LOCUS       sequence-373937-        7894 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 7894)
  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 7894)
  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..7894
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    17..35
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    53..71
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    89..107
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    125..143
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    161..179
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    197..215
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    233..251
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     278..302
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    368..392
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1776..1800)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1802..1828
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     gap             1891..1910
                     /estimated_length=20
     polyA_signal    1995..2129
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   2401..2435
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2446..2463)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2446..2462)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2455..2477)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2671..2690)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2792..2814
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2853..2871)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2939..3043
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3044..3112,3113..3904)
                     /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(3262..3281)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4075..4663
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4564..4583
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4817..4834
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5040..5061
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5076..5081,5082..5099,5100..5106)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5114..5130
                     /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     5119..5141
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5138..5154
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5138..5154
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   5180..5242
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    5418..5499
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(6216..6272)
                     /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(6282..7025)
                     /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(7026..7049)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      7049..7058
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(7078..7289)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        7290..7669
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 atcacctcga gtttactccc tatcagtgat agagaacgta tgaagagttt actccctatc
       61 agtgatagag aacgtatgca gactttactc cctatcagtg atagagaacg tataaggagt
      121 ttactcccta tcagtgatag agaacgtatg accagtttac tccctatcag tgatagagaa
      181 cgtatctaca gtttactccc tatcagtgat agagaacgta tatccagttt actccctatc
      241 agtgatagag aacgtataag cttgcctata taagcagagc tcgtttagtg aaccgtcaga
      301 tcgcctggag acgccatcca cgctgttttg acctccatag aagacaccgg gaccgatcca
      361 gcctaccaca agtttgtaca aaaaagttgg catggactat tcgtacgacg aggacctgga
      421 cgagctgtgc cccgtgtgcg gggacaaggt gtccggctac cactacggac tgctcacgtg
      481 tgagagctgc aagggcttct tcaagcgcac ggtgcagaac aacaagcact acacgtgcac
      541 cgagagccag agctgcaaga tcgacaagac gcagcgcaag cgctgtccct tctgccgctt
      601 ccagaaatgc ctgacggtgg ggatgcgcct ggaagccgtg cgcgctgacc gtatgagggg
      661 tggccggaac aagtttgggc cgatgtacaa gcgggaccgg gccctgaaac agcagaagaa
      721 ggcacagatt cgggccaatg gcttcaagct ggagacaggg cccccgatgg gggtgccccc
      781 gccgccccct cccgcaccgg actacgtgct gcctcccagc ctgcatgggc ctgagcccaa
      841 gggcctggcc gccggtccac ctgctgggcc actgggcgac tttggggccc cagcactgcc
