pTYB21

Bacterial expression vector that encodes a thiol-cleavable N-terminal intein with a chitin-binding domain.

Sequence Author: New England Biolabs

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SpeI (7372) PstI - SbfI (7359) EcoRI (7348) BamHI (7342) SalI - SgrDI (7336) EcoRV (7332) NotI (7323) NcoI (7316) NdeI (7310) SacI (7308) Eco53kI (7306) BspQI - SapI (7296) SacII (7188) PmlI (7177) StuI (7152) BstBI (7123) Sce VMA intein 3' region MscI (6752) MfeI (6646) BmtI (6593) NheI (6589) BglII (6255) HindIII (6224) Sce VMA intein 5' region KpnI (5786) Acc65I (5782) RBS XbaI (5683) lac operator T7 promoter PmeI (5609) rrnB T1 terminator rrnB T1 terminator rrnB T1 terminator rrnB T1 terminator rrnB T1 terminator EcoNI (4566) lacI promoter MluI (4098) BstEII (3916) BlpI (7431) T7 terminator BspEI (7508) PvuI (558) FspI (704) BsaI (861) SwaI (1099) PsiI (1196) AvaI - BsoBI (1213) DraIII (1324) PciI (2310) BstZ17I (2482) AfeI (3196) KasI (3459) NarI * (3460) SfoI (3461) PluTI (3463) BssHII (3687) PspOMI (3891) ApaI (3895) pTYB21 7514 bp
SpeI  (7372)
1 site
A C T A G T T G A T C A
PstI  (7359)
1 site
C T G C A G G A C G T C
SbfI  (7359)
1 site
C C T G C A G G G G A C G T C C
EcoRI  (7348)
1 site
G A A T T C C T T A A G
BamHI  (7342)
1 site
G G A T C C C C T A G G

After cleavage, BamHI-HF® (but not the original BamHI) can remain bound to DNA and alter its electrophoretic mobility.
SalI  (7336)
1 site
G T C G A C C A G C T G
SgrDI  (7336)
1 site
C G T C G A C G G C A G C T G C
EcoRV  (7332)
1 site
G A T A T C C T A T A G

EcoRV is reportedly more prone than its isoschizomer Eco32I to delete a base after cleavage.
NotI  (7323)
1 site
G C G G C C G C C G C C G G C G
NcoI  (7316)
1 site
C C A T G G G G T A C C
NdeI  (7310)
1 site
C A T A T G G T A T A C

Prolonged incubation with NdeI may lead to removal of additional nucleotides.
SacI  (7308)
1 site
G A G C T C C T C G A G
Eco53kI  (7306)
1 site
G A G C T C C T C G A G
BspQI  (7296)
1 site
G C T C T T C N C G A G A A G N N N N

Sticky ends from different BspQI sites may not be compatible.
SapI  (7296)
1 site
G C T C T T C N C G A G A A G N N N N

Sticky ends from different SapI sites may not be compatible.
SapI gradually settles in solution, so a tube of SapI should be mixed before removing an aliquot.
SacII  (7188)
1 site
C C G C G G G G C G C C

Efficient cleavage requires at least two copies of the SacII recognition sequence.
PmlI  (7177)
1 site
C A C G T G G T G C A C
StuI  (7152)
1 site
A G G C C T T C C G G A
BstBI  (7123)
1 site
T T C G A A A A G C T T
MscI  (6752)
1 site
T G G C C A A C C G G T
MfeI  (6646)
1 site
C A A T T G G T T A A C
BmtI  (6593)
1 site
G C T A G C C G A T C G
NheI  (6589)
1 site
G C T A G C C G A T C G
BglII  (6255)
1 site
A G A T C T T C T A G A
HindIII  (6224)
1 site
A A G C T T T T C G A A
KpnI  (5786)
1 site
G G T A C C C C A T G G
Acc65I  (5782)
1 site
G G T A C C C C A T G G
XbaI  (5683)
1 site
T C T A G A A G A T C T
PmeI  (5609)
1 site
G T T T A A A C C A A A T T T G
EcoNI  (4566)
1 site
C C T N N N N N A G G G G A N N N N N T C C

The 1-base overhangs produced by EcoNI may be hard to ligate.
Sticky ends from different EcoNI sites may not be compatible.
MluI  (4098)
1 site
A C G C G T T G C G C A
BstEII  (3916)
1 site
G G T N A C C C C A N T G G

Sticky ends from different BstEII sites may not be compatible.
BstEII is typically used at 60°C, but is 50% active at 37°C.
BlpI  (7431)
1 site
G C T N A G C C G A N T C G

Sticky ends from different BlpI sites may not be compatible.
BspEI  (7508)
1 site
T C C G G A A G G C C T
PvuI  (558)
1 site
C G A T C G G C T A G C
FspI  (704)
1 site
T G C G C A A C G C G T
BsaI  (861)
1 site
G G T C T C N C C A G A G N ( N ) 4

Sticky ends from different BsaI sites may not be compatible.
BsaI can be used between 37°C and 50°C.
SwaI  (1099)
1 site
A T T T A A A T T A A A T T T A

SwaI is typically used at 25°C, but is 50% active at 37°C.
PsiI  (1196)
1 site
T T A T A A A A T A T T
AvaI  (1213)
1 site
C Y C G R G G R G C Y C

Sticky ends from different AvaI sites may not be compatible.
BsoBI  (1213)
1 site
C Y C G R G G R G C Y C

Sticky ends from different BsoBI sites may not be compatible.
BsoBI is typically used at 37°C, but can be used at temperatures up to 65°C.
DraIII  (1324)
1 site
C A C N N N G T G G T G N N N C A C

