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
ORIGIN
        1 agttacgcta gggataacag ggtaatatag caactttatt atacatagtt gataattcac
       61 tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc
      121 ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc
      181 cttcccaaca gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat tttctcctta
      241 cgcatctgtg cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg
      301 ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
      361 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
      421 agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat
      481 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg
      541 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
      601 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
      661 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
      721 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
      781 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
      841 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
      901 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
      961 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     1021 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     1081 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     1141 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     1201 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     1261 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     1321 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     1381 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     1441 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     1501 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     1561 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     1621 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     1681 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     1741 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     1801 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     1861 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     1921 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     1981 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     2041 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     2101 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     2161 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     2221 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     2281 gcaacgcggc ctttttacgg ttcctggtct tttgctggcc ttttgctcac atgttctttc
     2341 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgatacca
     2401 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag ctatcaactt
     2461 tgtatagaaa agttgcagca gctgttctga gtacttgcta tttgaacata aactaggcca
     2521 acttattaaa taactgatgc tttctaaaat cttctttatt aaaaataaaa gaggagggcc
     2581 ttactaatta cttagtatca gttgtggtat agtgggactc tgtagggacc agaacaaagt
     2641 aaacattgaa gggagatgga agaaggaact ctagccagag tcttgcattt ctcagtccta
     2701 aacagggtaa tggactgggg ctgaatcaca tgaaggcaag gtcagatttt tattattatg
     2761 cacatctagc ttgaaaattt tctgttaagt caattacagt gaaaaacctt acctggtatt
     2821 gaatgcttgc attgtatgtc tggctattct gtgtttttat tttaaaatta taatatcaaa
     2881 atatttgtgt tataaaatat tctaactatg gaggccataa acaagaagac taaagttctc
     2941 tcctttcagc cttctgtaca catttcttct caagcactgg ctatgcatgt atactatatg
     3001 caaaagtaca tatatacatt tatattttaa cgtatgagta tagttttaaa tgttattgga
     3061 cacttttaat attagtgtgt ctagagctat ctaatatatt ttaaaggttg catagcattc
     3121 tgtcttatgg agataccata actgatttaa ccagtccact attgatagac actattttgt
     3181 tcttaccgac tgtactagaa gaaacattct tttacatgtt tggtacttgt tcagctttat
     3241 tcaagtggaa tttctgggtc aaggggaaag agtttattga atattttggt attgccaaat
     3301 tttcctctaa gaagttgaat cattttatac tcctgatgtt atatgagagt acctttctct
     3361 tcacaatttg tctctttttt tttttttttt gagacaaggt ctctgttgcc caggctgggg
     3421 tgcagtgcag cagaatgatc acagttcact gcagtctcaa cctcctgggt tcaagcgatc
     3481 cttccacctc agcctcctga gtagctggga ctataggtgt gcgccaccac tcccagctaa
     3541 tatttttatt ttgtagaaac agggttcgcc atgttaccca gcctcccaaa gtgctgggat
     3601 tacaggcatg agccactggc ccagtttcta cagtctctct taatattgta tattatccaa
     3661 gaaatttcat ttaatcagaa cctgccagtc tgataggtga aaatggtatc ttgtttttat
     3721 ttgcatttaa aaaaaattat gatagtggta tgcttggttt ttttgaaggt atcaaatttt
     3781 ttaccttatg aaacatgagg gcaaaggatg tgttacgtgg aagatttaaa aaaaattttt
     3841 aatgcatttt tttgagacaa ggtcttgctc tattgtccag gctggagtgc agtggcacaa
     3901 tcacagttca ctccagcctc aacatcctgc actaaagtga ttttcccacc tcacctctca
     3961 agtagctggg actacaggta catgctacca tgcctggcta attttttttt ttttgcaggc
     4021 atggggtctc actgtattgc ccaggttggt gtggaagttt aatgactaag aggtgtttgt
     4081 tataaagttt aatgtatgaa actttctatt aaattcctga ttttatttct gtaggactga
     4141 acgtcttgct cgagatgtgc aagtttgtac aaaaaagcag gctgcggccg ctaccgttcg
     4201 tatagcatac attatacgaa gttatggcgc gcccggggag ggcagaggaa gtcttctaac
     4261 atgcggtgac gtggaggaga atcccggccc tatggatcca ccggtcgcca ccatggtgag
     4321 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt
     4381 aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct
     4441 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac
     4501 caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga
