LOCUS       sequence-373942-        7903 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Barcoded piggybac transposon vector with Dox-inducible expression of
            TOX2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7903)
  AUTHORS   Pierson Smela MD, Kramme CC, Fortuna PRJ, Adams JL, Su R, Dong E, 
            Kobayashi M, Brixi G, Kavirayuni VS, Tysinger E, Kohman RE, Shioda 
            T, Chatterjee P, Church GM
  TITLE     Directed differentiation of human iPSCs to functional ovarian 
            granulosa-like cells via transcription factor overexpression.
  JOURNAL   Elife. 2023 Feb 21;12:e83291. doi: 10.7554/eLife.83291.
  PUBMED    36803359
REFERENCE   2  (bases 1 to 7903)
  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: "Elife."; 
            date: "2023-02-21"; volume: "12:e83291. doi"; pages: " 
            10.7554/eLife.83291"
COMMENT     Sequence Label: pPB-cT3G-cERP2-TOX2
FEATURES             Location/Qualifiers
     source          1..7903
                     /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"
     protein_bind    complement(1785..1809)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1811..1837
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     gap             1900..1919
                     /estimated_length=20
     polyA_signal    2004..2138
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   2410..2444
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2455..2472)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2455..2471)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2464..2486)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2680..2699)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2801..2823
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2862..2880)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2948..3052
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3053..3121,3122..3913)
                     /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(3271..3290)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4084..4672
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4573..4592
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4826..4843
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5049..5070
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5085..5090,5091..5108,5109..5115)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5123..5139
                     /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     5128..5150
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5147..5163
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5147..5163
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   5189..5251
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    5427..5508
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(6225..6281)
                     /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(6291..7034)
                     /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(7035..7058)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      7058..7067
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(7087..7298)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        7299..7678
                     /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 aaaaagttgg catgagtgac ggaaacccag agctcctgtc
      421 aaccagccag acctacaacg gccagagcga gaacaacgaa gactatgaga tccccccgat
      481 aacacctccc aacctcccgg agccatccct cctgcacctg ggggaccacg aagccagcta
      541 ccactcgctg tgccacggcc tcacccccaa cggtctgctc cctgcctact cctatcaggc
      601 catggacctc ccagccatca tggtgtccaa catgctagca caggacagcc acctgctgtc
      661 gggccagctg cccacgatcc aggagatggt ccactcggaa gtggctgcct atgactcggg
      721 ccggcccggg cccctgctgg gtcgcccggc aatgctggcc agccacatga gtgccctcag
      781 ccagtcccag ctcatctcgc agatgggcat ccggagcagc atcgcccaca gctccccatc
      841 accgccgggg agcaagtcag cgaccccctc tccctccagc tccactcagg aagaggagtc
      901 ggaagtgcat ttcaagatct cgggagaaaa gagaccttca gccgacccag gaaaaaaggc
      961 caagaacccg aagaagaaga aaaagaagga ccccaatgag ccgcagaagc ctgtgtcggc
