LOCUS       sequence-377930-        7897 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Barcoded piggybac transposon vector with Dox-inducible expression of
            NR5A1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7897)
  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 7897)
  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-NR5A1
FEATURES             Location/Qualifiers
     source          1..7897
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    21..39
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    57..75
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    93..111
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    129..147
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    165..183
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    201..219
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    237..255
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     282..306
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    372..396
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1780..1804)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1806..1832
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1999..2133
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   2405..2439
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2450..2467)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2450..2466)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2459..2481)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2675..2694)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2796..2818
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2857..2875)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2943..3047
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3048..3116,3117..3908)
                     /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(3266..3285)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4079..4667
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4568..4587
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4821..4838
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5044..5065
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5080..5085,5086..5103,5104..5110)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5118..5134
                     /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     5123..5145
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5142..5158
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5142..5158
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   5184..5246
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    5422..5503
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(6220..6276)
                     /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(6286..7029)
                     /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(7030..7053)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      7053..7062
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(7082..7293)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        7294..7673
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     protein_bind    join(7882..7897,1..3)
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
ORIGIN
        1 agaacgtatg aagagtttac tccctatcag tgatagagaa cgtatgmaga stttactccc
       61 tatcagtgat agagaacgta tgcagacttt actccctatc agtgatagag aacgtataag
      121 gagtttactc cctatcagtg atagagaacg tatgaccagt ttactcccta tcagtgatag
      181 agaacgtatc tacagtttac tccctatcag tgatagagaa cgtatatcca gtttactccc
      241 tatcagtgat agagaacgta taagcttgcc tatataagca gagctcgttt agtgaaccgt
      301 cagatcgcct ggagacgcca tccacgctgt tttgacctcc atagaagaca ccgggaccga
      361 tccagcctac cacaagtttg tacaaaaaag ttggcatgga ctattcgtac gacgaggacc
      421 tggacgagct gtgccccgtg tgcggggaca aggtgtccgg ctaccactac ggactgctca
      481 cgtgtgagag ctgcaagggc ttcttcaagc gcacggtgca gaacaacaag cactacacgt
      541 gcaccgagag ccagagctgc aagatcgaca agacgcagcg caagcgctgt cccttctgcc
      601 gcttccagaa atgcctgacg gtggggatgc gcctggaagc cgtgcgcgct gaccgtatga
      661 ggggtggccg gaacaagttt gggccgatgt acaagcggga ccgggccctg aaacagcaga
      721 agaaggcaca gattcgggcc aatggcttca agctggagac agggcccccg atgggggtgc
