pSB1AK3X

BBa_J18905 Version 1

Component

Source:
http://parts.igem.org/Part:BBa_J18905
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

pSB1AK3X Freiburg Fusion -> BioFusion conversion plasmid



Types
DnaRegion

Roles
plasmid

Plasmid

Sequences BBa_J18905_sequence (Version 1)

Description

pSB1AK3X shares the same backbone as pSB1AK3 but uses modified prefix and suffix sequences (that do not formally adhere to any BioBrick standard):











5' GAATTC GCGGCCGC T TCTAGA GCCGGC
EcoRI NotI XbaI NgoMIV
...part...

ACCGGT ACTAGT A GCGGCCG CTGCAG 3'
AgeI SpeI NotI PstI


These prefix / suffix sequences are designed to translate protein parts from the proposed Fusion (aka Freiburg) format to the older BioFusion format from the Silver lab. Construction vectors with this modified Freiburg prefix / suffix could bring a protein part in frame with BioFusion parts and remove the STOP between the AgeI and SpeI site. Restriction / ligation with AgeI + NaeI can (theoretically) transfer Expression parts into this conversion vector which can then be used for normal BioFusion cloning (at the cost of adding a T G before and after the part).
NaeI is an isoschizomer to NgoMIV but generates blunt ends which should allow for a directional transfer.

See BB formats.

pSB1AK3X 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 modified suffix and ends with the modified prefix. The complete vector sequence is therefore obtained from: part + vector.

See also:







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.

All 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 pSB1AK3. Only insert and flanks have been verified by sequencing.

Source

constructed from pSB1AK3

Sequence Annotation Location Component / Role(s)
crossFusion suffix
crossFusion prefix
1,26
3173,3199
feature/misc sequence_feature
sequence_feature feature/misc
igem#experience
None
 
igem#sampleStatus
Not in stock
igem#status
Unavailable
 
synbiohub#ownedBy
user/james
 
synbiohub#ownedBy
user/myers
 
synbiohub#topLevel
BBa_J18905/1