LOCUS       sequence-377907-        7519 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Barcoded piggybac transposon vector with Dox-inducible expression of
            JUNB.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7519)
  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 7519)
  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-JUNB
FEATURES             Location/Qualifiers
     source          1..7519
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     23..47
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    113..137
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1179..1203)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1205..1231
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1398..1532
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   1804..1838
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1849..1866)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1849..1865)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1858..1880)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2074..2093)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2195..2217
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2256..2274)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2342..2446
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2447..2515,2516..3307)
                     /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(2665..2684)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3478..4066
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3967..3986
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4220..4237
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4443..4464
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4479..4484,4485..4502,4503..4509)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4517..4533
                     /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     4522..4544
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4541..4557
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4541..4557
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4583..4645
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    4821..4902
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5619..5675)
                     /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(5685..6428)
                     /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(6429..6452)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6452..6461
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6481..6692)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        6693..7072
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     protein_bind    7281..7299
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7317..7335
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7353..7371
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7389..7407
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7425..7443
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7461..7479
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7497..7515
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
ORIGIN
        1 ataagcttgc ctatataagc agagctcgtt tagtgaaccg tcagatcgcc tggagacgcc
       61 atccacgctg ttttgacctc catagaagac accgggaccg atccagccta ccacaagttt
      121 gtacaaaaaa gttggcatgt gcactaaaat ggaacagccc ttctaccacg acgactcata
      181 cacagctacg ggatacggcc gggcccctgg tggcctctct ctacacgact acaaactcct
      241 gaaaccgagc ctggcggtca acctggccga cccctaccgg agtctcaaag cgcctggggc
      301 tcgcggaccc ggcccagagg gcggcggtgg cggcagctac ttttctggtc agggctcgga
      361 caccggcgcg tctctcaagc tcgcctcttc ggagctggaa cgcctgattg tccccaacag
      421 caacggcgtg atcacgacga cgcctacacc cccgggacag tacttttacc cccgcggggg
      481 tggcagcggt ggaggtgcag ggggcgcagg gggcggcgtc accgaggagc aggagggctt
      541 cgccgacggc tttgtcaaag ccctggacga tctgcacaag atgaaccacg tgacaccccc
      601 caacgtgtcc ctgggcgcta ccggggggcc cccggctggg cccgggggcg tctacgccgg
      661 cccggagcca cctcccgttt acaccaacct cagcagctac tccccagcct ctgcgtcctc
      721 gggaggcgcc ggggctgccg tcgggaccgg gagctcgtac ccgacgacca ccatcagcta
      781 cctcccacac gcgccgccct tcgccggtgg ccacccggcg cagctgggct tgggccgcgg
      841 cgcctccacc ttcaaggagg aaccgcagac cgtgccggag gcgcgcagcc gggacgccac
      901 gccgccggtg tcccccatca acatggaaga ccaagagcgc atcaaagtgg agcgcaagcg
      961 gctgcggaac cggctggcgg ccaccaagtg ccggaagcgg aagctggagc gcatcgcgcg
     1021 cctggaggac aaggtgaaga cgctcaaggc cgagaacgcg gggctgtcga gtaccgccgg
     1081 cctcctccgg gagcaggtgg cccagctcaa acagaaggtc atgacccacg tcagcaacgg
     1141 ctgtcagctg ctgcttgggg tcaagggaca cgccttctgc ccaactttct tgtacaaagt
     1201 ggtataccca tacgatgttc cagattacgc ttagtaatga gaaatactca gaagatgtca
     1261 ctcaccatag tccgcagtga aagataaatg atcctaattt ccgccccacg tgcgttttag
     1321 agctagaaat agcaagtagg ccactgagca agaactatga atgagatctt tagacatatt
     1381 gcggccgctt tctccgcgat ccagacatga taagatacat tgatgagttt ggacaaacca
     1441 aaactagaat gcagtgaaaa aaatgcctta tttgtgaaat ttgtgatgct attgccttat
     1501 ttgtaaccat tataagctgc aataaacaag ttatctataa caagaaaata tatatataat
     1561 aagttatcac gtaagtagaa catgaaataa caatataatt atcgtatgag ttaaatctta
     1621 aaagtcacgt aaaagataat catgcgtcat tttgactcac gcggtcgtta tagttcaaaa
     1681 tcagtgacac ttaccgcatt gacaagcacg cctcacggga gctccaagcg gcgactgaga
     1741 tgtcctaaat gcacagcgac ggattcgcgc tatttagaaa gagagagcaa tatttcaaga
     1801 atgcatgcgt caattttacg cagactatct ttctagggtt aagaattcac tggccgtcgt
     1861 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca
     1921 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
     1981 gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat tttctcctta cgcatctgtg
     2041 cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     2101 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     2161 ggcatccgct tacagacaag ctgtgaccat gtctccggga gctgcatgtg tcagaggttt
     2221 tcaccgtcat caccgaaaca cgcgagttcg aaagggcctc gtgatacgcc tatttttata
     2281 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
