LOCUS       sequence-442471-        7951 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac transposon plasmid for doxycycline inducible expression of 
            HDAC2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7951)
  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 7951)
  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-HDAC2
FEATURES             Location/Qualifiers
     source          1..7951
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    17..35
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    53..71
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    89..107
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    125..143
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    161..179
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    197..215
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    233..251
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     278..302
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    368..392
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      393..402
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(1866..1890)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1892..1918
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    2085..2219
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   2491..2525
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2536..2553)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2536..2552)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2545..2567)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2761..2780)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2882..2904
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2943..2961)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3029..3133
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3134..3202,3203..3994)
                     /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(3352..3371)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4165..4753
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4654..4673
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4907..4924
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5130..5151
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5166..5171,5172..5189,5190..5196)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5204..5220
                     /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     5209..5231
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5228..5244
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5228..5244
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   5270..5332
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    5508..5589
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(6306..6362)
                     /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(6372..7115)
                     /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(7116..7139)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      7139..7148
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(7168..7379)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        7380..7759
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 atcacctcga gtttactccc tatcagtgat agagaacgta tgaagagttt actccctatc
       61 agtgatagag aacgtatgca gactttactc cctatcagtg atagagaacg tataaggagt
      121 ttactcccta tcagtgatag agaacgtatg accagtttac tccctatcag tgatagagaa
      181 cgtatctaca gtttactccc tatcagtgat agagaacgta tatccagttt actccctatc
      241 agtgatagag aacgtataag cttgcctata taagcagagc tcgtttagtg aaccgtcaga
      301 tcgcctggag acgccatcca cgctgttttg acctccatag aagacaccgg gaccgatcca
      361 gcctaccaca agtttgtaca aaaaagcagg ctgccaccat ggcgtacagt caaggaggcg
      421 gcaaaaaaaa agtctgctac tactacgacg gtgatattgg aaattattat tatggacagg
      481 gtcatcccat gaagcctcat agaatccgca tgacccataa cttgctgtta aattatggct
      541 tatacagaaa aatggaaata tataggcccc ataaagccac tgccgaagaa atgacaaaat
      601 atcacagtga tgagtatatc aaatttctac ggtcaataag accagataac atgtctgagt
      661 atagtaagca gatgcagaga tttaatgttg gagaagattg tccagtgttt gatggactct
      721 ttgagttttg tcagctctca actggcggtt cagttgctgg agctgtgaag ttaaaccgac
      781 aacagactga tatggctgtt aattgggctg gaggattaca tcatgctaag aaatcagaag
