LOCUS       sequence-377018-        7615 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Barcoded piggybac transposon vector with Dox-inducible expression of
            FOXL2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7615)
  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 7615)
  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-FOXL2
FEATURES             Location/Qualifiers
     source          1..7615
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     21..45
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    111..135
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1273..1297)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1299..1325
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1492..1626
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   1898..1932
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1943..1960)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1943..1959)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1952..1974)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2168..2187)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2289..2311
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2350..2368)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2436..2540
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2541..2609,2610..3401)
                     /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(2759..2778)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3572..4160
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4061..4080
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4314..4331
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4537..4558
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4573..4578,4579..4596,4597..4603)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4611..4627
                     /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     4616..4638
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4635..4651
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4635..4651
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4677..4739
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    4915..4996
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5713..5769)
                     /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(5779..6522)
                     /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(6523..6546)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6546..6555
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6575..6786)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        6787..7166
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     protein_bind    7375..7393
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7411..7429
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7447..7465
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7483..7501
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7519..7537
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7555..7573
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7591..7609
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
ORIGIN
        1 aagcttgcct atataagcag agctcgttta gtgaaccgtc agatcgcctg gagacgccat
       61 ccacgctgtt ttgacctcca tagaagacac cgggaccgat ccagcctacc acaagtttgt
      121 acaaaaaagc aggctgccac catgatggcc agctaccccg agcccgagga cgcggcgggg
      181 gccctgctgg ccccagagac cggtcgcaca gtcaaggagc cagaagggcc gccgccgagc
      241 ccaggcaagg gcggtggggg tggcggcggg acagccccgg agaagccgga cccggcgcag
      301 aagcccccgt actcgtacgt ggcgctcatc gccatggcga tccgcgagag cgcggagaag
      361 aggctcacgc tgtccggcat ctaccagtac atcatcgcga agttcccgtt ctacgagaag
      421 aataagaagg gctggcaaaa tagcatccgc cacaacctca gcctcaacga gtgcttcatc
      481 aaggtgccgc gcgagggcgg cggcgagcgc aagggcaact actggacgct ggacccggcc
      541 tgcgaagaca tgttcgagaa gggcaactac cggcgccgcc gccgcatgaa gaggcccttc
      601 cggccgccgc ccgcgcactt ccagcccggc aaggggctct tcggggccgg aggcgccgca
      661 ggcgggtgcg gcgtggcggg cgccggggcc gacggctacg gctacctggc gccccccaag
      721 tacctgcagt ctggcttcct caacaactcg tggccgctac cgcagcctcc ctcacccatg
      781 ccctatgcct cctgccagat ggcggcagcc gcagcggctg cagcagctgc ggctgcagcc
      841 gcgggccccg gtagccctgg cgcggccgct gtggtcaagg ggctggcggg cccggccgcc
      901 tcgtacgggc cgtacacacg cgtgcagagc atggcgctgc cccccggcgt agtgaactcg
      961 tacaatggcc tgggaggccc gccggccgca cccccgcctc cgccgcaccc ccacccgcat
     1021 ccgcacgcac accatctgca cgcggccgcc gcaccgccgc ctgccccacc gcaccacggg
     1081 gccgccgcgc cgccgccggg ccagctcagc cctgccagcc cagccaccgc cgcgcccccg
     1141 gcgcccgcgc ccaccagtgc gccgggcctg cagttcgctt gtgcccggca gcccgagctc
     1201 gccatgatgc attgctctta ctgggaccac gacagcaaga ccggcgcgct gcattcgcgc
     1261 ctcgatctct gaacccagct ttcttgtaca aagtggtata cccatacgat gttccagatt
     1321 acgcttagta atgagaaata ctcagaagat gtcactcacc atagtccgca gtgaaagata
     1381 aatgatcact tctacacgta attgccagtt ttagagctag aaatagcaag taggccactg
     1441 agcaagaact atgaatgaga tctttagaca tattgcggcc gctttctccg cgatccagac
     1501 atgataagat acattgatga gtttggacaa accaaaacta gaatgcagtg aaaaaaatgc
     1561 cttatttgtg aaatttgtga tgctattgcc ttatttgtaa ccattataag ctgcaataaa
     1621 caagttatct ataacaagaa aatatatata taataagtta tcacgtaagt agaacatgaa
     1681 ataacaatat aattatcgta tgagttaaat cttaaaagtc acgtaaaaga taatcatgcg
     1741 tcattttgac tcacgcggtc gttatagttc aaaatcagtg acacttaccg cattgacaag
     1801 cacgcctcac gggagctcca agcggcgact gagatgtcct aaatgcacag cgacggattc
     1861 gcgctattta gaaagagaga gcaatatttc aagaatgcat gcgtcaattt tacgcagact
     1921 atctttctag ggttaagaat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc
     1981 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata
     2041 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc
     2101 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatggtgca
     2161 ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac
     2221 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     2281 ccatgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacacgcgag
