LOCUS       sequence-377019-        7504 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Barcoded piggybac transposon vector with Dox-inducible expression of
            FOSB.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7504)
  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 7504)
  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-FOSB
FEATURES             Location/Qualifiers
     source          1..7504
                     /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(1162..1186)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1188..1214
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1381..1515
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   1787..1821
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1832..1849)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1832..1848)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1841..1863)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2057..2076)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2178..2200
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2239..2257)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2325..2429
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2430..2498,2499..3290)
                     /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(2648..2667)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3461..4049
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3950..3969
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4203..4220
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4426..4447
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4462..4467,4468..4485,4486..4492)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4500..4516
                     /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     4505..4527
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4524..4540
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4524..4540
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4566..4628
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    4804..4885
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5602..5658)
                     /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(5668..6411)
                     /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(6412..6435)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6435..6444
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6464..6675)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        6676..7055
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     protein_bind    7264..7282
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7300..7318
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7336..7354
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7372..7390
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7408..7426
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7444..7462
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7480..7498
                     /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 aggctccacc atgtttcagg ctttccccgg agactacgac tccggctccc
      181 ggtgcagctc ctcaccctct gccgagtctc aatatctgtc ttcggtggac tccttcggca
      241 gtccacccac cgccgcggcc tcccaggagt gcgccggtct cggggaaatg cccggttcct
      301 tcgtgcccac ggtcaccgcg atcacaacca gccaggacct ccagtggctt gtgcaaccca
      361 ccctcatctc ttccatggcc cagtcccagg ggcagccact ggcctcccag cccccggtcg
      421 tcgaccccta cgacatgccg ggaaccagct actccacacc aggcatgagt ggctacagca
      481 gtggcggagc gagtggcagt ggtgggcctt ccaccagcgg aactaccagt gggcctgggc
      541 ctgcccgccc agcccgagcc cggcctagga gaccccgaga ggagacgctc accccagagg
      601 aagaggagaa gcgaagggtg cgccgggaac gaaataaact agcagcagct aaatgcagga
      661 accggcggag ggagctgacc gaccgactcc aggcggagac agatcagttg gaggaagaaa
      721 aagcagagct ggagtcggag atcgccgagc tccaaaagga gaaggaacgt ctggagtttg
      781 tgctggtggc ccacaaaccg ggctgcaaga tcccctacga agaggggccc gggccgggcc
      841 cgctggcgga ggtgagagat ttgccgggct cagcaccggc taaggaagat ggcttcagct
      901 ggctgctgcc gcccccgcca ccaccgcccc tgcccttcca gaccagccaa gacgcacccc
      961 ccaacctgac ggcttctctc tttacacaca gtgaagttca agtcctcggc gaccccttcc
     1021 ccgttgttaa cccttcgtac acttcttcgt ttgtcctcac ctgcccggag gtctccgcgt
     1081 tcgccggcgc ccaacgcacc agcggcagtg accagccttc cgatcccctg aactcgccct
     1141 ccctcctcgc tctgtggatc cacccagctt tcttgtacaa agtggtatac ccatacgatg
     1201 ttccagatta cgcttagtaa tgagaaatac tcagaagatg tcactcacca tagtccgcag
     1261 tgaaagataa atgatccgaa caagttatga atttttgttt tagagctaga aatagcaagt
     1321 aggccactga gcaagaacta tgaatgagat ctttagacat attgcggccg ctttctccgc
     1381 gatccagaca tgataagata cattgatgag tttggacaaa ccaaaactag aatgcagtga
     1441 aaaaaatgcc ttatttgtga aatttgtgat gctattgcct tatttgtaac cattataagc
     1501 tgcaataaac aagttatcta taacaagaaa atatatatat aataagttat cacgtaagta
     1561 gaacatgaaa taacaatata attatcgtat gagttaaatc ttaaaagtca cgtaaaagat
     1621 aatcatgcgt cattttgact cacgcggtcg ttatagttca aaatcagtga cacttaccgc
     1681 attgacaagc acgcctcacg ggagctccaa gcggcgactg agatgtccta aatgcacagc
     1741 gacggattcg cgctatttag aaagagagag caatatttca agaatgcatg cgtcaatttt
     1801 acgcagacta tctttctagg gttaagaatt cactggccgt cgttttacaa cgtcgtgact
     1861 gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct
     1921 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg
     1981 gcgaatggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     2041 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc
     2101 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     2161 aagctgtgac catgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     2221 acacgcgagt tcgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
