Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Raik Gruenberg
Date created: 2008-08-10 11:00:00
Date modified: 2015-08-31 04:08:35
pSB1AT3F construction plasmid for protein fusion BioBricks
| Types | DnaRegion |
| Roles | Plasmid plasmid |
| Sequences | BBa_J18903_sequence (Version 1) |
Description
pSB1AT3F is almost identical to pSB1AT3 but uses the "Freiburg" extension of the standard BioBrick prefix and suffix and features silent mutations in the tetracycline resistance gene. The Freiburg extension adds a new pair of inner restriction enzymes -- NgoMIV (prefix) and AgeI (suffix) -- which allows the in-frame fusion of protein parts. See BB formats.pSB1AT3F is a high copy number plasmid carrying ampicillin and tetracyclin resistance. The replication origin is a pUC19-derived pMB1 (copy number of 100-300 per cell). For the version of this plasmid with a CcdB insert, see BBa_P1010.
The given sequence starts with the Freiburg BB suffix and ends with the Freiburg BB prefix. The complete vector sequence is therefore obtained from:
part + vector
Notes
This plasmid was constructed by PCR and InFusion recombination.1) The pSB1AK3 vector backbone was linearized by PCR, introducing the NgoMIV / AgeI restriction sites
2) The P1010 insert was amplified by PCR, introducing the NgoMIV / AgeI restriction sites
3) The two overlapping PCR products were recombined using the Clonetech InFusion kit.
4) Two NgoMIV restriction sites in the original pSB1AT3 (within the TcR) were deleted following the QuickChange protocol (from Qiagen).
PCR reactions were performed with a proof-reading polymerase featuring a very low mutation rate. Nevertheless, the vector backbone may contain mutations with respect to pSB1AT3. Only insert and flanks have been verified by sequencing. The deletion of NgoMIV sites has been verified by restriction digests.
Source
constructed from pSB1AT3| Sequence Annotation | Location | Component / Role(s) |
| extended BioBrick suffix extended BioBrick prefix BBVR BBVF2 AmpR TetR replication origin (MB1) | 1,30 3434,3462 166,185 3315,3335 2333,3193 1136,2326 270,884 | feature/misc sequence_feature feature/misc sequence_feature feature/primer_binding primer_binding_site feature/primer_binding primer_binding_site feature/cds CDS CDS feature/cds sequence_feature feature/misc |