LOCUS       sequence-439855-        7960 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac transposon plasmid for doxycycline inducible expression of 
            3xFLAG-SMAD9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7960)
  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 7960)
  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-cERP2-3xFLAG-SMAD9-active
FEATURES             Location/Qualifiers
     source          1..7960
                     /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..472
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
     protein_bind    complement(1875..1899)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1901..1927
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    2094..2228
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   2500..2534
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2545..2562)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2545..2561)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2554..2576)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2770..2789)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2891..2913
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2952..2970)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3038..3142
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3143..3211,3212..4003)
                     /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(3361..3380)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4174..4762
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4663..4682
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4916..4933
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5139..5160
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5175..5180,5181..5198,5199..5205)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5213..5229
                     /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     5218..5240
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5237..5253
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5237..5253
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   5279..5341
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    5517..5598
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(6315..6371)
                     /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(6381..7124)
                     /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(7125..7148)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      7148..7157
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(7177..7388)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        7389..7768
                     /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 gattataagg acgatgatga
      421 caaggactat aaagacgatg atgataaaga ctacaaagat gacgacgata agcatagtac
      481 aactcctatc agcagtctgt tttcctttac ttcacctgcc gttaagaggc tcctgggctg
      541 gaagcaagga gatgaggaag agaaatgggc cgagaaggct gtagatagcc tggtcaagaa
      601 gttgaagaag aagaagggag ccatggacga acttgagcgc gccctcagct gcccaggcca
      661 gcctagcaag tgtgtcacca tcccaaggag cctggacggg agactccagg tctcacatcg
      721 aaagggactg ccccatgtca tctactgccg cgtgtggagg tggcccgacc tgcagagcca
      781 ccacgagttg aagcccctgg agtgctgcga attcccattt ggatccaagc agaaagaggt
      841 ttgtatcaat ccataccatt accggcgcgt ggaaactcct gtgttgcctc ccgtgctggt
      901 gcctagacac agtgaataca atccacagct cagtctgctg gctaagttcc gctctgcctc
      961 acttcattcc gagcccctga tgccgcacaa cgctacatat ccagatagct ttcagcagcc
     1021 accttgttcc gccctcccac cttctccttc ccatgccttt tctcagtctc cctgtacagc
     1081 atcctatccc cattcccccg ggagccccag tgaacctgaa agcccgtacc agcacagtgt
     1141 ggacacccca cctctgccgt atcacgctac cgaggcctca gaaacccaga gcggccagcc
     1201 agtagatgca actgcggaca gacacgtcgt gctcagtatc ccgaacggcg actttcggcc
     1261 agtatgctat gaagagcccc aacattggtg ttcagtcgct tactacgaac tgaacaatcg
     1321 cgtcggcgag acttttcagg ctagctcaag gtctgtgctt atcgatggct tcacggatcc
     1381 ttccaacaat aggaaccggt tctgcctggg gctgctgtct aatgtgaata ggaactccac
     1441 tatagaaaat actagaagac acatcggtaa gggtgttcat ctctattatg tgggaggcga
     1501 ggtttacgca gagtgcgtaa gcgactcctc catcttcgtg cagtccagaa actgtaacta
     1561 ccagcacggg ttccatcccg ccacagtgtg caaaattcca tctgggtgta gtctgaaggt
     1621 ctttaataac cagctcttcg cgcagctcct ggcacaatcc gtccaccatg ggtttgaagt
     1681 tgtatacgaa ttgactaaga tgtgcacaat cagaatgagt ttcgtgaagg gctggggggc
     1741 cgaatatcat cgacaggatg tgacatctac tccctgttgg atagaaatcc atctccatgg
     1801 gcctctgcag tggctggaca aggttttgac ccagatggga tcaccacaca accctatcag
     1861 tgaagtggaa tgagacccag ctttcttgta caaagtggta tacccatacg atgttccaga
     1921 ttacgcttag taatgagaaa tactcagaag atgtcactca ccatagtccg cagtgaaaga
     1981 taaatgatcc acagaggtcg gacgaagttg ttttagagct agaaatagca agtaggccac
     2041 tgagcaagaa ctatgaatga gatctttaga catattgcgg ccgctttctc cgcgatccag
     2101 acatgataag atacattgat gagtttggac aaaccaaaac tagaatgcag tgaaaaaaat
     2161 gccttatttg tgaaatttgt gatgctattg ccttatttgt aaccattata agctgcaata
     2221 aacaagttat ctataacaag aaaatatata tataataagt tatcacgtaa gtagaacatg
     2281 aaataacaat ataattatcg tatgagttaa atcttaaaag tcacgtaaaa gataatcatg
     2341 cgtcattttg actcacgcgg tcgttatagt tcaaaatcag tgacacttac cgcattgaca
     2401 agcacgcctc acgggagctc caagcggcga ctgagatgtc ctaaatgcac agcgacggat
     2461 tcgcgctatt tagaaagaga gagcaatatt tcaagaatgc atgcgtcaat tttacgcaga
     2521 ctatctttct agggttaaga attcactggc cgtcgtttta caacgtcgtg actgggaaaa
     2581 ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa
     2641 tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg
     2701 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatggtg
     2761 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac
     2821 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     2881 gaccatgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacacgcg
