Basic Vector Information
- Vector Name:
- pPKm-227
- Antibiotic Resistance:
- Ampicillin
- Length:
- 8600 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.
- Promoter:
- CMV
pPKm-227 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-227 vector Sequence
LOCUS V004067 8600 bp DNA circular SYN 18-DEC-2018
DEFINITION Exported.
ACCESSION V004067
VERSION V004067
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 8600)
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 8600)
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 8600)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 8600)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "ACS Synth
Biol (2018) In press"
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"
SGRef: number: 3; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..8600
/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"
CDS 732..881
/label="VP64"
/note="tetrameric repeat of the minimal activation domain
of herpes simplex virus VP16 (Beerli et al., 1998)"
misc_feature 888..905
/label="linker 1"
/note="linker 1"
CDS 906..926
/label="SV40 NLS"
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
CDS 1356..1712
/label="RelA (p65) AD"
/note="transcriptional activation domain of human RelA,
also known as p65 (O'Shea and Perkins, 2008)"
misc_feature 1713..1730
/label="linker 2"
/note="linker 2"
CDS 1731..2300
/label="Rta AD"
/note="transcriptional activation domain from the human
herpesvirus 4 (Epstein-Barr virus) replication and
transcription activator Rta/BRLF1 (Hardwick et al., 1992;
Chavez et al., 2015)"
CDS 2340..3908
/gene="PIF3"
/label="Transcription factor PIF3"
/note="Transcription factor PIF3 from Arabidopsis thaliana.
Accession#: O80536"
promoter complement(3944..3962)
/label="SP6 promoter"
/note="promoter for bacteriophage SP6 RNA polymerase"
polyA_signal 3988..4212
/label="bGH poly(A) signal"
/note="bovine growth hormone polyadenylation signal"
rep_origin 4258..4686
/label="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 4700..5030
/label="SV40 promoter"
/note="SV40 enhancer and early promoter"
CDS 5097..5888
/label="NeoR/KanR"
/note="aminoglycoside phosphotransferase"
polyA_signal 6065..6198
/label="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
primer_bind complement(6235..6251)
/label="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(6259..6275)
/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(6283..6313)
/label="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind complement(6328..6349)
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(6637..7225)
/direction=LEFT
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(7399..8256)
/label="AmpR"
/note="beta-lactamase"
promoter complement(8257..8361)
/label="AmpR promoter"
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