Radeck et al J Biol Eng. 2013 Dec 2;7(1):29. doi: 10.1186/1754-1611-7-29.

Backbone

• Vector backbone: pAX01
• Backbone manufacturer: Hartl et al 2001
• Modifications to backbone: pAX01 was cut with SacI. The 6.3 kb fragment was religated to remove the xylR-PxylA-fragment. The PstI site in bla was removed. The vector was cut with XbaI and the 6 kb fragment religated to reduce the amount of forbidden restriction sites. The lost terminator of erm was replaced by the PCR-amplified terminator with BsaI-overhangs. The vector was opened with XbaI and ligated with the PCR product cut with BsaI (XbaI-compatible overhang). The correct direction was checked by sequencing and removal of the XbaI-site was confirmed. Finally, the MCS was amplified from pSB1C3 with PstI and NsiI overhangs, cut with PstI and NsiI and ligated into the PstI-cut vector. Correct orientation of the insert was confirmed by restriction digest and sequencing. The remaining NgoMIV sites were removed by subsequent site-directed mutagenesis.
• Vector type: Synthetic Biology ; Bacillus BioBrick Box
• Promoter: none
• Selectable markers: erm: specifies resistance to macrolide, lincosamide and streptogramin B antibiotics if induced by erythromycin

Growth in Bacteria

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

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ sequencing primer: na

Resource Information

• Supplemental Documents:
  • pBS2E genbank file

Terms and Licenses

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

Depositor Comments

This is an “empty” vector that lacks promoters and reporter genes. The integrative part contains the flanking homology regions, a resistance cassette for selection in B. subtilis and the multiple cloning site (MCS), containing an rfp-cassette flanked by the restriction sites EcoRI, NotI, XbaI (upstream) and SpeI, NotI and PstI (downstream). They allow cloning in BioBrick standard with selection for white colonies as a result of the removal of the rfp-insert, which – if still present – leads to formation of red colonies in E. coli.
For sequencing of inserts, use the following primers:
fwd: GGCAACCGAGCGTTCTG
rev: CTGACAGCGTTTCGATCC