     2281 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     2341 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     2401 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     2461 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     2521 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     2581 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     2641 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     2701 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     2761 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     2821 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     2881 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     2941 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     3001 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     3061 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     3121 ataaatctgg agccggtgag cgtggttcac gcggtatcat tgcagcactg gggccagatg
     3181 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     3241 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     3301 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     3361 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     3421 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     3481 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     3541 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     3601 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     3661 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     3721 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     3781 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     3841 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     3901 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     3961 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     4021 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     4081 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     4141 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     4201 gcagcgagtc agtgagcgag gaagcggatg agcgcccaat acgcaaaccg cctctcaccg
     4261 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
     4321 gtgagcgcaa cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
     4381 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
     4441 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
     4501 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaaggtcga
     4561 cttaacccta gaaagataat catattgtga cgtacgttaa agataatcat gcgtaaaatt
     4621 gacgcatgtg ttttatcggt ctgtatatcg aggtttattt attaatttga atagatatta
     4681 agttttatta tatttacact tacatactaa taataaattc aacaaacaat ttatttatgt
     4741 ttatttattt attaaaaaaa aacaaaaact caaaatttct tctataaagt aacaaaactt
     4801 ttagcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac
     4861 cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt
     4921 tcagggggag gtgtgggagg ttttttaaag caagtaaaac ctctacaaat gtggtatggc
     4981 tgattatgat cctctggaga ttcatgcgcc aggttttctg gtcatgcacc acgttcgtgg
     5041 gccctcagga acctccacgt cagcggtaac tgtgaagccc agtcgctcgt aaaaaggaag
     5101 attccgggga gcactggttt cgaggaacgc cggcacgcca gcccgttccg cggcctcaac
     5161 accaggaaga acgacagcgc ttcccaaacc tttaccttgg tgatctgggc tgacaccgac
     5221 agtggcaaga aaccacgcag gctcctttgg tctatgagga gccagcaagc cctccatttg
     5281 ctgttgtgcc gccaacctac tccctgacag ctcggccatc cgcggaccta tttccgcgaa
     5341 cactgcgccg gcttcgacgc tttccggagt agtccagacc gcaactgcgg caccatcgtc
     5401 cgcaacccaa actttaccaa tgtccagacc cacgcgcgta agaaacagct cttgcaactc
     5461 agttaccctc tctatgtgtc tgtccggatc tacggtatgg cgggtcgctg gataatctgc
     5521 aaatgctgcg gcaagtgttc ggacagcccg tggtacatcg tcacgggtag cgagcctaac
     5581 agtgggctta tattccgtca tggggccggg gttctcctcc acgtcgccgg cctgcttcag
     5641 caggctgaag ttggtggcgc cgctgccccc ggggagcatg tcaaggtcaa aatcgtcaag
     5701 agcgtcagca ggcagcatat caaggtcaaa gtcgtcaagg gcatcggctg ggagcatgtc
     5761 taagtcaaaa tcgtcaaggg cgtcggtcgg cccgccgctt tcgcacttta gctgtttctc
     5821 caggccacat atgattagtt ccaggccgaa aaggaaggca ggttcggctc cctgccggtc
     5881 gaacagctca attgcttgtt tcagaagtgg gggcatagaa tcggtggtag gtgtctctct
     5941 ttcctctttt gctacttgat gctcctgttc ctccaatacg cagcccagtg taaagtggcc
     6001 cacggcggac agagcgtaca gtgcgttctc cagggagaag ccttgctgac acaggaacgc
     6061 gagctgattt tccagggttt cgtactgttt ctctgttggg cgggtgccga gatgcacttt
     6121 agccccgtca cgatgtgaga ggagagcaca gcggtatgac ttggcgttgt tccgcagaaa
     6181 gtcttgccat gactcgcctt ccagggggca ggagtgggta tgatgcctgt ccagcatctc
     6241 gattggcagg gcatcgagca gggcccgctt gttcttcacg tgccagtaca gggtaggctg
     6301 ctcaactccc agcttttgag cgagtttcct tgtcgtcagg ccttcgatac cgactccatt
     6361 gagtaattcc agagcagagt ttatgacttt gctcttgtcc agtctagaca tcttatcgtc
     6421 atcgtctttg taatccatgg tggcggatcc cgcgtcacga cacctgtgtt ctggcggcaa
     6481 acccgttgcg aaaaagaacg ttcacggcga ctactgcact tatatacggt tctcccccac
     6541 cctcgggaaa aaggcggagc cagtacacga catcactttc ccagtttacc ccgcgccacc
     6601 ttctctaggc accggttcaa ttgccgaccc ctccccccaa cttctcgggc actgtgggcc
     6661 atgtgcgctc tgccccatgg taatagccat gactaatacg tagatgtact gccaagtagg
     6721 aaagtgccat aaggtcatgt actgggcata atgccaggcg ggccatttac cgtcattgac
     6781 gtcaataggg ggcgtacttg gcatatgata cacttgatgt actgccaagt gggcagttta
     6841 ccgtaaatac tccacccatt gacgtcaatg gaaagtgcct attggcgtta ctatgggaac
     6901 atacgtcatt attgacgtca atgggcgggg gtcgttgggc ggtcagccag gcgggccatt
     6961 taccgtaagt tatgtaacgc ggaactccat atatgggcta tgaactaatg accccgtaat
     7021 tgattactat taataactag tcaataatca atgtcataag ctctaatcgc ataatcgagc
     7081 taagagcgat taatatgctt agctagctta gctaagctag atttctctat tctcccacaa
     7141 tatatagcag ctattagctt agcagtccga atacgatcgt ccacgatcca taagcgtaac
     7201 acatttgagt agcgactgac gggcaccgga gccactcgag tggaattatc acctcgagtt
     7261 tactccctat cagtgataga gaacgtatga agagtttact ccctatcagt gatagagaac
     7321 gtatgcagac tttactccct atcagtgata gagaacgtat aaggagttta ctccctatca
     7381 gtgatagaga acgtatgacc agtttactcc ctatcagtga tagagaacgt atctacagtt
     7441 tactccctat cagtgataga gaacgtatat ccagtttact ccctatcagt gatagagaac
     7501 gtat
//
