LOCUS       Exported                8905 bp ds-DNA     circular SYN 17-DEC-2018
DEFINITION  Targeting vector p4.5 HPRT-2A-GFP-2A-Puro DNA, complete sequence.
ACCESSION   LC270125
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)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..8905
                     /organism="Targeting vector p4.5 HPRT-2A-GFP-2A-Puro"
                     /mol_type="other DNA"
                     /db_xref="taxon:2003190"
     protein_bind    complement(31..51)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /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
                     /gene="bla"
                     /label=AmpR promoter
     CDS             654..1514
                     /codon_start=1
                     /product="beta-lactamase"
                     /label=beta-lactamase
                     /note="ampicillin-resistance gene"
                     /protein_id="BAX73947.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      1685..2273
                     /label=pBR 322 origin
                     /note="pBR 322 origin"
     primer_bind     2419..2435
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2455..2475
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     misc_feature    2476..4159
                     /gene="HPRT"
                     /label=5 arm
                     /note="5 arm"
     gene            2476..4159
                     /gene="HPRT"
                     /label=HPRT
     CDS             2645..2656
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     protein_bind    4159..4183
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    4192..4225
                     /label=lox71
                     /bound_moiety="Cre recombinase"
                     /note="Left element (LE) mutant of loxP (Araki et al., 
                     2010). Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     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"
     regulatory      4307..4316
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     misc_feature    4313..5026
                     /gene="EGFP"
                     /label=enhanced green fluorescent protein
                     /note="enhanced green fluorescent protein"
     gene            4313..5026
                     /gene="EGFP"
                     /label=EGFP
     CDS             4313..5026
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /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"
     regulatory      5783..5965
                     /regulatory_class="polyA_signal_sequence"
                     /note="SV40 poly(A) signal"
     polyA_signal    5884..5965
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    6066..6099
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     protein_bind    complement(6110..6134)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /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