LOCUS       sequence-198848-        7645 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A piggyBac transposon based dual directional gene trap vector, in 
            which, the mutagens are two non-selectable gene-trap cassettes in 
            opposite orientations..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7645)
  AUTHORS   Liu G, Wang X, Liu Y, Zhang M, Cai T, Shen Z, Jia Y, Huang Y
  TITLE     Arrayed mutant haploid embryonic stem cell libraries facilitate 
            phenotype-driven genetic screens.
  JOURNAL   Nucleic Acids Res. 2017 Sep 28. doi: 10.1093/nar/gkx857.
  PUBMED    29036617
REFERENCE   2  (bases 1 to 7645)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nucleic 
            Acids Res. 2017 Sep 28. doi: 10.1093/nar/gkx857."
COMMENT     Sequence Label: PBDGTV
FEATURES             Location/Qualifiers
     source          1..7645
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        24..128
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(129..197,198..989)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(347..366)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1160..1748
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1649..1668
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1902..1919
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2036..2057
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2072..2077,2078..2095,2096..2102)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2110..2126
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /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)."
     primer_bind     2115..2137
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2134..2150
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2134..2150
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2171..2189
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     repeat_region   2252..2286
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        3325..3824
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3360..3383)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3757..3776
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     regulatory      3901..3910
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3907..4506
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MGTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIE
                     RVTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRL
                     AAQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLET
                     SAPRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     4406..4426
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             4513..5508
                     /codon_start=1
                     /product="truncated herpes simplex virus thymidine kinase 
                     (Salomon et al., 1995)"
                     /label=Delta-TK
                     /note="confers sensitivity to nucleoside analogs such as 
                     acyclovir or ganciclovir"
                     /translation="MPTLLRVYIDGPHGMGKTTTTQLLVALGSRDDIVYVPEPMTYWQV
                     LGASETIANIYTTQHRLDQGEISAGDAAVVMTSAQITMGMPYAVTDAVLAPHIGGEAGS
                     SHAPPPALTLIFDRHPIAALLCYPAARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGAL
                     PEDRHIDRLAKRQRPGERLDLAMLAAIRRVYGLLANTVRYLQGGGSWREDWGQLSGTAV
                     PPQGAEPQSNAGPRPHIGDTLFTLFRAPELLAPNGDLYNVFAWALDVLAKRLRPMHVFI
                     LDYDQSPAGCRDALLQLTSGMVQTHVTTPGSIPTICDLARTFAREMGEAN"
     primer_bind     complement(5530..5547)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5536..5760
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6859..6921
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(7004..7023)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7005..7023)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(7030..7047)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7030..7046)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7039..7061)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      7188..7643
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7275..7294)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7485..7506
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
       61 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
      121 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
      181 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
      241 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
      301 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
      361 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
      421 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
      481 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
      541 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
      601 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
      661 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
      721 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
      781 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
      841 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
      901 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
      961 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     1021 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     1081 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     1141 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     1201 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     1261 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     1321 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     1381 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     1441 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     1501 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     1561 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     1621 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     1681 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     1741 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     1801 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     1861 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     1921 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     1981 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     2041 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
     2101 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg
     2161 ccaagctcga aattaaccct cactaaaggg aacaaaagct ggagctccac cgcggaaagt
     2221 ttaggtcgag taaagcgcaa atctttttta accctagaaa gatagtctgc gtaaaattga
