LOCUS       sequence-437986-        7501 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac transposon plasmid for doxycycline inducible expression of 
            DMC1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7501)
  AUTHORS   Pierson Smela M, Adams J, Ma C, Breimann L, Widocki U, Shioda T, 
            Church GM
  TITLE     Induction of Meiosis from Human Pluripotent Stem Cells
  JOURNAL   bioRxiv 2024.05.31.596483
REFERENCE   2  (bases 1 to 7501)
  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: "bioRxiv 
            2024.05.31.596483"
COMMENT     Sequence Label: pPB-cT3G-cERP2-DMC1
FEATURES             Location/Qualifiers
     source          1..7501
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(657..681)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             683..709
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    876..1010
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   1282..1316
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1327..1344)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1327..1343)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1336..1358)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1552..1571)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1673..1695
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1734..1752)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1820..1924
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1925..1993,1994..2785)
                     /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(2143..2162)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2956..3544
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3445..3464
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3698..3715
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3921..3942
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3957..3962,3963..3980,3981..3987)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3995..4011
                     /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     4000..4022
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4019..4035
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4019..4035
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4061..4123
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    4299..4380
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5097..5153)
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             complement(5163..5906)
                     /codon_start=1
                     /product="modified rtTA protein that binds tightly to 
                     promoters containing the tet operator in the presence of 
                     doxycycline"
                     /label=Tet-On(R) 3G
                     /translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     CDS             complement(5907..5930)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      5930..5939
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(5959..6170)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        6171..6550
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     protein_bind    6759..6777
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6795..6813
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6831..6849
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6867..6885
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6903..6921
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6939..6957
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6975..6993
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     7020..7044
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    7110..7134
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
ORIGIN
        1 agaagctttt ggagaatttc gtactggaaa aacccagctt tctcataccc tctgtgtgac
       61 agctcaactt ccaggagctg gtggctaccc aggaggaaag attatcttca ttgatacaga
      121 aaatactttc cgtccagatc gccttaggga cattgctgat cgctttaatg tagaccatga
      181 tgcagtactg gacaacgtac tttatgcacg tgcatatact agtgaacatc agatggagct
      241 acttgattat gtagcagcaa agttccatga agaagctggc atcttcaagc tattgattat
      301 cgattcaata atggcacttt ttcgagtgga tttcagtggc cgtggggagt tggccgaacg
      361 gcagcaaaaa ttggcccaga tgttgtcacg actccaaaaa atctcagaag aatataacgt
