LOCUS       sequence-377021-        7927 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Barcoded piggybac transposon vector with Dox-inducible expression of
            RUNX1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7927)
  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 7927)
  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-RUNX1
FEATURES             Location/Qualifiers
     source          1..7927
                     /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"
     regulatory      136..145
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(1585..1609)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1611..1637
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1804..1938
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   2210..2244
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2255..2272)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2255..2271)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2264..2286)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2480..2499)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2601..2623
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2662..2680)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2748..2852
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2853..2921,2922..3713)
                     /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(3071..3090)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3884..4472
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4373..4392
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4626..4643
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4849..4870
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4885..4890,4891..4908,4909..4915)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4923..4939
                     /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     4928..4950
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4947..4963
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4947..4963
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4989..5051
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    5227..5308
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(6025..6081)
                     /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(6091..6834)
                     /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(6835..6858)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6858..6867
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6887..7098)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        7099..7478
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     protein_bind    7687..7705
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7723..7741
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7759..7777
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7795..7813
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7831..7849
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     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"
ORIGIN
        1 aagcttgcct atataagcag agctcgttta gtgaaccgtc agatcgcctg gagacgccat
       61 ccacgctgtt ttgacctcca tagaagacac cgggaccgat ccagcctacc acaagtttgt
      121 acaaaaaagc aggctgccac catggcttca gacagcatat ttgagtcatt tccttcgtac
      181 ccacagtgct tcatgagaga atgcatactt ggaatgaatc cttctagaga cgtccacgat
      241 gccagcacga gccgccgctt cacgccgcct tccaccgcgc tgagcccagg caagatgagc
      301 gaggcgttgc cgctgggcgc cccggacgcc ggcgctgccc tggccggcaa gctgaggagc
      361 ggcgaccgca gcatggtgga ggtgctggcc gaccacccgg gcgagctggt gcgcaccgac
      421 agccccaact tcctctgctc cgtgctgcct acgcactggc gctgcaacaa gaccctgccc
      481 atcgctttca aggtggtggc cctaggggat gttccagatg gcactctggt cactgtgatg
      541 gctggcaatg atgaaaacta ctcggctgag ctgagaaatg ctaccgcagc catgaagaac
      601 caggttgcaa gatttaatga cctcaggttt gtcggtcgaa gtggaagagg gaaaagcttc
      661 actctgacca tcactgtctt cacaaaccca ccgcaagtcg ccacctacca cagagccatc
      721 aaaatcacag tggatgggcc ccgagaacct cgaagacatc ggcagaaact agatgatcag
      781 accaagcccg ggagcttgtc cttttccgag cggctcagtg aactggagca gctgcggcgc
      841 acagccatga gggtcagccc acaccaccca gcccccacgc ccaaccctcg tgcctccctg
