LOCUS       sequence-377025-        8107 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Barcoded piggybac transposon vector with Dox-inducible expression of
            EGR1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8107)
  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 8107)
  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-EGR1
FEATURES             Location/Qualifiers
     source          1..8107
                     /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(1765..1789)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1791..1817
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1984..2118
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   2390..2424
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2435..2452)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2435..2451)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2444..2466)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2660..2679)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2781..2803
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2842..2860)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2928..3032
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3033..3101,3102..3893)
                     /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(3251..3270)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4064..4652
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4553..4572
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4806..4823
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5029..5050
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5065..5070,5071..5088,5089..5095)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5103..5119
                     /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     5108..5130
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5127..5143
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5127..5143
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   5169..5231
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    5407..5488
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(6205..6261)
                     /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(6271..7014)
                     /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(7015..7038)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      7038..7047
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(7067..7278)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        7279..7658
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     protein_bind    7867..7885
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7903..7921
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7939..7957
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7975..7993
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    8011..8029
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    8047..8065
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    8083..8101
                     /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 acaaaaaagt tggcatggcc gcggccaagg ccgagatgca gctgatgtcc ccgctgcaga
      181 tctctgaccc gttcggatcc tttcctcact cgcccaccat ggacaactac cctaagctgg
      241 aggagatgat gctgctgagc aacggggctc cccagttcct cggcgccgcc ggggccccag
      301 agggcagcgg cagcaacagc agcagcagca gcagcggggg cggtggaggc ggcgggggcg
      361 gcagcaacag cagcagcagc agcagcacct tcaaccctca ggcggacacg ggcgagcagc
      421 cctacgagca cctgaccgca gagtcttttc ctgacatctc tctgaacaac gagaaggtgc
      481 tggtggagac cagttacccc agccaaacca ctcgactgcc ccccatcacc tatactggcc
      541 gcttttccct ggagcctgca cccaacagtg gcaacacctt gtggcccgag cccctcttca
      601 gcttggtcag tggcctagtg agcatgacca acccaccggc ctcctcgtcc tcagcaccat
      661 ctccagcggc ctcctccgcc tccgcctccc agagcccacc cctgagctgc gcagtgccat
      721 ccaacgacag cagtcccatt tactcagcgg cacccacctt ccccacgccg aacactgaca
      781 ttttccctga gccacaaagc caggccttcc cgggctcggc agggacagcg ctccagtacc
      841 cgcctcctgc ctaccctgcc gccaagggtg gcttccaggt tcccatgatc cccgactacc
      901 tgtttccaca gcagcagggg gatctgggcc tgggcacccc agaccagaag cccttccagg
      961 gcctggagag ccgcacccag cagccttcgc taacccctct gtctactatt aaggcctttg