     2281 cgcatgcatt cttgaaatat tgctctctct ttctaaatag cgcgaatccg tcgctgtgca
     2341 tttaggacat ctcagtcgcc gcttggagct cccgtgaggc gtgcttgtca atgcggtaag
     2401 tgtcactgat tttgaactat aacgaccgcg tgagtcaaaa tgacgcatga ttatctttta
     2461 cgtgactttt aagatttaac tcatacgata attatattgt tatttcatgt tctacttacg
     2521 tgataactta ttatatatat attttcttgt tatagatgcc taccggccga gccaaggaga
     2581 cagactgggc agccccctga gtgggtattt ctacagggtg acgacttagg ttgaaaccca
     2641 tatggaactt ctagacacaa aactaaagac catccaccac cttctacaag actgccagcc
     2701 tggcacagga gtggaagttc cagaaccagg taaaaggttg ggaggtttct atggctggag
     2761 agccatggct gtgaaactag agcaagttgg tctctccttc acagaggcca gcataccctt
     2821 tactgaggac ctggggaaag aagaccaaga agctggaaac agtcaggcct gggaagagcc
     2881 cataacctgg agcccagaaa cacggaacct agctccttcc tgggaggtgg agcaggggct
     2941 gaggagaagg tggcacaaga cggtgacaaa gggtccagca gtaaatgggg aacaaccact
     3001 gaaggtttag taaaaagagt cccctcgtac tgggtgttcc tctgtgcttt ttccactccc
     3061 tgctacacac acacacacgg tgcaaaagcc aagcactctt cccttattag gaagagagag
     3121 actgccttcc agaatgtgaa tggagaagac agagagagag ccacagttct gcccttgtgt
     3181 ttctgaggct accgagatgc cagccatcaa ggctcaaact ccattttctc gtgatttaga
     3241 caggaaggct tttgcaaaaa caacccttta ttaagatcca gtggggaaat aaaggcgcga
     3301 gcttgatatc gaattaattc taccgggtag gggaggcgct tttcccaagg cagtctggag
     3361 catgcgcttt agcagccccg ctgggcactt ggcgctacac aagtggcctc tggcctcgca
     3421 cacattccac atccaccggt aggcgccaac cggctccgtt ctttggtggc cccttcgcgc
     3481 caccttctac tcctccccta gtcaggaagt tcccccccgc cccgcagctc gcgtcgtgca
     3541 ggacgtgaca aatggaagta gcacgtctca ctagtctcgt gcagatggac agcaccgctg
     3601 agcaatggaa gcgggtaggc ctttggggca gcggccaata gcagctttgc tccttcgctt
     3661 tctgggctca gaggctggga aggggtgggt ccgggggcgg gctcaggggc gggctcaggg
     3721 gcggggcggg cgcccgaagg tcctccggag gcccggcatt ctgcacgctt caaaagcgca
     3781 cgtctgccgc gctgttctcc tcttcctcat ctccgggcct ttcgacctgc agataacttc
     3841 gtatagcata cattatacga agcggggggg cccggtacgg atcctcgagc agctgccaac
     3901 gccaccatgg ggaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc
     3961 cgggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc
     4021 gacccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc
     4081 gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc
     4141 acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag
     4201 ttgagcggtt cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg
     4261 cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca ccagggcaag
     4321 ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc
     4381 gccttcctgg agacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc
     4441 gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc
     4501 ggtgccggat ccatgcccac gctactgcgg gtttatatag acggtcctca cgggatgggg
     4561 aaaaccacca ccacgcaact gctggtggcc ctgggttcgc gcgacgatat cgtctacgta
     4621 cccgagccga tgacttactg gcaggtgctg ggggcttccg agacaatcgc gaacatctac
     4681 accacacaac accgcctcga ccagggtgag atatcggccg gggacgcggc ggtggtaatg
     4741 acaagcgccc agataacaat gggcatgcct tatgccgtga ccgacgccgt tctggctcct
     4801 catatcgggg gggaggctgg gagctcacat gccccgcccc cggccctcac cctcatcttc
     4861 gaccgccatc ccatcgccgc cctcctgtgc tacccggccg cgcgatacct tatgggcagc
     4921 atgacccccc aggccgtgct ggcgttcgtg gccctcatcc cgccgacctt gcccggcaca
     4981 aacatcgtgt tgggggccct tccggaggac agacacatcg accgcctggc caaacgccag
     5041 cgccccggcg agcggcttga cctggctatg ctggccgcga ttcgccgcgt ttacgggctg
     5101 cttgccaata cggtgcggta tctgcagggc ggcgggtcgt ggcgggagga ttggggacag
     5161 ctttcgggga cggccgtgcc gccccagggt gccgagcccc agagcaacgc gggcccacga
     5221 ccccatatcg gggacacgtt atttaccctg tttcgggccc ccgagttgct ggcccccaac
     5281 ggcgacctgt acaacgtgtt tgcctgggcc ttggacgtct tggccaaacg cctccgtccc
     5341 atgcacgtct ttatcctgga ttacgaccaa tcgcccgccg gctgccggga cgccctgctg
     5401 caacttacct ccgggatggt ccagacccac gtcaccaccc ccggctccat accgacgatc
     5461 tgcgacctgg cgcgcacgtt tgcccgggag atgggggagg ctaactgagc tctagagctc
     5521 gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
     5581 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
     5641 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
     5701 gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg
     5761 cttctgaggc ggaaagaacc agctggggct cgagatccac tagttctagc ctcgagcaag
     5821 tctttcagtc ctatagaaat aaaatcagga attaaaaaga agattctgaa tatttatctt
     5881 ttataacaaa tacttcttac acattaaact tttacaggac acattccttg tctctatgaa
     5941 gggaattcgc gccgccagcc tctacaccca gtaaaggatg tgtggataga aatgtaacaa
     6001 taagctgggt tgtagtagca gcgaaggggc agataaatct ctttgagttt gatgccaacc
     6061 atgttttgaa aaaactaaaa acaaacaaaa aaaaccccct aaaaacccaa aaatataaat
     6121 aaagagcaaa ccagagtgct tggagtatgg taacttggaa cagaggtgac tccattcaga
     6181 cacagcaaga gacccactta gaaacacgcc tttattatct gcacggagcc cagacttgtc
     6241 ctcagtcctt gggagaggga gtctttgaga gtggtgggag gtcttgggtc atagtctcca
     6301 cataccatga gggcaggaag gcatagggtg cgtcacgatg gacagacaca gtgacagggc
     6361 caccaggttc ccaggagaag aggtggacaa gcctgtggaa gacagaggtg tggggtgaag
     6421 ccagagattg agccgggatc cagaggcagt gcacaaacat gaaggcctta cctgggatcg
     6481 tcctggacta ctgtgctctg ggcaaagcta aggaaggcag cacagaagtt catgcacaca
     6541 gagggaattc agccttctcg gtcattctgg agggggcaca agaggcagaa gactgggggt
     6601 ggaaagagga aaaggcaggt ggcaaagggg cactgggtct gaggacttgg ggccggccgg
     6661 atttttgtaa aagagagaat gtttaaaagt tttgttactt tatagaagaa attttgagtt
     6721 tttgtttttt tttaataaat aaataaacat aaataaattg tttgttgaat ttattattag
     6781 tatgtaagtg taaatataat aaaacttaat atctattcaa attaataaat aaacctcgat
     6841 atacagaccg ataaaacaca tgcgtcaatt ttacgcatga ttatctttaa cgtacgtcac
     6901 aatatgatta tctttctagg gttaaataat agtttctaat ttttttatta ttcaggcgaa
     6961 gttacatgta tcgatctcga gggggggccc ggtacccaat tcgccctata gtgagtcgta
     7021 ttacaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
     7081 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
     7141 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatgggacg cgccctgtag
     7201 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     7261 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     7321 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     7381 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     7441 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     7501 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     7561 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     7621 caaaatatta acgcttacaa tttag
//
