Basic Vector Information
pTYB4 is an E. coli expression vector (7,474 bp) used in the IMPACT™ Kit (NEB #E6901) (1). This C-terminal fusion vector is designed for the in-frame insertion of a target gene into a polylinker upstream of an intein tag (the Sce VMA intein/chitin binding domain, 55 kDa). 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.
pTYB4 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 ℃.
pTYB4 vector Sequence
LOCUS pTYB4. 7474 bp DNA circular SYN 18-SEP-2021 DEFINITION synthetic circular DNA. ACCESSION . VERSION . KEYWORDS pTYB4. 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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