pAmCyan1-N1 encodes a human codon-optimized variant of wild-type Anemonia majano cyan fluorescent protein, AmCyan1 (1). The AmCyan1 coding sequence contains a series of silent base-pair changes, which correspond to human codon-usage preferences, for optimal expression in mammalian cells (2). Additionally, an upstream sequence—located just 5 to the AmCyan1 start codon—has been converted to a Kozak consensus translation initiation site (3) to further increase the translation efficiency in eukaryotic cells. Two amino acid substitutions (Asn-34 to Ser; Lys-68 to Met) have been made to enhance the emission characteristics of AmCyan1 (excitation maximum = 458 nm; emission maximum = 489 nm).The multiple cloning site (MCS) in pAmCyan1-N1 is positioned between the immediate-early promoter of CMV (PCMV IE) and the AmCyan1 coding sequence. Thus, genes cloned into the MCS will be expressed as fusions to the N-terminus of AmCyan1 if they are in the same reading frame as AmCyan1 and there are no intervening stop codons. The SV40 polyadenylation signals (SV40 polyA) downstream of the AmCyan1 gene direct proper processing of the 3 end of AmCyan1 mRNA.The vector backbone contains an SV40 origin (SV40 ori) for replication in mammalian cells that express the SV40 T antigen, a pUC origin of replication (pUC ori) for propagation in E. coli, and an f1 origin (f1 ori) for single-stranded DNA production. In addition, a neomycin-resistance cassette— consisting of the SV40 early promoter (PSV40e), the neomycin/kanamycin resistance gene of Tn5 (Neor/Kanr), and polyadenylation signals from the Herpes simplex virus thymidine kinase (HSV TK poly A) gene—allows stably transfected eukaryotic cells to be selected using G418 (4). A bacterial promoter (P) upstream of this cassette drives expression of the Neor/ Kanr gene in E. coli host cells, which can be selected with kanamycin.Fusions to the N terminus of AmCyan1 retain the fluorescent properties of the native protein allowing the localization of the fusion protein in vivo . The target gene should be cloned into pAmCyan1-N1 so that it is in frame with the AmCyan1 coding sequence, with no intervening, in-frame stop codons. The inserted gene should include the initiating ATG codon. The recombinant pAmCyan1-N1 vector can be transfected into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (available from Clontech; Cat. Nos. 631307 & 631308). We recommend selecting mammalian cell cultures in 500–1,300 μg/ml G418, depending on the cell line. Be sure to establish a kill curve for each cell line and each lot of G418 to determine the optimal selection concentration. Unmodified (i.e., non-recombinant) pAmCyan1-N1 can also be used simply to express AmCyan1 in a cell line of interest (e.g., as a transfection marker).
| 载体基本信息 | |||
| 载体名称 | pAmCyan-N1 | 载体抗性 | Kanamycin |
|---|---|---|---|
| 载体长度 | 4703 bp | 载体类型 | Fluorescent Protein Reporter Vectors |
| 筛选标记 | Neomycin | 启动子 | CMV |
| 载体标签 | AmCyan1 | ||
pAmCyan-N1 载体载体图谱
pAmCyan-N1 载体载体序列
LOCUS Exported 4703 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 4703)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..4703
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 61..364
/note="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 365..568
/note="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
misc_feature 591..671
/note="MCS"
/note="multiple cloning site"
CDS 679..1368
/codon_start=1
/product="Anemonia majano cyan fluorescent protein"
/note="AmCyan1"
/note="mammalian codon-optimized"
/translation="MALSNKFIGDDMKMTYHMDGCVNGHYFTVKGEGSGKPYEGTQTST
FKVTMANGGPLAFSFDILSTVFMYGNRCFTAYPTSMPDYFKQAFPDGMSYERTFTYEDG
GVATASWEISLKGNCFEHKSTFHGVNFPADGPVMAKKTTGWDPSFEKMTVCDGILKGDV
TAFLMLQGGGNYRCQFHTSYKTKKPVTMPPNHVVEHRIARTDLDKGGNSVQLTEHAVAH
ITSVVPF"
polyA_signal 1491..1612
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
rep_origin complement(1619..2074)
/direction=LEFT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 2101..2205
/gene="bla"
/note="AmpR promoter"
promoter 2207..2564
/note="SV40 promoter"
/note="SV40 enhancer and early promoter"
rep_origin 2415..2550
/note="SV40 ori"
/note="SV40 origin of replication"
CDS 2599..3393
/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
GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
polyA_signal 3625..3672
/note="HSV TK poly(A) signal"
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
rep_origin 4001..4589
/direction=RIGHT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