     1021 ctacgcactc ttcttcagag acactcaggc cgccatcaag ggtcagaacc ccagtgccac
     1081 tttcggtgac gtgtccaaaa tcgtggcctc catgtgggac agcctgggag aggaacagaa
     1141 gcaggcctac aagaggaaga cagaagcagc aaagaaggaa tatctgaagg ccctggcagc
     1201 ctaccgggct agcctcgtct ccaagagctc cccagatcaa ggtgagacca agagcactca
     1261 ggcaaaccca ccagccaaaa tgctcccacc caagcagccc atgtatgcca tgccaggcct
     1321 ggcctccttc ctgacgccgt cggacctgca ggccttccgc agtggggcct cccctgccag
     1381 cctcgcccgg acgctgggct ccaagtctct gctgccaggc ctcagtgcgt ccccgccgcc
     1441 gccaccctcc ttcccgctca gccccacact gcaccagcag ctgtcactgc cccctcacgc
     1501 ccagggcgcc ctcctcagtc cacctgttag catgtcccca gccccccagc cccctgtcct
     1561 gcccaccccc atggcactcc aggtgcagct ggcgatgagc ccctcacctc cagggccaca
     1621 ggacttcccg cacatctctg agttccccag cagctcggga tcctgctcac ctggcccatc
     1681 caaccccacc agcagcgggg actgggacag cagctacccc agtggggagt gtggcatcag
     1741 cacctgcagc ctgctcccca gggacaaatc gctctacctc acctacccag ctttcttgta
     1801 caaagtggta tacccatacg atgttccaga ttacgcttag taatgagaaa tactcagaag
     1861 atgtcactca ccatagtccg cagtgaaaga taaatgatcn nnnnnnnnnn nnnnnnnnng
     1921 ttttagagct agaaatagca agtaggccac tgagcaagaa ctatgaatga gatctttaga
     1981 catattgcgg ccgctttctc cgcgatccag acatgataag atacattgat gagtttggac
     2041 aaaccaaaac tagaatgcag tgaaaaaaat gccttatttg tgaaatttgt gatgctattg
     2101 ccttatttgt aaccattata agctgcaata aacaagttat ctataacaag aaaatatata
     2161 tataataagt tatcacgtaa gtagaacatg aaataacaat ataattatcg tatgagttaa
     2221 atcttaaaag tcacgtaaaa gataatcatg cgtcattttg actcacgcgg tcgttatagt
     2281 tcaaaatcag tgacacttac cgcattgaca agcacgcctc acgggagctc caagcggcga
     2341 ctgagatgtc ctaaatgcac agcgacggat tcgcgctatt tagaaagaga gagcaatatt
     2401 tcaagaatgc atgcgtcaat tttacgcaga ctatctttct agggttaaga attcactggc
     2461 cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc
     2521 agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc
     2581 ccaacagttg cgcagcctga atggcgaatg gcgcctgatg cggtattttc tccttacgca
     2641 tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct ctgatgccgc
     2701 atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     2761 gctcccggca tccgcttaca gacaagctgt gaccatgtct ccgggagctg catgtgtcag
     2821 aggttttcac cgtcatcacc gaaacacgcg agttcgaaag ggcctcgtga tacgcctatt
     2881 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg
     2941 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     3001 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     3061 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     3121 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     3181 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     3241 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     3301 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     3361 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     3421 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     3481 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     3541 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     3601 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     3661 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     3721 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtggtt cacgcggtat
     3781 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     3841 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     3901 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     3961 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     4021 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     4081 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     4141 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     4201 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     4261 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     4321 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     4381 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     4441 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     4501 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     4561 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     4621 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     4681 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     4741 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     4801 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg atgagcgccc