     2341 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     2401 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     2461 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     2521 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     2581 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     2641 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     2701 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     2761 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     2821 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     2881 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     2941 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     3001 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     3061 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     3121 tggctggttt attgctgata aatctggagc cggtgagcgt ggttcacgcg gtatcattgc
     3181 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     3241 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     3301 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     3361 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     3421 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     3481 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     3541 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     3601 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     3661 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     3721 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     3781 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     3841 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     3901 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     3961 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     4021 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     4081 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     4141 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     4201 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggatgagc gcccaatacg
     4261 caaaccgcct ctcaccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
     4321 cgactggaaa gcgggcagtg agcgcaacgc aaaccgcctc tccccgcgcg ttggccgatt
     4381 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
     4441 attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct
     4501 cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat
     4561 gattacgcca aggtcgactt aaccctagaa agataatcat attgtgacgt acgttaaaga
     4621 taatcatgcg taaaattgac gcatgtgttt tatcggtctg tatatcgagg tttatttatt
     4681 aatttgaata gatattaagt tttattatat ttacacttac atactaataa taaattcaac
     4741 aaacaattta tttatgttta tttatttatt aaaaaaaaac aaaaactcaa aatttcttct
     4801 ataaagtaac aaaactttta gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct
     4861 attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt
     4921 cattttatgt ttcaggttca gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc
     4981 tacaaatgtg gtatggctga ttatgatcct ctggagattc atgcgccagg ttttctggtc
     5041 atgcaccacg ttcgtgggcc ctcaggaacc tccacgtcag cggtaactgt gaagcccagt
     5101 cgctcgtaaa aaggaagatt ccggggagca ctggtttcga ggaacgccgg cacgccagcc
     5161 cgttccgcgg cctcaacacc aggaagaacg acagcgcttc ccaaaccttt accttggtga
     5221 tctgggctga caccgacagt ggcaagaaac cacgcaggct cctttggtct atgaggagcc
     5281 agcaagccct ccatttgctg ttgtgccgcc aacctactcc ctgacagctc ggccatccgc
     5341 ggacctattt ccgcgaacac tgcgccggct tcgacgcttt ccggagtagt ccagaccgca
     5401 actgcggcac catcgtccgc aacccaaact ttaccaatgt ccagacccac gcgcgtaaga
     5461 aacagctctt gcaactcagt taccctctct atgtgtctgt ccggatctac ggtatggcgg
     5521 gtcgctggat aatctgcaaa tgctgcggca agtgttcgga cagcccgtgg tacatcgtca
     5581 cgggtagcga gcctaacagt gggcttatat tccgtcatgg ggccggggtt ctcctccacg
     5641 tcgccggcct gcttcagcag gctgaagttg gtggcgccgc tgcccccggg gagcatgtca
     5701 aggtcaaaat cgtcaagagc gtcagcaggc agcatatcaa ggtcaaagtc gtcaagggca
     5761 tcggctggga gcatgtctaa gtcaaaatcg tcaagggcgt cggtcggccc gccgctttcg
     5821 cactttagct gtttctccag gccacatatg attagttcca ggccgaaaag gaaggcaggt
     5881 tcggctccct gccggtcgaa cagctcaatt gcttgtttca gaagtggggg catagaatcg
     5941 gtggtaggtg tctctctttc ctcttttgct acttgatgct cctgttcctc caatacgcag
     6001 cccagtgtaa agtggcccac ggcggacaga gcgtacagtg cgttctccag ggagaagcct
     6061 tgctgacaca ggaacgcgag ctgattttcc agggtttcgt actgtttctc tgttgggcgg
     6121 gtgccgagat gcactttagc cccgtcacga tgtgagagga gagcacagcg gtatgacttg
     6181 gcgttgttcc gcagaaagtc ttgccatgac tcgccttcca gggggcagga gtgggtatga
     6241 tgcctgtcca gcatctcgat tggcagggca tcgagcaggg cccgcttgtt cttcacgtgc
     6301 cagtacaggg taggctgctc aactcccagc ttttgagcga gtttccttgt cgtcaggcct
     6361 tcgataccga ctccattgag taattccaga gcagagttta tgactttgct cttgtccagt
     6421 ctagacatct tatcgtcatc gtctttgtaa tccatggtgg cggatcccgc gtcacgacac
     6481 ctgtgttctg gcggcaaacc cgttgcgaaa aagaacgttc acggcgacta ctgcacttat
     6541 atacggttct cccccaccct cgggaaaaag gcggagccag tacacgacat cactttccca
     6601 gtttaccccg cgccaccttc tctaggcacc ggttcaattg ccgacccctc cccccaactt
     6661 ctcgggcact gtgggccatg tgcgctctgc cccatggtaa tagccatgac taatacgtag
     6721 atgtactgcc aagtaggaaa gtgccataag gtcatgtact gggcataatg ccaggcgggc
     6781 catttaccgt cattgacgtc aatagggggc gtacttggca tatgatacac ttgatgtact
     6841 gccaagtggg cagtttaccg taaatactcc acccattgac gtcaatggaa agtgcctatt
     6901 ggcgttacta tgggaacata cgtcattatt gacgtcaatg ggcgggggtc gttgggcggt
     6961 cagccaggcg ggccatttac cgtaagttat gtaacgcgga actccatata tgggctatga
     7021 actaatgacc ccgtaattga ttactattaa taactagtca ataatcaatg tcataagctc
     7081 taatcgcata atcgagctaa gagcgattaa tatgcttagc tagcttagct aagctagatt
     7141 tctctattct cccacaatat atagcagcta ttagcttagc agtccgaata cgatcgtcca
     7201 cgatccataa gcgtaacaca tttgagtagc gactgacggg caccggagcc actcgagtgg
     7261 aattatcacc tcgagtttac tccctatcag tgatagagaa cgtatgaaga gtttactccc
     7321 tatcagtgat agagaacgta tgcagacttt actccctatc agtgatagag aacgtataag
     7381 gagtttactc cctatcagtg atagagaacg tatgaccagt ttactcccta tcagtgatag
     7441 agaacgtatc tacagtttac tccctatcag tgatagagaa cgtatatcca gtttactccc
     7501 tatcagtgat agagaacgt
//
