TET promoter (minimal CMV promoter containing seven upstream Tet-operator sites) transcribes GFP and miR30-based shRNA targeting FF3. The FF3 hairpin can be replaced with any other hairpin by cloning into the EcoR1/Xho1 site.Please note that the full plasmid sequence shown here is for the empty vector only and does not contain the FF3 targeting sequence (AGCTCCCGTGAATTGGAATCCTAGTGAAGCCACAGATGTAGGATTCCAATTCAGCGGGAGCC)
| 载体基本信息 | |||
| 载体名称 | pPRIME-TET-GFP-FF3 | 载体抗性 | Ampicillin |
|---|---|---|---|
| 载体长度 | 8226 bp | 载体类型 | Lentiviral vectors |
| 拷贝数 | Low copy number | 启动子 | TET |
| 克隆方法 | Enzyme digestion and ligation | 5引物 | EGFP-C Forward: CATGGTCCTGCTGGAGTTCGTG |
| 载体标签 | GFP | ||
pPRIME-TET-GFP-FF3 载体载体图谱
pPRIME-TET-GFP-FF3 载体载体序列
LOCUS Exported 8226 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 8226)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..8226
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 238..617
/note="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
misc_feature 1023..1148
/note="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1645..1878
/note="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
misc_feature 2405..2522
/note="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2597..2911
/note="tight TRE promoter"
/note="Tet-responsive promoter PTight, consisting of seven
tet operator sequences followed by the minimal CMV
promoter"
protein_bind 2605..2623
/gene="tetO"
/bound_moiety="tetracycline repressor TetR"
/note="tet operator"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 2641..2659
/gene="tetO"
/bound_moiety="tetracycline repressor TetR"
/note="tet operator"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 2676..2694
/gene="tetO"
/bound_moiety="tetracycline repressor TetR"
/note="tet operator"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 2712..2730
/gene="tetO"
/bound_moiety="tetracycline repressor TetR"
/note="tet operator"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 2748..2766
/gene="tetO"
/bound_moiety="tetracycline repressor TetR"
/note="tet operator"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 2783..2801
/gene="tetO"
/bound_moiety="tetracycline repressor TetR"
/note="tet operator"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 2819..2837
/gene="tetO"
/bound_moiety="tetracycline repressor TetR"
/note="tet operator"
/note="bacterial operator O2 for the tetR and tetA genes"
CDS 2928..3647
/codon_start=1
/product="enhanced GFP"
/note="EGFP"
/note="mammalian codon-optimized"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
ncRNA 3697..3790
/note="5 miR-30a"
/note="sequence upstream of the 71-nt precursor of the
human miR-30a microRNA (Zeng et al., 2002)"
/ncRNA_class="miRNA"
ncRNA 3804..3916
/note="3 miR-30a"
/note="sequence downstream of the 71-nt precursor of the
human miR-30a microRNA (Zeng et al., 2002)"
/ncRNA_class="miRNA"
promoter 4069..4171
/note="cat promoter"
/note="promoter of the E. coli cat gene encoding
chloramphenicol acetyltransferase"
CDS 4172..4831
/codon_start=1
/gene="cat"
/product="chloramphenicol acetyltransferase"
/note="CmR"
/note="confers resistance to chloramphenicol"
/translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
CDS 4963..4992
/codon_start=1
/product="Myc (human c-Myc oncogene) epitope tag"
/note="Myc"
/translation="EQKLISEEDL"
misc_feature 5115..5703
/note="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
CDS complement(5586..5597)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/note="Factor Xa site"
/translation="IEGR"
primer_bind complement(5706..5722)
/note="KS primer"
/note="common sequencing primer, one of multiple similar
variants"
LTR 6232..6412
/note="5 LTR (truncated)"
/note="truncated 5 long terminal repeat (LTR) from HIV-1"
rep_origin complement(6474..7062)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(7233..8093)
/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(8094..8198)
/gene="bla"
/note="AmpR promoter"
