General GABI-Kat sequencing strategy

 

The figure below gives an overview on how we create the sequences which are primed from the T-DNA present in pAC161 (a map and the sequence of the binary vector pAC161 is available from the download page). To orient a (potential) insertion sequence relative to YFG (your favorite gene), you need to figure out which primer has been used in the sequencing reaction. The name of the primer is given at the end of the name of the sequence (displayed in FASTA format in the sequence window).
Example: primer o8409 was used to generate the sequence 65-K012471-022-054-A09-8409

 Sequencing Strategy

Info on the oligos used to build the adapter which is ligated to the BfaI overhang

 

sequence (5' to 3')

comment

long adapter oligo
GAGTAATACGACTCACTATAGGGAGGATCCCA
contains annealing site for "green" primer
short adapter oligo
TATGGGATCACATTAA(3dC)
blocked 3'-end to avoid extension
BfaI site: C'TA_G
(only 7 bases form ds-DNA, plus 2 from overhang)
 

 

Info on the oligos used as primers in the sequence reaction (dark-red in the picture above)

sequencing primer name

sequence (5' to 3')

reading out of which border?

o8409
ATATTGACCATCATACTCATTGC
Left
o3144
GTGGATTGATGTGATATCTCC
Right
oriL
ATATTGACCATCATACTCATTGC
Left
35St
GTGGATTGATGTGATATCTCC
Right