Basic Vector Information
- Vector Name:
- pPKm-195
- Antibiotic Resistance:
- Ampicillin
- Length:
- 7477 bp
- Type:
- Cloning vector
- Replication origin:
- ori
- Source/Author:
- Kyriakakis P, Catanho M, Hoffner N, Thavarajah W, Jian-Yu V, Chao SS, Hsu A, Pham V, Naghavian L, Dozier LE, Patrick G, Coleman T.
pPKm-195 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 ℃.
pPKm-195 vector Sequence
LOCUS 40924_34774 7477 bp DNA circular SYN 18-DEC-2018 DEFINITION Cloning vector pPKm-195, complete sequence. ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7477) AUTHORS Kyriakakis P, Catanho M, Hoffner N, Thavarajah W, Jian-Yu V, Chao SS, Hsu A, Pham V, Naghavian L, Dozier LE, Patrick G, Coleman T. TITLE Biosynthesis of Orthogonal Molecules Using Ferredoxin and Ferredoxin-NADP+ Reductase Systems Enables Genetically Encoded PhyB Optogenetics JOURNAL ACS Synth Biol (2018) In press PUBMED 29301067 REFERENCE 2 (bases 1 to 7477) AUTHORS Kyriakakis P. TITLE Direct Submission JOURNAL Submitted (15-NOV-2017) Bioengineering, UCSD, 9500 Gilman drive, PFBH room 251 MC0412, La Jolla, CA 92093, United States of America REFERENCE 3 (bases 1 to 7477) TITLE Direct Submission REFERENCE 4 (bases 1 to 7477) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "ACS Synth Biol (2018) In press" COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted (15-NOV-2017) Bioengineering, UCSD, 9500 Gilman drive, PFBH room 251 MC0412, La Jolla, CA 92093, United States of America" COMMENT SGRef: number: 3; type: "Journal Article" FEATURES Location/Qualifiers source 1..7477 /mol_type="other DNA" /organism="synthetic DNA construct" enhancer 27..406 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 407..610 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" promoter 655..673 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" misc_feature 693..2555 /label=PhyB /note="PhyB" misc_feature 2562..2737 /label=MTAD /note="MTAD" CDS 2742..2753 /codon_start=1 /label=Factor Xa site /note="Factor Xa recognition and cleavage site" /translation="IEGR" CDS 2773..2793 /codon_start=1 /label=SV40 NLS /note="nuclear localization signal of SV40 (simian virus 40) large T antigen" /translation="PKKKRKV" promoter complement(2821..2839) /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" polyA_signal 2865..3089 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 3135..3563 /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" promoter 3577..3907 /label=SV40 promoter /note="SV40 enhancer and early promoter" CDS 3974..4765 /codon_start=1 /label=NeoR/KanR /note="aminoglycoside phosphotransferase" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" polyA_signal 4942..5075 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(5112..5128) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" protein_bind complement(5136..5152) /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(5160..5190) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind complement(5205..5226) /label=CAP binding site /note="CAP binding activates transcription in the presence of cAMP." rep_origin complement(5514..6102) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(6276..7133) /codon_start=1 /label=AmpR /note="beta-lactamase" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" promoter complement(7134..7238) /label=AmpR promoter
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