Basic Vector Information
p4.5 HPRT-2A-GFP-2A-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 ℃.
p4.5 HPRT-2A-GFP-2A-Puro vector Sequence
LOCUS 40924_2747 8905 bp DNA circular SYN 17-DEC-2018 DEFINITION Targeting vector p4.5 HPRT-2A-GFP-2A-Puro DNA, complete sequence. ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8905) AUTHORS Saito S, Ura K, Kodama M, Adachi N. TITLE Construction and applications of exon-trapping gene-targeting vectors with a novel strategy for negative selection JOURNAL BMC Res Notes 8, 278 (2015) PUBMED 26123730 REFERENCE 2 (bases 1 to 8905) AUTHORS Saito S, Adachi N. TITLE Direct Submission JOURNAL Submitted (15-MAY-2017) Contact:Shinta Saito Yokohama City University, Graduate school of Nanobioscience; 22-2 Seto, Kanazawa-ku, Yokohama, Kanagawa 236-0027, Japan REFERENCE 3 (bases 1 to 8905) TITLE Direct Submission REFERENCE 4 (bases 1 to 8905) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "BMC Res Notes 8, 278 (2015)" COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted (15-MAY-2017) Contact:Shinta Saito Yokohama City University, Graduate school of Nanobioscience; 22-2 Seto, Kanazawa-ku, Yokohama, Kanagawa 236-0027, Japan" COMMENT SGRef: number: 3; type: "Journal Article" FEATURES Location/Qualifiers source 1..8905 /mol_type="other DNA" /organism="synthetic DNA construct" protein_bind complement(31..51) /label=attB3 /note="core recombination site for the Gateway(R) BP reaction" primer_bind complement(59..75) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" promoter 549..653 /label=AmpR promoter CDS 654..1511 /codon_start=1 /label=AmpR /note="beta-lactamase" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" rep_origin 1685..2273 /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 2419..2435 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" protein_bind 2455..2475 /label=attB4 /note="core recombination site for the Gateway(R) BP reaction" CDS 2645..2656 /codon_start=1 /label=Factor Xa site /note="Factor Xa recognition and cleavage site" /translation="IEGR" protein_bind 4159..4183 /label=attB1 /note="recombination site for the Gateway(R) BP reaction" protein_bind complement(4192..4225) /label=lox71 /note="Left element (LE) mutant of loxP (Araki et al., 2010). Cre-mediated recombination occurs in the 8-bp core sequence (ATGTATGC) (Shaw et al., 2021)." misc_feature 4238..4291 /label=T2A /note="T2A" CDS 4238..4291 /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 4313..5026 /codon_start=1 /label=EGFP /note="enhanced GFP" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELY" misc_feature 5030..5083 /label=T2A /note="T2A" CDS 5030..5083 /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 5111..5710 /codon_start=1 /product="puromycin-N-acetyl-transferase" /label=puromycin-N-acetyl-transferase /note="puromycin-resistance gene" /protein_id="BAX73948.1" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" polyA_signal 5884..5965 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" protein_bind complement(6066..6099) /label=loxP /note="Cre-mediated recombination occurs in the 8-bp core sequence (ATGTATGC) (Shaw et al., 2021)." protein_bind complement(6110..6134) /label=attB2 /note="recombination site for the Gateway(R) BP reaction" misc_feature 6134..8905 /gene="HPRT" /label=3' arm /note="3' arm" gene 6134..8905 /gene="HPRT" /label=HPRT
This page is informational only.