Basic Vector Information
pTYB2 is an E. coli cloning and expression vector (7474 bp) used in the IMPACT™ Kit (NEB #E6901) which allows the overexpression of a target protein as a fusion to a self-cleavable affinity tag (1,2). This C-terminal fusion vector is designed for the in-frame insertion of a target gene into the polylinker upstream of an intein tag (the Sce VMA intein/chitin binding domain, 55 kDa) (1,2). This results in the fusion of the C-terminus of the target protein to the N-terminus of the intein tag. Thiol-induced self-cleavage of the intein releases the target protein from the chitin-bound intein tag, resulting in a single column purification of the target protein. For fusion of the N-terminus of the target protein to the intein tag, use pTYB21 (NEB #N6709) or pTYB22 (NEB #N6710). This vector can be used in conjuction with pTYB22 (NEB #N6710) to test which fusion constructin (N-terminal or C-terminal) maximizes the expression and yield of a target protein (3).
- Vector Name:
- pTYB2
- Antibiotic Resistance:
- Ampicillin
- Length:
- 7474 bp
- Type:
- E.coli expression plasmid
- Replication origin:
- ori
- Copy Number:
- High copy number
- Promoter:
- T7
- Cloning Method:
- Enzyme Cut
pTYB2 vector Map
pTYB2 vector Sequence
LOCUS pTYB2. 7474 bp DNA circular SYN 18-SEP-2021
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS pTYB2
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7474)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 7474)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..7474
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 35..139
/label=AmpR promoter
CDS 140..997
/label=AmpR
/note="beta-lactamase"
rep_origin complement(1042..1555)
/direction=LEFT
/label=M13 ori
/note="M13 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
rep_origin 1666..2254
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(2626..2814)
/label=rop
/note="Rop protein, which maintains plasmids at low copy
number"
protein_bind complement(3337..3358)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
CDS complement(3374..4453)
/label=lacI
/note="lac repressor"
promoter complement(4454..4531)
/label=lacI promoter
terminator 4684..4770
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 4867..4953
/gene="Escherichia coli rrnB"
/label=Escherichia coli rrnB terminator
/note="rrnB T1 terminator"
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 5050..5136
/gene="Escherichia coli rrnB"
/label=Escherichia coli rrnB terminator
/note="rrnB T1 terminator"
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 5233..5319
/gene="Escherichia coli rrnB"
/label=Escherichia coli rrnB terminator
/note="rrnB T1 terminator"
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator complement(5419..5462)
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
promoter 5637..5655
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 5656..5680
/label=lac operator
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
RBS 5695..5717
/label=RBS
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 5773..6591
/label=Sce VMA intein 5' region
/note="5' region of the intein from the yeast Vma1 subunit
of the vacuolar ATPase (Chong et al., 1998)"
CDS 6595..7128
/label=Sce VMA intein 3' region
/note="modified 3' region of the intein from the yeast Vma1
subunit of the vacuolar ATPase (Chong et al., 1998)"
CDS 7168..7323
/label=CBD
/note="chitin binding domain from chitinase A1 (Watanabe et
al., 1994)"
terminator 7402..7449
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
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