      421 ggctgttttt gtgaccaatc aaatgactgc cgatccagga gcaactatga cctttcaggc
      481 agatcccaaa aaacccattg ggggacacat tctggctcat gcttcaacaa caagaataag
      541 cttgcgaaag ggaagaggag agctcagaat tgccaagatt tatgacagtc ctgagatgcc
      601 tgaaaatgaa gccaccttcg caataactgc tggaggaatt ggggatgcca aggagttgcc
      661 aactttcttg tacaaagtgg tatacccata cgatgttcca gattacgctt agtaatgaga
      721 aatactcaga agatgtcact caccatagtc cgcagtgaaa gataaatgat cgccggtaac
      781 gtcggtgacg agttttagag ctagaaatag caagtaggcc actgagcaag aactatgaat
      841 gagatcttta gacatattgc ggccgctttc tccgcgatcc agacatgata agatacattg
      901 atgagtttgg acaaaccaaa actagaatgc agtgaaaaaa atgccttatt tgtgaaattt
      961 gtgatgctat tgccttattt gtaaccatta taagctgcaa taaacaagtt atctataaca
     1021 agaaaatata tatataataa gttatcacgt aagtagaaca tgaaataaca atataattat
     1081 cgtatgagtt aaatcttaaa agtcacgtaa aagataatca tgcgtcattt tgactcacgc
     1141 ggtcgttata gttcaaaatc agtgacactt accgcattga caagcacgcc tcacgggagc
     1201 tccaagcggc gactgagatg tcctaaatgc acagcgacgg attcgcgcta tttagaaaga
     1261 gagagcaata tttcaagaat gcatgcgtca attttacgca gactatcttt ctagggttaa
     1321 gaattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact
     1381 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac
     1441 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt
     1501 tctccttacg catctgtgcg gtatttcaca ccgcatatgg tgcactctca gtacaatctg
     1561 ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg
     1621 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccatgt ctccgggagc
     1681 tgcatgtgtc agaggttttc accgtcatca ccgaaacacg cgagttcgaa agggcctcgt
     1741 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
     1801 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     1861 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     1921 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     1981 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     2041 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     2101 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     2161 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     2221 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     2281 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     2341 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     2401 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     2461 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     2521 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     2581 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     2641 ttcacgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     2701 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     2761 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     2821 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     2881 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     2941 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     3001 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     3061 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     3121 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     3181 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     3241 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     3301 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     3361 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     3421 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     3481 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     3541 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     3601 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     3661 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     3721 ggatgagcgc ccaatacgca aaccgcctct caccgcgcgt tggccgattc attaatgcag
     3781 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa accgcctctc
     3841 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
     3901 ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta
     3961 cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca
     4021 ggaaacagct atgaccatga ttacgccaag gtcgacttaa ccctagaaag ataatcatat
     4081 tgtgacgtac gttaaagata atcatgcgta aaattgacgc atgtgtttta tcggtctgta
