LOCUS       sequence-442473-        8170 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac transposon plasmid for doxycycline inducible expression of 
            SMAD4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8170)
  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 8170)
  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-SMAD4
FEATURES             Location/Qualifiers
     source          1..8170
                     /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"
     CDS             401..427
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    complement(2085..2109)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2111..2137
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    2304..2438
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   2710..2744
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2755..2772)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2755..2771)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2764..2786)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2980..2999)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3101..3123
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3162..3180)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3248..3352
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3353..3421,3422..4213)
                     /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(3571..3590)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4384..4972
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4873..4892
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5126..5143
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5349..5370
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5385..5390,5391..5408,5409..5415)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5423..5439
                     /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     5428..5450
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5447..5463
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5447..5463
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   5489..5551
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    5727..5808
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(6525..6581)
                     /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(6591..7334)
                     /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(7335..7358)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      7358..7367
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(7387..7598)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        7599..7978
                     /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 ctgcaccatg tacccctatg atgtgccaga
      421 ctatgccgat aacatgagta tcaccaacac tcctacgtct aacgatgcat gtctgagtat
      481 agtgcattct ctcatgtgcc atcggcaggg gggtgagtcc gagacgttcg ccaaacgggc
      541 aattgaatcc ctcgtgaaga aacttaagga aaagaaggat gagctcgatt cactcatcac
      601 ggctataaca actaacggag cccatccgtc taagtgtgtc accatacagc gcaccctgga
      661 cggccggctt caggtcgccg gccgaaaagg ctttccacac gtgatttatg ccaggctgtg
      721 gagatggccc gaccttcaca agaatgagct caaacacgtg aaatactgtc agtatgcttt
      781 cgaccttaaa tgcgactccg tgtgcgtcaa tccataccat tatgaaaggg tggtgagtcc
      841 agggatcgac ctttccggcc tgacactcca gtccaatgcc ccgtcatcaa tgatggtgaa
      901 ggacgagtat gtgcatgatt ttgaaggaca accctccctg tcaacagagg gacactctat
      961 acaaacaatc cagcatcccc caagcaacag ggcctcaacc gagacatatt ctacccccgc
     1021 cctcctggct ccctccgaga gcaatgccac atctaccgct aacttcccta acatccctgt
     1081 cgccagtact tcacagcctg ccagcatact cggtggctca cattccgaag gcctgctcca
     1141 gattgccagc gggcctcaac cagggcagca gcagaatggg tttaccggcc agcctgccac
     1201 ctaccaccac aactccacaa caacttggac gggcagtaga actgctccct acacccctaa
     1261 cctgcctcac caccagaacg gccatctcca gcatcaccct ccgatgcccc ctcacccagg
     1321 gcactactgg ccagttcata acgagttggc attccagccc cccatttcca atcatccagc
     1381 tccagagtac tggtgtagca tcgcatattt cgagatggat gtgcaagtag gcgaaacatt