      841 catcaggatt ctgttacgtt aatgatattg tgcttgccat ccttgaatta ctaaagtatc
      901 atcagagagt cttatatatt gatatagata ttcatcatgg tgatggtgtt gaagaagctt
      961 tttatacaac agatcgtgta atgacggtat cattccataa atatggggaa tactttcctg
     1021 gcacaggaga cttgagggat attggtgctg gaaaaggcaa atactatgct gtcaattttc
     1081 caatgagaga tggtatagat gatgagtcat atgggcagat atttaagcct attatctcaa
     1141 aggtgatgga gatgtatcaa cctagtgctg tggtattaca gtgtggtgca gactcattat
     1201 ctggtgatag actgggttgt ttcaatctaa cagtcaaagg tcatgctaaa tgtgtagaag
     1261 ttgtaaaaac ttttaactta ccattactga tgcttggagg aggtggctac acaatccgta
     1321 atgttgctcg atgttggaca tatgagactg cagttgccct tgattgtgag attcccaatg
     1381 agttgccata taatgattac tttgagtatt ttggaccaga cttcaaactg catattagtc
     1441 cttcaaacat gacaaaccag aacactccag aatatatgga aaagataaaa cagcgtttgt
     1501 ttgaaaattt gcgcatgtta cctcatgcac ctggtgtcca gatgcaagct attccagaag
     1561 atgctgttca tgaagacagt ggagatgaag atggagaaga tccagacaag agaatttcta
     1621 ttcgagcatc agacaagcgg atagcttgtg atgaagaatt ctcagattct gaggatgaag
     1681 gagaaggagg tcgaagaaat gtggctgatc ataagaaagg agcaaagaaa gctagaattg
     1741 aagaagataa gaaagaaaca gaggacaaaa aaacagacgt taaggaagaa gataaatcca
     1801 aggacaacag tggtgaaaaa acagatacca aaggaaccaa atcagaacag ctcagcaacc
     1861 cctgaaccca gctttcttgt acaaagtggt atacccatac gatgttccag attacgctta
     1921 gtaatgagaa atactcagaa gatgtcactc accatagtcc gcagtgaaag ataaatgatc
     1981 atgcaatgct aagttggaga gttttagagc tagaaatagc aagtaggcca ctgagcaaga
     2041 actatgaatg agatctttag acatattgcg gccgctttct ccgcgatcca gacatgataa
     2101 gatacattga tgagtttgga caaaccaaaa ctagaatgca gtgaaaaaaa tgccttattt
     2161 gtgaaatttg tgatgctatt gccttatttg taaccattat aagctgcaat aaacaagtta
     2221 tctataacaa gaaaatatat atataataag ttatcacgta agtagaacat gaaataacaa
     2281 tataattatc gtatgagtta aatcttaaaa gtcacgtaaa agataatcat gcgtcatttt
     2341 gactcacgcg gtcgttatag ttcaaaatca gtgacactta ccgcattgac aagcacgcct
     2401 cacgggagct ccaagcggcg actgagatgt cctaaatgca cagcgacgga ttcgcgctat
     2461 ttagaaagag agagcaatat ttcaagaatg catgcgtcaa ttttacgcag actatctttc
     2521 tagggttaag aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
     2581 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga
     2641 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat
     2701 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag
     2761 tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga
     2821 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccatgtc
     2881 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacacgc gagttcgaaa
     2941 gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac
     3001 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     3061 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     3121 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     3181 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     3241 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     3301 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     3361 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     3421 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     3481 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     3541 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     3601 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     3661 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     3721 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     3781 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     3841 tgagcgtggt tcacgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     3901 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     3961 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     4021 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     4081 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     4141 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     4201 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     4261 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     4321 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     4381 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     4441 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     4501 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     4561 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     4621 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     4681 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     4741 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     4801 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     4861 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     4921 cgaggaagcg gatgagcgcc caatacgcaa accgcctctc accgcgcgtt ggccgattca
