LOCUS       sequence-377026-        6970 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Barcoded piggybac transposon vector with Dox-inducible expression of
            MAFF.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6970)
  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 6970)
  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-MAFF
FEATURES             Location/Qualifiers
     source          1..6970
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    8..26
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    44..62
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    80..98
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    116..134
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    152..170
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    188..206
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    224..242
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     269..293
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    359..383
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(876..900)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             902..928
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1095..1229
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   1501..1535
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1546..1563)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1546..1562)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1555..1577)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1771..1790)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1892..1914
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1953..1971)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2039..2143
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2144..2212,2213..3004)
                     /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(2362..2381)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3175..3763
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3664..3683
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3917..3934
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4140..4161
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4176..4181,4182..4199,4200..4206)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4214..4230
                     /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     4219..4241
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4238..4254
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4238..4254
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4280..4342
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    4518..4599
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5316..5372)
                     /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(5382..6125)
                     /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(6126..6149)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6149..6158
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6178..6389)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        6390..6769
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 agtttactcc ctatcagtga tagagaacgt atgaagagtt tactccctat cagtgataga
       61 gaacgtatgc agactttact ccctatcagt gatagagaac gtataaggag tttactccct
      121 atcagtgata gagaacgtat gaccagttta ctccctatca gtgatagaga acgtatctac
      181 agtttactcc ctatcagtga tagagaacgt atatccagtt tactccctat cagtgataga
      241 gaacgtataa gcttgcctat ataagcagag ctcgtttagt gaaccgtcag atcgcctgga
      301 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctaccac
      361 aagtttgtac aaaaaagttg gcatgtctgt ggatccccta tccagcaaag ctctaaagat
      421 caagcgagag ctgagcgaga acacgccgca cctgtcggac gaggcgctga tggggctgtc
      481 ggtgcgcgag ctgaaccggc atctgcgcgg gctctccgcc gaggaggtga cacggctcaa
      541 gcagcggcgc cgcacactca aaaaccgtgg ctacgccgcc agctgccgcg tgaagcgcgt
      601 gtgccagaag gaggagctgc agaagcagaa gtcggagctg gagcgcgagg tggacaagct
      661 ggcgcgcgag aacgccgcca tgcgcctgga gctcgacgcg ctgcgcggca agtgcgaggc
      721 gctgcagggc ttcgcgcgct ccgtggccgc cgcccgcggg cccgccacgc tcgtggcgcc
      781 ggccagcgtc atcaccatcg tcaagtccac cccgggctcg gggtctggcc ccgcccacgg
      841 cccggacccc gcccacggcc cggcctcctg ctcctaccca actttcttgt acaaagtggt
      901 atacccatac gatgttccag attacgctta gtaatgagaa atactcagaa gatgtcactc
      961 accatagtcc gcagtgaaag ataaatgatc ccctgagcac ccgttaagtc gttttagagc
     1021 tagaaatagc aagtaggcca ctgagcaaga actatgaatg agatctttag acatattgcg
     1081 gccgctttct ccgcgatcca gacatgataa gatacattga tgagtttgga caaaccaaaa
     1141 ctagaatgca gtgaaaaaaa tgccttattt gtgaaatttg tgatgctatt gccttatttg
     1201 taaccattat aagctgcaat aaacaagtta tctataacaa gaaaatatat atataataag
     1261 ttatcacgta agtagaacat gaaataacaa tataattatc gtatgagtta aatcttaaaa
     1321 gtcacgtaaa agataatcat gcgtcatttt gactcacgcg gtcgttatag ttcaaaatca
     1381 gtgacactta ccgcattgac aagcacgcct cacgggagct ccaagcggcg actgagatgt
     1441 cctaaatgca cagcgacgga ttcgcgctat ttagaaagag agagcaatat ttcaagaatg
     1501 catgcgtcaa ttttacgcag actatctttc tagggttaag aattcactgg ccgtcgtttt
     1561 acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc
     1621 ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt
     1681 gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     1741 tatttcacac cgcatatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     1801 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     1861 atccgcttac agacaagctg tgaccatgtc tccgggagct gcatgtgtca gaggttttca
     1921 ccgtcatcac cgaaacacgc gagttcgaaa gggcctcgtg atacgcctat ttttataggt
     1981 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
