pTCK303 Plasmid
PVT3203 2ug
pTCK303 Plasmid information
Promoter: Ubi, CaMV 35S
Replicon: ori
Terminator: NOS
Plasmid classification: plant series, RNAi vector
Plasmid size: 14621bp
Prokaryotic resistance: Kan
Screening markers: HygR
Cloned strain: DH5 alpha
Culture conditions: 37 centigrade, aerobic LB
Expression host: plant cells
5'sequencing primers: primers designed according to sequence
3'sequencing primers: primers designed according to sequence
Use: RNAi
pTCK303 Plasmid Description
RNA interferences (RNAi) has proven to be an effective strategy to knock out homologous genes in a wide range of species. Based on its principle, a new generation of vectors containing an inverted target sequence separated by an intron as a loop, developing simplifications to the procedure of RNAi construction are required to improve the efficiency of gene inactivation techniques. Here, a novel polymerase chain reaction (PCR)—based RNAi vector pTCK303 with a maize ubiquitin promoter, 2 specific multiple enzyme sites, and a rice intron was constructed for monocot gene silencing. With this vector, only 1 PCR product amplified by a single pair of primers and 2 ligation reactions were needed to create an RNAi construct, which shortened the time span before being transformed into the plant. To test the efficiency of vector pTCK303, a rice geneOsGAS1 was used, and its RNAi construct was introduced into rice calli. Southern blot analysis of the transgenic rice confirmed the presence of theOsGAS1 RNAi structure. The decrease inOsGAS1 level in the transgenic rice was detected by Northern blot probed with anOsGAS1-specific sequence. Moreover, the rate of inhibition of the RNA expression level in RNAi transgenic rice was approximately 85% according to our real-time PCR. Therefore, the RNAi vector pTCK303 based on the homology-dependent gene-silencing mechanisms facilitated the inhibition of endogenous genes in a monocot and was proven to be a practical and efficient platform for silencing a rice gene.
pTCK303 Plasmid Sequence
LOCUS Exported 14621 bp ds-DNA circular SYN 19-SEP-2016
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS Untitled 6
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 14621)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Monday, September 19, 2016 from SnapGene Viewer 3.2.1
FEATURES Location/Qualifiers
source 1..14621
/organism="synthetic DNA construct"
/mol_type="other DNA"
intron 10..199
/note="cat1 intron"
/note="castor bean catalase intron, modified"
CDS 2024..2041
/codon_start=1
/product="6xHis affinity tag"
/note="6xHis"
/translation="HHHHHH"
terminator 2076..2328
/note="NOS terminator"
/note="nopaline synthase terminator and poly(A) signal"
misc_feature 2350..2374
/note="RB T-DNA repeat"
/note="right border repeat from nopaline C58 T-DNA"
CDS 3674..4303
/codon_start=1
/product="stability protein from the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
/note="pVS1 StaA"
/translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
misc_feature 6408..6548
/note="bom"
/note="basis of mobility region from pBR322"
rep_origin complement(6734..7322)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(7409..8203)
/codon_start=1
/gene="aphA-3"
/product="aminoglycoside phosphotransferase"
/note="KanR"
/note="confers resistance to kanamycin"
/translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
misc_feature 8628..8652
/note="LB T-DNA repeat"
/note="left border repeat from nopaline C58 T-DNA"
polyA_signal 8730..8904
/note="CaMV poly(A) signal"
/note="cauliflower mosaic virus polyadenylation signal"
CDS complement(8944..9969)
/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
K"
promoter complement(10036..10713)
/note="CaMV 35S promoter (enhanced)"
/note="cauliflower mosaic virus 35S promoter with a
duplicated enhancer region"
protein_bind 10905..10926
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 10941..10971
/note="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 10979..10995
/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)."
primer_bind 11003..11019
