Basic Vector Information
pTet-O-Ngn2-puro 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 ℃.
pTet-O-Ngn2-puro vector Sequence
LOCUS pTet-O-Ngn2-puro 9886 bp DNA circular SYN 13-MAY-2021 DEFINITION 3rd generation lentiviral vector, expresses Ngn2 and puromycin resistent gene under control of TetON promoter. ACCESSION . VERSION . KEYWORDS pTet-O-Ngn2-puro. SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9886) AUTHORS Zhang Y, Pak C, Han Y, Ahlenius H, Zhang Z, Chanda S, Marro S, Patzke C, Acuna C, Covy J, Xu W, Yang N, Danko T, Chen L, Wernig M, Sudhof TC TITLE Rapid single-step induction of functional neurons from human pluripotent stem cells. JOURNAL Neuron. 2013 Jun 5;78(5):785-98. doi: 10.1016/j.neuron.2013.05.029. PUBMED 23764284 REFERENCE 2 (bases 1 to 9886) TITLE Direct Submission REFERENCE 3 (bases 1 to 9886) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; doi: "10.1016/j.neuron.2013.05"; journalName: "Neuron"; date: "2013-06-5- 5"; volume: "78"; issue: "5"; pages: "785-98" COMMENT SGRef: number: 2; type: "Journal Article" FEATURES Location/Qualifiers source 1..9886 /mol_type="other DNA" /organism="synthetic DNA construct" enhancer 100..479 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 480..682 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" LTR 697..877 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 924..1049 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1542..1775 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 1960..2004 /label=gp41 peptide /note="antigenic peptide corresponding to amino acids 655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik et al., 2013)" CDS 2153..2194 /note="Protein Tat from Human immunodeficiency virus type 1 group M subtype B (isolate WMJ22). Accession#: P12509" misc_feature 2302..2419 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" protein_bind 2476..2746 /label=tetracycline response element /note="contains seven copies of the tetracycline operator tetO" promoter 2779..2817 /label=minimal CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 2814..2838 /label=LNCX /note="Human CMV promoter, forward primer" regulatory 2917..2926 /label=Kozak sequence /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" /regulatory_class="other" CDS 2926..2949 /label=FLAG /note="FLAG(R) epitope tag, followed by an enterokinase cleavage site" CDS 2950..3735 /gene="Neurog2" /label=Neurog2 /note="Neurogenin-2 from Mus musculus. Accession#: P70447" CDS 3742..3795 /label=T2A /note="2A peptide from Thosea asigna virus capsid protein" CDS 3802..4398 /label=PuroR /note="puromycin N-acetyltransferase" misc_feature 4440..5028 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(5031..5047) /label=KS primer /note="common sequencing primer, one of multiple similar variants" LTR 5553..5733 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" polyA_signal 5765..5989 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 6035..6463 /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" promoter 6477..6806 /label=SV40 promoter /note="SV40 enhancer and early promoter" promoter 6854..6901 /label=EM7 promoter /note="synthetic bacterial promoter" CDS 6920..7291 /label=BleoR /note="antibiotic-binding protein" polyA_signal 7424..7557 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(7594..7610) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" protein_bind complement(7618..7634) /label=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(7642..7672) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind complement(7687..7708) /label=CAP binding site /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(7825..7842) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(7996..8584) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(8758..9615) /label=AmpR /note="beta-lactamase" promoter complement(9616..9720) /label=AmpR promoter primer_bind complement(9795..9814) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker"
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