      901 catggccgtg cccggtgccc acgggccact ggctggctac ctctaccctg cctttcctgg
      961 ccgtgccatc aagtctgagt acccggagcc ttatgccagc cccccacagc ctgggctgcc
     1021 gtacggctac ccagagccct tctctggagg gcccaacgtg cctgagctca tcctgcagct
     1081 gctgcagctg gagccggatg aggaccaggt gcgggcccgc atcttgggct gcctgcagga
     1141 gcccaccaaa agccgccccg accagccggc ggccttcggc ctcctgtgca gaatggccga
     1201 ccagaccttc atctccatcg tggactgggc acgcaggtgc atggtcttca aggagctgga
     1261 ggtggccgac cagatgacgc tgctgcagaa ctgctggagc gagctgctgg tgttcgacca
     1321 catctaccgc caggtccagc acggcaagga gggcagcatc ctgctggtca ccgggcagga
     1381 ggtggagctg accacagtgg ccacccaggc gggctcgctg ctgcacagcc tggtgttgcg
     1441 ggcgcaggag ctggtgctgc agctgcttgc gctgcagctg gaccggcagg agtttgtctg
     1501 cctcaagttc atcatcctct tcagcctgga tttgaagttc ctgaataacc acatcctggt
     1561 gaaagacgct caggagaagg ccaacgccgc cctgcttgac tacaccctgt gccactaccc
     1621 gcactgcggg gacaaattcc agcagctgct gctgtgcctg gtggaggtgc gggccctgag
     1681 catgcaggcc aaggagtacc tgtaccacaa gcacctgggc aacgagatgc cccgcaacaa
     1741 cctgctcatc gaaatgctgc aagccaagca gacttgccca gctttcttgt acaaagtggt
     1801 atacccatac gatgttccag attacgctta gtaatgagaa atactcagaa gatgtcactc
     1861 accatagtcc gcagtgaaag ataaatgatc nnnnnnnnnn nnnnnnnnnn gttttagagc
     1921 tagaaatagc aagtaggcca ctgagcaaga actatgaatg agatctttag acatattgcg
     1981 gccgctttct ccgcgatcca gacatgataa gatacattga tgagtttgga caaaccaaaa
     2041 ctagaatgca gtgaaaaaaa tgccttattt gtgaaatttg tgatgctatt gccttatttg
     2101 taaccattat aagctgcaat aaacaagtta tctataacaa gaaaatatat atataataag
     2161 ttatcacgta agtagaacat gaaataacaa tataattatc gtatgagtta aatcttaaaa
     2221 gtcacgtaaa agataatcat gcgtcatttt gactcacgcg gtcgttatag ttcaaaatca
     2281 gtgacactta ccgcattgac aagcacgcct cacgggagct ccaagcggcg actgagatgt
     2341 cctaaatgca cagcgacgga ttcgcgctat ttagaaagag agagcaatat ttcaagaatg
     2401 catgcgtcaa ttttacgcag actatctttc tagggttaag aattcactgg ccgtcgtttt
     2461 acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc
     2521 ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt
     2581 gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     2641 tatttcacac cgcatatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     2701 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     2761 atccgcttac agacaagctg tgaccatgtc tccgggagct gcatgtgtca gaggttttca
     2821 ccgtcatcac cgaaacacgc gagttcgaaa gggcctcgtg atacgcctat ttttataggt
     2881 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
     2941 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     3001 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     3061 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     3121 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     3181 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     3241 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     3301 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     3361 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     3421 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     3481 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     3541 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     3601 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     3661 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     3721 ctggtttatt gctgataaat ctggagccgg tgagcgtggt tcacgcggta tcattgcagc
     3781 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     3841 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     3901 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     3961 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     4021 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     4081 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     4141 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     4201 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     4261 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     4321 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     4381 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     4441 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     4501 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     4561 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     4621 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     4681 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     4741 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     4801 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gatgagcgcc caatacgcaa