     4561 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga
     4621 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg
     4681 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga
     4741 gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa
     4801 ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta
     4861 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag
     4921 cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga
     4981 gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacgggg agggcagagg
     5041 aagtcttcta acatgcggtg acgtggagga gaatcccggc cctatggatc tcgagcagct
     5101 gaagcttacc atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc
     5161 cagggccgta cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt
     5221 cgatccggac cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt
     5281 cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac
     5341 cacgccggag agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga
     5401 gttgagcggt tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg
     5461 gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc accagggcaa
     5521 gggtctgggc agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc
     5581 cgccttcctg gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac
     5641 cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc
     5701 cggtgcctga cgcccgcccc acgacccgca gcgcccgacc gaaaggagcg cacgacccat
     5761 gcatcgatga tctagaggat cataatcagc cataccacat ttgtagaggt tttacttgct
     5821 ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt
     5881 gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     5941 acaaataaag catttttttc actgcctcga gcttcctcgc tcactgactc gctgcgctcg
     6001 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg taacgatgcg
     6061 atcgcataac ttcgtatagc atacattata cgaagttata aggccgctta cccagctttc
     6121 ttgtacaaag tggatcacat tgtagccctc tgtgtgctca aggggggcta taaattcttt
     6181 gctgacctgc tggattacat caaagcactg aatagaaata gtgatagatc cattcctatg
     6241 actgtagatt ttatcagact gaagagctat tgtgtgagta tatttaatat atgattcttt
     6301 ttagtggcaa cagtaggttt tcttatattt tctttgaatc tctgcaaacc atacttgctt
     6361 tcatttcact tggttacagt gagatttttc taacatattc actagtactt tacatcaaag
     6421 ccaatactgt ttttttaaaa ctagtcacct tggaggatat atacttattt tacaggtgtg
     6481 tgtggttttt taaataaact ccttttagga attgctgttg ggacttggga tacttttttc
     6541 actatacata ctggtgacag ataccctctc ttgagctaca tcggtttgtg gggagtcaaa
     6601 agtcctttgg agctaggttt gacaaataag gtgggttaac acttgtttcc tagaaagcac
     6661 atggagagct agagtattgg cgaattgaag aaatccccct ttttttttaa cacacttaag
     6721 aaaggggact gcaggtatac tcaagagagt aagtcgcacc agaaaccact tttgatccac
     6781 agtctgcctg tgtcacacaa ttgaaatgca tcacaacatt gacactgtgg atgaaacaaa
     6841 atcagtgtga attttagtag tgaatttcat tcataatttg atcgtgcaaa cgtttgattt
     6901 ttattacttt agactattgt ttctgatttt atgttgggtt ggtatttcct gtgagttact
     6961 gttttccttt aaaataggaa tttttcatac tcttcaaaga ttagaacaaa tgtccagttt
     7021 ttgctgtttc atgaatgagt cctgtccatc tttgtagaaa ctcgccttat gttcacattt
     7081 ttattgagaa taagaccact tatctacatt taactatcaa cctcatcctc tccattaatc
     7141 atctatttta gtgacccaag tttttgacct tttccatgtt tacatcaatc ctgtaggtga
     7201 ttgggcagcc atttaagtat tattatagac attttcacta tcccattaaa accctttatg
     7261 cccatacatc ataacactac ttcctaccca taagctcctt ttaacttgtt aaagtcttgc
     7321 ttgaattaaa gacttgttta aacacaaaat ttagactttt actcaacaaa agtgattgat
     7381 tgattgattg attgattgat ggtttacagt aggacttcat tctagtcatt atagctgctg
     7441 gcagtataac tggccagcct ttaatacatt gctgcttaga gtcaaagcat gtacttagag
     7501 ttggtatgat ttatcttttt ggtcttctat agcctccttc cccatcccca tcagtcttaa
     7561 tcagtcttgt tacgttatga ctaatctttg gggatttgtg cagaatgtta ttttagataa
     7621 gcaaaaacga gcaaaatagg ggagtttaac tttaatattt tcttttaaaa agcatttcat
     7681 gttataagat caattctgag tggtagaaat gctttgacat tttatttcca ttttctactt
     7741 ttagtttttt tcctatttgt ttaagatctt agaggattat taagctgaac tcctcaactg
     7801 ataaaaagca tgacatctta aacataagca aagcatattt ttaggttaat tttcacatag
     7861 aaaacagttt attttatgtg aaattctatg tagatatact atttttttgg tatttattga
     7921 tatgtttatt ttattttatt ttattttatt ttattttatt ttattttatt tatttatttt
     7981 tttttttgag acagagtctc actctgttgc ccaggctgga gtgcagtggc atgatcgtag
     8041 ctcactgcaa cctccacctc ccgggttcaa gcaattcttc tgtctcagcc tcccgagtag
     8101 ctgggactac aggtgcctgc cactatgccc ggctaatttt tgtgttttta gtagagatgg
     8161 ggtttcacct tgttggtcag gctggtctcg aacccctgac ctcaggtgat ccacccacct
     8221 cagcctccca aagtgctggg attataggca tgagccaccg tgcccggccg acatgttaat
     8281 tttttaaaaa aggctttact ggggtatatt ttatataata taataatcac atgttttaac
     8341 tatacaattc caagcttttt agtatattta tagggctatg caaggaagat atactgttaa
     8401 acagtagaaa ttgagaaagc tcttctgata atatctcttg atttgatgat ggctcatgcc
     8461 tgtaatctca gtgctttgga aggccaagac agcagaatca cttgaggcca ggggttcgag
     8521 accagcctgg gcaacacagc aataccctat ctttacaaat aataaaaata tctgttgatt
     8581 tgaagtaaag ttttttttta aagacaaggt ctcattctgt cacccaggct ggaatgcagt
     8641 agcaagatca cagctcactg tggccttgac cttctgggct caagtgattc tcccacttcg
     8701 gcctcccgag tagctgggac taacaggtgt gcaccaccat ggctggctaa ttttttttta
     8761 tgtttgtaga gattgggtct tactgtgttg cccaggctga tcccgaactc ctgggctcaa
     8821 gcagtcttcc tgcctcagcc tctaaaattg ctgggattac aggcttgagt caccatgccc
     8881 agcctgaagt agcatttcta ccctg
//