     1441 taaagtccct tcttcatgcc ctatcgtcac agtggacggc tatgtcgatc catccggggg
     1501 cgatcgcttc tgccttgggc agctgtccaa cgtacataga accgaggcca tcgaacgcgc
     1561 ccgcctgcac attggcaagg gcgtgcaact ggagtgcaaa ggagaggggg atgtgtgggt
     1621 gcgatgcctt agtgatcacg ccgtgtttgt ccaatcctat tatctcgata gggaagctgg
     1681 tcgggcaccg ggggatgccg tacacaagat ctatccatcc gcatatatca aagtctttga
     1741 tctgagacag tgccatcgcc aaatgcagca gcaggctgcc accgcacagg cggcagccgc
     1801 tgcgcaagca gcagctgtgg cggggaacat tccagggcct ggcagcgtgg gaggcatcgc
     1861 tcctgccatc tcactgtcag ccgccgctgg tatcggagtg gacgacctgc gacgactctg
     1921 tatcctgaga atgagtttcg taaaggggtg gggcccagat taccccagac agtccatcaa
     1981 agagacccct tgctggattg aaatccacct gcacagggcc ctccagctct tggatgaagt
     2041 actgcacact atgcctatcg cagatcctca accccttgac taagacccag ctttcttgta
     2101 caaagtggta tacccatacg atgttccaga ttacgcttag taatgagaaa tactcagaag
     2161 atgtcactca ccatagtccg cagtgaaaga taaatgatct tcatatcgat ctttagcgcg
     2221 ttttagagct agaaatagca agtaggccac tgagcaagaa ctatgaatga gatctttaga
     2281 catattgcgg ccgctttctc cgcgatccag acatgataag atacattgat gagtttggac
     2341 aaaccaaaac tagaatgcag tgaaaaaaat gccttatttg tgaaatttgt gatgctattg
     2401 ccttatttgt aaccattata agctgcaata aacaagttat ctataacaag aaaatatata
     2461 tataataagt tatcacgtaa gtagaacatg aaataacaat ataattatcg tatgagttaa
     2521 atcttaaaag tcacgtaaaa gataatcatg cgtcattttg actcacgcgg tcgttatagt
     2581 tcaaaatcag tgacacttac cgcattgaca agcacgcctc acgggagctc caagcggcga
     2641 ctgagatgtc ctaaatgcac agcgacggat tcgcgctatt tagaaagaga gagcaatatt
     2701 tcaagaatgc atgcgtcaat tttacgcaga ctatctttct agggttaaga attcactggc
     2761 cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc
     2821 agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc
     2881 ccaacagttg cgcagcctga atggcgaatg gcgcctgatg cggtattttc tccttacgca
     2941 tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct ctgatgccgc
     3001 atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     3061 gctcccggca tccgcttaca gacaagctgt gaccatgtct ccgggagctg catgtgtcag
     3121 aggttttcac cgtcatcacc gaaacacgcg agttcgaaag ggcctcgtga tacgcctatt
     3181 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg
     3241 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     3301 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     3361 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     3421 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     3481 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     3541 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     3601 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     3661 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     3721 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     3781 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     3841 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     3901 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     3961 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     4021 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtggtt cacgcggtat
     4081 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     4141 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     4201 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     4261 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     4321 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     4381 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     4441 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     4501 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     4561 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     4621 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     4681 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     4741 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     4801 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     4861 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     4921 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     4981 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     5041 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     5101 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg atgagcgccc
