LOCUS       sequence-376978-        7804 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Barcoded piggybac transposon vector with Dox-inducible expression of
            GATA4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7804)
  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 7804)
  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-GATA4
FEATURES             Location/Qualifiers
     source          1..7804
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     22..46
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    112..136
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1463..1487)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1489..1515
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1682..1816
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   2088..2122
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2133..2150)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2133..2149)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2142..2164)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2358..2377)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2479..2501
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2540..2558)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2626..2730
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2731..2799,2800..3591)
                     /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(2949..2968)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3762..4350
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4251..4270
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4504..4521
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4727..4748
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4763..4768,4769..4786,4787..4793)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4801..4817
                     /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     4806..4828
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4825..4841
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4825..4841
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4867..4929
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    5105..5186
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5903..5959)
                     /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(5969..6712)
                     /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(6713..6736)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6736..6745
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6765..6976)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        6977..7356
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     protein_bind    7565..7583
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7601..7619
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7637..7655
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7673..7691
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7709..7727
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7745..7763
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7781..7799
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
ORIGIN
        1 taagcttgcc tatataagca gagctcgttt agtgaaccgt cagatcgcct ggagacgcca
       61 tccacgctgt tttgacctcc atagaagaca ccgggaccga tccagcctac cacaagtttg
      121 tacaaaaaag ttggcatgta ccagagcctg gctatggctg ctaatcatgg acctccccct
      181 ggagcctatg aagccggagg acctggcgct tttatgcatg gagctggcgc cgcttcttct
      241 cccgtgtatg tgcctacacc tagagtgccc agcagcgtgc tgggcctttc ttatcttcag
      301 ggaggaggag caggatctgc ttctggcgga gcttcaggcg gatcttctgg aggcgctgct
      361 tcaggtgctg gacctggaac tcaacaggga tctcctggat ggtcacaggc aggagctgat
      421 ggagccgctt atacccctcc tcctgtgagc cccaggttta gctttcctgg cacaacaggc
      481 tctttagctg ccgctgctgc tgcagccgca gctagagaag cagctgcata ttctagtggc
      541 ggaggagctg ctggagccgg cttagctgga agagagcagt acggaagagc cggatttgcc
      601 ggaagctata gcagccctta ccctgcctat atggccgatg ttggcgcatc ttgggcagcc
      661 gccgcagcag cttctgcagg accttttgac tcacctgtgc ttcactctct gcctggcaga
      721 gctaatcctg ccgccagaca tcccaacctg gacatgttcg acgacttcag cgagggcaga
      781 gaatgcgtga actgcggagc catgagcacc cccctttgga gaagagacgg caccggccac
      841 tacctttgca atgcctgtgg cctgtaccac aagatgaacg gcatcaacag acccctgatc
      901 aagccccaga gaagactgag cgctagcaga agagtgggcc tgtcctgcgc caattgccag
      961 accacaacca ccacactgtg gaggagaaat gccgagggcg agcctgtgtg taacgcctgt
     1021 ggactgtaca tgaagctgca cggcgtgccc agacctctgg ccatgagaaa ggagggcatc
     1081 cagaccagaa agagaaagcc caagaacctg aacaagagca agacccccgc tgctccttct
     1141 ggaagcgaga gcctgcctcc agcctctgga gccagcagca atagctctaa cgccaccaca
     1201 tcttcttctg aggagatgag gcccatcaaa accgagccag gcctgagcag ccactacggc
     1261 cacagctcta gcgtgagcca gacttttagc gtgtctgcca tgtcaggcca cggacctagc
     1321 attcaccctg tgctgagcgc cctgaagttg agcccacagg gctatgcttc tcctgtgtct
     1381 cagagccctc agacctccag caagcaggac tcctggaatt ctctggtgct ggccgacagc
     1441 cacggcgata tcatcaccgc ctgcccaact ttcttgtaca aagtggtata cccatacgat
     1501 gttccagatt acgcttagta atgagaaata ctcagaagat gtcactcacc atagtccgca
     1561 gtgaaagata aatgatccgt ccagaaagaa cccattagtt ttagagctag aaatagcaag
     1621 taggccactg agcaagaact atgaatgaga tctttagaca tattgcggcc gctttctccg
     1681 cgatccagac atgataagat acattgatga gtttggacaa accaaaacta gaatgcagtg
     1741 aaaaaaatgc cttatttgtg aaatttgtga tgctattgcc ttatttgtaa ccattataag
     1801 ctgcaataaa caagttatct ataacaagaa aatatatata taataagtta tcacgtaagt
     1861 agaacatgaa ataacaatat aattatcgta tgagttaaat cttaaaagtc acgtaaaaga
     1921 taatcatgcg tcattttgac tcacgcggtc gttatagttc aaaatcagtg acacttaccg
     1981 cattgacaag cacgcctcac gggagctcca agcggcgact gagatgtcct aaatgcacag
     2041 cgacggattc gcgctattta gaaagagaga gcaatatttc aagaatgcat gcgtcaattt
     2101 tacgcagact atctttctag ggttaagaat tcactggccg tcgttttaca acgtcgtgac
     2161 tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc
     2221 tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat
     2281 ggcgaatggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     2341 atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac
     2401 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     2461 caagctgtga ccatgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     2521 aacacgcgag ttcgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     2581 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     2641 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     2701 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     2761 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     2821 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     2881 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     2941 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     3001 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     3061 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     3121 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     3181 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     3241 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     3301 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     3361 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     3421 gataaatctg gagccggtga gcgtggttca cgcggtatca ttgcagcact ggggccagat
     3481 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     3541 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     3601 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     3661 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     3721 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     3781 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     3841 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     3901 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     3961 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     4021 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     4081 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     4141 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     4201 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     4261 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     4321 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     4381 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     4441 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     4501 cgcagcgagt cagtgagcga ggaagcggat gagcgcccaa tacgcaaacc gcctctcacc
     4561 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     4621 agtgagcgca acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
     4681 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
     4741 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
     4801 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaaggtcg
     4861 acttaaccct agaaagataa tcatattgtg acgtacgtta aagataatca tgcgtaaaat
     4921 tgacgcatgt gttttatcgg tctgtatatc gaggtttatt tattaatttg aatagatatt
     4981 aagttttatt atatttacac ttacatacta ataataaatt caacaaacaa tttatttatg
     5041 tttatttatt tattaaaaaa aaacaaaaac tcaaaatttc ttctataaag taacaaaact
     5101 tttagcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa
     5161 ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg
     5221 ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtatgg
     5281 ctgattatga tcctctggag attcatgcgc caggttttct ggtcatgcac cacgttcgtg
     5341 ggccctcagg aacctccacg tcagcggtaa ctgtgaagcc cagtcgctcg taaaaaggaa
     5401 gattccgggg agcactggtt tcgaggaacg ccggcacgcc agcccgttcc gcggcctcaa
     5461 caccaggaag aacgacagcg cttcccaaac ctttaccttg gtgatctggg ctgacaccga
     5521 cagtggcaag aaaccacgca ggctcctttg gtctatgagg agccagcaag ccctccattt
     5581 gctgttgtgc cgccaaccta ctccctgaca gctcggccat ccgcggacct atttccgcga
     5641 acactgcgcc ggcttcgacg ctttccggag tagtccagac cgcaactgcg gcaccatcgt
     5701 ccgcaaccca aactttacca atgtccagac ccacgcgcgt aagaaacagc tcttgcaact
     5761 cagttaccct ctctatgtgt ctgtccggat ctacggtatg gcgggtcgct ggataatctg
     5821 caaatgctgc ggcaagtgtt cggacagccc gtggtacatc gtcacgggta gcgagcctaa
     5881 cagtgggctt atattccgtc atggggccgg ggttctcctc cacgtcgccg gcctgcttca
     5941 gcaggctgaa gttggtggcg ccgctgcccc cggggagcat gtcaaggtca aaatcgtcaa
     6001 gagcgtcagc aggcagcata tcaaggtcaa agtcgtcaag ggcatcggct gggagcatgt
     6061 ctaagtcaaa atcgtcaagg gcgtcggtcg gcccgccgct ttcgcacttt agctgtttct
     6121 ccaggccaca tatgattagt tccaggccga aaaggaaggc aggttcggct ccctgccggt
     6181 cgaacagctc aattgcttgt ttcagaagtg ggggcataga atcggtggta ggtgtctctc
     6241 tttcctcttt tgctacttga tgctcctgtt cctccaatac gcagcccagt gtaaagtggc
     6301 ccacggcgga cagagcgtac agtgcgttct ccagggagaa gccttgctga cacaggaacg
     6361 cgagctgatt ttccagggtt tcgtactgtt tctctgttgg gcgggtgccg agatgcactt
     6421 tagccccgtc acgatgtgag aggagagcac agcggtatga cttggcgttg ttccgcagaa
     6481 agtcttgcca tgactcgcct tccagggggc aggagtgggt atgatgcctg tccagcatct
     6541 cgattggcag ggcatcgagc agggcccgct tgttcttcac gtgccagtac agggtaggct
     6601 gctcaactcc cagcttttga gcgagtttcc ttgtcgtcag gccttcgata ccgactccat
     6661 tgagtaattc cagagcagag tttatgactt tgctcttgtc cagtctagac atcttatcgt
     6721 catcgtcttt gtaatccatg gtggcggatc ccgcgtcacg acacctgtgt tctggcggca
     6781 aacccgttgc gaaaaagaac gttcacggcg actactgcac ttatatacgg ttctccccca
     6841 ccctcgggaa aaaggcggag ccagtacacg acatcacttt cccagtttac cccgcgccac
     6901 cttctctagg caccggttca attgccgacc cctcccccca acttctcggg cactgtgggc
     6961 catgtgcgct ctgccccatg gtaatagcca tgactaatac gtagatgtac tgccaagtag
     7021 gaaagtgcca taaggtcatg tactgggcat aatgccaggc gggccattta ccgtcattga
     7081 cgtcaatagg gggcgtactt ggcatatgat acacttgatg tactgccaag tgggcagttt
     7141 accgtaaata ctccacccat tgacgtcaat ggaaagtgcc tattggcgtt actatgggaa
     7201 catacgtcat tattgacgtc aatgggcggg ggtcgttggg cggtcagcca ggcgggccat
     7261 ttaccgtaag ttatgtaacg cggaactcca tatatgggct atgaactaat gaccccgtaa
     7321 ttgattacta ttaataacta gtcaataatc aatgtcataa gctctaatcg cataatcgag
     7381 ctaagagcga ttaatatgct tagctagctt agctaagcta gatttctcta ttctcccaca
     7441 atatatagca gctattagct tagcagtccg aatacgatcg tccacgatcc ataagcgtaa
     7501 cacatttgag tagcgactga cgggcaccgg agccactcga gtggaattat cacctcgagt
     7561 ttactcccta tcagtgatag agaacgtatg aagagtttac tccctatcag tgatagagaa
     7621 cgtatgcaga ctttactccc tatcagtgat agagaacgta taaggagttt actccctatc
     7681 agtgatagag aacgtatgac cagtttactc cctatcagtg atagagaacg tatctacagt
     7741 ttactcccta tcagtgatag agaacgtata tccagtttac tccctatcag tgatagagaa
     7801 cgta
//
