Vector information sheet.
Vector Name
pFB-Sec-NH
Source
Sequence accession/link
Grazyna Kochan
(SGC)
Description
Baculovirus transfer vector with a signal peptide (from
baculovirus gp64), a His6 tag and a TEV protease cleavage site
fused to the N-terminal of the cloned protein. Used for secreted
or integral membrane proteins. Includes sites for LIC cloning,
and a “stuffer” fragment that includes the SacB gene, allowing
negative selection of transformed bacteria on 5% sucrose
Antibiotic resistance
Promoter
Cloning
Initiation codon
N-terminal fusion –
seq.
N-terminal fusion –
MW
Termination codons
Protease cleavage
Additional features
Preferred host
5’ sequencing
primer
3’ sequencing
primer
Ampicillin, 100 g/ml
Polyhedrin
LIC. (vector treated with BseRI, then with T4 DNA polymerase in
presence of dGTP)
Supplied in PCR primer
MVSAIVLYVLLAAAAHSAFAAAMGHHHHHHSSGVDLGTENLYFQ*SM
(* - TEV cleavage site)
5012 Da including Met (4682.9 Da removed by TEV cleavage)
supplied in PCR primer
TEV
Tn7 sequences for in vivo recombination into bacmid DNA in DH10Bac
(using Invitrogen’s Bac-to-bac system).
Initial transformation into any cloning strain, then transform purified
plasmid into DH10Bac to generate recombinant bacmid DNA
FBAC1: TATTCATACCGTCCCACCA
FBAC2: GGGAGGTTTTTTAAAGCAAGTAAA
Tn7L
pFBac-2 primer
HindIII (6848)
Pst I (6839)
F1 origin
BamHI (6785)
Bse RI (6769)
3'LIC
AmpR
SacB
pFB-Se c-NH
Bse RI (4756)
7386 bp
pUC origin
5'LIC
His/TEV
6xHIS
gp64 sig seq
pFBac-1 primer
PH promoter
Tn7R
GentR
PH promoter
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
pFBac-1 primer
~~~~~~~~~~~~~~~~~~~~~
4561
4621
4681
4741
gp64 sig seq
~~~~
M V ·
CCTATAAATA TTCCGGATTA TTCATACCGT CCCACCATCG GGCGCGGATC TAAAACATGG
GGATATTTAT AAGGCCTAAT AAGTATGGCA GGGTGGTAGC CCGCGCCTAG ATTTTGTACC
gp64 sig seq
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
· S A I
V L Y
V L L A
A A A
H S A
F A A A ·
TAAGCGCTAT TGTTTTATAT GTGCTTTTGG CGGCGGCGGC GCATTCTGCC TTTGCGGCCG
ATTCGCGATA ACAAAATATA CACGAAAACC GCCGCCGCCG CGTAAGACGG AAACGCCGGC
His/TEV
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
6xHIS
5'LIC
~~~~~~~~~~~~~~~~~~~~
~
· M G H
H H H
H H S S
G V D
L G T
E N L Y ·
CCATGGGCCA CCATCATCAT CATCATTCTT CTGGTGTAGA TCTGGGTACC GAGAACCTGT
GGTACCCGGT GGTAGTAGTA GTAGTAAGAA GACCACATCT AGACCCATGG CTCTTGGACA
His/TEV
~~~~~~~~
5'LIC
~~~~~~~~~~~~~~
BseRI
~~~~~~
· F Q
ACTTCCAATC CATAAGCTAG CTTCTCCTCC
TGAAGGTTAG GTATTCGATC GAAGAGGAGG
SacB linker--
--
3'LIC
~~~~~~~~~~~~~
6721
6781
BseRI
~~~~~~
CCTATTGGCA TTGACGTCAG GTGGCACTTT TCGAGGAGTT TACTAGTAAG TAAAGGTGGA
GGATAACCGT AACTGCAGTC CACCGTGAAA AGCTCCTCAA ATGATCATTC ATTTCCACCT
3'LIC
~~
BamHI
PstI
~~~~~~
~~~~~~
TACGGATCCG AATTCGAGAA TCGAATTCCC GCGGCCGCTT TCGAATCTAG AGCCTGCAGT
ATGCCTAGGC TTAAGCTCTT AGCTTAAGGG CGCCGGCGAA AGCTTAGATC TCGGACGTCA
Primers for LIC cloning:
Upstream: add TACTTCCAATCCATG to the 5’ end (ATG in-frame with the desired coding
sequence).
Downstream: add TATCCACCTTTACTG to 5’ end of downstream primer; add termination
codon, if necessary.