LOCUS       sequence-442456-        6964 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac transposon plasmid for doxycycline inducible expression of 
            DPPA3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6964)
  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 6964)
  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-DPPA3
FEATURES             Location/Qualifiers
     source          1..6964
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      142..730
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     631..650
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     884..901
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1107..1128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1143..1148,1149..1166,1167..1173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1181..1197
                     /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     1186..1208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1205..1221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1205..1221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   1247..1309
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    1485..1566
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(2283..2339)
                     /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(2349..3092)
                     /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(3093..3116)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      3116..3125
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(3145..3356)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        3357..3736
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     protein_bind    3945..3963
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3981..3999
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4017..4035
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4053..4071
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4089..4107
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4125..4143
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4161..4179
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     4206..4230
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    4296..4320
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(4807..4831)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             4833..4859
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    5026..5160
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   5432..5466
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5477..5494)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5477..5493)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5486..5508)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(5702..5721)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5823..5845
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5884..5902)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5970..6074
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6075..6143,6144..6935)
                     /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(6293..6312)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
       61 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
      121 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
      181 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
      241 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
      301 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
      361 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
      421 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
      481 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
      541 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
      601 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
      661 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
      721 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
      781 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
      841 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
      901 ggaagcggat gagcgcccaa tacgcaaacc gcctctcacc gcgcgttggc cgattcatta
      961 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaaaccg
     1021 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
     1081 aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag
     1141 gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt
     1201 cacacaggaa acagctatga ccatgattac gccaaggtcg acttaaccct agaaagataa
     1261 tcatattgtg acgtacgtta aagataatca tgcgtaaaat tgacgcatgt gttttatcgg
     1321 tctgtatatc gaggtttatt tattaatttg aatagatatt aagttttatt atatttacac
     1381 ttacatacta ataataaatt caacaaacaa tttatttatg tttatttatt tattaaaaaa
     1441 aaacaaaaac tcaaaatttc ttctataaag taacaaaact tttagcagtg aaaaaaatgc
     1501 tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa
     1561 caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag
     1621 gttttttaaa gcaagtaaaa cctctacaaa tgtggtatgg ctgattatga tcctctggag
     1681 attcatgcgc caggttttct ggtcatgcac cacgttcgtg ggccctcagg aacctccacg
     1741 tcagcggtaa ctgtgaagcc cagtcgctcg taaaaaggaa gattccgggg agcactggtt
     1801 tcgaggaacg ccggcacgcc agcccgttcc gcggcctcaa caccaggaag aacgacagcg
     1861 cttcccaaac ctttaccttg gtgatctggg ctgacaccga cagtggcaag aaaccacgca
     1921 ggctcctttg gtctatgagg agccagcaag ccctccattt gctgttgtgc cgccaaccta
     1981 ctccctgaca gctcggccat ccgcggacct atttccgcga acactgcgcc ggcttcgacg
     2041 ctttccggag tagtccagac cgcaactgcg gcaccatcgt ccgcaaccca aactttacca
     2101 atgtccagac ccacgcgcgt aagaaacagc tcttgcaact cagttaccct ctctatgtgt
     2161 ctgtccggat ctacggtatg gcgggtcgct ggataatctg caaatgctgc ggcaagtgtt
     2221 cggacagccc gtggtacatc gtcacgggta gcgagcctaa cagtgggctt atattccgtc
