LOCUS       sequence-376976-        7480 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Barcoded piggybac transposon vector with Dox-inducible expression of
            JUN.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7480)
  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 7480)
  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-JUN
FEATURES             Location/Qualifiers
     source          1..7480
                     /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"
     CDS             complement(159..170)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     protein_bind    complement(1138..1162)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1164..1190
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1357..1491
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   1763..1797
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1808..1825)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1808..1824)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1817..1839)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2033..2052)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2154..2176
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2215..2233)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2301..2405
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2406..2474,2475..3266)
                     /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(2624..2643)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3437..4025
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3926..3945
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4179..4196
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4402..4423
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4438..4443,4444..4461,4462..4468)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4476..4492
                     /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     4481..4503
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4500..4516
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4500..4516
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4542..4604
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    4780..4861
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5578..5634)
                     /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(5644..6387)
                     /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(6388..6411)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6411..6420
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6440..6651)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        6652..7031
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     protein_bind    7240..7258
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7276..7294
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7312..7330
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7348..7366
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7384..7402
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7420..7438
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7456..7474
                     /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 atgactgcaa agatggaaac gaccttctat gacgatgccc
      181 tcaacgcctc gttcctcccg tccgagagcg gaccttatgg ctacagtaac cccaagatcc
      241 tgaaacagag catgaccctg aacctggccg acccagtggg gagcctgaag ccgcacctcc
      301 gcgccaagaa ctcggacctc ctcacctcgc ccgacgtggg gctgctcaag ctggcgtcgc
      361 ccgagctgga gcgcctgata atccagtcca gcaacgggca catcaccacc acgccgaccc
      421 ccacccagtt cctgtgcccc aagaacgtga cagatgagca ggagggcttc gccgagggct
      481 tcgtgcgcgc cctggccgaa ctgcacagcc agaacacgct gcccagcgtc acgtcggcgg
      541 cgcagccggt caacggggca ggcatggtgg ctcccgcggt agcctcggtg gcagggggca
      601 gcggcagcgg cggcttcagc gccagcctgc acagcgagcc gccggtctac gcaaacctca
      661 gcaacttcaa cccaggcgcg ctgagcagcg gcggcggggc gccctcctac ggcgcggccg
      721 gcctggcctt tcccgcgcaa ccccagcagc agcagcagcc gccgcaccac ctgccccagc
      781 agatgcccgt gcagcacccg cggctgcagg ccctgaagga ggagcctcag acagtgcccg
      841 agatgcccgg cgagacaccg cccctgtccc ccatcgacat ggagtcccag gagcggatca
      901 aggcggagag gaagcgcatg aggaaccgca tcgctgcctc caagtgccga aaaaggaagc
      961 tggagagaat cgcccggctg gaggaaaaag tgaaaacctt gaaagctcag aactcggagc
     1021 tggcgtccac ggccaacatg ctcagggaac aggtggcaca gcttaaacag aaagtcatga
     1081 accacgttaa cagtgggtgc caactcatgc taacgcagca gttgcaaaca tttttggacc
     1141 cagctttctt gtacaaagtg gtatacccat acgatgttcc agattacgct tagtaatgag
     1201 aaatactcag aagatgtcac tcaccatagt ccgcagtgaa agataaatga tcctcattgc
     1261 ggggcttcga gtgttttaga gctagaaata gcaagtaggc cactgagcaa gaactatgaa
     1321 tgagatcttt agacatattg cggccgcttt ctccgcgatc cagacatgat aagatacatt
     1381 gatgagtttg gacaaaccaa aactagaatg cagtgaaaaa aatgccttat ttgtgaaatt
     1441 tgtgatgcta ttgccttatt tgtaaccatt ataagctgca ataaacaagt tatctataac
     1501 aagaaaatat atatataata agttatcacg taagtagaac atgaaataac aatataatta
     1561 tcgtatgagt taaatcttaa aagtcacgta aaagataatc atgcgtcatt ttgactcacg
     1621 cggtcgttat agttcaaaat cagtgacact taccgcattg acaagcacgc ctcacgggag
     1681 ctccaagcgg cgactgagat gtcctaaatg cacagcgacg gattcgcgct atttagaaag
     1741 agagagcaat atttcaagaa tgcatgcgtc aattttacgc agactatctt tctagggtta
     1801 agaattcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac
     1861 ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca
     1921 ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggcgcctg atgcggtatt
     1981 ttctccttac gcatctgtgc ggtatttcac accgcatatg gtgcactctc agtacaatct
     2041 gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct
     2101 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccatg tctccgggag
     2161 ctgcatgtgt cagaggtttt caccgtcatc accgaaacac gcgagttcga aagggcctcg
     2221 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg
     2281 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     2341 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     2401 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     2461 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     2521 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     2581 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     2641 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     2701 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     2761 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     2821 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     2881 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     2941 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     3001 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     3061 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     3121 gttcacgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     3181 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     3241 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     3301 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     3361 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     3421 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     3481 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     3541 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     3601 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     3661 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     3721 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     3781 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     3841 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     3901 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     3961 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     4021 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     4081 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     4141 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     4201 cggatgagcg cccaatacgc aaaccgcctc tcaccgcgcg ttggccgatt cattaatgca
     4261 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca aaccgcctct
     4321 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc
     4381 gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt
     4441 acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac
     4501 aggaaacagc tatgaccatg attacgccaa ggtcgactta accctagaaa gataatcata
     4561 ttgtgacgta cgttaaagat aatcatgcgt aaaattgacg catgtgtttt atcggtctgt
     4621 atatcgaggt ttatttatta atttgaatag atattaagtt ttattatatt tacacttaca
     4681 tactaataat aaattcaaca aacaatttat ttatgtttat ttatttatta aaaaaaaaca
     4741 aaaactcaaa atttcttcta taaagtaaca aaacttttag cagtgaaaaa aatgctttat
     4801 ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt
     4861 taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt gggaggtttt
     4921 ttaaagcaag taaaacctct acaaatgtgg tatggctgat tatgatcctc tggagattca
     4981 tgcgccaggt tttctggtca tgcaccacgt tcgtgggccc tcaggaacct ccacgtcagc
     5041 ggtaactgtg aagcccagtc gctcgtaaaa aggaagattc cggggagcac tggtttcgag
     5101 gaacgccggc acgccagccc gttccgcggc ctcaacacca ggaagaacga cagcgcttcc
     5161 caaaccttta ccttggtgat ctgggctgac accgacagtg gcaagaaacc acgcaggctc
     5221 ctttggtcta tgaggagcca gcaagccctc catttgctgt tgtgccgcca acctactccc
     5281 tgacagctcg gccatccgcg gacctatttc cgcgaacact gcgccggctt cgacgctttc
     5341 cggagtagtc cagaccgcaa ctgcggcacc atcgtccgca acccaaactt taccaatgtc
     5401 cagacccacg cgcgtaagaa acagctcttg caactcagtt accctctcta tgtgtctgtc
     5461 cggatctacg gtatggcggg tcgctggata atctgcaaat gctgcggcaa gtgttcggac
     5521 agcccgtggt acatcgtcac gggtagcgag cctaacagtg ggcttatatt ccgtcatggg
     5581 gccggggttc tcctccacgt cgccggcctg cttcagcagg ctgaagttgg tggcgccgct
     5641 gcccccgggg agcatgtcaa ggtcaaaatc gtcaagagcg tcagcaggca gcatatcaag
     5701 gtcaaagtcg tcaagggcat cggctgggag catgtctaag tcaaaatcgt caagggcgtc
     5761 ggtcggcccg ccgctttcgc actttagctg tttctccagg ccacatatga ttagttccag
     5821 gccgaaaagg aaggcaggtt cggctccctg ccggtcgaac agctcaattg cttgtttcag
     5881 aagtgggggc atagaatcgg tggtaggtgt ctctctttcc tcttttgcta cttgatgctc
     5941 ctgttcctcc aatacgcagc ccagtgtaaa gtggcccacg gcggacagag cgtacagtgc
     6001 gttctccagg gagaagcctt gctgacacag gaacgcgagc tgattttcca gggtttcgta
     6061 ctgtttctct gttgggcggg tgccgagatg cactttagcc ccgtcacgat gtgagaggag
     6121 agcacagcgg tatgacttgg cgttgttccg cagaaagtct tgccatgact cgccttccag
     6181 ggggcaggag tgggtatgat gcctgtccag catctcgatt ggcagggcat cgagcagggc
     6241 ccgcttgttc ttcacgtgcc agtacagggt aggctgctca actcccagct tttgagcgag
     6301 tttccttgtc gtcaggcctt cgataccgac tccattgagt aattccagag cagagtttat
     6361 gactttgctc ttgtccagtc tagacatctt atcgtcatcg tctttgtaat ccatggtggc
     6421 ggatcccgcg tcacgacacc tgtgttctgg cggcaaaccc gttgcgaaaa agaacgttca
     6481 cggcgactac tgcacttata tacggttctc ccccaccctc gggaaaaagg cggagccagt
     6541 acacgacatc actttcccag tttaccccgc gccaccttct ctaggcaccg gttcaattgc
     6601 cgacccctcc ccccaacttc tcgggcactg tgggccatgt gcgctctgcc ccatggtaat
     6661 agccatgact aatacgtaga tgtactgcca agtaggaaag tgccataagg tcatgtactg
     6721 ggcataatgc caggcgggcc atttaccgtc attgacgtca atagggggcg tacttggcat
     6781 atgatacact tgatgtactg ccaagtgggc agtttaccgt aaatactcca cccattgacg
     6841 tcaatggaaa gtgcctattg gcgttactat gggaacatac gtcattattg acgtcaatgg
     6901 gcgggggtcg ttgggcggtc agccaggcgg gccatttacc gtaagttatg taacgcggaa
     6961 ctccatatat gggctatgaa ctaatgaccc cgtaattgat tactattaat aactagtcaa
     7021 taatcaatgt cataagctct aatcgcataa tcgagctaag agcgattaat atgcttagct
     7081 agcttagcta agctagattt ctctattctc ccacaatata tagcagctat tagcttagca
     7141 gtccgaatac gatcgtccac gatccataag cgtaacacat ttgagtagcg actgacgggc
     7201 accggagcca ctcgagtgga attatcacct cgagtttact ccctatcagt gatagagaac
     7261 gtatgaagag tttactccct atcagtgata gagaacgtat gcagacttta ctccctatca
     7321 gtgatagaga acgtataagg agtttactcc ctatcagtga tagagaacgt atgaccagtt
     7381 tactccctat cagtgataga gaacgtatct acagtttact ccctatcagt gatagagaac
     7441 gtatatccag tttactccct atcagtgata gagaacgtat
//
