LOCUS       sequence-377936-        7741 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Barcoded piggybac transposon vector with Dox-inducible expression of
            LHX1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7741)
  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 7741)
  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-LHX1
FEATURES             Location/Qualifiers
     source          1..7741
                     /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(1624..1648)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1650..1676
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1843..1977
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   2249..2283
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2294..2311)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2294..2310)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2303..2325)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2519..2538)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2640..2662
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2701..2719)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2787..2891
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2892..2960,2961..3752)
                     /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(3110..3129)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3923..4511
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4412..4431
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4665..4682
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4888..4909
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4924..4929,4930..4947,4948..4954)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4962..4978
                     /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     4967..4989
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4986..5002
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4986..5002
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   5028..5090
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    5266..5347
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(6064..6120)
                     /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(6130..6873)
                     /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(6874..6897)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6897..6906
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6926..7137)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        7138..7517
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     protein_bind    join(7726..7741,1..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 stttactccc
       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 caggctccac catggttcac tgtgccggct
      421 gcaaaaggcc catcctggac cgctttctct tgaacgtgct ggacagggcc tggcacgtca
      481 agtgcgtcca gtgctgtgaa tgtaaatgca acctgaccga gaagtgcttc tccagggaag
      541 gcaaactcta ctgcaagaac gacttcttcc ggtgtttcgg taccaaatgc gcaggctgcg
      601 ctcagggcat ctcccctagc gacctggtgc ggagagcgcg gagcaaagtg tttcacctga
      661 actgcttcac ctgcatgatg tgtaacaagc agctctccac tggcgaggaa ctctacatca
      721 tcgacgagaa taagttcgtc tgcaaagagg attacctaag taacagcagt gttgccaaag
      781 agaacagcct tcactcggcc accacgggca gtgaccccag tttgtctccg gattcccaag
      841 acccgtcgca ggacgacgcc aaggactcgg agagcgccaa cgtgtcggac aaggaagcgg
      901 gtagcaacga gaatgacgac cagaacctgg gcgccaagcg gcggggaccg cgcaccacca
      961 tcaaagccaa gcagctggag acgctgaagg ccgccttcgc tgctacaccc aagcccaccc
     1021 gccacatccg cgagcagctg gcgcaggaga ccggcctcaa catgcgcgtc attcaggtct
     1081 ggttccagaa ccggcgctcc aaggagcgga ggatgaagca gctgagcgcc ctgggcgccc
     1141 ggcgccacgc cttcttccgc agtccgcgcc ggatgcggcc gctggtggac cgcctggagc
     1201 cgggcgagct catccccaat ggtcccttct ccttctacgg agattaccag agcgagtact
     1261 acgggcccgg gggcaactac gacttcttcc cgcaaggccc cccgtcctcg caggcccaga
     1321 caccagtgga cctacccttc gtgccgtcat ctgggccgtc cgggacgccc ctgggtggcc
     1381 tggagcaccc gctgccgggc caccacccgt cgagcgaggc gcagcggttt accgacatcc
     1441 tggcgcaccc acccggggac tcgcccagcc ccgagcccag cctgcccggg cctctgcact
     1501 ccatgtcggc cgaggtcttc ggacccagcc cgcccttctc gtcgctgtcg gtcaacggtg
     1561 gggcgagcta cggaaaccac ctgtcccacc cccccgaaat gaacgaggcg gccgtgtggt
     1621 tggacccagc tttcttgtac aaagtggtat acccatacga tgttccagat tacgcttagt
     1681 aatgagaaat actcagaaga tgtcactcac catagtccgc agtgaaagat aaatgatcat
     1741 tggaggactt ttcagttagt tttagagcta gaaatagcaa gtaggccact gagcaagaac
     1801 tatgaatgag atctttagac atattgcggc cgctttctcc gcgatccaga catgataaga
     1861 tacattgatg agtttggaca aaccaaaact agaatgcagt gaaaaaaatg ccttatttgt
     1921 gaaatttgtg atgctattgc cttatttgta accattataa gctgcaataa acaagttatc
     1981 tataacaaga aaatatatat ataataagtt atcacgtaag tagaacatga aataacaata
     2041 taattatcgt atgagttaaa tcttaaaagt cacgtaaaag ataatcatgc gtcattttga
     2101 ctcacgcggt cgttatagtt caaaatcagt gacacttacc gcattgacaa gcacgcctca
     2161 cgggagctcc aagcggcgac tgagatgtcc taaatgcaca gcgacggatt cgcgctattt
     2221 agaaagagag agcaatattt caagaatgca tgcgtcaatt ttacgcagac tatctttcta
     2281 gggttaagaa ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta
     2341 cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg
     2401 cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgcctgatgc
     2461 ggtattttct ccttacgcat ctgtgcggta tttcacaccg catatggtgc actctcagta
     2521 caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg
     2581 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accatgtctc
     2641 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacacgcga gttcgaaagg
     2701 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt
     2761 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     2821 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     2881 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     2941 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     3001 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     3061 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     3121 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     3181 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     3241 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     3301 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     3361 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     3421 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     3481 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     3541 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     3601 agcgtggttc acgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     3661 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     3721 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     3781 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     3841 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     3901 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     3961 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     4021 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     4081 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     4141 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     4201 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     4261 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     4321 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     4381 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     4441 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     4501 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     4561 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     4621 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     4681 aggaagcgga tgagcgccca atacgcaaac cgcctctcac cgcgcgttgg ccgattcatt
     4741 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaaacc
     4801 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
     4861 gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca
     4921 ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt
     4981 tcacacagga aacagctatg accatgatta cgccaaggtc gacttaaccc tagaaagata
     5041 atcatattgt gacgtacgtt aaagataatc atgcgtaaaa ttgacgcatg tgttttatcg
     5101 gtctgtatat cgaggtttat ttattaattt gaatagatat taagttttat tatatttaca
     5161 cttacatact aataataaat tcaacaaaca atttatttat gtttatttat ttattaaaaa
     5221 aaaacaaaaa ctcaaaattt cttctataaa gtaacaaaac ttttagcagt gaaaaaaatg
     5281 ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa
     5341 acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga
     5401 ggttttttaa agcaagtaaa acctctacaa atgtggtatg gctgattatg atcctctgga
     5461 gattcatgcg ccaggttttc tggtcatgca ccacgttcgt gggccctcag gaacctccac
     5521 gtcagcggta actgtgaagc ccagtcgctc gtaaaaagga agattccggg gagcactggt
     5581 ttcgaggaac gccggcacgc cagcccgttc cgcggcctca acaccaggaa gaacgacagc
     5641 gcttcccaaa cctttacctt ggtgatctgg gctgacaccg acagtggcaa gaaaccacgc
     5701 aggctccttt ggtctatgag gagccagcaa gccctccatt tgctgttgtg ccgccaacct
     5761 actccctgac agctcggcca tccgcggacc tatttccgcg aacactgcgc cggcttcgac
     5821 gctttccgga gtagtccaga ccgcaactgc ggcaccatcg tccgcaaccc aaactttacc
     5881 aatgtccaga cccacgcgcg taagaaacag ctcttgcaac tcagttaccc tctctatgtg
     5941 tctgtccgga tctacggtat ggcgggtcgc tggataatct gcaaatgctg cggcaagtgt
     6001 tcggacagcc cgtggtacat cgtcacgggt agcgagccta acagtgggct tatattccgt
     6061 catggggccg gggttctcct ccacgtcgcc ggcctgcttc agcaggctga agttggtggc
     6121 gccgctgccc ccggggagca tgtcaaggtc aaaatcgtca agagcgtcag caggcagcat
     6181 atcaaggtca aagtcgtcaa gggcatcggc tgggagcatg tctaagtcaa aatcgtcaag
     6241 ggcgtcggtc ggcccgccgc tttcgcactt tagctgtttc tccaggccac atatgattag
     6301 ttccaggccg aaaaggaagg caggttcggc tccctgccgg tcgaacagct caattgcttg
     6361 tttcagaagt gggggcatag aatcggtggt aggtgtctct ctttcctctt ttgctacttg
     6421 atgctcctgt tcctccaata cgcagcccag tgtaaagtgg cccacggcgg acagagcgta
     6481 cagtgcgttc tccagggaga agccttgctg acacaggaac gcgagctgat tttccagggt
     6541 ttcgtactgt ttctctgttg ggcgggtgcc gagatgcact ttagccccgt cacgatgtga
     6601 gaggagagca cagcggtatg acttggcgtt gttccgcaga aagtcttgcc atgactcgcc
     6661 ttccaggggg caggagtggg tatgatgcct gtccagcatc tcgattggca gggcatcgag
     6721 cagggcccgc ttgttcttca cgtgccagta cagggtaggc tgctcaactc ccagcttttg
     6781 agcgagtttc cttgtcgtca ggccttcgat accgactcca ttgagtaatt ccagagcaga
     6841 gtttatgact ttgctcttgt ccagtctaga catcttatcg tcatcgtctt tgtaatccat
     6901 ggtggcggat cccgcgtcac gacacctgtg ttctggcggc aaacccgttg cgaaaaagaa
     6961 cgttcacggc gactactgca cttatatacg gttctccccc accctcggga aaaaggcgga
     7021 gccagtacac gacatcactt tcccagttta ccccgcgcca ccttctctag gcaccggttc
     7081 aattgccgac ccctcccccc aacttctcgg gcactgtggg ccatgtgcgc tctgccccat
     7141 ggtaatagcc atgactaata cgtagatgta ctgccaagta ggaaagtgcc ataaggtcat
     7201 gtactgggca taatgccagg cgggccattt accgtcattg acgtcaatag ggggcgtact
     7261 tggcatatga tacacttgat gtactgccaa gtgggcagtt taccgtaaat actccaccca
     7321 ttgacgtcaa tggaaagtgc ctattggcgt tactatggga acatacgtca ttattgacgt
     7381 caatgggcgg gggtcgttgg gcggtcagcc aggcgggcca tttaccgtaa gttatgtaac
     7441 gcggaactcc atatatgggc tatgaactaa tgaccccgta attgattact attaataact
     7501 agtcaataat caatgtcata agctctaatc gcataatcga gctaagagcg attaatatgc
     7561 ttagctagct tagctaagct agatttctct attctcccac aatatatagc agctattagc
     7621 ttagcagtcc gaatacgatc gtccacgatc cataagcgta acacatttga gtagcgactg
     7681 acgggcaccg gagccactcg agtggaatta tcacctcgag tttactccct atcagtgata
     7741 g
//