      901 aaccactcca ctgcctttaa ccctcagcct cagagtcaga tgcaggatac aaggcagatc
      961 caaccatccc caccgtggtc ctacgatcag tcctaccaat acctgggatc cattgcctct
     1021 ccttctgtgc acccagcaac gcccatttca cctggacgtg ccagcggcat gacaaccctc
     1081 tctgcagaac tttccagtcg actctcaacg gcacccgacc tgacagcgtt cagcgacccg
     1141 cgccagttcc ccgcgctgcc ctccatctcc gacccccgca tgcactatcc aggcgccttc
     1201 acctactccc cgacgccggt cacctcgggc atcggcatcg gcatgtcggc catgggctcg
     1261 gccacgcgct accacaccta cctgccgccg ccctaccccg gctcgtcgca agcgcaggga
     1321 ggcccgttcc aagccagctc gccctcctac cacctgtact acggcgcctc ggccggctcc
     1381 taccagttct ccatggtggg cggcgagcgc tcgccgccgc gcatcctgcc gccctgcacc
     1441 aacgcctcca ccggctccgc gctgctcaac cccagcctcc cgaaccagag cgacgtggtg
     1501 gaggccgagg gcagccacag caactccccc accaacatgg cgccctccgc gcgcctggag
     1561 gaggccgtgt ggaggcccta ctgaacccag ctttcttgta caaagtggta tacccatacg
     1621 atgttccaga ttacgcttag taatgagaaa tactcagaag atgtcactca ccatagtccg
     1681 cagtgaaaga taaatgatcc ttctcatatg cagaaacaag ttttagagct agaaatagca
     1741 agtaggccac tgagcaagaa ctatgaatga gatctttaga catattgcgg ccgctttctc
     1801 cgcgatccag acatgataag atacattgat gagtttggac aaaccaaaac tagaatgcag
     1861 tgaaaaaaat gccttatttg tgaaatttgt gatgctattg ccttatttgt aaccattata
     1921 agctgcaata aacaagttat ctataacaag aaaatatata tataataagt tatcacgtaa
     1981 gtagaacatg aaataacaat ataattatcg tatgagttaa atcttaaaag tcacgtaaaa
     2041 gataatcatg cgtcattttg actcacgcgg tcgttatagt tcaaaatcag tgacacttac
     2101 cgcattgaca agcacgcctc acgggagctc caagcggcga ctgagatgtc ctaaatgcac
     2161 agcgacggat tcgcgctatt tagaaagaga gagcaatatt tcaagaatgc atgcgtcaat
     2221 tttacgcaga ctatctttct agggttaaga attcactggc cgtcgtttta caacgtcgtg
     2281 actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca
     2341 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga
     2401 atggcgaatg gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     2461 gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
     2521 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     2581 gacaagctgt gaccatgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     2641 gaaacacgcg agttcgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     2701 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     2761 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     2821 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     2881 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     2941 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     3001 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     3061 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     3121 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     3181 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     3241 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     3301 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     3361 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     3421 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     3481 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     3541 ctgataaatc tggagccggt gagcgtggtt cacgcggtat cattgcagca ctggggccag
     3601 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     3661 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     3721 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     3781 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     3841 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     3901 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     3961 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     4021 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     4081 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     4141 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     4201 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     4261 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     4321 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     4381 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     4441 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     4501 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     4561 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     4621 agcgcagcga gtcagtgagc gaggaagcgg atgagcgccc aatacgcaaa ccgcctctca