Sticky ends from different DraIII sites may not be compatible.
PciI  (2310)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BstZ17I  (2482)
1 site
G T A T A C C A T A T G
AfeI  (3196)
1 site
A G C G C T T C G C G A
KasI  (3459)
1 site
G G C G C C C C G C G G
NarI  (3460)
1 site
G G C G C C C C G C G G
* Blocked by Dcm methylation.
Efficient cleavage requires at least two copies of the NarI recognition sequence.
SfoI  (3461)
1 site
G G C G C C C C G C G G
PluTI  (3463)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the PluTI recognition sequence.
BssHII  (3687)
1 site
G C G C G C C G C G C G

BssHII is typically used at 50°C, but is 75% active at 37°C.
PspOMI  (3891)
1 site
G G G C C C C C C G G G
ApaI  (3895)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
MBP peptide
5725 .. 5754  =  30 bp
10 amino acids  =  1.2 kDa
Product: N-terminal peptide from mature maltose-binding protein
MBP peptide
5725 .. 5754  =  30 bp
10 amino acids  =  1.2 kDa
Product: N-terminal peptide from mature maltose-binding protein
Sce VMA intein 5' region
5770 .. 6588  =  819 bp
273 amino acids  =  31.2 kDa
Product: 5' region of the intein from the yeast Vma1 subunit of the vacuolar ATPase (Chong et al., 1998)
Sce VMA intein 5' region
5770 .. 6588  =  819 bp
273 amino acids  =  31.2 kDa
Product: 5' region of the intein from the yeast Vma1 subunit of the vacuolar ATPase (Chong et al., 1998)
CBD
6595 .. 6747  =  153 bp
51 amino acids  =  5.6 kDa
Product: chitin binding domain from chitinase A1 (Watanabe et al., 1994)
CBD
6595 .. 6747  =  153 bp
51 amino acids  =  5.6 kDa
Product: chitin binding domain from chitinase A1 (Watanabe et al., 1994)
Sce VMA intein 3' region
6751 .. 7299  =  549 bp
183 amino acids  =  19.9 kDa
Product: modified 3' region of the intein from the yeast Vma1 subunit of the vacuolar ATPase (Chong et al., 1998)
C-terminal cleavage of this intein can be induced by thiols.
Sce VMA intein 3' region
6751 .. 7299  =  549 bp
183 amino acids  =  19.9 kDa
Product: modified 3' region of the intein from the yeast Vma1 subunit of the vacuolar ATPase (Chong et al., 1998)
C-terminal cleavage of this intein can be induced by thiols.
lacI
3371 .. 4453  =  1083 bp
360 amino acids  =  38.6 kDa
Product: lac repressor
The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-β-D-thiogalactopyranoside (IPTG).
lacI
3371 .. 4453  =  1083 bp
360 amino acids  =  38.6 kDa
Product: lac repressor
The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-β-D-thiogalactopyranoside (IPTG).
AmpR
140 .. 1000  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 1:  signal sequence  
   140 .. 208  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
140 .. 1000  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 2:  
   209 .. 1000  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
140 .. 1000  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
1666 .. 2254  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
1666 .. 2254  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
M13 ori
1042 .. 1555  =  514 bp
M13 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
M13 ori
1042 .. 1555  =  514 bp
M13 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
rop
2623 .. 2814  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
rop
2623 .. 2814  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
AmpR promoter
35 .. 139  =  105 bp
AmpR promoter
35 .. 139  =  105 bp
rrnB T1 terminator
4684 .. 4770  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
rrnB T1 terminator
4684 .. 4770  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
rrnB T1 terminator
4867 .. 4953  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
rrnB T1 terminator
4867 .. 4953  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
rrnB T1 terminator
5050 .. 5136  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
rrnB T1 terminator
5050 .. 5136  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
rrnB T1 terminator
5233 .. 5319  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
rrnB T1 terminator
5233 .. 5319  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
rrnB T1 terminator
5416 .. 5502  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
rrnB T1 terminator
5416 .. 5502  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
lacI promoter
4454 .. 4531  =  78 bp
lacI promoter
4454 .. 4531  =  78 bp
MCS
7300 .. 7377  =  78 bp
multiple cloning site
MCS
7300 .. 7377  =  78 bp
multiple cloning site
T7 terminator
7442 .. 7489  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 terminator
7442 .. 7489  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
lac operator
5656 .. 5680  =  25 bp
The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-β-D-thiogalactopyranoside (IPTG).
lac operator
5656 .. 5680  =  25 bp
The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-β-D-thiogalactopyranoside (IPTG).
RBS
5695 .. 5717  =  23 bp
efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)
RBS
5695 .. 5717  =  23 bp
efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)
T7 promoter
5637 .. 5655  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
5637 .. 5655  =  19 bp
promoter for bacteriophage T7 RNA polymerase
ORF:  2953 .. 3588  =  636 bp
ORF:  211 amino acids  =  23.1 kDa
ORF:  4492 .. 4839  =  348 bp
ORF:  115 amino acids  =  12.9 kDa
ORF:  5725 .. 7368  =  1644 bp
ORF:  547 amino acids  =  60.7 kDa
ORF:  140 .. 1000  =  861 bp
ORF:  286 amino acids  =  31.5 kDa
ORF:  3351 .. 3614  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  4203 .. 4568  =  366 bp
ORF:  121 amino acids  =  13.1 kDa
ORF:  3371 .. 4330  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
ORF:  604 .. 870  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  2623 .. 2847  =  225 bp
ORF:  74 amino acids  =  8.5 kDa
ORF:  2989 .. 3315  =  327 bp
ORF:  108 amino acids  =  11.8 kDa
ORF:  4654 .. 5415  =  762 bp
ORF:  253 amino acids  =  28.4 kDa
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