Basic Vector Information
pTWIN2 is an E. coli expression vector which can be used with the IMPACT™ Kit. A polylinker in the vector is designed for the in-frame fusion of a target gene between the modified Ssp DnaB and Mth RIR1 inteins. The presence of the chitin binding domain from Bacillus circulans facilitates purification. pTWIN vectors are designed for protein purification or for the isolation of proteins with an N-terminal cysteine and/or a C-terminal thioester. The double-stranded vector is 7192 base pairs in length.
pTWIN2 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 ℃.
pTWIN2 vector Sequence
LOCUS pTWIN2. 7192 bp DNA circular SYN 09-DEC-2020
DEFINITION Cloning vector pTWIN2, complete sequence.
ACCESSION .
VERSION .
KEYWORDS pTWIN2.
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7192)
AUTHORS Evans TC Jr., Benner J, Xu M-Q.
TITLE The in vitro ligation of bacterially expressed protein using an
intein from Methanobacterium thermoautotrophicum
JOURNAL J Biol Chem 274, 3923-3926 (1999)
PUBMED 9933578
REFERENCE 2 (bases 1 to 7192)
AUTHORS Mathys S, Evans TC Jr., Chute IC, Wu H, Chong S, Benner J, Liu X-Q.,
Xu M-Q.
TITLE Characterization of a self-splicing mini-intein and its conversion
into autocatalytic N- and C-terminal cleavage elements: facile
production of protein building blocks for protein ligation
JOURNAL Gene 231, 1-13 (1999)
PUBMED 10231563
REFERENCE 3 (bases 1 to 7192)
AUTHORS Evans TC Jr., Benner J, Xu M-Q.
TITLE The cyclization and polymerization of bacterially-expressed proteins
using modified self-splicing inteins
JOURNAL J Biol Chem 274, 18359-18363 (1999)
PUBMED 10373440
REFERENCE 4 (bases 1 to 7192)
AUTHORS Perler FB.
TITLE InBase, the intein database
JOURNAL Nucleic Acids Res 30 (1), 383-384 (2002)
PUBMED 11752343
REFERENCE 5 (bases 1 to 7192)
AUTHORS Ghosh I, Xu MQ.
TITLE Direct Submission
JOURNAL Submitted (23-OCT-2007) Research Department, New England Biolabs,
240 County Road, Ipswich, MA 01938, USA
REFERENCE 6 (bases 1 to 7192)
TITLE Direct Submission
REFERENCE 7 (bases 1 to 7192)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "J Biol Chem
274, 3923-3926 (1999)"
COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Gene 231,
1-13 (1999)"
COMMENT SGRef: number: 3; type: "Journal Article"; journalName: "J Biol Chem
274, 18359-18363 (1999)"
COMMENT SGRef: number: 4; type: "Journal Article"; journalName: "Nucleic
Acids Res"; date: "2002"; volume: "30"; issue: "1"; pages: "383-384"
COMMENT SGRef: number: 5; type: "Journal Article"; journalName: "Submitted
(23-OCT-2007) Research Department, New England Biolabs, 240 County
Road, Ipswich, MA 01938, USA"
COMMENT SGRef: number: 6; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..7192
/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 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)"
primer_bind 6296..6315
/note="Ssp DnaB intein forward primer sequence (NEB
#S1269S)"
misc_feature 6403..6444
/gene="expression CDS"
/label=multiple cloning site (SapI-SapI)
/note="multiple cloning site (SapI-SapI)"
primer_bind complement(6505..6522)
/note="Mth RIR1 intein reverse primer sequence (NEB
#S1270S)"
terminator 7120..7167
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
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