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.
| Basic Vector Information | |||
|---|---|---|---|
| Vector Name | pSecTag2/Hygro A | Antibiotic Resistance | Ampicillin |
| Length | 5745 bp | Type | Mammalian Expression Vectors |
| Source | Thermo Fisher Scientific | Selection Marker | Hygromycin |
| Copy Number | High copy number | Promoter | CMV |
| Cloning Method | Enzyme Cut | 5' Primer | TAATACGACTCACTATAGGG |
| Fusion Tag | His Tag (6x), c-Myc Epitope Tag, IgK Leader Sequence | ||
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 Exported 5745 bp ds-DNA circular SYN 13-DEC-2021
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS Untitled
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5745)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..5745
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 235..614
/note="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 615..818
/note="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
promoter 863..881
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 905..967
/codon_start=1
/product="leader sequence from mouse immunoglobulin kappa
light chain"
/note="Ig-kappa leader"
/translation="METDTLLLWVLLLWVPGSTGD"
CDS 1082..1111
/codon_start=1
/product="Myc (human c-Myc oncogene) epitope tag"
/note="Myc"
/translation="EQKLISEEDL"
CDS 1127..1144
/codon_start=1
/product="6xHis affinity tag"
/note="6xHis"
/translation="HHHHHH"
polyA_signal 1173..1397
/note="bGH poly(A) signal"
/note="bovine growth hormone polyadenylation signal"
rep_origin 1443..1871
/direction=RIGHT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 1885..2214
/note="SV40 promoter"
/note="SV40 enhancer and early promoter"
rep_origin 2065..2200
/note="SV40 ori"
/note="SV40 origin of replication"
CDS 2263..3288
/codon_start=1
/gene="aph(4)-Ia"
/product="aminoglycoside phosphotransferase from E. coli"
/note="HygR"
/note="confers resistance to hygromycin"
/translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
E"
polyA_signal 3418..3539
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
primer_bind complement(3588..3604)
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 3612..3628
/bound_moiety="lac repressor encoded by lacI"
/note="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)
/note="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 3681..3702
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(3990..4578)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(4749..5609)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(5610..5714)
/gene="bla"
/note="AmpR promoter"
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