      781 ccccgccgcc ccctcccgca ccggactacg tgctgcctcc cagcctgcat gggcctgagc
      841 ccaagggcct ggccgccggt ccacctgctg ggccactggg cgactttggg gccccagcac
      901 tgcccatggc cgtgcccggt gcccacgggc cactggctgg ctacctctac cctgcctttc
      961 ctggccgtgc catcaagtct gagtacccgg agccttatgc cagcccccca cagcctgggc
     1021 tgccgtacgg ctacccagag cccttctctg gagggcccaa cgtgcctgag ctcatcctgc
     1081 agctgctgca gctggagccg gatgaggacc aggtgcgggc ccgcatcttg ggctgcctgc
     1141 aggagcccac caaaagccgc cccgaccagc cggcggcctt cggcctcctg tgcagaatgg
     1201 ccgaccagac cttcatctcc atcgtggact gggcacgcag gtgcatggtc ttcaaggagc
     1261 tggaggtggc cgaccagatg acgctgctgc agaactgctg gagcgagctg ctggtgttcg
     1321 accacatcta ccgccaggtc cagcacggca aggagggcag catcctgctg gtcaccgggc
     1381 aggaggtgga gctgaccaca gtggccaccc aggcgggctc gctgctgcac agcctggtgt
     1441 tgcgggcgca ggagctggtg ctgcagctgc ttgcgctgca gctggaccgg caggagtttg
     1501 tctgcctcaa gttcatcatc ctcttcagcc tggatttgaa gttcctgaat aaccacatcc
     1561 tggtgaaaga cgctcaggag aaggccaacg ccgccctgct tgactacacc ctgtgccact
     1621 acccgcactg cggggacaaa ttccagcagc tgctgctgtg cctggtggag gtgcgggccc
     1681 tgagcatgca ggccaaggag tacctgtacc acaagcacct gggcaacgag atgccccgca
     1741 acaacctgct catcgaaatg ctgcaagcca agcagacttg cccaactttc ttgtacaaag
     1801 tggtataccc atacgatgtt ccagattacg cttagtaatg agaaatactc agaagatgtc
     1861 actcaccata gtccgcagtg aaagataaat gatcagcaat ttctgtgacg ctcggtttta
     1921 gagctagaaa tagcaagtag gccactgagc aagaactatg aatgagatct ttagacatat
     1981 tgcggccgct ttctccgcga tccagacatg ataagataca ttgatgagtt tggacaaacc
     2041 aaaactagaa tgcagtgaaa aaaatgcctt atttgtgaaa tttgtgatgc tattgcctta
     2101 tttgtaacca ttataagctg caataaacaa gttatctata acaagaaaat atatatataa
     2161 taagttatca cgtaagtaga acatgaaata acaatataat tatcgtatga gttaaatctt
     2221 aaaagtcacg taaaagataa tcatgcgtca ttttgactca cgcggtcgtt atagttcaaa
     2281 atcagtgaca cttaccgcat tgacaagcac gcctcacggg agctccaagc ggcgactgag
     2341 atgtcctaaa tgcacagcga cggattcgcg ctatttagaa agagagagca atatttcaag
     2401 aatgcatgcg tcaattttac gcagactatc tttctagggt taagaattca ctggccgtcg
     2461 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac
     2521 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac
     2581 agttgcgcag cctgaatggc gaatggcgcc tgatgcggta ttttctcctt acgcatctgt
     2641 gcggtatttc acaccgcata tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     2701 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     2761 cggcatccgc ttacagacaa gctgtgacca tgtctccggg agctgcatgt gtcagaggtt
     2821 ttcaccgtca tcaccgaaac acgcgagttc gaaagggcct cgtgatacgc ctatttttat
     2881 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     2941 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     3001 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     3061 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     3121 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     3181 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     3241 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     3301 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     3361 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     3421 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     3481 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     3541 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     3601 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     3661 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     3721 ctggctggtt tattgctgat aaatctggag ccggtgagcg tggttcacgc ggtatcattg
     3781 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     3841 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     3901 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     3961 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     4021 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     4081 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     4141 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     4201 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     4261 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     4321 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     4381 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     4441 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     4501 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     4561 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     4621 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     4681 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     4741 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     4801 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggatgag cgcccaatac
