LOCUS       sequence-442470-        7579 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac transposon plasmid for doxycycline inducible expression of 
            DMRTC2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7579)
  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 7579)
  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-DMRTC2
FEATURES             Location/Qualifiers
     source          1..7579
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    16..34
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    52..70
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    88..106
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    124..142
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    160..178
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    196..214
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    232..250
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     277..301
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    367..391
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1493..1517)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1519..1545
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1712..1846
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   2118..2152
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2163..2180)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2163..2179)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2172..2194)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2388..2407)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2509..2531
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2570..2588)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2656..2760
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2761..2829,2830..3621)
                     /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(2979..2998)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3792..4380
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4281..4300
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4534..4551
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4757..4778
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4793..4798,4799..4816,4817..4823)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4831..4847
                     /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     4836..4858
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4855..4871
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4855..4871
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4897..4959
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    5135..5216
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5933..5989)
                     /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(5999..6742)
                     /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(6743..6766)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6766..6775
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6795..7006)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        7007..7386
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 tcacctcgag tttactccct atcagtgata gagaacgtat gaagagttta ctccctatca
       61 gtgatagaga acgtatgcag actttactcc ctatcagtga tagagaacgt ataaggagtt
      121 tactccctat cagtgataga gaacgtatga ccagtttact ccctatcagt gatagagaac
      181 gtatctacag tttactccct atcagtgata gagaacgtat atccagttta ctccctatca
      241 gtgatagaga acgtataagc ttgcctatat aagcagagct cgtttagtga accgtcagat
      301 cgcctggaga cgccatccac gctgttttga cctccataga agacaccggg accgatccag
      361 cctaccacaa gtttgtacaa aaaagttggc atggaaccca gtgacatgcc tgctggctac
      421 cactgcccct tagactctgc cccctgggat gagaccagag acccccagag cacagagctg
      481 atccccagga gagccatcag ccgctctcca acctgcgccc gctgccgcaa ccatggtgtc
      541 accgcccatc tcaagggcca caagcgcctc tgcctcttcc aggcttgcga gtgtcacaaa
      601 tgtgtcctca tcctggagcg ccgcagggtc atggctgccc aggtggcctt gcgtaggcag
      661 caggaggcgc agctaaagaa gcacctgatg aggagagggg aagcctctcc caaagctccc
      721 aaccacttca gaaagggaac cactcagcca caggtcccct ctggaaagga gaacatagca
      781 ccccagcctc agacccccca tggggcagtc ctgctggcac cgacaccccc cgggaagaac
      841 tcctgtgggc ctctgctgct cagccatccc ccggaagcct cgcccttgtc ctggactccg
      901 gtgcctcctg gcccttgggt ccctggacac tggctgcctc aaggcttctc catgccacca
      961 ccagtggtgt gccgcctgct gtaccaagaa cctgctgtct ctctgcctcc cttccctggc
     1021 tttgaccctg gcacctccct ccagctgccc actcatgggc ccttcaccac ctgcccagga
     1081 tctcacccag tactgacagc tcctctttct ggagagcccc aagggccccc tagccagccc
     1141 cgcacacact caactctgat actccagccc tgtggcaccc cagaccctct tcagctacag
     1201 ccacaggcct ctggagcctc gtgcctggcc cggacatctg gcccctcaga gtggcagctg
     1261 cagcaagagg cagctgaagc cctcgtgggg ctgaaagatt catcccaggc tcctcgtgtg
     1321 accccttctg tgccccccaa ccctgcctgg atctccctgc ttcacccctg tggcccacca
     1381 gctcctgctg gaggaagagg attccagcct gttggcccct gtcttcgacc cagcccagcc
     1441 ccctctgttg ctctgcatat tggccgtctg gggtccatct ccctcctgag ctacccaact
     1501 ttcttgtaca aagtggtata cccatacgat gttccagatt acgcttagta atgagaaata
     1561 ctcagaagat gtcactcacc atagtccgca gtgaaagata aatgatcttt gtatattccg
     1621 gcaatgtgtt ttagagctag aaatagcaag taggccactg agcaagaact atgaatgaga
     1681 tctttagaca tattgcggcc gctttctccg cgatccagac atgataagat acattgatga
     1741 gtttggacaa accaaaacta gaatgcagtg aaaaaaatgc cttatttgtg aaatttgtga
     1801 tgctattgcc ttatttgtaa ccattataag ctgcaataaa caagttatct ataacaagaa
     1861 aatatatata taataagtta tcacgtaagt agaacatgaa ataacaatat aattatcgta
     1921 tgagttaaat cttaaaagtc acgtaaaaga taatcatgcg tcattttgac tcacgcggtc
     1981 gttatagttc aaaatcagtg acacttaccg cattgacaag cacgcctcac gggagctcca
     2041 agcggcgact gagatgtcct aaatgcacag cgacggattc gcgctattta gaaagagaga
     2101 gcaatatttc aagaatgcat gcgtcaattt tacgcagact atctttctag ggttaagaat
     2161 tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat
     2221 cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat
     2281 cgcccttccc aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg gtattttctc
     2341 cttacgcatc tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct
