pRNAT-U6.1/Neo is designed for mammalian transfection. It carries a Neomycin resistance gene as the selectable marker, which can be used for establishing stable cell line. The GFP marker (coral GFP, cGFP) under CMV promoter control can be used to track the transfection efficiency. It uses U6 promoter for siRNA expression.
| 载体基本信息 | |||
| 载体名称 | pRNAT-U6.1/neo | 载体抗性 | Ampicillin |
|---|---|---|---|
| 载体长度 | 6380 bp | 载体类型 | RNAi vectors |
| 筛选标记 | Neomycin | 启动子 | U6 |
| 克隆方法 | Enzyme digestion and ligation | 5引物 | pRNA-U6 Forward (TACGATACAAGGCTGTTAGAGAG) |
| 3引物 | pRNA Reverse (TAGAAGGCACAGTCGAGG) | 载体标签 | cGFP |
pRNAT-U6.1/neo 载体载体图谱
pRNAT-U6.1/neo 载体载体序列
LOCUS Exported 6380 bp ds-DNA circular SYN 13-JAN-2022
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6380)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..6380
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 192..495
/note="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 496..699
/note="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
polyA_signal 1478..1599
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
rep_origin 1700..2128
/direction=RIGHT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 2142..2471
/note="SV40 promoter"
/note="SV40 enhancer and early promoter"
rep_origin 2322..2457
/note="SV40 ori"
/note="SV40 origin of replication"
CDS 2538..3332
/codon_start=1
/gene="aph(3)-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/note="NeoR/KanR"
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
polyA_signal 3506..3627
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
primer_bind complement(3676..3692)
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 3700..3716
/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)."
promoter complement(3724..3754)
/note="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 3769..3790
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(4078..4663)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(4834..5694)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(5695..5799)
/gene="bla"
/note="AmpR promoter"
promoter 6065..6083
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
promoter 6214..74
/note="U6 promoter"
/note="RNA polymerase III promoter for human U6 snRNA"
