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Restriction Maps
TECHNICAL SUPPORT
pTYB1
pTYB1 is an E. coli plasmid cloning vector designed for
recombinant protein expression and purification using the
IMPACT™ Kit (NEB #E6901) (1,2). It contains the pMB1 origin
of replication from pBR322 and is maintained at a similar copy
number to pBR322; in addition, pTYB1 also contains an M13
origin of replication.
The multiple cloning site (MCS) is positioned to allow
translational fusion of the Sce VMA intein tag to the C-terminus
of the cloned target protein (1). The chitin binding domain
(CBD) from B. circulans, fused to the C-terminus of the intein,
facilitates purification of the intein-target protein precursor.
overlapping the initiating methionine codon of the intein fusion
gene. The N-terminal cysteine residue (“Cys1”) of the intein is
shaded.
Sequence file available at www.neb.com
See page 164 for ordering information.
Enzymes with unique restriction sites are shown in bold type.
Location of sites of all NEB restriction enzymes can be found on
the NEB web site (choose Technical Reference > DNA Sequences
and Maps). Restriction site coordinates refer to the position of the
5´-most base on the top strand in each recognition sequence.
Feature
bla (ApR)
M13 origin
origin
rop
lacI
T7 promoter
expression ORF
MCS
Sce VMA intein
CBD
Open reading frame (ORF) coordinates are in the form
"translational start – translational stop”; numbers refer to
positions on the top (clockwise) strand, regardless of the direction
of transcription and include the start and stop codons. Component
genes or regions of fusion ORFs are indented below the ORF itself.
Transcription of the gene fusion is controlled by the inducible
T7 promoter, requiring E. coli strains containing integrated
copies of the T7 RNA polymerase gene [e.g., C2566, C2833
or BL21(DE3)] for expression. Basal expression from the T7
promoter is minimized by the binding of the Lac repressor,
encoded by the lacI gene, to the lac operator immediately
downstream of the T7 promoter (3). Translation of the fusion
utilizes the translation initiation signal (Shine Dalgarno
sequence) from the strongly expressed T7 gene 10 protein (φ10).
pMB1 origin of replication coordinates include the region from the
-35 promoter sequence of the RNAII transcript to the RNA/DNA
switch point. For the M13 origin, the arrow shows the direction
of synthesis of the (+) strand, which gets packaged into phage
particles. bla (ApR) gene coordinates include the signal sequence.
Coordinates
140-1000
1042-1555
1666-2254
2814-2623
4453-3371
5637-5654
5725-7329
5722-5775
5776-7137
7171-7329
Source
Tn3
M13
pMB1
pMB1
E. coli
T7
–
–
S. cerevisiae
B. circulans
ori = origin of replication
Ap = ampicillin
There are no restriction sites for the following
enzymes: AarI(x), AatII, AflII, AscI, AsiSI, AvrII,
BbvCI, BmgBI, BseRI, BsiWI, BsmI, BspDI,
Bsu36I, ClaI, CspCI, FseI, FspAI(x), I-CeuI,
I-SceI, MscI, NcoI, PI-PspI, PI-SceI, PacI,
PpuMI, RsrII, SacI, SanDI(x), SbfI, SexAI,
SfiI, SgrAI, SmaI, SnaBI, SpeI, SrfI(x), TspMI,
XmaI, ZraI
pTYB1, pTYB2, pTYB3, and pTYB4 are identical except for the
MCS regions (opposite page). All four vectors contain either
an NdeI (pTYB1 and pTYB2) or NcoI (pTYB3 and pTYB4) site
Sce
Sce
pTYB1
ori
7,477 bp
XbaI 5683
PmeI 5606
PciI 2310
PciI 2310
ro
p
ro
p
BstZ17I 2480
BstZ17I 2480
lacI
EcoNI 4562
EcoNI 4562
MluI 4098
10
ori
PaeR7I -MCS
TliI - XhoI 5761
MCS
PspXI 5760
EcoRI 5755
NotI 5747
XbaI 5683
SalI 5740
NruI 5734
PmeI 5606
BmtI - NheI 5728
NdeI 5722
lacI
APPENDIX
PspXI 5760
EcoRI 5755
NotI 5747
SalI 5740
NruI 5734
BmtI - NheI 5728
NdeI 5722
ori
ori
13
13
–M
–M
int
e
int
e
PstI 7330
PstI 7330
MfeI 7225
MfeI 7225
AgeI 7150
AgeI BlpI
7150 7393
BlpI 7393
NsiI 7133
NsiI 7133 BspEI 7471
BspEI 7471
(x) = enzyme not available from NEB
BtgI - SacII 7023
BtgI - SacII 7023
PvuI
555
PmlI 7013
PvuI 555
PmlI 7013
FspI 702
BstBI 6960
FspI 702
BstBI 6960
CBD
BsaI 855
D
BsaI 855
ACB
R
p
Ap R
BglII 6261
BglII 6261
SwaI 1096
SwaI 1096
n
HindIII 6230
i
HindIII 6230
in
PsiI 1194
PsiI 1194
BsrGI 5915
BsrGI 5915
Acc65I - KpnI 5788
DraIII 1319
Acc65I - KpnI 5788
DraIII 1319
BaeI 5788
BaeI 5788
BspQI - SapI 5768
+
BspQI - SapI 5768
+
PaeR7I - TliI - XhoI 5761
AfeI 3194
KasI - NarI - SfoI 3459
BstEII 3916 MluI 4098
AfeI 3194
ApaI - PspOMI 3891
EcoRV
KasI - NarI - SfoI 3459
BstEII
39163650
BssHII 3687
ApaI - PspOMI 3891
EcoRV 3650
BssHII 3687
T7 promoter
lac operator
Shine Dalgarno
T7 promoter
XbaI
...CGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATA
lac operator
Shine Dalgarno
XbaI
.
