LOCUS       sequence-382104-        7654 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Barcoded piggybac transposon vector with Dox-inducible expression of
            FOS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7654)
  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 7654)
  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-FOS
FEATURES             Location/Qualifiers
     source          1..7654
                     /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(1537..1561)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1563..1589
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1756..1890
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   2162..2196
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2207..2224)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2207..2223)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2216..2238)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2432..2451)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2553..2575
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2614..2632)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2700..2804
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2805..2873,2874..3665)
                     /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(3023..3042)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3836..4424
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4325..4344
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4578..4595
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4801..4822
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4837..4842,4843..4860,4861..4867)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4875..4891
                     /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     4880..4902
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4899..4915
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4899..4915
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4941..5003
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    5179..5260
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5977..6033)
                     /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(6043..6786)
                     /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(6787..6810)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6810..6819
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6839..7050)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        7051..7430
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     protein_bind    join(7639..7654,1..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 cgtatgcaga ctttactccc
       61 tatcagtgat agagaacgta trmrgasttt 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 ttggcatgat gttctcgggc ttcaacgcag
      421 actacgaggc gtcatcctcc cgctgcagca gcgcgtcccc ggccggggat agcctctctt
      481 actaccactc acccgcagac tccttctcca gcatgggctc gcctgtcaac gcgcaggact
      541 tctgcacgga cctggccgtc tccagtgcca acttcattcc cacggtcact gccatctcga
      601 ccagtccgga cctgcagtgg ctggtgcagc ccgccctcgt ctcctctgtg gccccatcgc
      661 agaccagagc ccctcaccct ttcggagtcc ccgccccctc cgctggggct tactccaggg
      721 ctggcgttgt gaagaccatg acaggaggcc gagcgcagag cattggcagg aggggcaagg
      781 tggaacagtt atctccagaa gaagaagaga aaaggagaat ccgaagggaa aggaataaga
      841 tggctgcagc caaatgccgc aaccggagga gggagctgac tgatacactc caagcggaga
      901 cagaccaact agaagatgag aagtctgctt tgcagaccga gattgccaac ctgctgaagg
      961 agaaggaaaa actagagttc atcctggcag ctcaccgacc tgcctgcaag atccctgatg
     1021 acctgggctt cccagaagag atgtctgtgg cttcccttga tctgactggg ggcctgccag
     1081 aggttgccac cccggagtct gaggaggcct tcaccctgcc tctcctcaat gaccctgagc
     1141 ccaagccctc agtggaacct gtcaagagca tcagcagcat ggagctgaag accgagccct
     1201 ttgatgactt cctgttccca gcatcatcca ggcccagtgg ctctgagaca gcccgctccg
     1261 tgccagacat ggacctatct gggtccttct atgcagcaga ctgggagcct ctgcacagtg
     1321 gctccctggg gatggggccc atggccacag agctggagcc cctgtgcact ccggtggtca
     1381 cctgtactcc cagctgcact gcttacacgt cttccttcgt cttcacctac cccgaggctg
     1441 actccttccc cagctgtgca gctgcccacc gcaagggcag cagcagcaat gagccttcct
     1501 ctgactcgct cagctcaccc acgctgctgg ccctgtgccc aactttcttg tacaaagtgg
     1561 tatacccata cgatgttcca gattacgctt agtaatgaga aatactcaga agatgtcact
     1621 caccatagtc cgcagtgaaa gataaatgat catcctgatt ttgaaaatat tgttttagag
     1681 ctagaaatag caagtaggcc actgagcaag aactatgaat gagatcttta gacatattgc
     1741 ggccgctttc tccgcgatcc agacatgata agatacattg atgagtttgg acaaaccaaa
     1801 actagaatgc agtgaaaaaa atgccttatt tgtgaaattt gtgatgctat tgccttattt
     1861 gtaaccatta taagctgcaa taaacaagtt atctataaca agaaaatata tatataataa
     1921 gttatcacgt aagtagaaca tgaaataaca atataattat cgtatgagtt aaatcttaaa
     1981 agtcacgtaa aagataatca tgcgtcattt tgactcacgc ggtcgttata gttcaaaatc
     2041 agtgacactt accgcattga caagcacgcc tcacgggagc tccaagcggc gactgagatg
     2101 tcctaaatgc acagcgacgg attcgcgcta tttagaaaga gagagcaata tttcaagaat
     2161 gcatgcgtca attttacgca gactatcttt ctagggttaa gaattcactg gccgtcgttt
     2221 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc
     2281 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt
     2341 tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt tctccttacg catctgtgcg
     2401 gtatttcaca ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     2461 gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     2521 catccgctta cagacaagct gtgaccatgt ctccgggagc tgcatgtgtc agaggttttc
     2581 accgtcatca ccgaaacacg cgagttcgaa agggcctcgt gatacgccta tttttatagg
     2641 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