     4861 aatacgcaaa ccgcctctca ccgcgcgttg gccgattcat taatgcagct ggcacgacag
     4921 gtttcccgac tggaaagcgg gcagtgagcg caacgcaaac cgcctctccc cgcgcgttgg
     4981 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
     5041 aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt
     5101 ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat
     5161 gaccatgatt acgccaaggt cgacttaacc ctagaaagat aatcatattg tgacgtacgt
     5221 taaagataat catgcgtaaa attgacgcat gtgttttatc ggtctgtata tcgaggttta
     5281 tttattaatt tgaatagata ttaagtttta ttatatttac acttacatac taataataaa
     5341 ttcaacaaac aatttattta tgtttattta tttattaaaa aaaaacaaaa actcaaaatt
     5401 tcttctataa agtaacaaaa cttttagcag tgaaaaaaat gctttatttg tgaaatttgt
     5461 gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat
     5521 tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta aagcaagtaa
     5581 aacctctaca aatgtggtat ggctgattat gatcctctgg agattcatgc gccaggtttt
     5641 ctggtcatgc accacgttcg tgggccctca ggaacctcca cgtcagcggt aactgtgaag
     5701 cccagtcgct cgtaaaaagg aagattccgg ggagcactgg tttcgaggaa cgccggcacg
     5761 ccagcccgtt ccgcggcctc aacaccagga agaacgacag cgcttcccaa acctttacct
     5821 tggtgatctg ggctgacacc gacagtggca agaaaccacg caggctcctt tggtctatga
     5881 ggagccagca agccctccat ttgctgttgt gccgccaacc tactccctga cagctcggcc
     5941 atccgcggac ctatttccgc gaacactgcg ccggcttcga cgctttccgg agtagtccag
     6001 accgcaactg cggcaccatc gtccgcaacc caaactttac caatgtccag acccacgcgc
     6061 gtaagaaaca gctcttgcaa ctcagttacc ctctctatgt gtctgtccgg atctacggta
     6121 tggcgggtcg ctggataatc tgcaaatgct gcggcaagtg ttcggacagc ccgtggtaca
     6181 tcgtcacggg tagcgagcct aacagtgggc ttatattccg tcatggggcc ggggttctcc
     6241 tccacgtcgc cggcctgctt cagcaggctg aagttggtgg cgccgctgcc cccggggagc
     6301 atgtcaaggt caaaatcgtc aagagcgtca gcaggcagca tatcaaggtc aaagtcgtca
     6361 agggcatcgg ctgggagcat gtctaagtca aaatcgtcaa gggcgtcggt cggcccgccg
     6421 ctttcgcact ttagctgttt ctccaggcca catatgatta gttccaggcc gaaaaggaag
     6481 gcaggttcgg ctccctgccg gtcgaacagc tcaattgctt gtttcagaag tgggggcata
     6541 gaatcggtgg taggtgtctc tctttcctct tttgctactt gatgctcctg ttcctccaat
     6601 acgcagccca gtgtaaagtg gcccacggcg gacagagcgt acagtgcgtt ctccagggag
     6661 aagccttgct gacacaggaa cgcgagctga ttttccaggg tttcgtactg tttctctgtt
     6721 gggcgggtgc cgagatgcac tttagccccg tcacgatgtg agaggagagc acagcggtat
     6781 gacttggcgt tgttccgcag aaagtcttgc catgactcgc cttccagggg gcaggagtgg
     6841 gtatgatgcc tgtccagcat ctcgattggc agggcatcga gcagggcccg cttgttcttc
     6901 acgtgccagt acagggtagg ctgctcaact cccagctttt gagcgagttt ccttgtcgtc
     6961 aggccttcga taccgactcc attgagtaat tccagagcag agtttatgac tttgctcttg
     7021 tccagtctag acatcttatc gtcatcgtct ttgtaatcca tggtggcgga tcccgcgtca
     7081 cgacacctgt gttctggcgg caaacccgtt gcgaaaaaga acgttcacgg cgactactgc
     7141 acttatatac ggttctcccc caccctcggg aaaaaggcgg agccagtaca cgacatcact
     7201 ttcccagttt accccgcgcc accttctcta ggcaccggtt caattgccga cccctccccc
     7261 caacttctcg ggcactgtgg gccatgtgcg ctctgcccca tggtaatagc catgactaat
     7321 acgtagatgt actgccaagt aggaaagtgc cataaggtca tgtactgggc ataatgccag
     7381 gcgggccatt taccgtcatt gacgtcaata gggggcgtac ttggcatatg atacacttga
     7441 tgtactgcca agtgggcagt ttaccgtaaa tactccaccc attgacgtca atggaaagtg
     7501 cctattggcg ttactatggg aacatacgtc attattgacg tcaatgggcg ggggtcgttg
     7561 ggcggtcagc caggcgggcc atttaccgta agttatgtaa cgcggaactc catatatggg
     7621 ctatgaacta atgaccccgt aattgattac tattaataac tagtcaataa tcaatgtcat
     7681 aagctctaat cgcataatcg agctaagagc gattaatatg cttagctagc ttagctaagc
     7741 tagatttctc tattctccca caatatatag cagctattag cttagcagtc cgaatacgat
     7801 cgtccacgat ccataagcgt aacacatttg agtagcgact gacgggcacc ggagccactc
     7861 gagtggaatt actgacgggc accggagcca ctcgagtgga att
//