.
.
.
.
.
.
T7
Universal Primer
...CGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATA
568O
57OO
572O
.
.
.
.
.
.
.
T7 Universal Primer
568O SapI
57OO
572O
NdeI NheI NruI SalI
NotI
EcoRI XhoI
pTYB1 MCS
pTYB2 MCS
CATATGGCTAGCTCGCGAGTCGACGGCGGCCGCGAATTCCTCGAGGGCTCTTCC TGC...
M A S S NdeI
R V NheI
D G NruI
G R SalI
E F LNotI
E G EcoRI
S S XhoI
C
SapI
pTYB1 MCS
CATATGGCTAGCTCGCGAGTCGACGGCGGCCGCGAATTCCTCGAGGGCTCTTCC TGC...
NdeI
NheI NruI
M A SalI
S S NotI
R V EcoRI
D G XhoI
G R SmaI
E F L E G S S
C
CATATGGCTAGCTCGCGAGTCGACGGCGGCCGCGAATTCCTCGAGCCCGGG TGC...
M A S SNdeI
R V NheI
D G NruI
G R ESalI
F L NotI
E P EcoRI
G
C XhoI SmaI
pTYB2 MCS
pTYB3 MCS
pTYB4 MCS
CATATGGCTAGCTCGCGAGTCGACGGCGGCCGCGAATTCCTCGAGCCCGGG TGC...
NcoI NheI NruI SalI
M A NotI
S S EcoRI
R V DXhoI
G GSapI
R E F L E P G
C
ACCATGGCTAGCTCGCGAGTCGACGGCGGCCGCGAATTCCTCGAGGGCTCTTCC TGC...
M A S S R V D G G R E F L E G S S
C
NcoI NheI NruI SalI
NotI
EcoRI XhoI
SapI
ACCATGGCTAGCTCGCGAGTCGACGGCGGCCGCGAATTCCTCGAGGGCTCTTCC TGC...
pTYB3 MCS
NcoI NheI NruI SalI
NotI
EcoRI XhoI SmaI
M A S S R V D G G R E F TGC...
L E G S S
C
References
ACCATGGCTAGCTCGCGAGTCGACGGCGGCCGCGAATTCCTCGAGCCCGGG
R V D G G R E F L E P G
C
(1)Chong et al. (1996) J. Biol. Chem., 271,
NcoI NheI NruI SalI
NotI
EcoRI XhoI SmaI
22159–22168
ACCATGGCTAGCTCGCGAGTCGACGGCGGCCGCGAATTCCTCGAGCCCGGG
TGC...
Sce VMA intein
(2)Chong et al. (1997) Gene, 192, 277–281.
M A S S R V D G G R E F L E P G
C
KpnI
(3)Dubendorff, J.W. and Studier, F.W. (1991)
TTTGCCAAGGGTACCAATGTTTTAATGGCGGATGGGTCTATTGAATGTATTGAAAACATTGAGGTTGGTAATAAGGTCATGGGT...
M
A
S
S
pTYB4 MCS
Intein Reverse Sequencing Primer
Sce VMABiol.,
intein
J. Mol.
219, 45–59.
KpnI
TTTGCCAAGGGTACCAATGTTTTAATGGCGGATGGGTCTATTGAATGTATTGAAAACATTGAGGTTGGTAATAAGGTCATGGGT...
Intein Reverse Sequencing Primer
357