     2341 ttcgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     2401 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     2461 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     2521 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     2581 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     2641 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     2701 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     2761 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     2821 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     2881 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     2941 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     3001 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     3061 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     3121 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     3181 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     3241 gagccggtga gcgtggttca cgcggtatca ttgcagcact ggggccagat ggtaagccct
     3301 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     3361 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     3421 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     3481 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     3541 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     3601 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     3661 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     3721 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     3781 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     3841 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     3901 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     3961 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     4021 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     4081 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     4141 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     4201 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     4261 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     4321 cagtgagcga ggaagcggat gagcgcccaa tacgcaaacc gcctctcacc gcgcgttggc
     4381 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     4441 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
     4501 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
     4561 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
     4621 ataacaattt cacacaggaa acagctatga ccatgattac gccaaggtcg acttaaccct
     4681 agaaagataa tcatattgtg acgtacgtta aagataatca tgcgtaaaat tgacgcatgt
     4741 gttttatcgg tctgtatatc gaggtttatt tattaatttg aatagatatt aagttttatt
     4801 atatttacac ttacatacta ataataaatt caacaaacaa tttatttatg tttatttatt
     4861 tattaaaaaa aaacaaaaac tcaaaatttc ttctataaag taacaaaact tttagcagtg
     4921 aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag
     4981 ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga
     5041 ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtatgg ctgattatga
     5101 tcctctggag attcatgcgc caggttttct ggtcatgcac cacgttcgtg ggccctcagg
     5161 aacctccacg tcagcggtaa ctgtgaagcc cagtcgctcg taaaaaggaa gattccgggg
     5221 agcactggtt tcgaggaacg ccggcacgcc agcccgttcc gcggcctcaa caccaggaag
     5281 aacgacagcg cttcccaaac ctttaccttg gtgatctggg ctgacaccga cagtggcaag
     5341 aaaccacgca ggctcctttg gtctatgagg agccagcaag ccctccattt gctgttgtgc
     5401 cgccaaccta ctccctgaca gctcggccat ccgcggacct atttccgcga acactgcgcc
     5461 ggcttcgacg ctttccggag tagtccagac cgcaactgcg gcaccatcgt ccgcaaccca
     5521 aactttacca atgtccagac ccacgcgcgt aagaaacagc tcttgcaact cagttaccct
     5581 ctctatgtgt ctgtccggat ctacggtatg gcgggtcgct ggataatctg caaatgctgc
     5641 ggcaagtgtt cggacagccc gtggtacatc gtcacgggta gcgagcctaa cagtgggctt
     5701 atattccgtc atggggccgg ggttctcctc cacgtcgccg gcctgcttca gcaggctgaa
     5761 gttggtggcg ccgctgcccc cggggagcat gtcaaggtca aaatcgtcaa gagcgtcagc
     5821 aggcagcata tcaaggtcaa agtcgtcaag ggcatcggct gggagcatgt ctaagtcaaa
     5881 atcgtcaagg gcgtcggtcg gcccgccgct ttcgcacttt agctgtttct ccaggccaca
     5941 tatgattagt tccaggccga aaaggaaggc aggttcggct ccctgccggt cgaacagctc
     6001 aattgcttgt ttcagaagtg ggggcataga atcggtggta ggtgtctctc tttcctcttt
     6061 tgctacttga tgctcctgtt cctccaatac gcagcccagt gtaaagtggc ccacggcgga
     6121 cagagcgtac agtgcgttct ccagggagaa gccttgctga cacaggaacg cgagctgatt
     6181 ttccagggtt tcgtactgtt tctctgttgg gcgggtgccg agatgcactt tagccccgtc
     6241 acgatgtgag aggagagcac agcggtatga cttggcgttg ttccgcagaa agtcttgcca
     6301 tgactcgcct tccagggggc aggagtgggt atgatgcctg tccagcatct cgattggcag
     6361 ggcatcgagc agggcccgct tgttcttcac gtgccagtac agggtaggct gctcaactcc
     6421 cagcttttga gcgagtttcc ttgtcgtcag gccttcgata ccgactccat tgagtaattc
     6481 cagagcagag tttatgactt tgctcttgtc cagtctagac atcttatcgt catcgtcttt
     6541 gtaatccatg gtggcggatc ccgcgtcacg acacctgtgt tctggcggca aacccgttgc
     6601 gaaaaagaac gttcacggcg actactgcac ttatatacgg ttctccccca ccctcgggaa
     6661 aaaggcggag ccagtacacg acatcacttt cccagtttac cccgcgccac cttctctagg
     6721 caccggttca attgccgacc cctcccccca acttctcggg cactgtgggc catgtgcgct
     6781 ctgccccatg gtaatagcca tgactaatac gtagatgtac tgccaagtag gaaagtgcca
     6841 taaggtcatg tactgggcat aatgccaggc gggccattta ccgtcattga cgtcaatagg
     6901 gggcgtactt ggcatatgat acacttgatg tactgccaag tgggcagttt accgtaaata
     6961 ctccacccat tgacgtcaat ggaaagtgcc tattggcgtt actatgggaa catacgtcat
     7021 tattgacgtc aatgggcggg ggtcgttggg cggtcagcca ggcgggccat ttaccgtaag
     7081 ttatgtaacg cggaactcca tatatgggct atgaactaat gaccccgtaa ttgattacta
     7141 ttaataacta gtcaataatc aatgtcataa gctctaatcg cataatcgag ctaagagcga
     7201 ttaatatgct tagctagctt agctaagcta gatttctcta ttctcccaca atatatagca
     7261 gctattagct tagcagtccg aatacgatcg tccacgatcc ataagcgtaa cacatttgag
     7321 tagcgactga cgggcaccgg agccactcga gtggaattat cacctcgagt ttactcccta
     7381 tcagtgatag agaacgtatg aagagtttac tccctatcag tgatagagaa cgtatgcaga
     7441 ctttactccc tatcagtgat agagaacgta taaggagttt actccctatc agtgatagag
     7501 aacgtatgac cagtttactc cctatcagtg atagagaacg tatctacagt ttactcccta
     7561 tcagtgatag agaacgtata tccagtttac tccctatcag tgatagagaa cgtat
//
