Basic Vector Information
pSecTag2 and pSecTag2/Hygro are mammalian expression vectors designed for the secretion, purification, and detection of fusion proteins. Each vector has a large multiple cloning site in three reading frames to simplify cloning in frame with the N-terminal secretion signal. The vectors (Figure 1) offer the following features:1.Secretion signal from the V-J2-C region of the mouse Ig kappa-chain for efficient secretion of recombinant proteins; 2. Cytomegalovirus (CMV) promoter for high-level constitutive expression; 3.C-terminal polyhistidine (6xHis) tag for rapid purification with nickel-chelating resin and detection with an Anti-His(C-term) Antibody; 4. C-terminal c-myc epitope for detection with an Anti-myc Antibody; 5. Bovine Growth Hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability; 5. SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen (e.g., COS-1, COS-7). The pSecTag2 vectors carry the Zeocin™ resistance gene for cost-effective selection in mammalian cells. Zeocin™ selection can also be used in E. coli.The pSecTag2/Hygro vectors have the Hygromycin B resistance gene for selection of stable mammalian cell lines.
pSecTag2/Hygro A 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 ℃.
pSecTag2/Hygro A vector Sequence
LOCUS 40924_39143 5745 bp DNA circular SYN 13-DEC-2021 DEFINITION synthetic circular DNA. ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5745) TITLE Direct Submission REFERENCE 2 (bases 1 to 5745) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article" FEATURES Location/Qualifiers source 1..5745 /mol_type="other DNA" /organism="synthetic DNA construct" enhancer 235..614 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 615..818 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" promoter 863..881 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" sig_peptide 905..967 /label=Ig-kappa leader /note="leader sequence from mouse immunoglobulin kappa light chain" CDS 1082..1111 /codon_start=1 /label=Myc /note="Myc (human c-Myc proto-oncogene) epitope tag" /translation="EQKLISEEDL" CDS 1127..1144 /codon_start=1 /label=6xHis /note="6xHis affinity tag" /translation="HHHHHH" polyA_signal 1173..1397 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 1443..1871 /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" promoter 1885..2214 /label=SV40 promoter /note="SV40 enhancer and early promoter" CDS 2263..3285 /codon_start=1 /label=HygR /note="aminoglycoside phosphotransferase from E. coli" /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK E" polyA_signal 3418..3551 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3588..3604) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" protein_bind complement(3612..3628) /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)." promoter complement(3636..3666) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind complement(3681..3702) /label=CAP binding site /note="CAP binding activates transcription in the presence of cAMP." rep_origin complement(3990..4578) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(4752..5609) /codon_start=1 /label=AmpR /note="beta-lactamase" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" promoter complement(5610..5714) /label=AmpR promoter
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