     2701 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     2761 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     2821 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     2881 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     2941 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     3001 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     3061 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     3121 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     3181 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     3241 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     3301 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     3361 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     3421 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     3481 gctggtttat tgctgataaa tctggagccg gtgagcgtgg ttcacgcggt atcattgcag
     3541 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     3601 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     3661 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     3721 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     3781 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     3841 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     3901 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     3961 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     4021 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     4081 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     4141 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     4201 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     4261 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     4321 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     4381 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     4441 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     4501 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     4561 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggatgagcgc ccaatacgca
     4621 aaccgcctct caccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
     4681 actggaaagc gggcagtgag cgcaacgcaa accgcctctc cccgcgcgtt ggccgattca
     4741 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat
     4801 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
     4861 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
     4921 ttacgccaag gtcgacttaa ccctagaaag ataatcatat tgtgacgtac gttaaagata
     4981 atcatgcgta aaattgacgc atgtgtttta tcggtctgta tatcgaggtt tatttattaa
     5041 tttgaataga tattaagttt tattatattt acacttacat actaataata aattcaacaa
     5101 acaatttatt tatgtttatt tatttattaa aaaaaaacaa aaactcaaaa tttcttctat
     5161 aaagtaacaa aacttttagc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat
     5221 tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca
     5281 ttttatgttt caggttcagg gggaggtgtg ggaggttttt taaagcaagt aaaacctcta
     5341 caaatgtggt atggctgatt atgatcctct ggagattcat gcgccaggtt ttctggtcat
     5401 gcaccacgtt cgtgggccct caggaacctc cacgtcagcg gtaactgtga agcccagtcg
     5461 ctcgtaaaaa ggaagattcc ggggagcact ggtttcgagg aacgccggca cgccagcccg
     5521 ttccgcggcc tcaacaccag gaagaacgac agcgcttccc aaacctttac cttggtgatc
     5581 tgggctgaca ccgacagtgg caagaaacca cgcaggctcc tttggtctat gaggagccag
     5641 caagccctcc atttgctgtt gtgccgccaa cctactccct gacagctcgg ccatccgcgg
     5701 acctatttcc gcgaacactg cgccggcttc gacgctttcc ggagtagtcc agaccgcaac
     5761 tgcggcacca tcgtccgcaa cccaaacttt accaatgtcc agacccacgc gcgtaagaaa
     5821 cagctcttgc aactcagtta ccctctctat gtgtctgtcc ggatctacgg tatggcgggt
     5881 cgctggataa tctgcaaatg ctgcggcaag tgttcggaca gcccgtggta catcgtcacg
     5941 ggtagcgagc ctaacagtgg gcttatattc cgtcatgggg ccggggttct cctccacgtc
     6001 gccggcctgc ttcagcaggc tgaagttggt ggcgccgctg cccccgggga gcatgtcaag
     6061 gtcaaaatcg tcaagagcgt cagcaggcag catatcaagg tcaaagtcgt caagggcatc
     6121 ggctgggagc atgtctaagt caaaatcgtc aagggcgtcg gtcggcccgc cgctttcgca
     6181 ctttagctgt ttctccaggc cacatatgat tagttccagg ccgaaaagga aggcaggttc
     6241 ggctccctgc cggtcgaaca gctcaattgc ttgtttcaga agtgggggca tagaatcggt
     6301 ggtaggtgtc tctctttcct cttttgctac ttgatgctcc tgttcctcca atacgcagcc
     6361 cagtgtaaag tggcccacgg cggacagagc gtacagtgcg ttctccaggg agaagccttg
     6421 ctgacacagg aacgcgagct gattttccag ggtttcgtac tgtttctctg ttgggcgggt
     6481 gccgagatgc actttagccc cgtcacgatg tgagaggaga gcacagcggt atgacttggc
     6541 gttgttccgc agaaagtctt gccatgactc gccttccagg gggcaggagt gggtatgatg
     6601 cctgtccagc atctcgattg gcagggcatc gagcagggcc cgcttgttct tcacgtgcca
     6661 gtacagggta ggctgctcaa ctcccagctt ttgagcgagt ttccttgtcg tcaggccttc
     6721 gataccgact ccattgagta attccagagc agagtttatg actttgctct tgtccagtct
     6781 agacatctta tcgtcatcgt ctttgtaatc catggtggcg gatcccgcgt cacgacacct
     6841 gtgttctggc ggcaaacccg ttgcgaaaaa gaacgttcac ggcgactact gcacttatat
     6901 acggttctcc cccaccctcg ggaaaaaggc ggagccagta cacgacatca ctttcccagt
     6961 ttaccccgcg ccaccttctc taggcaccgg ttcaattgcc gacccctccc cccaacttct
     7021 cgggcactgt gggccatgtg cgctctgccc catggtaata gccatgacta atacgtagat
     7081 gtactgccaa gtaggaaagt gccataaggt catgtactgg gcataatgcc aggcgggcca
     7141 tttaccgtca ttgacgtcaa tagggggcgt acttggcata tgatacactt gatgtactgc
     7201 caagtgggca gtttaccgta aatactccac ccattgacgt caatggaaag tgcctattgg
     7261 cgttactatg ggaacatacg tcattattga cgtcaatggg cgggggtcgt tgggcggtca
     7321 gccaggcggg ccatttaccg taagttatgt aacgcggaac tccatatatg ggctatgaac
     7381 taatgacccc gtaattgatt actattaata actagtcaat aatcaatgtc ataagctcta
     7441 atcgcataat cgagctaaga gcgattaata tgcttagcta gcttagctaa gctagatttc
     7501 tctattctcc cacaatatat agcagctatt agcttagcag tccgaatacg atcgtccacg
     7561 atccataagc gtaacacatt tgagtagcga ctgacgggca ccggagccac tcgagtggaa
     7621 ttatcacctc gagtttactc cctatcagtg atag
//