     4141 tatcgaggtt tatttattaa tttgaataga tattaagttt tattatattt acacttacat
     4201 actaataata aattcaacaa acaatttatt tatgtttatt tatttattaa aaaaaaacaa
     4261 aaactcaaaa tttcttctat aaagtaacaa aacttttagc agtgaaaaaa atgctttatt
     4321 tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt
     4381 aacaacaaca attgcattca ttttatgttt caggttcagg gggaggtgtg ggaggttttt
     4441 taaagcaagt aaaacctcta caaatgtggt atggctgatt atgatcctct ggagattcat
     4501 gcgccaggtt ttctggtcat gcaccacgtt cgtgggccct caggaacctc cacgtcagcg
     4561 gtaactgtga agcccagtcg ctcgtaaaaa ggaagattcc ggggagcact ggtttcgagg
     4621 aacgccggca cgccagcccg ttccgcggcc tcaacaccag gaagaacgac agcgcttccc
     4681 aaacctttac cttggtgatc tgggctgaca ccgacagtgg caagaaacca cgcaggctcc
     4741 tttggtctat gaggagccag caagccctcc atttgctgtt gtgccgccaa cctactccct
     4801 gacagctcgg ccatccgcgg acctatttcc gcgaacactg cgccggcttc gacgctttcc
     4861 ggagtagtcc agaccgcaac tgcggcacca tcgtccgcaa cccaaacttt accaatgtcc
     4921 agacccacgc gcgtaagaaa cagctcttgc aactcagtta ccctctctat gtgtctgtcc
     4981 ggatctacgg tatggcgggt cgctggataa tctgcaaatg ctgcggcaag tgttcggaca
     5041 gcccgtggta catcgtcacg ggtagcgagc ctaacagtgg gcttatattc cgtcatgggg
     5101 ccggggttct cctccacgtc gccggcctgc ttcagcaggc tgaagttggt ggcgccgctg
     5161 cccccgggga gcatgtcaag gtcaaaatcg tcaagagcgt cagcaggcag catatcaagg
     5221 tcaaagtcgt caagggcatc ggctgggagc atgtctaagt caaaatcgtc aagggcgtcg
     5281 gtcggcccgc cgctttcgca ctttagctgt ttctccaggc cacatatgat tagttccagg
     5341 ccgaaaagga aggcaggttc ggctccctgc cggtcgaaca gctcaattgc ttgtttcaga
     5401 agtgggggca tagaatcggt ggtaggtgtc tctctttcct cttttgctac ttgatgctcc
     5461 tgttcctcca atacgcagcc cagtgtaaag tggcccacgg cggacagagc gtacagtgcg
     5521 ttctccaggg agaagccttg ctgacacagg aacgcgagct gattttccag ggtttcgtac
     5581 tgtttctctg ttgggcgggt gccgagatgc actttagccc cgtcacgatg tgagaggaga
     5641 gcacagcggt atgacttggc gttgttccgc agaaagtctt gccatgactc gccttccagg
     5701 gggcaggagt gggtatgatg cctgtccagc atctcgattg gcagggcatc gagcagggcc
     5761 cgcttgttct tcacgtgcca gtacagggta ggctgctcaa ctcccagctt ttgagcgagt
     5821 ttccttgtcg tcaggccttc gataccgact ccattgagta attccagagc agagtttatg
     5881 actttgctct tgtccagtct agacatctta tcgtcatcgt ctttgtaatc catggtggcg
     5941 gatcccgcgt cacgacacct gtgttctggc ggcaaacccg ttgcgaaaaa gaacgttcac
     6001 ggcgactact gcacttatat acggttctcc cccaccctcg ggaaaaaggc ggagccagta
     6061 cacgacatca ctttcccagt ttaccccgcg ccaccttctc taggcaccgg ttcaattgcc
     6121 gacccctccc cccaacttct cgggcactgt gggccatgtg cgctctgccc catggtaata
     6181 gccatgacta atacgtagat gtactgccaa gtaggaaagt gccataaggt catgtactgg
     6241 gcataatgcc aggcgggcca tttaccgtca ttgacgtcaa tagggggcgt acttggcata
     6301 tgatacactt gatgtactgc caagtgggca gtttaccgta aatactccac ccattgacgt
     6361 caatggaaag tgcctattgg cgttactatg ggaacatacg tcattattga cgtcaatggg
     6421 cgggggtcgt tgggcggtca gccaggcggg ccatttaccg taagttatgt aacgcggaac
     6481 tccatatatg ggctatgaac taatgacccc gtaattgatt actattaata actagtcaat
     6541 aatcaatgtc ataagctcta atcgcataat cgagctaaga gcgattaata tgcttagcta
     6601 gcttagctaa gctagatttc tctattctcc cacaatatat agcagctatt agcttagcag
     6661 tccgaatacg atcgtccacg atccataagc gtaacacatt tgagtagcga ctgacgggca
     6721 ccggagccac tcgagtggaa ttatcacctc gagtttactc cctatcagtg atagagaacg
     6781 tatgaagagt ttactcccta tcagtgatag agaacgtatg cagactttac tccctatcag
     6841 tgatagagaa cgtataagga gtttactccc tatcagtgat agagaacgta tgaccagttt
     6901 actccctatc agtgatagag aacgtatcta cagtttactc cctatcagtg atagagaacg
     6961 tatatccagt ttactcccta tcagtgatag agaacgtata agcttgccta tataagcaga
     7021 gctcgtttag tgaaccgtca gatcgcctgg agacgccatc cacgctgttt tgacctccat
     7081 agaagacacc gggaccgatc cagcctacca caagtttgta caaaaaagtt ggcaccatga
     7141 aggaggatca agttgtggcg gaagaaccag gattccaaga tgaagaggaa tctttgtttc
     7201 aagatattga cctgttacag aaacatggaa ttaacgtggc tgacattaag aaactgaaat
     7261 cagtaggaat ctgtaccatc aaaggtatac agatgacaac aagaagagct ctatgcaatg
     7321 tcaaaggact ctcagaagcc aaagtagaca agattaaaga ggcagcgaac aaactaattg
     7381 aaccaggatt cttgactgca tttgagtata gtgaaaagag gaaaatggtt ttccatatca
     7441 ccaccgggag ccaggaattt gataagttac taggaggtgg aattgaaagt atggcaatta
     7501 c
//