     2281 atggggccgg ggttctcctc cacgtcgccg gcctgcttca gcaggctgaa gttggtggcg
     2341 ccgctgcccc cggggagcat gtcaaggtca aaatcgtcaa gagcgtcagc aggcagcata
     2401 tcaaggtcaa agtcgtcaag ggcatcggct gggagcatgt ctaagtcaaa atcgtcaagg
     2461 gcgtcggtcg gcccgccgct ttcgcacttt agctgtttct ccaggccaca tatgattagt
     2521 tccaggccga aaaggaaggc aggttcggct ccctgccggt cgaacagctc aattgcttgt
     2581 ttcagaagtg ggggcataga atcggtggta ggtgtctctc tttcctcttt tgctacttga
     2641 tgctcctgtt cctccaatac gcagcccagt gtaaagtggc ccacggcgga cagagcgtac
     2701 agtgcgttct ccagggagaa gccttgctga cacaggaacg cgagctgatt ttccagggtt
     2761 tcgtactgtt tctctgttgg gcgggtgccg agatgcactt tagccccgtc acgatgtgag
     2821 aggagagcac agcggtatga cttggcgttg ttccgcagaa agtcttgcca tgactcgcct
     2881 tccagggggc aggagtgggt atgatgcctg tccagcatct cgattggcag ggcatcgagc
     2941 agggcccgct tgttcttcac gtgccagtac agggtaggct gctcaactcc cagcttttga
     3001 gcgagtttcc ttgtcgtcag gccttcgata ccgactccat tgagtaattc cagagcagag
     3061 tttatgactt tgctcttgtc cagtctagac atcttatcgt catcgtcttt gtaatccatg
     3121 gtggcggatc ccgcgtcacg acacctgtgt tctggcggca aacccgttgc gaaaaagaac
     3181 gttcacggcg actactgcac ttatatacgg ttctccccca ccctcgggaa aaaggcggag
     3241 ccagtacacg acatcacttt cccagtttac cccgcgccac cttctctagg caccggttca
     3301 attgccgacc cctcccccca acttctcggg cactgtgggc catgtgcgct ctgccccatg
     3361 gtaatagcca tgactaatac gtagatgtac tgccaagtag gaaagtgcca taaggtcatg
     3421 tactgggcat aatgccaggc gggccattta ccgtcattga cgtcaatagg gggcgtactt
     3481 ggcatatgat acacttgatg tactgccaag tgggcagttt accgtaaata ctccacccat
     3541 tgacgtcaat ggaaagtgcc tattggcgtt actatgggaa catacgtcat tattgacgtc
     3601 aatgggcggg ggtcgttggg cggtcagcca ggcgggccat ttaccgtaag ttatgtaacg
     3661 cggaactcca tatatgggct atgaactaat gaccccgtaa ttgattacta ttaataacta
     3721 gtcaataatc aatgtcataa gctctaatcg cataatcgag ctaagagcga ttaatatgct
     3781 tagctagctt agctaagcta gatttctcta ttctcccaca atatatagca gctattagct
     3841 tagcagtccg aatacgatcg tccacgatcc ataagcgtaa cacatttgag tagcgactga
     3901 cgggcaccgg agccactcga gtggaattat cacctcgagt ttactcccta tcagtgatag
     3961 agaacgtatg aagagtttac tccctatcag tgatagagaa cgtatgcaga ctttactccc
     4021 tatcagtgat agagaacgta taaggagttt actccctatc agtgatagag aacgtatgac
     4081 cagtttactc cctatcagtg atagagaacg tatctacagt ttactcccta tcagtgatag
     4141 agaacgtata tccagtttac tccctatcag tgatagagaa cgtataagct tgcctatata
     4201 agcagagctc gtttagtgaa ccgtcagatc gcctggagac gccatccacg ctgttttgac
     4261 ctccatagaa gacaccggga ccgatccagc ctaccacaag tttgtacaaa aaagcaggct
     4321 gcaccatgga cccatcacag tttaatccaa cctacatccc agggtctcca caaatgctca
     4381 ccgaagaaaa ttcccgggac gattcagggg cctctcaaat ctcctccgag acgttgataa
     4441 agaaccttag taacttgact atcaacgcta gtagcgaatc tgtttcccct ctatcggaag
     4501 ctttactccg tcgagagtct gtaggagcag cagtcctcag ggaaatcgaa gatgagtggc
     4561 tttacagcag gagaggagta agaacattgc tgtctgtgca gagagaaaag atggcaagat
     4621 tgagatacat gttactcggc ggagttcgta cgcatgaaag aagaccaaca aacaaggagc
     4681 ctaagggagt taagaaggaa tcaagaccat tcaaatgtcc ctgcagtttc tgcgtgtcta
     4741 atggatggga tccttctgag aatgctagaa tagggaatca agacaccaag ccacttcagc
     4801 cataagaccc agctttcttg tacaaagtgg tatacccata cgatgttcca gattacgctt
     4861 agtaatgaga aatactcaga agatgtcact caccatagtc cgcagtgaaa gataaatgat
     4921 cgatagcatg acttatgaaa tgttttagag ctagaaatag caagtaggcc actgagcaag
     4981 aactatgaat gagatcttta gacatattgc ggccgctttc tccgcgatcc agacatgata
     5041 agatacattg atgagtttgg acaaaccaaa actagaatgc agtgaaaaaa atgccttatt
     5101 tgtgaaattt gtgatgctat tgccttattt gtaaccatta taagctgcaa taaacaagtt
     5161 atctataaca agaaaatata tatataataa gttatcacgt aagtagaaca tgaaataaca
     5221 atataattat cgtatgagtt aaatcttaaa agtcacgtaa aagataatca tgcgtcattt
     5281 tgactcacgc ggtcgttata gttcaaaatc agtgacactt accgcattga caagcacgcc
     5341 tcacgggagc tccaagcggc gactgagatg tcctaaatgc acagcgacgg attcgcgcta
     5401 tttagaaaga gagagcaata tttcaagaat gcatgcgtca attttacgca gactatcttt
     5461 ctagggttaa gaattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg
     5521 ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag
     5581 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga
     5641 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatatgg tgcactctca
     5701 gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg
     5761 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccatgt
     5821 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacacg cgagttcgaa
     5881 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     5941 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     6001 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     6061 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     6121 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     6181 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     6241 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     6301 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     6361 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     6421 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     6481 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     6541 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     6601 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     6661 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     6721 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     6781 gtgagcgtgg ttcacgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     6841 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     6901 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     6961 tact
//
