p4.5 HPRT-2A-GFP-2A-Puro vector (V009731)

Basic Vector Information

      • Vector Name:
      • p4.5 HPRT-2A-GFP-2A-Puro
      • Antibiotic Resistance:
      • Ampicillin
      • Length:
      • 8905 bp
      • Type:
      • Targeting vector
      • Source/Author:
      • Saito S, Ura K, Kodama M, Adachi N.

p4.5 HPRT-2A-GFP-2A-Puro vector Vector Map

p4.5 HPRT-2A-GFP-2A-Puro8905 bp40080012001600200024002800320036004000440048005200560060006400680072007600800084008800attB3M13 fwdAmpR promoterAmpRoriM13 revattB4Factor Xa siteattB1lox71T2AEGFPT2Apuromycin-resistance geneSV40 poly(A) signalloxPattB23' arm

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

Copy Sequence

Download GeneBank File(.gb)

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

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