     4681 ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg
     4741 gcagtgagcg caacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     4801 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
     4861 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
     4921 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaaggt
     4981 cgacttaacc ctagaaagat aatcatattg tgacgtacgt taaagataat catgcgtaaa
     5041 attgacgcat gtgttttatc ggtctgtata tcgaggttta tttattaatt tgaatagata
     5101 ttaagtttta ttatatttac acttacatac taataataaa ttcaacaaac aatttattta
     5161 tgtttattta tttattaaaa aaaaacaaaa actcaaaatt tcttctataa agtaacaaaa
     5221 cttttagcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt
     5281 aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca
     5341 ggttcagggg gaggtgtggg aggtttttta aagcaagtaa aacctctaca aatgtggtat
     5401 ggctgattat gatcctctgg agattcatgc gccaggtttt ctggtcatgc accacgttcg
     5461 tgggccctca ggaacctcca cgtcagcggt aactgtgaag cccagtcgct cgtaaaaagg
     5521 aagattccgg ggagcactgg tttcgaggaa cgccggcacg ccagcccgtt ccgcggcctc
     5581 aacaccagga agaacgacag cgcttcccaa acctttacct tggtgatctg ggctgacacc
     5641 gacagtggca agaaaccacg caggctcctt tggtctatga ggagccagca agccctccat
     5701 ttgctgttgt gccgccaacc tactccctga cagctcggcc atccgcggac ctatttccgc
     5761 gaacactgcg ccggcttcga cgctttccgg agtagtccag accgcaactg cggcaccatc
     5821 gtccgcaacc caaactttac caatgtccag acccacgcgc gtaagaaaca gctcttgcaa
     5881 ctcagttacc ctctctatgt gtctgtccgg atctacggta tggcgggtcg ctggataatc
     5941 tgcaaatgct gcggcaagtg ttcggacagc ccgtggtaca tcgtcacggg tagcgagcct
     6001 aacagtgggc ttatattccg tcatggggcc ggggttctcc tccacgtcgc cggcctgctt
     6061 cagcaggctg aagttggtgg cgccgctgcc cccggggagc atgtcaaggt caaaatcgtc
     6121 aagagcgtca gcaggcagca tatcaaggtc aaagtcgtca agggcatcgg ctgggagcat
     6181 gtctaagtca aaatcgtcaa gggcgtcggt cggcccgccg ctttcgcact ttagctgttt
     6241 ctccaggcca catatgatta gttccaggcc gaaaaggaag gcaggttcgg ctccctgccg
     6301 gtcgaacagc tcaattgctt gtttcagaag tgggggcata gaatcggtgg taggtgtctc
     6361 tctttcctct tttgctactt gatgctcctg ttcctccaat acgcagccca gtgtaaagtg
     6421 gcccacggcg gacagagcgt acagtgcgtt ctccagggag aagccttgct gacacaggaa
     6481 cgcgagctga ttttccaggg tttcgtactg tttctctgtt gggcgggtgc cgagatgcac
     6541 tttagccccg tcacgatgtg agaggagagc acagcggtat gacttggcgt tgttccgcag
     6601 aaagtcttgc catgactcgc cttccagggg gcaggagtgg gtatgatgcc tgtccagcat
     6661 ctcgattggc agggcatcga gcagggcccg cttgttcttc acgtgccagt acagggtagg
     6721 ctgctcaact cccagctttt gagcgagttt ccttgtcgtc aggccttcga taccgactcc
     6781 attgagtaat tccagagcag agtttatgac tttgctcttg tccagtctag acatcttatc
     6841 gtcatcgtct ttgtaatcca tggtggcgga tcccgcgtca cgacacctgt gttctggcgg
     6901 caaacccgtt gcgaaaaaga acgttcacgg cgactactgc acttatatac ggttctcccc
     6961 caccctcggg aaaaaggcgg agccagtaca cgacatcact ttcccagttt accccgcgcc
     7021 accttctcta ggcaccggtt caattgccga cccctccccc caacttctcg ggcactgtgg
     7081 gccatgtgcg ctctgcccca tggtaatagc catgactaat acgtagatgt actgccaagt
     7141 aggaaagtgc cataaggtca tgtactgggc ataatgccag gcgggccatt taccgtcatt
     7201 gacgtcaata gggggcgtac ttggcatatg atacacttga tgtactgcca agtgggcagt
     7261 ttaccgtaaa tactccaccc attgacgtca atggaaagtg cctattggcg ttactatggg
     7321 aacatacgtc attattgacg tcaatgggcg ggggtcgttg ggcggtcagc caggcgggcc
     7381 atttaccgta agttatgtaa cgcggaactc catatatggg ctatgaacta atgaccccgt
     7441 aattgattac tattaataac tagtcaataa tcaatgtcat aagctctaat cgcataatcg
     7501 agctaagagc gattaatatg cttagctagc ttagctaagc tagatttctc tattctccca
     7561 caatatatag cagctattag cttagcagtc cgaatacgat cgtccacgat ccataagcgt
     7621 aacacatttg agtagcgact gacgggcacc ggagccactc gagtggaatt atcacctcga
     7681 gtttactccc tatcagtgat agagaacgta tgaagagttt actccctatc agtgatagag
     7741 aacgtatgca gactttactc cctatcagtg atagagaacg tataaggagt ttactcccta
     7801 tcagtgatag agaacgtatg accagtttac tccctatcag tgatagagaa cgtatctaca
     7861 gtttactccc tatcagtgat agagaacgta tatccagttt actccctatc agtgatagag
     7921 aacgtat
//
