Basic Vector Information
- Vector Name:
- pNGWA
- Antibiotic Resistance:
- Ampicillin
- Length:
- 8593 bp
- Type:
- Expression vector
- Replication origin:
- ori
- Source/Author:
- Busso D, Delagoutte-Busso B, Moras D.
pNGWA 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 ℃.
pNGWA vector Sequence
LOCUS 40924_33177 8593 bp DNA circular SYN 18-DEC-2018
DEFINITION Expression vector pNGWA, complete sequence.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8593)
AUTHORS Busso D, Delagoutte-Busso B, Moras D.
TITLE Construction of a set Gateway-based destination vectors for
high-throughput cloning and expression screening in Escherichia coli
JOURNAL Anal. Biochem. 343 (2), 313-321 (2005)
PUBMED 15993367
REFERENCE 2 (bases 1 to 8593)
AUTHORS Busso D, Delagoutte-Busso B, Salim L, Moras D.
TITLE Direct Submission
JOURNAL Submitted (25-APR-2008) Structural Biology and Genomics Platform,
IGBMC, 1, rue Laurent Fries, Illkirch 67404, France
REFERENCE 3 (bases 1 to 8593)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 8593)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Anal.
Biochem."; date: "2005"; volume: "343"; issue: "2"; pages: "313-321"
COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(25-APR-2008) Structural Biology and Genomics Platform, IGBMC, 1,
rue Laurent Fries, Illkirch 67404, France"
COMMENT SGRef: number: 3; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..8593
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 27..45
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 46..70
/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 85..107
/label=RBS
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 123..1604
/codon_start=1
/label=NusA
/note="transcription elongation protein (N utilization
substance protein A)"
/translation="NKEILAVVEAVSNEKALPREKIFEALESALATATKKKYEQEIDVR
VQIDRKSGDFDTFRRWLVVDEVTQPTKEITLEAARYEDESLNLGDYVEDQIESVTFDRI
TTQTAKQVIVQKVREAERAMVVDQFREHEGEIITGVVKKVNRDNISLDLGNNAEAVILR
EDMLPRENFRPGDRVRGVLYSVRPEARGAQLFVTRSKPEMLIELFRIEVPEIGEEVIEI
KAAARDPGSRAKIAVKTNDKRIDPVGACVGMRGARVQAVSTELGGERIDIVLWDDNPAQ
FVINAMAPADVASIVVDEDKHTMDIAVEAGNLAQAIGRNGQNVRLASQLSGWELNVMTV
DDLQAKHQAEAHAAIDTFTKYLDIDEDFATVLVEEGFSTLEELAYVPMKELLEIEGLDE
PTVEALRERAKNALATIAQAQEESLGDNKPADDLLNLEGVDRDLAFKLAARGVCTLEDL
AEQGIDDLADIEGLTDEKAGALIMAARNICWFGDEA"
protein_bind 1623..1747
/label=attR1
/note="recombination site for the Gateway(R) LR reaction"
promoter 1772..1802
/label=lac UV5 promoter
/note="E. coli lac promoter with an 'up' mutation"
CDS 1856..2512
/codon_start=1
/label=CmR
/note="chloramphenicol acetyltransferase"
/translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
CDS 2857..3159
/codon_start=1
/label=ccdB
/note="CcdB, a bacterial toxin that poisons DNA gyrase"
/translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
protein_bind complement(3203..3327)
/label=attR2
/note="recombination site for the Gateway(R) LR reaction"
CDS 3341..3358
/codon_start=1
/label=6xHis
/note="6xHis affinity tag"
/translation="HHHHHH"
terminator 3425..3472
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
rep_origin 3509..3964
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 3991..4095
/label=AmpR promoter
CDS 4096..4953
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 5127..5715
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
misc_feature complement(5901..6043)
/label=bom
/note="basis of mobility region from pBR322"
CDS complement(6148..6336)
/codon_start=1
/label=rop
/note="Rop protein, which maintains plasmids at low copy
number"
/translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
protein_bind complement(7111..7132)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
CDS complement(7148..8227)
/codon_start=1
/label=lacI
/note="lac repressor"
/translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
ALADSLMQLARQVSRLESGQ"
promoter complement(8228..8305)
/label=lacI promoter
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