     5161 aatacgcaaa ccgcctctca ccgcgcgttg gccgattcat taatgcagct ggcacgacag
     5221 gtttcccgac tggaaagcgg gcagtgagcg caacgcaaac cgcctctccc cgcgcgttgg
     5281 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
     5341 aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt
     5401 ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat
     5461 gaccatgatt acgccaaggt cgacttaacc ctagaaagat aatcatattg tgacgtacgt
     5521 taaagataat catgcgtaaa attgacgcat gtgttttatc ggtctgtata tcgaggttta
     5581 tttattaatt tgaatagata ttaagtttta ttatatttac acttacatac taataataaa
     5641 ttcaacaaac aatttattta tgtttattta tttattaaaa aaaaacaaaa actcaaaatt
     5701 tcttctataa agtaacaaaa cttttagcag tgaaaaaaat gctttatttg tgaaatttgt
     5761 gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat
     5821 tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta aagcaagtaa
     5881 aacctctaca aatgtggtat ggctgattat gatcctctgg agattcatgc gccaggtttt
     5941 ctggtcatgc accacgttcg tgggccctca ggaacctcca cgtcagcggt aactgtgaag
     6001 cccagtcgct cgtaaaaagg aagattccgg ggagcactgg tttcgaggaa cgccggcacg
     6061 ccagcccgtt ccgcggcctc aacaccagga agaacgacag cgcttcccaa acctttacct
     6121 tggtgatctg ggctgacacc gacagtggca agaaaccacg caggctcctt tggtctatga
     6181 ggagccagca agccctccat ttgctgttgt gccgccaacc tactccctga cagctcggcc
     6241 atccgcggac ctatttccgc gaacactgcg ccggcttcga cgctttccgg agtagtccag
     6301 accgcaactg cggcaccatc gtccgcaacc caaactttac caatgtccag acccacgcgc
     6361 gtaagaaaca gctcttgcaa ctcagttacc ctctctatgt gtctgtccgg atctacggta
     6421 tggcgggtcg ctggataatc tgcaaatgct gcggcaagtg ttcggacagc ccgtggtaca
     6481 tcgtcacggg tagcgagcct aacagtgggc ttatattccg tcatggggcc ggggttctcc
     6541 tccacgtcgc cggcctgctt cagcaggctg aagttggtgg cgccgctgcc cccggggagc
     6601 atgtcaaggt caaaatcgtc aagagcgtca gcaggcagca tatcaaggtc aaagtcgtca
     6661 agggcatcgg ctgggagcat gtctaagtca aaatcgtcaa gggcgtcggt cggcccgccg
     6721 ctttcgcact ttagctgttt ctccaggcca catatgatta gttccaggcc gaaaaggaag
     6781 gcaggttcgg ctccctgccg gtcgaacagc tcaattgctt gtttcagaag tgggggcata
     6841 gaatcggtgg taggtgtctc tctttcctct tttgctactt gatgctcctg ttcctccaat
     6901 acgcagccca gtgtaaagtg gcccacggcg gacagagcgt acagtgcgtt ctccagggag
     6961 aagccttgct gacacaggaa cgcgagctga ttttccaggg tttcgtactg tttctctgtt
     7021 gggcgggtgc cgagatgcac tttagccccg tcacgatgtg agaggagagc acagcggtat
     7081 gacttggcgt tgttccgcag aaagtcttgc catgactcgc cttccagggg gcaggagtgg
     7141 gtatgatgcc tgtccagcat ctcgattggc agggcatcga gcagggcccg cttgttcttc
     7201 acgtgccagt acagggtagg ctgctcaact cccagctttt gagcgagttt ccttgtcgtc
     7261 aggccttcga taccgactcc attgagtaat tccagagcag agtttatgac tttgctcttg
     7321 tccagtctag acatcttatc gtcatcgtct ttgtaatcca tggtggcgga tcccgcgtca
     7381 cgacacctgt gttctggcgg caaacccgtt gcgaaaaaga acgttcacgg cgactactgc
     7441 acttatatac ggttctcccc caccctcggg aaaaaggcgg agccagtaca cgacatcact
     7501 ttcccagttt accccgcgcc accttctcta ggcaccggtt caattgccga cccctccccc
     7561 caacttctcg ggcactgtgg gccatgtgcg ctctgcccca tggtaatagc catgactaat
     7621 acgtagatgt actgccaagt aggaaagtgc cataaggtca tgtactgggc ataatgccag
     7681 gcgggccatt taccgtcatt gacgtcaata gggggcgtac ttggcatatg atacacttga
     7741 tgtactgcca agtgggcagt ttaccgtaaa tactccaccc attgacgtca atggaaagtg
     7801 cctattggcg ttactatggg aacatacgtc attattgacg tcaatgggcg ggggtcgttg
     7861 ggcggtcagc caggcgggcc atttaccgta agttatgtaa cgcggaactc catatatggg
     7921 ctatgaacta atgaccccgt aattgattac tattaataac tagtcaataa tcaatgtcat
     7981 aagctctaat cgcataatcg agctaagagc gattaatatg cttagctagc ttagctaagc
     8041 tagatttctc tattctccca caatatatag cagctattag cttagcagtc cgaatacgat
     8101 cgtccacgat ccataagcgt aacacatttg agtagcgact gacgggcacc ggagccactc
     8161 gagtggaatt
//