     4861 accgcctctc accgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga
     4921 ctggaaagcg ggcagtgagc gcaacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
     4981 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
     5041 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt
     5101 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat
     5161 tacgccaagg tcgacttaac cctagaaaga taatcatatt gtgacgtacg ttaaagataa
     5221 tcatgcgtaa aattgacgca tgtgttttat cggtctgtat atcgaggttt atttattaat
     5281 ttgaatagat attaagtttt attatattta cacttacata ctaataataa attcaacaaa
     5341 caatttattt atgtttattt atttattaaa aaaaaacaaa aactcaaaat ttcttctata
     5401 aagtaacaaa acttttagca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt
     5461 gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat
     5521 tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta aaacctctac
     5581 aaatgtggta tggctgatta tgatcctctg gagattcatg cgccaggttt tctggtcatg
     5641 caccacgttc gtgggccctc aggaacctcc acgtcagcgg taactgtgaa gcccagtcgc
     5701 tcgtaaaaag gaagattccg gggagcactg gtttcgagga acgccggcac gccagcccgt
     5761 tccgcggcct caacaccagg aagaacgaca gcgcttccca aacctttacc ttggtgatct
     5821 gggctgacac cgacagtggc aagaaaccac gcaggctcct ttggtctatg aggagccagc
     5881 aagccctcca tttgctgttg tgccgccaac ctactccctg acagctcggc catccgcgga
     5941 cctatttccg cgaacactgc gccggcttcg acgctttccg gagtagtcca gaccgcaact
     6001 gcggcaccat cgtccgcaac ccaaacttta ccaatgtcca gacccacgcg cgtaagaaac
     6061 agctcttgca actcagttac cctctctatg tgtctgtccg gatctacggt atggcgggtc
     6121 gctggataat ctgcaaatgc tgcggcaagt gttcggacag cccgtggtac atcgtcacgg
     6181 gtagcgagcc taacagtggg cttatattcc gtcatggggc cggggttctc ctccacgtcg
     6241 ccggcctgct tcagcaggct gaagttggtg gcgccgctgc ccccggggag catgtcaagg
     6301 tcaaaatcgt caagagcgtc agcaggcagc atatcaaggt caaagtcgtc aagggcatcg
     6361 gctgggagca tgtctaagtc aaaatcgtca agggcgtcgg tcggcccgcc gctttcgcac
     6421 tttagctgtt tctccaggcc acatatgatt agttccaggc cgaaaaggaa ggcaggttcg
     6481 gctccctgcc ggtcgaacag ctcaattgct tgtttcagaa gtgggggcat agaatcggtg
     6541 gtaggtgtct ctctttcctc ttttgctact tgatgctcct gttcctccaa tacgcagccc
     6601 agtgtaaagt ggcccacggc ggacagagcg tacagtgcgt tctccaggga gaagccttgc
     6661 tgacacagga acgcgagctg attttccagg gtttcgtact gtttctctgt tgggcgggtg
     6721 ccgagatgca ctttagcccc gtcacgatgt gagaggagag cacagcggta tgacttggcg
     6781 ttgttccgca gaaagtcttg ccatgactcg ccttccaggg ggcaggagtg ggtatgatgc
     6841 ctgtccagca tctcgattgg cagggcatcg agcagggccc gcttgttctt cacgtgccag
     6901 tacagggtag gctgctcaac tcccagcttt tgagcgagtt tccttgtcgt caggccttcg
     6961 ataccgactc cattgagtaa ttccagagca gagtttatga ctttgctctt gtccagtcta
     7021 gacatcttat cgtcatcgtc tttgtaatcc atggtggcgg atcccgcgtc acgacacctg
     7081 tgttctggcg gcaaacccgt tgcgaaaaag aacgttcacg gcgactactg cacttatata
     7141 cggttctccc ccaccctcgg gaaaaaggcg gagccagtac acgacatcac tttcccagtt
     7201 taccccgcgc caccttctct aggcaccggt tcaattgccg acccctcccc ccaacttctc
     7261 gggcactgtg ggccatgtgc gctctgcccc atggtaatag ccatgactaa tacgtagatg
     7321 tactgccaag taggaaagtg ccataaggtc atgtactggg cataatgcca ggcgggccat
     7381 ttaccgtcat tgacgtcaat agggggcgta cttggcatat gatacacttg atgtactgcc
     7441 aagtgggcag tttaccgtaa atactccacc cattgacgtc aatggaaagt gcctattggc
     7501 gttactatgg gaacatacgt cattattgac gtcaatgggc gggggtcgtt gggcggtcag
     7561 ccaggcgggc catttaccgt aagttatgta acgcggaact ccatatatgg gctatgaact
     7621 aatgaccccg taattgatta ctattaataa ctagtcaata atcaatgtca taagctctaa
     7681 tcgcataatc gagctaagag cgattaatat gcttagctag cttagctaag ctagatttct
     7741 ctattctccc acaatatata gcagctatta gcttagcagt ccgaatacga tcgtccacga
     7801 tccataagcg taacacattt gagtagcgac tgacgggcac cggagccact cgagtggaat
     7861 tactgacggg caccggagcc actcgagtgg aatt
//
