Part:BBa_K187001:Design
pBA Universal Plasmid for BioBytes Gene Assembly System
- 10INCOMPATIBLE WITH RFC[10]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 750
Illegal XbaI site found at 792
Illegal PstI site found at 837 - 12INCOMPATIBLE WITH RFC[12]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 750
Illegal PstI site found at 837
Illegal NotI site found at 756
Illegal NotI site found at 861 - 21INCOMPATIBLE WITH RFC[21]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 750 - 23INCOMPATIBLE WITH RFC[23]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 750
Illegal XbaI site found at 792
Illegal PstI site found at 837 - 25INCOMPATIBLE WITH RFC[25]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 750
Illegal XbaI site found at 792
Illegal PstI site found at 837 - 1000INCOMPATIBLE WITH RFC[1000]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal BsaI site found at 1727
Illegal SapI.rc site found at 846
Design Notes
The boundary sequences required for sticky end production for the BioBytes gene assembly system are acquired by inserting gene sequences destined to become bytes into either of the host plasmids pAB or pBA. Both are derivatives of pUC19 with the following key modifications:
1. Replacement of the pUC19 MCS with the MCS shown in figure 1.
2. Elimination of the Z alpha fragment
3. Elimination of the restriction sites Bts1 and BspQ1
Functional features include:
1. The ability to adapt biobricks to the Byte format using the Xba1 and Pst1 sites contained in the MCS.
2. The ability to adapt the in the ASKA collection (Kitagawa 2005) to the Byte format (4000+ E. coli K12 ORFs). ORFs are cloned into an expression vector, accompanied by a 6xHis tag at the N-terminus and a GFP fusion on the C-terminus. ASKA parts can be freed by digestion with SfiI, leaving noncomplementary 3 bp 3' sticky ends, which can be cloned directionally into the MCS after digestion with BstAPI. Since the ASKA parts are freed from the interior of the CDS, the MCS provides a start codon 10 bp downstream of the RBS, and a stop codon (TGA) downstream of the insertion. ORFs cloned from ASKA plasmids replace the N-terminal methionine of the predicted ORF with Met-Ala-Leu-Arg-Ala, and append Cys-Leu at the C-terminus.
3. Other potentially useful restriction sites have been included in the MCS as well as an RBS consensus positioned 8 bp upstream of the ATG used for ORF’s derived from 1. And 2.
4. The ability to select for insert-containing constructs through the linearization of parental contaminants by cleavage with the rare- cutters SwaI and PmeI contained in the MCS.
5. Two routes for the production of 12 base overhangs by PCR using universal primers: A) PCR followed by nicking using the engineered enzymes Nb.BbvCI and Nb.BtsI. B) PCR followed by removal of a primer-encoded uracil residue using BioLab’s UserTM system. Primer positions and sequences are indicated for both. Note that only the User method is compatible with RFC #47
Source
Derivative of pUC19