     1021 ccactcagtc gggctcccag gacctgaagg ccctcaatac cagctaccag tcccagctca
     1081 tcaaacccag ccgcatgcgc aagtacccca accggcccag caagacgccc ccccacgaac
     1141 gcccttacgc ttgcccagtg gagtcctgtg atcgccgctt ctcccgctcc gacgagctca
     1201 cccgccacat ccgcatccac acaggccaga agcccttcca gtgccgcatc tgcatgcgca
     1261 acttcagccg cagcgaccac ctcaccaccc acatccgcac ccacacaggc gaaaagccct
     1321 tcgcctgcga catctgtgga agaaagtttg ccaggagcga tgaacgcaag aggcatacca
     1381 agatccactt gcggcagaag gacaagaaag cagacaaaag tgttgtggcc tcttcggcca
     1441 cctcctctct ctcttcctac ccgtccccgg ttgctacctc ttacccgtcc ccggttacta
     1501 cctcttatcc atccccggcc accacctcat acccatcccc tgtgcccacc tccttctcct
     1561 ctcccggctc ctcgacctac ccatcccctg tgcacagtgg cttcccctcc ccgtcggtgg
     1621 ccaccacgta ctcctctgtt ccccctgctt tcccggccca ggtcagcagc ttcccttcct
     1681 cagctgtcac caactccttc agcgcctcca cagggctttc ggacatgaca gcaacctttt
     1741 ctcccaggac aattgaaatt tgctacccaa ctttcttgta caaagtggta tacccatacg
     1801 atgttccaga ttacgcttag taatgagaaa tactcagaag atgtcactca ccatagtccg
     1861 cagtgaaaga taaatgatca attcttcaaa aaataacaag ttttagagct agaaatagca
     1921 agtaggccac tgagcaagaa ctatgaatga gatctttaga catattgcgg ccgctttctc
     1981 cgcgatccag acatgataag atacattgat gagtttggac aaaccaaaac tagaatgcag
     2041 tgaaaaaaat gccttatttg tgaaatttgt gatgctattg ccttatttgt aaccattata
     2101 agctgcaata aacaagttat ctataacaag aaaatatata tataataagt tatcacgtaa
     2161 gtagaacatg aaataacaat ataattatcg tatgagttaa atcttaaaag tcacgtaaaa
     2221 gataatcatg cgtcattttg actcacgcgg tcgttatagt tcaaaatcag tgacacttac
     2281 cgcattgaca agcacgcctc acgggagctc caagcggcga ctgagatgtc ctaaatgcac
     2341 agcgacggat tcgcgctatt tagaaagaga gagcaatatt tcaagaatgc atgcgtcaat
     2401 tttacgcaga ctatctttct agggttaaga attcactggc cgtcgtttta caacgtcgtg
     2461 actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca
     2521 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga
     2581 atggcgaatg gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     2641 gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
     2701 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     2761 gacaagctgt gaccatgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     2821 gaaacacgcg agttcgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     2881 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     2941 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     3001 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     3061 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     3121 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     3181 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     3241 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     3301 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     3361 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     3421 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     3481 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     3541 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     3601 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     3661 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     3721 ctgataaatc tggagccggt gagcgtggtt cacgcggtat cattgcagca ctggggccag
     3781 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     3841 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     3901 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     3961 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     4021 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     4081 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     4141 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     4201 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     4261 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     4321 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     4381 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     4441 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     4501 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     4561 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     4621 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     4681 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     4741 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     4801 agcgcagcga gtcagtgagc gaggaagcgg atgagcgccc aatacgcaaa ccgcctctca
