Basic Vector Information
pTWIN1 is an E. coli plasmid cloning vector designed for recombinant protein expression, labeling, and cyclization using the IMPACT-TWIN Kit (NEB #E6901) (1). It contains the pMB1 origin of replication from pBR322 and is maintained at a similar copy number to pBR322; in addition, pTWIN1 also contains an M13 origin of replication. The multiple cloning site (MCS) is positioned to allow translational fusion of an intein tag to the N-terminus, C-terminus, or both, of the cloned target protein. The mini-inteins encoded by pTWIN1, the Ssp DnaB intein and the Mxe GyrA intein, cleave the peptide bond at their C- and N-termini, respectively (1-4). The chitin binding domain (CBD) from B. circulans, fused to each intein, facilitates purification of the intein-target protein precursor. Transcription of the intein tags is controlled by the inducible T7 promoter, requiring E. coli strains containing integrated copies of the T7 RNA polymerase gene [e.g., NEB #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 (5). Translation of the intein tags utilizes the translation initiation signal (Shine Dalgarno sequence) from the strongly expressed T7 gene 10 protein (φ10).
- Vector Name:
- pTWIN1
- Antibiotic Resistance:
- Ampicillin
- Length:
- 7375 bp
- Type:
- Bacterial expression vector
- Replication origin:
- ori
- Source/Author:
- NEB
- Copy Number:
- High copy number
- Promoter:
- T7
- Cloning Method:
- Enzyme Cut
- Fusion Tag:
- intein
- Expression Method:
- IPTG induced
pTWIN1 vector Vector Map
Plasmid Resuspension Protocol:
1. Centrifuge at 5,000×g for 5 min.
2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.
3. Close the tube and incubate for 10 minutes at room temperature.
4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.
5.Store the plasmid at -20 ℃.
pTWIN1 vector Sequence
LOCUS pTWIN1. 7375 bp DNA circular SYN 09-DEC-2020
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS pTWIN1.
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7375)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 7375)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..7375
/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 5749..5901
/label=CBD
/note="chitin binding domain from chitinase A1 (Watanabe et
al., 1994)"
CDS 5941..6402
/codon_start=1
/label=Ssp DnaB intein
/note="Ssp DnaB intein"
/translation="AISGDSLISLASTGKRVSIKDLLDEKDFEIWAINEQTMKLESAKV
SRVFCTGKKLVYILKTRLGRTIKATANHRFLTIDGWKRLDELSLKEHIALPRKLESSSL
QLSPEIEKLSQSDIYWDSIVSITETGVEEVFDLTVPGPHNFVANDIIVHN"
CDS 6445..7038
/label=Mxe GyrA intein
/note="modified GyrA intein (Southworth et al., 1999)"
CDS 7069..7224
/codon_start=1
/gene="Bacillus circulans chiA"
/product="chitin binding domain from chitinase A1 (Watanabe
et al., 1994)"
/label=Bacillus circulans chiA
/note="CBD"
/translation="TTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNV
PALWQLQ"
terminator 7303..7350
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
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