     4981 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaaa
     5041 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
     5101 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc
     5161 caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa
     5221 tttcacacag gaaacagcta tgaccatgat tacgccaagg tcgacttaac cctagaaaga
     5281 taatcatatt gtgacgtacg ttaaagataa tcatgcgtaa aattgacgca tgtgttttat
     5341 cggtctgtat atcgaggttt atttattaat ttgaatagat attaagtttt attatattta
     5401 cacttacata ctaataataa attcaacaaa caatttattt atgtttattt atttattaaa
     5461 aaaaaacaaa aactcaaaat ttcttctata aagtaacaaa acttttagca gtgaaaaaaa
     5521 tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat
     5581 aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg
     5641 gaggtttttt aaagcaagta aaacctctac aaatgtggta tggctgatta tgatcctctg
     5701 gagattcatg cgccaggttt tctggtcatg caccacgttc gtgggccctc aggaacctcc
     5761 acgtcagcgg taactgtgaa gcccagtcgc tcgtaaaaag gaagattccg gggagcactg
     5821 gtttcgagga acgccggcac gccagcccgt tccgcggcct caacaccagg aagaacgaca
     5881 gcgcttccca aacctttacc ttggtgatct gggctgacac cgacagtggc aagaaaccac
     5941 gcaggctcct ttggtctatg aggagccagc aagccctcca tttgctgttg tgccgccaac
     6001 ctactccctg acagctcggc catccgcgga cctatttccg cgaacactgc gccggcttcg
     6061 acgctttccg gagtagtcca gaccgcaact gcggcaccat cgtccgcaac ccaaacttta
     6121 ccaatgtcca gacccacgcg cgtaagaaac agctcttgca actcagttac cctctctatg
     6181 tgtctgtccg gatctacggt atggcgggtc gctggataat ctgcaaatgc tgcggcaagt
     6241 gttcggacag cccgtggtac atcgtcacgg gtagcgagcc taacagtggg cttatattcc
     6301 gtcatggggc cggggttctc ctccacgtcg ccggcctgct tcagcaggct gaagttggtg
     6361 gcgccgctgc ccccggggag catgtcaagg tcaaaatcgt caagagcgtc agcaggcagc
     6421 atatcaaggt caaagtcgtc aagggcatcg gctgggagca tgtctaagtc aaaatcgtca
     6481 agggcgtcgg tcggcccgcc gctttcgcac tttagctgtt tctccaggcc acatatgatt
     6541 agttccaggc cgaaaaggaa ggcaggttcg gctccctgcc ggtcgaacag ctcaattgct
     6601 tgtttcagaa gtgggggcat agaatcggtg gtaggtgtct ctctttcctc ttttgctact
     6661 tgatgctcct gttcctccaa tacgcagccc agtgtaaagt ggcccacggc ggacagagcg
     6721 tacagtgcgt tctccaggga gaagccttgc tgacacagga acgcgagctg attttccagg
     6781 gtttcgtact gtttctctgt tgggcgggtg ccgagatgca ctttagcccc gtcacgatgt
     6841 gagaggagag cacagcggta tgacttggcg ttgttccgca gaaagtcttg ccatgactcg
     6901 ccttccaggg ggcaggagtg ggtatgatgc ctgtccagca tctcgattgg cagggcatcg
     6961 agcagggccc gcttgttctt cacgtgccag tacagggtag gctgctcaac tcccagcttt
     7021 tgagcgagtt tccttgtcgt caggccttcg ataccgactc cattgagtaa ttccagagca
     7081 gagtttatga ctttgctctt gtccagtcta gacatcttat cgtcatcgtc tttgtaatcc
     7141 atggtggcgg atcccgcgtc acgacacctg tgttctggcg gcaaacccgt tgcgaaaaag
     7201 aacgttcacg gcgactactg cacttatata cggttctccc ccaccctcgg gaaaaaggcg
     7261 gagccagtac acgacatcac tttcccagtt taccccgcgc caccttctct aggcaccggt
     7321 tcaattgccg acccctcccc ccaacttctc gggcactgtg ggccatgtgc gctctgcccc
     7381 atggtaatag ccatgactaa tacgtagatg tactgccaag taggaaagtg ccataaggtc
     7441 atgtactggg cataatgcca ggcgggccat ttaccgtcat tgacgtcaat agggggcgta
     7501 cttggcatat gatacacttg atgtactgcc aagtgggcag tttaccgtaa atactccacc
     7561 cattgacgtc aatggaaagt gcctattggc gttactatgg gaacatacgt cattattgac
     7621 gtcaatgggc gggggtcgtt gggcggtcag ccaggcgggc catttaccgt aagttatgta
     7681 acgcggaact ccatatatgg gctatgaact aatgaccccg taattgatta ctattaataa
     7741 ctagtcaata atcaatgtca taagctctaa tcgcataatc gagctaagag cgattaatat
     7801 gcttagctag cttagctaag ctagatttct ctattctccc acaatatata gcagctatta
     7861 gcttagcagt ccgaatacga tcgtccacga tccataagcg taacacattt gagtagcgac
     7921 tgacgggcac cggagccact cgagtggaat t
//