     2401 gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     2461 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccatgtctcc gggagctgca
     2521 tgtgtcagag gttttcaccg tcatcaccga aacacgcgag ttcgaaaggg cctcgtgata
     2581 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact
     2641 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     2701 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     2761 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     2821 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     2881 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     2941 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     3001 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     3061 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     3121 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     3181 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     3241 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     3301 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     3361 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     3421 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtggttca
     3481 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     3541 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     3601 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     3661 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     3721 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     3781 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     3841 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     3901 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     3961 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     4021 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     4081 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     4141 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     4201 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     4261 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     4321 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     4381 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     4441 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     4501 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggat
     4561 gagcgcccaa tacgcaaacc gcctctcacc gcgcgttggc cgattcatta atgcagctgg
     4621 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaaaccg cctctccccg
     4681 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
     4741 gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact
     4801 ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa
     4861 acagctatga ccatgattac gccaaggtcg acttaaccct agaaagataa tcatattgtg
     4921 acgtacgtta aagataatca tgcgtaaaat tgacgcatgt gttttatcgg tctgtatatc
     4981 gaggtttatt tattaatttg aatagatatt aagttttatt atatttacac ttacatacta
     5041 ataataaatt caacaaacaa tttatttatg tttatttatt tattaaaaaa aaacaaaaac
     5101 tcaaaatttc ttctataaag taacaaaact tttagcagtg aaaaaaatgc tttatttgtg
     5161 aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca
     5221 acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaaa
     5281 gcaagtaaaa cctctacaaa tgtggtatgg ctgattatga tcctctggag attcatgcgc
     5341 caggttttct ggtcatgcac cacgttcgtg ggccctcagg aacctccacg tcagcggtaa
     5401 ctgtgaagcc cagtcgctcg taaaaaggaa gattccgggg agcactggtt tcgaggaacg
     5461 ccggcacgcc agcccgttcc gcggcctcaa caccaggaag aacgacagcg cttcccaaac
     5521 ctttaccttg gtgatctggg ctgacaccga cagtggcaag aaaccacgca ggctcctttg
     5581 gtctatgagg agccagcaag ccctccattt gctgttgtgc cgccaaccta ctccctgaca
     5641 gctcggccat ccgcggacct atttccgcga acactgcgcc ggcttcgacg ctttccggag
     5701 tagtccagac cgcaactgcg gcaccatcgt ccgcaaccca aactttacca atgtccagac
     5761 ccacgcgcgt aagaaacagc tcttgcaact cagttaccct ctctatgtgt ctgtccggat
     5821 ctacggtatg gcgggtcgct ggataatctg caaatgctgc ggcaagtgtt cggacagccc
     5881 gtggtacatc gtcacgggta gcgagcctaa cagtgggctt atattccgtc atggggccgg
     5941 ggttctcctc cacgtcgccg gcctgcttca gcaggctgaa gttggtggcg ccgctgcccc
     6001 cggggagcat gtcaaggtca aaatcgtcaa gagcgtcagc aggcagcata tcaaggtcaa
     6061 agtcgtcaag ggcatcggct gggagcatgt ctaagtcaaa atcgtcaagg gcgtcggtcg
     6121 gcccgccgct ttcgcacttt agctgtttct ccaggccaca tatgattagt tccaggccga
     6181 aaaggaaggc aggttcggct ccctgccggt cgaacagctc aattgcttgt ttcagaagtg
     6241 ggggcataga atcggtggta ggtgtctctc tttcctcttt tgctacttga tgctcctgtt
     6301 cctccaatac gcagcccagt gtaaagtggc ccacggcgga cagagcgtac agtgcgttct
     6361 ccagggagaa gccttgctga cacaggaacg cgagctgatt ttccagggtt tcgtactgtt
     6421 tctctgttgg gcgggtgccg agatgcactt tagccccgtc acgatgtgag aggagagcac
     6481 agcggtatga cttggcgttg ttccgcagaa agtcttgcca tgactcgcct tccagggggc
     6541 aggagtgggt atgatgcctg tccagcatct cgattggcag ggcatcgagc agggcccgct
     6601 tgttcttcac gtgccagtac agggtaggct gctcaactcc cagcttttga gcgagtttcc
     6661 ttgtcgtcag gccttcgata ccgactccat tgagtaattc cagagcagag tttatgactt
     6721 tgctcttgtc cagtctagac atcttatcgt catcgtcttt gtaatccatg gtggcggatc
     6781 ccgcgtcacg acacctgtgt tctggcggca aacccgttgc gaaaaagaac gttcacggcg
     6841 actactgcac ttatatacgg ttctccccca ccctcgggaa aaaggcggag ccagtacacg
     6901 acatcacttt cccagtttac cccgcgccac cttctctagg caccggttca attgccgacc
     6961 cctcccccca acttctcggg cactgtgggc catgtgcgct ctgccccatg gtaatagcca
     7021 tgactaatac gtagatgtac tgccaagtag gaaagtgcca taaggtcatg tactgggcat
     7081 aatgccaggc gggccattta ccgtcattga cgtcaatagg gggcgtactt ggcatatgat
     7141 acacttgatg tactgccaag tgggcagttt accgtaaata ctccacccat tgacgtcaat
     7201 ggaaagtgcc tattggcgtt actatgggaa catacgtcat tattgacgtc aatgggcggg
     7261 ggtcgttggg cggtcagcca ggcgggccat ttaccgtaag ttatgtaacg cggaactcca
     7321 tatatgggct atgaactaat gaccccgtaa ttgattacta ttaataacta gtcaataatc
     7381 aatgtcataa gctctaatcg cataatcgag ctaagagcga ttaatatgct tagctagctt
     7441 agctaagcta gatttctcta ttctcccaca atatatagca gctattagct tagcagtccg
     7501 aatacgatcg tccacgatcc ataagcgtaa cacatttgag tagcgactga cgggcaccgg
     7561 agccactcga gtggaatta
//