     2941 agttcgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
     3001 ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     3061 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     3121 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     3181 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     3241 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     3301 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     3361 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     3421 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     3481 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     3541 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     3601 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     3661 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     3721 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     3781 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     3841 tggagccggt gagcgtggtt cacgcggtat cattgcagca ctggggccag atggtaagcc
     3901 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     3961 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     4021 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     4081 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     4141 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     4201 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     4261 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     4321 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     4381 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     4441 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     4501 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     4561 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     4621 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     4681 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     4741 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     4801 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     4861 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     4921 gtcagtgagc gaggaagcgg atgagcgccc aatacgcaaa ccgcctctca ccgcgcgttg
     4981 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg
     5041 caacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
     5101 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat
     5161 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc
     5221 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaaggt cgacttaacc
     5281 ctagaaagat aatcatattg tgacgtacgt taaagataat catgcgtaaa attgacgcat
     5341 gtgttttatc ggtctgtata tcgaggttta tttattaatt tgaatagata ttaagtttta
     5401 ttatatttac acttacatac taataataaa ttcaacaaac aatttattta tgtttattta
     5461 tttattaaaa aaaaacaaaa actcaaaatt tcttctataa agtaacaaaa cttttagcag
     5521 tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata
     5581 agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg
     5641 gaggtgtggg aggtttttta aagcaagtaa aacctctaca aatgtggtat ggctgattat
     5701 gatcctctgg agattcatgc gccaggtttt ctggtcatgc accacgttcg tgggccctca
     5761 ggaacctcca cgtcagcggt aactgtgaag cccagtcgct cgtaaaaagg aagattccgg
     5821 ggagcactgg tttcgaggaa cgccggcacg ccagcccgtt ccgcggcctc aacaccagga
     5881 agaacgacag cgcttcccaa acctttacct tggtgatctg ggctgacacc gacagtggca
     5941 agaaaccacg caggctcctt tggtctatga ggagccagca agccctccat ttgctgttgt
     6001 gccgccaacc tactccctga cagctcggcc atccgcggac ctatttccgc gaacactgcg
     6061 ccggcttcga cgctttccgg agtagtccag accgcaactg cggcaccatc gtccgcaacc
     6121 caaactttac caatgtccag acccacgcgc gtaagaaaca gctcttgcaa ctcagttacc
     6181 ctctctatgt gtctgtccgg atctacggta tggcgggtcg ctggataatc tgcaaatgct
     6241 gcggcaagtg ttcggacagc ccgtggtaca tcgtcacggg tagcgagcct aacagtgggc
     6301 ttatattccg tcatggggcc ggggttctcc tccacgtcgc cggcctgctt cagcaggctg
     6361 aagttggtgg cgccgctgcc cccggggagc atgtcaaggt caaaatcgtc aagagcgtca
     6421 gcaggcagca tatcaaggtc aaagtcgtca agggcatcgg ctgggagcat gtctaagtca
     6481 aaatcgtcaa gggcgtcggt cggcccgccg ctttcgcact ttagctgttt ctccaggcca
     6541 catatgatta gttccaggcc gaaaaggaag gcaggttcgg ctccctgccg gtcgaacagc
     6601 tcaattgctt gtttcagaag tgggggcata gaatcggtgg taggtgtctc tctttcctct
     6661 tttgctactt gatgctcctg ttcctccaat acgcagccca gtgtaaagtg gcccacggcg
     6721 gacagagcgt acagtgcgtt ctccagggag aagccttgct gacacaggaa cgcgagctga
     6781 ttttccaggg tttcgtactg tttctctgtt gggcgggtgc cgagatgcac tttagccccg
     6841 tcacgatgtg agaggagagc acagcggtat gacttggcgt tgttccgcag aaagtcttgc
     6901 catgactcgc cttccagggg gcaggagtgg gtatgatgcc tgtccagcat ctcgattggc
     6961 agggcatcga gcagggcccg cttgttcttc acgtgccagt acagggtagg ctgctcaact
     7021 cccagctttt gagcgagttt ccttgtcgtc aggccttcga taccgactcc attgagtaat
     7081 tccagagcag agtttatgac tttgctcttg tccagtctag acatcttatc gtcatcgtct
     7141 ttgtaatcca tggtggcgga tcccgcgtca cgacacctgt gttctggcgg caaacccgtt
     7201 gcgaaaaaga acgttcacgg cgactactgc acttatatac ggttctcccc caccctcggg
     7261 aaaaaggcgg agccagtaca cgacatcact ttcccagttt accccgcgcc accttctcta
     7321 ggcaccggtt caattgccga cccctccccc caacttctcg ggcactgtgg gccatgtgcg
     7381 ctctgcccca tggtaatagc catgactaat acgtagatgt actgccaagt aggaaagtgc
     7441 cataaggtca tgtactgggc ataatgccag gcgggccatt taccgtcatt gacgtcaata
     7501 gggggcgtac ttggcatatg atacacttga tgtactgcca agtgggcagt ttaccgtaaa
     7561 tactccaccc attgacgtca atggaaagtg cctattggcg ttactatggg aacatacgtc
     7621 attattgacg tcaatgggcg ggggtcgttg ggcggtcagc caggcgggcc atttaccgta
     7681 agttatgtaa cgcggaactc catatatggg ctatgaacta atgaccccgt aattgattac
     7741 tattaataac tagtcaataa tcaatgtcat aagctctaat cgcataatcg agctaagagc
     7801 gattaatatg cttagctagc ttagctaagc tagatttctc tattctccca caatatatag
     7861 cagctattag cttagcagtc cgaatacgat cgtccacgat ccataagcgt aacacatttg
     7921 agtagcgact gacgggcacc ggagccactc gagtggaatt
//
