pET28a-LIC Vector (GenBank accession EF442785)

Source Constructed by Peter Loppnau

Company Structural Genomics Consortium, Toronto

Description The pET28a-LIC vector was derived from expression
plasmid pET28a (Novagen). It is used for T7 promoter
driven expression of recombinant proteins with the addition
of a 19 amino acid N-terminal fusion tag containing a 6X
His-tag followed by a thrombin protease cleavage site. Two
stop codons are included in the vector at the C-terminal
cloning site.

Antibiotic resistance Kanamycin, 50 ug/ml
Promoter T7 - lacO
Cloning Methods Insertion of DNA sequence into the cloning/expression
region is preformed using BD-Biosciences Infusion enzyme
mediated directional recombination between complementary
15 nucleotide DNA sequences at the ends of the insert (PCR
product) and BseRI linearized vector. Insertion of target
sequence involves replacement of a SacB gene stuffer
sequence, which provides for negative selection of the
original plasmid on 5% sucrose.
Initiation Codon NcoI site in vector at 5071 bp
N terminal fusion
sequence
MGSSHHHHHHSSGLVPRGS

Termination codons TGATGA included in 3 PCR primer and vector cloning
site. No amino acid residues added at cloning junction
Additional features
5 primer for amplification
of insert
5 GTT CCG CGT GGT AGT --- 3

5 primer for amplification
of insert without tag for
NcoI/BseRI cloning
5 AAG AAG GAG ATA TAC CAT G --- 3
3 primer for amplification
of insert
5 CAA GCT TCG TCA TCA --- 3

5 sequencing primer T7-
Fwd
5 AATTAATACGACTCACTATAGGG 3
3 sequencing primer T7-
Rev
5 ATGCTAGTTATTGCTCAGCGG 3

pET28a-LIC vector map

T7 promoter 4984-5000
N-terminal tag coding sequence 5071-5127
N-terminal cloning site 5113-5127
C-terminal cloning site 7143-7157
T7 terminator 7257-7303
f1 origin 12-467
aph coding sequence 563-1375
pBR322 origin 2084
lacI coding sequence 3518-4597
sacB coding sequence 5649-7067



T7 promoter
T7 Terminator
Kan Resistance
ori
lacI
SacB gene
NcoI (5069)
BglII (4964)
BseRI (5138)
BseRI (7127)
HindIII (7151)
XhoI (7166)
XbaI (5030)
f1 origin
N-terminal tag
pET28a-LIC
7328 bp

pET28a-LIC cloning/expression region

T7 FWD lac operator
~~~~~~~~~~~~~~~
4968 ct cgat cccg cgaaat t aat acgact cact at aggggaat t gt gagcgga
gagct agggc gct t t aat t a t gct gagt ga t at cccct t a acact cgcct

~~~~~~~~~~
5018 t aacaat t cc cct ct agaaa t aat t t t gt t t aact t t aag aaggagat at
at t gt t aagg ggagat ct t t at t aaaacaa at t gaaat t c t t cct ct at a

NcoI
M G S S H H H H H H S S G L V P
5068 accat gggca gcagccat ca t cat cat cat cacagcagcg gcct ggt t cc
t ggt acccgt cgt cggt agt agt agt agt a gt gt cgt cgc cggaccaagg


R G S BseRI
5118 gcgt ggt agt / at t at gagt t ct cct c- - - - - SACB casset t e( 2 kb) - - -
cgcaccat ca/ t aat act caa gaggag


BseRI st op Hi ndI I I XhoI
7127 gaggagat ca t gcaca/ t gat gacgaagct t gcggccgcac t cgagcacca
ct cct ct agt acgt gt / act a ct gct t cgaa cgccggcgt g agct cgt gga


7174 ccaccaccac cact gagat c cggct gct aa caaagcccga aaggaagct g
ggt ggt ggt g gt gact ct ag gccgacgat t gt t t cgggct t t cct t cgac

T7 REV

7227 agt t ggct gc t gccaccgct gagcaat aac t agcat aacc cct t ggggcc
t caaccgacg acggt ggcga ct cgt t at t g at cgt at t gg ggaaccccgg