It is a 3rd gen lenti-vector. The 3rd generation LV packaging plasmid includes gag, coding for the virion main structural proteins; pol, responsible for the retrovirus-specific enzymes; and RRE, a binding site for the Rev protein which facilitates export of the RNA from the nucleus. This plasmid does not include Rev. The third-generation packaging system offers maximal biosafety but is more cumbersome, as it involves the transfection of four different plasmids in the producer cells. If you wish to use the 3rd gen lentivector, you need to have a lentiviral vector with a chimeric 5 LTR in which the HIV promoter is replaced with CMV or RSV, thus making it TAT-independent. Examples of these vectors include pLKO.1, pLL3.7, pLB, pLenti6, pSico, pCL and pCS. Most Aebischer and Trono lab lentiviral vectors CANNOT be used with this system. A lentiviral vector carrying a chimeric 5 LTR can be packaged with either the 2nd or 3rd generation packaging system.
| 载体基本信息 | |||
| 载体名称 | pCDH-EF1-copGFP-T2A-Puro | 载体抗性 | Ampicillin |
|---|---|---|---|
| 载体长度 | 8549 bp | 载体类型 | Mammalian Expression, Lentiviral |
| 筛选标记 | Puromycin | 拷贝数 | High Copy |
| 启动子 | elongation factor-1 | 克隆方法 | Restriction Enzyme |
| 5引物 | GCACTTGATGTAATTCTCC | 3引物 | CAACACCACGGAATTGTCAG |
pCDH-EF1-copGFP-T2A-Puro 载体载体图谱
pCDH-EF1-copGFP-T2A-Puro 载体载体序列
LOCUS Exported 8549 bp ds-DNA circular SYN 13-MAY-2021
DEFINITION Express copGFP and puromycin resistance gene under EF1 promoter.
ACCESSION .
VERSION .
KEYWORDS pCDH-EF1-copGFP-T2A-Puro
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8549)
TITLE lenti vector
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 8549)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName:
"Unpublished"
FEATURES Location/Qualifiers
source 1..8549
/organism="synthetic DNA construct"
/mol_type="other DNA"
LTR 234..414
/label=5 LTR (truncated)
/note="truncated 5 long terminal repeat (LTR) from HIV-1"
misc_feature 458..583
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1076..1309
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 1493..1537
/codon_start=1
/product="antigenic peptide corresponding to amino acids
655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik
et al., 2013)"
/label=gp41 peptide
/note="recognized by the 2H10 single-chain llama nanobody"
/translation="KNEQELLELDKWASL"
misc_feature 1804..1921
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2013..3191
/label=EF-1-alpha promoter
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
intron 2244..3182
/label=EF-1-alpha intron A
/note="intron upstream of the start codon of human
EF-1-alpha"
primer_bind 3139..3159
/label=EF1a-F
/note="Human elongation factor-1a promoter, forward primer"
CDS 3231..3893
/codon_start=1
/product="green fluorescent protein 2 from Pontellina
plumata, also known as ppluGFP2 (Shagin et al., 2004)"
/label=CopGFP
/translation="PAMEIECRITGTLNGVEFELVGGGEGTPKQGRMTNKMKSTKGALT
FSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTRIEKYEDGGVLHVSFSYRY
EAGRVIGDFKVVGTGFPEDSVIFTDKIIRSNATVEHLHPMGDNVLVGSFARTFSLRDGG
YYSFVVDSHMHFKSAIHPSILQNGGPMFAFRRVEELHSNTELGIVEYQHAFKTPIAFA"
CDS 3978..4031
/codon_start=1
/product="2A peptide from Thosea asigna virus capsid
protein"
/label=T2A
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="EGRGSLLTCGDVEENPGP"
CDS 4032..4631
/codon_start=1
/gene="pac from Streptomyces alboniger"
/product="puromycin N-acetyltransferase"
/label=PuroR
/note="confers resistance to puromycin"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
primer_bind complement(4032..4051)
/label=Puro-R
/note="Puromycin resistance gene, reverse primer. Also
called puro-variant-R"
primer_bind 4528..4548
/label=Puro-F
/note="Puromycin resistance gene, forward primer"
misc_feature 4644..5232
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(4697..4717)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(5115..5126)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
LTR 5306..5539
/label=3 LTR (Delta-U3)
/note="self-inactivating 3 long terminal repeat (LTR) from
HIV-1"
polyA_signal 5611..5732
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(5648..5667)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 5702..5721
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
rep_origin 5751..5886
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 5813..5832
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
primer_bind complement(5924..5940)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(5924..5940)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(5937..5959)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 5948..5964
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/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(5972..6002)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 6017..6038
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(6155..6172)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(6326..6914)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(6406..6425)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(7085..7945)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind 7708..7727
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(7946..8050)
/gene="bla"
/label=AmpR promoter
primer_bind 8118..8136
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
primer_bind complement(8174..8196)
/label=pGEX 3
/note="pGEX vectors, reverse primer"
primer_bind 8296..8315
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 8509..8531
/label=M13/pUC Forward
/note="In lacZ gene"
primer_bind 8523..8540
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind 8524..8540
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
