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 | 545 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
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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