     4861 gcaaaccgcc tctcaccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
     4921 ccgactggaa agcgggcagt gagcgcaacg caaaccgcct ctccccgcgc gttggccgat
     4981 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
     5041 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc
     5101 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca
     5161 tgattacgcc aaggtcgact taaccctaga aagataatca tattgtgacg tacgttaaag
     5221 ataatcatgc gtaaaattga cgcatgtgtt ttatcggtct gtatatcgag gtttatttat
     5281 taatttgaat agatattaag ttttattata tttacactta catactaata ataaattcaa
     5341 caaacaattt atttatgttt atttatttat taaaaaaaaa caaaaactca aaatttcttc
     5401 tataaagtaa caaaactttt agcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc
     5461 tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat
     5521 tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttaaagca agtaaaacct
     5581 ctacaaatgt ggtatggctg attatgatcc tctggagatt catgcgccag gttttctggt
     5641 catgcaccac gttcgtgggc cctcaggaac ctccacgtca gcggtaactg tgaagcccag
     5701 tcgctcgtaa aaaggaagat tccggggagc actggtttcg aggaacgccg gcacgccagc
     5761 ccgttccgcg gcctcaacac caggaagaac gacagcgctt cccaaacctt taccttggtg
     5821 atctgggctg acaccgacag tggcaagaaa ccacgcaggc tcctttggtc tatgaggagc
     5881 cagcaagccc tccatttgct gttgtgccgc caacctactc cctgacagct cggccatccg
     5941 cggacctatt tccgcgaaca ctgcgccggc ttcgacgctt tccggagtag tccagaccgc
     6001 aactgcggca ccatcgtccg caacccaaac tttaccaatg tccagaccca cgcgcgtaag
     6061 aaacagctct tgcaactcag ttaccctctc tatgtgtctg tccggatcta cggtatggcg
     6121 ggtcgctgga taatctgcaa atgctgcggc aagtgttcgg acagcccgtg gtacatcgtc
     6181 acgggtagcg agcctaacag tgggcttata ttccgtcatg gggccggggt tctcctccac
     6241 gtcgccggcc tgcttcagca ggctgaagtt ggtggcgccg ctgcccccgg ggagcatgtc
     6301 aaggtcaaaa tcgtcaagag cgtcagcagg cagcatatca aggtcaaagt cgtcaagggc
     6361 atcggctggg agcatgtcta agtcaaaatc gtcaagggcg tcggtcggcc cgccgctttc
     6421 gcactttagc tgtttctcca ggccacatat gattagttcc aggccgaaaa ggaaggcagg
     6481 ttcggctccc tgccggtcga acagctcaat tgcttgtttc agaagtgggg gcatagaatc
     6541 ggtggtaggt gtctctcttt cctcttttgc tacttgatgc tcctgttcct ccaatacgca
     6601 gcccagtgta aagtggccca cggcggacag agcgtacagt gcgttctcca gggagaagcc
     6661 ttgctgacac aggaacgcga gctgattttc cagggtttcg tactgtttct ctgttgggcg
     6721 ggtgccgaga tgcactttag ccccgtcacg atgtgagagg agagcacagc ggtatgactt
     6781 ggcgttgttc cgcagaaagt cttgccatga ctcgccttcc agggggcagg agtgggtatg
     6841 atgcctgtcc agcatctcga ttggcagggc atcgagcagg gcccgcttgt tcttcacgtg
     6901 ccagtacagg gtaggctgct caactcccag cttttgagcg agtttccttg tcgtcaggcc
     6961 ttcgataccg actccattga gtaattccag agcagagttt atgactttgc tcttgtccag
     7021 tctagacatc ttatcgtcat cgtctttgta atccatggtg gcggatcccg cgtcacgaca
     7081 cctgtgttct ggcggcaaac ccgttgcgaa aaagaacgtt cacggcgact actgcactta
     7141 tatacggttc tcccccaccc tcgggaaaaa ggcggagcca gtacacgaca tcactttccc
     7201 agtttacccc gcgccacctt ctctaggcac cggttcaatt gccgacccct ccccccaact
     7261 tctcgggcac tgtgggccat gtgcgctctg ccccatggta atagccatga ctaatacgta
     7321 gatgtactgc caagtaggaa agtgccataa ggtcatgtac tgggcataat gccaggcggg
     7381 ccatttaccg tcattgacgt caataggggg cgtacttggc atatgataca cttgatgtac
     7441 tgccaagtgg gcagtttacc gtaaatactc cacccattga cgtcaatgga aagtgcctat
     7501 tggcgttact atgggaacat acgtcattat tgacgtcaat gggcgggggt cgttgggcgg
     7561 tcagccaggc gggccattta ccgtaagtta tgtaacgcgg aactccatat atgggctatg
     7621 aactaatgac cccgtaattg attactatta ataactagtc aataatcaat gtcataagct
     7681 ctaatcgcat aatcgagcta agagcgatta atatgcttag ctagcttagc taagctagat
     7741 ttctctattc tcccacaata tatagcagct attagcttag cagtccgaat acgatcgtcc
     7801 acgatccata agcgtaacac atttgagtag cgactgacgg gcaccggagc cactcgagtg
     7861 gaattatcac ctcgagttta ctccctatca gtgatag
//