     4861 ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg
     4921 gcagtgagcg caacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     4981 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
     5041 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
     5101 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaaggt
     5161 cgacttaacc ctagaaagat aatcatattg tgacgtacgt taaagataat catgcgtaaa
     5221 attgacgcat gtgttttatc ggtctgtata tcgaggttta tttattaatt tgaatagata
     5281 ttaagtttta ttatatttac acttacatac taataataaa ttcaacaaac aatttattta
     5341 tgtttattta tttattaaaa aaaaacaaaa actcaaaatt tcttctataa agtaacaaaa
     5401 cttttagcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt
     5461 aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca
     5521 ggttcagggg gaggtgtggg aggtttttta aagcaagtaa aacctctaca aatgtggtat
     5581 ggctgattat gatcctctgg agattcatgc gccaggtttt ctggtcatgc accacgttcg
     5641 tgggccctca ggaacctcca cgtcagcggt aactgtgaag cccagtcgct cgtaaaaagg
     5701 aagattccgg ggagcactgg tttcgaggaa cgccggcacg ccagcccgtt ccgcggcctc
     5761 aacaccagga agaacgacag cgcttcccaa acctttacct tggtgatctg ggctgacacc
     5821 gacagtggca agaaaccacg caggctcctt tggtctatga ggagccagca agccctccat
     5881 ttgctgttgt gccgccaacc tactccctga cagctcggcc atccgcggac ctatttccgc
     5941 gaacactgcg ccggcttcga cgctttccgg agtagtccag accgcaactg cggcaccatc
     6001 gtccgcaacc caaactttac caatgtccag acccacgcgc gtaagaaaca gctcttgcaa
     6061 ctcagttacc ctctctatgt gtctgtccgg atctacggta tggcgggtcg ctggataatc
     6121 tgcaaatgct gcggcaagtg ttcggacagc ccgtggtaca tcgtcacggg tagcgagcct
     6181 aacagtgggc ttatattccg tcatggggcc ggggttctcc tccacgtcgc cggcctgctt
     6241 cagcaggctg aagttggtgg cgccgctgcc cccggggagc atgtcaaggt caaaatcgtc
     6301 aagagcgtca gcaggcagca tatcaaggtc aaagtcgtca agggcatcgg ctgggagcat
     6361 gtctaagtca aaatcgtcaa gggcgtcggt cggcccgccg ctttcgcact ttagctgttt
     6421 ctccaggcca catatgatta gttccaggcc gaaaaggaag gcaggttcgg ctccctgccg
     6481 gtcgaacagc tcaattgctt gtttcagaag tgggggcata gaatcggtgg taggtgtctc
     6541 tctttcctct tttgctactt gatgctcctg ttcctccaat acgcagccca gtgtaaagtg
     6601 gcccacggcg gacagagcgt acagtgcgtt ctccagggag aagccttgct gacacaggaa
     6661 cgcgagctga ttttccaggg tttcgtactg tttctctgtt gggcgggtgc cgagatgcac
     6721 tttagccccg tcacgatgtg agaggagagc acagcggtat gacttggcgt tgttccgcag
     6781 aaagtcttgc catgactcgc cttccagggg gcaggagtgg gtatgatgcc tgtccagcat
     6841 ctcgattggc agggcatcga gcagggcccg cttgttcttc acgtgccagt acagggtagg
     6901 ctgctcaact cccagctttt gagcgagttt ccttgtcgtc aggccttcga taccgactcc
     6961 attgagtaat tccagagcag agtttatgac tttgctcttg tccagtctag acatcttatc
     7021 gtcatcgtct ttgtaatcca tggtggcgga tcccgcgtca cgacacctgt gttctggcgg
     7081 caaacccgtt gcgaaaaaga acgttcacgg cgactactgc acttatatac ggttctcccc
     7141 caccctcggg aaaaaggcgg agccagtaca cgacatcact ttcccagttt accccgcgcc
     7201 accttctcta ggcaccggtt caattgccga cccctccccc caacttctcg ggcactgtgg
     7261 gccatgtgcg ctctgcccca tggtaatagc catgactaat acgtagatgt actgccaagt
     7321 aggaaagtgc cataaggtca tgtactgggc ataatgccag gcgggccatt taccgtcatt
     7381 gacgtcaata gggggcgtac ttggcatatg atacacttga tgtactgcca agtgggcagt
     7441 ttaccgtaaa tactccaccc attgacgtca atggaaagtg cctattggcg ttactatggg
     7501 aacatacgtc attattgacg tcaatgggcg ggggtcgttg ggcggtcagc caggcgggcc
     7561 atttaccgta agttatgtaa cgcggaactc catatatggg ctatgaacta atgaccccgt
     7621 aattgattac tattaataac tagtcaataa tcaatgtcat aagctctaat cgcataatcg
     7681 agctaagagc gattaatatg cttagctagc ttagctaagc tagatttctc tattctccca
     7741 caatatatag cagctattag cttagcagtc cgaatacgat cgtccacgat ccataagcgt
     7801 aacacatttg agtagcgact gacgggcacc ggagccactc gagtggaatt atcacctcga
     7861 gtttactccc tatcagtgat agagaacgta tgaagagttt actccctatc agtgatagag
     7921 aacgtatgca gactttactc cctatcagtg atagagaacg tataaggagt ttactcccta
     7981 tcagtgatag agaacgtatg accagtttac tccctatcag tgatagagaa cgtatctaca
     8041 gtttactccc tatcagtgat agagaacgta tatccagttt actccctatc agtgatagag
     8101 aacgtat
//
