Plasmid pFUGW was constructed by inserting the following into the multicloning site of HRCS-G: HIV-1 flap sequence PCR-amplified from the HIV NLA4.3 genome, the human polyubiquitin promoter-C (gift of L. Thiel, Amgen), the EGFP gene, and the WRE (woodchuck hepatitis virus posttranscriptional regulatory element) (gift of D. Trono, University of Geneva). Lentiviruses can be produced by cotransfecting the HIV-1 packaging vector Delta8.9 and the VSVG envelope glycoprotein into 293 fibroblasts.Order of elements: CMV LTR PstI flap PacI Ubiquitin promoter SpeI HindIII PstI SalI XbaI BamHI SmaI KpnI GFP NotI EagI XbaI EcoRI EcoRV HindIII ClaI WRE ClaI SalI XhoI KpnI 3LTR ApaI PmeI.Please note that there are 2 gaps upstream of the EGFP between authors provided sequence and Addgenes quality control sequence. These gaps are in the non-coding vector region and should not affect the expression of EGFP.
| 载体基本信息 | |||
| 载体名称 | FUGW | 载体抗性 | Ampicillin |
|---|---|---|---|
| 载体长度 | 9955 bp | 载体类型 | Lentiviral vectors |
| 拷贝数 | High copy number | 启动子 | hUbc |
| 克隆方法 | Enzyme digestion and ligation | 5引物 | hUBCpro-F: TGAAGCTCCGGTTTTGAACT |
| 3引物 | EGFP-N: CGTCGCCGTCCAGCTCGACCAG | 载体标签 | C-EGFP |
FUGW 载体载体图谱
FUGW 载体载体序列
LOCUS Exported 9941 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 9941)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..9941
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 238..617
/note="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 619..817
/note="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
LTR 835..1015
/note="5 LTR (truncated)"
/note="truncated 5 long terminal repeat (LTR) from HIV-1"
misc_feature 1062..1187
/note="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1680..1913
/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 2440..2557
/note="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
CDS 3873..4592
/codon_start=1
/product="enhanced GFP"
/note="EGFP"
/note="mammalian codon-optimized"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
misc_feature 4633..5221
/note="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
CDS complement(5104..5115)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/note="Factor Xa site"
/translation="IEGR"
primer_bind complement(5224..5240)
/note="KS primer"
/note="common sequencing primer, one of multiple similar
variants"
LTR 5746..5926
/note="5 LTR (truncated)"
/note="truncated 5 long terminal repeat (LTR) from HIV-1"
polyA_signal 5958..6182
/note="bGH poly(A) signal"
/note="bovine growth hormone polyadenylation signal"
rep_origin 6228..6656
/direction=RIGHT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 6670..6999
/note="SV40 promoter"
/note="SV40 enhancer and early promoter"
rep_origin 6850..6985
/note="SV40 ori"
/note="SV40 origin of replication"
promoter 7047..7094
/note="EM7 promoter"
/note="synthetic bacterial promoter "
CDS 7113..7487
/codon_start=1
/gene="Sh ble from Streptoalloteichus hindustanus"
/product="antibiotic-binding protein"
/note="BleoR"
/note="confers resistance to bleomycin, phleomycin, and
Zeocin(TM)"
/translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
EFALRDPAGNCVHFVAEEQD"
polyA_signal 7617..7738
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
primer_bind complement(7787..7803)
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 7811..7827
/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(7835..7865)
/note="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 7880..7901
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(8189..8777)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(8948..9808)
/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(9809..9913)
/gene="bla"
/note="AmpR promoter"