     2041 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     2101 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     2161 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     2221 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     2281 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     2341 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     2401 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     2461 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     2521 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     2581 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     2641 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     2701 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     2761 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     2821 ctggtttatt gctgataaat ctggagccgg tgagcgtggt tcacgcggta tcattgcagc
     2881 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     2941 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     3001 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     3061 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     3121 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     3181 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     3241 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     3301 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     3361 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     3421 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     3481 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     3541 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     3601 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     3661 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     3721 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     3781 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     3841 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     3901 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gatgagcgcc caatacgcaa
     3961 accgcctctc accgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga
     4021 ctggaaagcg ggcagtgagc gcaacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
     4081 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
     4141 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt
     4201 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat
     4261 tacgccaagg tcgacttaac cctagaaaga taatcatatt gtgacgtacg ttaaagataa
     4321 tcatgcgtaa aattgacgca tgtgttttat cggtctgtat atcgaggttt atttattaat
     4381 ttgaatagat attaagtttt attatattta cacttacata ctaataataa attcaacaaa
     4441 caatttattt atgtttattt atttattaaa aaaaaacaaa aactcaaaat ttcttctata
     4501 aagtaacaaa acttttagca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt
     4561 gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat
     4621 tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta aaacctctac
     4681 aaatgtggta tggctgatta tgatcctctg gagattcatg cgccaggttt tctggtcatg
     4741 caccacgttc gtgggccctc aggaacctcc acgtcagcgg taactgtgaa gcccagtcgc
     4801 tcgtaaaaag gaagattccg gggagcactg gtttcgagga acgccggcac gccagcccgt
     4861 tccgcggcct caacaccagg aagaacgaca gcgcttccca aacctttacc ttggtgatct
     4921 gggctgacac cgacagtggc aagaaaccac gcaggctcct ttggtctatg aggagccagc
     4981 aagccctcca tttgctgttg tgccgccaac ctactccctg acagctcggc catccgcgga
     5041 cctatttccg cgaacactgc gccggcttcg acgctttccg gagtagtcca gaccgcaact
     5101 gcggcaccat cgtccgcaac ccaaacttta ccaatgtcca gacccacgcg cgtaagaaac
     5161 agctcttgca actcagttac cctctctatg tgtctgtccg gatctacggt atggcgggtc
     5221 gctggataat ctgcaaatgc tgcggcaagt gttcggacag cccgtggtac atcgtcacgg
     5281 gtagcgagcc taacagtggg cttatattcc gtcatggggc cggggttctc ctccacgtcg
     5341 ccggcctgct tcagcaggct gaagttggtg gcgccgctgc ccccggggag catgtcaagg
     5401 tcaaaatcgt caagagcgtc agcaggcagc atatcaaggt caaagtcgtc aagggcatcg
     5461 gctgggagca tgtctaagtc aaaatcgtca agggcgtcgg tcggcccgcc gctttcgcac
     5521 tttagctgtt tctccaggcc acatatgatt agttccaggc cgaaaaggaa ggcaggttcg
     5581 gctccctgcc ggtcgaacag ctcaattgct tgtttcagaa gtgggggcat agaatcggtg
     5641 gtaggtgtct ctctttcctc ttttgctact tgatgctcct gttcctccaa tacgcagccc
     5701 agtgtaaagt ggcccacggc ggacagagcg tacagtgcgt tctccaggga gaagccttgc
     5761 tgacacagga acgcgagctg attttccagg gtttcgtact gtttctctgt tgggcgggtg
     5821 ccgagatgca ctttagcccc gtcacgatgt gagaggagag cacagcggta tgacttggcg
     5881 ttgttccgca gaaagtcttg ccatgactcg ccttccaggg ggcaggagtg ggtatgatgc
     5941 ctgtccagca tctcgattgg cagggcatcg agcagggccc gcttgttctt cacgtgccag
     6001 tacagggtag gctgctcaac tcccagcttt tgagcgagtt tccttgtcgt caggccttcg
     6061 ataccgactc cattgagtaa ttccagagca gagtttatga ctttgctctt gtccagtcta
     6121 gacatcttat cgtcatcgtc tttgtaatcc atggtggcgg atcccgcgtc acgacacctg
     6181 tgttctggcg gcaaacccgt tgcgaaaaag aacgttcacg gcgactactg cacttatata
     6241 cggttctccc ccaccctcgg gaaaaaggcg gagccagtac acgacatcac tttcccagtt
     6301 taccccgcgc caccttctct aggcaccggt tcaattgccg acccctcccc ccaacttctc
     6361 gggcactgtg ggccatgtgc gctctgcccc atggtaatag ccatgactaa tacgtagatg
     6421 tactgccaag taggaaagtg ccataaggtc atgtactggg cataatgcca ggcgggccat
     6481 ttaccgtcat tgacgtcaat agggggcgta cttggcatat gatacacttg atgtactgcc
     6541 aagtgggcag tttaccgtaa atactccacc cattgacgtc aatggaaagt gcctattggc
     6601 gttactatgg gaacatacgt cattattgac gtcaatgggc gggggtcgtt gggcggtcag
     6661 ccaggcgggc catttaccgt aagttatgta acgcggaact ccatatatgg gctatgaact
     6721 aatgaccccg taattgatta ctattaataa ctagtcaata atcaatgtca taagctctaa
     6781 tcgcataatc gagctaagag cgattaatat gcttagctag cttagctaag ctagatttct
     6841 ctattctccc acaatatata gcagctatta gcttagcagt ccgaatacga tcgtccacga
     6901 tccataagcg taacacattt gagtagcgac tgacgggcac cggagccact cgagtggaat
     6961 tatcacctcg
//
