Basic Vector Information
8xNFAT-ZsG-hCD8 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 ℃.
8xNFAT-ZsG-hCD8 vector Sequence
LOCUS 8xNFAT-ZsG-hCD8. 7850 bp DNA circular SYN 13-MAY-2021
DEFINITION NFAT-driven ZsGreen-1 reporter gene with human CD8ab gene.
ACCESSION .
VERSION .
KEYWORDS 8xNFAT-ZsG-hCD8.
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7850)
AUTHORS Mann SE, Zhou Z, Landry LG, Anderson AM, Alkanani AK, Fischer J,
Peakman M, Mallone R, Campbell K, Michels AW, Nakayama M
TITLE Multiplex T Cell Stimulation Assay Utilizing a T Cell Activation
Reporter-Based Detection System.
JOURNAL Front Immunol. 2020 Apr 9;11:633. doi: 10.3389/fimmu.2020.00633.
eCollection 2020.
PUBMED 32328071
REFERENCE 2 (bases 1 to 7850)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 7850)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Front
Immunol."; date: "2020-04-9"; pages: "
10.3389/fimmu.2020.00633. eCollection 2020"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..7850
/mol_type="other DNA"
/organism="synthetic DNA construct"
enhancer 1..380
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 381..584
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
LTR 585..760
/label=5' LTR (truncated)
/note="truncated long terminal repeat from Moloney murine
sarcoma virus"
misc_feature 823..1022
/label=MMLV Psi
/note="packaging signal of Moloney murine leukemia virus
(MMLV)"
CDS 1223..1639
/label=gag (truncated)
/note="truncated Moloney murine leukemia virus (MMLV) gag
gene lacking the start codon"
protein_bind 1673..1702
/label=NFAT binding site
/note="NFAT binding site from the human interleukin 2
(IL-2) gene (Mattila et al., 1990)"
protein_bind 1703..1732
/label=NFAT binding site
/bound_moiety="NFAT transcription factor"
/note="NFAT binding site from the human interleukin 2
(IL-2) gene (Mattila et al., 1990)"
protein_bind 1733..1762
/label=NFAT binding site
/bound_moiety="NFAT transcription factor"
/note="NFAT binding site from the human interleukin 2
(IL-2) gene (Mattila et al., 1990)"
protein_bind 1763..1792
/label=NFAT binding site
/bound_moiety="NFAT transcription factor"
/note="NFAT binding site from the human interleukin 2
(IL-2) gene (Mattila et al., 1990)"
protein_bind 1793..1822
/label=NFAT binding site
/bound_moiety="NFAT transcription factor"
/note="NFAT binding site from the human interleukin 2
(IL-2) gene (Mattila et al., 1990)"
protein_bind 1823..1852
/label=NFAT binding site
/bound_moiety="NFAT transcription factor"
/note="NFAT binding site from the human interleukin 2
(IL-2) gene (Mattila et al., 1990)"
protein_bind 1853..1882
/label=NFAT binding site
/bound_moiety="NFAT transcription factor"
/note="NFAT binding site from the human interleukin 2
(IL-2) gene (Mattila et al., 1990)"
protein_bind 1883..1912
/label=NFAT binding site
/bound_moiety="NFAT transcription factor"
/note="NFAT binding site from the human interleukin 2
(IL-2) gene (Mattila et al., 1990)"
promoter 1924..1955
/label=minP
/note="minimal TATA-box promoter with low basal activity"
CDS 1975..2667
/label=ZsGreen1
/note="Zoanthus green fluorescent protein"
promoter 2693..3192
/label=PGK promoter
/note="mouse phosphoglycerate kinase 1 promoter"
CDS 3210..3914
/gene="CD8A"
/label=CD8A
/note="T-cell surface glycoprotein CD8 alpha chain from
Homo sapiens. Accession#: P01732"
CDS 3924..3980
/label=P2A
/note="2A peptide from porcine teschovirus-1 polyprotein"
CDS 3981..4601
/gene="CD8B"
/label=CD8B
/note="T-cell surface glycoprotein CD8 beta chain from Homo
sapiens. Accession#: P10966"
LTR 4793..5218
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
Moloney murine leukemia virus"
rep_origin complement(5388..5976)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(6150..7007)
/label=AmpR
/note="beta-lactamase"
promoter complement(7008..7112)
/label=AmpR promoter
primer_bind 7180..7198
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
primer_bind complement(7236..7258)
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind 7358..7377
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 7571..7593
/label=M13/pUC Forward
/note="In lacZ gene"
primer_bind 7721..7740
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
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