pComb3X SS
Catalog No. PVT10572
Packing 2ug
pComb3X SS Description
pComb3X is the newest of the pComb vectors. Improvements over pComb3 include increased stability and introduction of an asymmetric SfiI cassette for directional cloning of full Fab, scFv, peptide and other protein for phage display. 6xHis and HA tags allow for purification and detection. An amber stop codon was introduced to turn-off expression of the pIII fusion protein by switching to a non-supressor strain of E. coli allowing production of soluble protein without subcloning. Alternatively, the gene for phage protein pIII can be removed by SpeI/NheI digest. pComb3XSS is recommended for preparation of vector for library cloning. The “SS” refers to the double stuffer, a 1200bp stuffer in the Fab light chain cloning region bounded by SacI and XbaI restriction sites and a 300bp stuffer in Fab heavy chain cloning region bound by XhoI and SpeI restriction sites. Also, the 1600bp double stuffer (both stuffers plus the leader sequence between the Fab light chain and heavy chain cloning regions) can be removed by SfiI digest so that non-Fab genes of interest can be cloned. Also available on Addgene: pComb3XTT and pComb3XLambda are only needed at templates for the construction of chimeric Fab libraries as described in Phage Display: A Laboratory Manual. pComb3XTT can also be used as an Fab expression control. 3rd generation plasmid for phage display on modified geneIII, contains stuffer fragment.
Promoter: Lac/lac promoter
Plasmid size: 4991bp
Plasmid Label: C-6*HIS, C-HA
Prokaryotic resistance: ampicillin Amp
Cloning strain: Escherichia coli Stbl3
Culture conditions: 37鈩? aerobic, LB
pComb3X SS Sequence
LOCUS Exported 4991 bp ds-DNA circular SYN 27-OCT-2017
DEFINITION synthetic circular DNA
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4991)
FEATURES Location/Qualifiers
source 1..4991
/organism="synthetic DNA construct"
/mol_type="other DNA"
protein_bind 109..130
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 145..175
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 183..199
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/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)."
misc_feature 294..1470
/label=Light Chain
CDS complement(574..591)
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
misc_feature 1500..1577
/label=PelB Leader
CDS 1584..1907
/codon_start=1
/gene="trxA"
/product="E. coli thioredoxin"
/label=Heavy Chain
/translation="MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDE
IADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFL
DANL"
CDS 1962..1979
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
CDS 1986..2012
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/label=HA
/translation="YPYDVPDYA"
CDS 2118..2552
/codon_start=1
/label=geneIII
/translation="MANANKGAMTENADENVLQSDAKGKLDSVATDYGAAIDGFIGDVS
GLANGNGATGDFAGSNSQMAQVGDGDNSPLMNNFRQYLPSLPQSVECRPFVFGAGKPYE
FSIDCDKINLFRGVFAFLLYVATFMYVFSTFANILRNKES"
rep_origin complement(2606..3061)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 3088..3192
/gene="bla"
/label=AmpR promoter
CDS 3193..4053
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 4224..4812
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
ORIGIN
1 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
61 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
121 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
181 aattgtgagc ggataacaat tgaattcagg aggaatttaa aatgaaaaag acagctatcg
241 cgattgcagt ggcactggct ggtttcgcta ccgtggccca ggcggccgag ctcgccatgg
301 ctggttgggc agcgagtaat aacaatccag cggctgccgt aggcaatagg tatttcatta
361 tgactgtctc cttggcgact agctagttta gaattcgtaa tcatggtcat agctgtttcc
421 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
481 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
541 cgctttccag tcgggaaacc tgtcgtgtta ctaatgatgg tgatggtgat ggctagtttt
601 gtcacaagat ttgggctcaa ctttcttgtc caccttggtg ttgctgggct tgtgattcac
661 gttgcagatg taggtctggg tgcccaagct gctggagggc acggtcacca cgctgctgag
721 ggagtagagt cctgaggact gtaggacagc cgggaaggtg tgcacgccgc tggtcagggc
781 gcctgagttc cacgacaccg tcgccggttc ggggaagtag tccttgacca ggcagcccag
841 ggccgctgtg cccccagagg tgctcttgga ggagggtgcc agggggaaga ccgatgggcc
901 cttggtggag gctgcggaga cggtgaccgt ggtaccagca gaaacctggc caggctccca
961 ggctcctcat ctatggtaca tccagcaggg ccactggcat cccagacagg ttcagtggca
1021 gtgggtctgg gacagacttc actctcacca tcagcagact ggagcctgaa gattttgcag
文献和实验
技术资料