LOCUS       sequence-377557-        8245 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Barcoded piggybac transposon vector with Dox-inducible expression of
            RUNX2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8245)
  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 8245)
  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-RUNX2
FEATURES             Location/Qualifiers
     source          1..8245
                     /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(1903..1927)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1929..1955
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    2122..2256
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   2528..2562
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2573..2590)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2573..2589)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2582..2604)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2798..2817)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2919..2941
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2980..2998)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3066..3170
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3171..3239,3240..4031)
                     /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(3389..3408)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4202..4790
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4691..4710
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4944..4961
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5167..5188
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5203..5208,5209..5226,5227..5233)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5241..5257
                     /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     5246..5268
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5265..5281
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5265..5281
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   5307..5369
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    5545..5626
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(6343..6399)
                     /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(6409..7152)
                     /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(7153..7176)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      7176..7185
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(7205..7416)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        7417..7796
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     protein_bind    8005..8023
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    8041..8059
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    8077..8095
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    8113..8131
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    8149..8167
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    8185..8203
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    8221..8239
                     /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 tggcatgctt cattcgcctc acaaacaacc acagaaccac aagtgcggtg
      181 caaactttct ccaggaggac agcaagaagt ctctggtttt taaatggtta atctccgcag
      241 gtcactacca gccaccgaga ccaacagagt catttaaggc tgcaagcagt atttacaaca
      301 gagggtacaa gttctatctg aaaaaaaaag gagggactat ggcatcaaac agcctcttca
      361 gcacagtgac accatgtcag caaaacttct tttgggatcc gagcaccagc cggcgcttca
      421 gccccccctc cagcagcctg cagcccggca aaatgagcga cgtgagcccg gtggtggctg
      481 cgcaacagca gcagcaacag cagcagcagc aacagcagca gcagcagcag caacagcagc
      541 agcagcagca ggaggcggcg gcggcggctg cggcggcggc ggcggctgcg gcggcggcag
      601 ctgcagtgcc ccggttgcgg ccgccccacg acaaccgcac catggtggag atcatcgccg
      661 accacccggc cgaactcgtc cgcaccgaca gccccaactt cctgtgctcg gtgctgccct
      721 cgcactggcg ctgcaacaag accctgcccg tggccttcaa ggtggtagcc ctcggagagg
      781 taccagatgg gactgtggtt actgtcatgg cgggtaacga tgaaaattat tctgctgagc
      841 tccggaatgc ctctgctgtt atgaaaaacc aagtagcaag gttcaacgat ctgagatttg
      901 tgggccggag tggacgaggc aagagtttca ccttgaccat aaccgtcttc acaaatcctc
      961 cccaagtagc tacctatcac agagcaatta aagttacagt agatggacct cgggaaccca
     1021 gaaggcacag acagaagctt gatgactcta aacctagttt gttctctgac cgcctcagtg
     1081 atttagggcg cattcctcat cccagtatga gagtaggtgt cccgcctcag aacccacggc
     1141 cctccctgaa ctctgcacca agtcctttta atccacaagg acagagtcag attacagacc
     1201 ccaggcaggc acagtcttcc ccgccgtggt cctatgacca gtcttacccc tcctacctga
     1261 gccagatgac gtccccgtcc atccactcta ccaccccgct gtcttccaca cggggcactg
     1321 ggcttcctgc catcaccgat gtgcctaggc gcatttcaga tgatgacact gccacctctg
     1381 acttctgcct ctggccttcc actctcagta agaagagcca ggcaggtgct tcagaactgg
     1441 gccctttttc agaccccagg cagttcccaa gcatttcatc cctcactgag agccgcttct
     1501 ccaacccacg aatgcactat ccagccacct ttacttacac cccgccagtc acctcaggca
     1561 tgtccctcgg tatgtccgcc accactcact accacaccta cctgccacca ccctaccccg
     1621 gctcttccca aagccagagt ggacccttcc agaccagcag cactccatat ctctactatg
     1681 gcacttcgtc aggatcctat cagtttccca tggtgccggg gggagaccgg tctccttcca
     1741 gaatgcttcc gccatgcacc accacctcga atggcagcac gctattaaat ccaaatttgc
     1801 ctaaccagaa tgatggtgtt gacgctgatg gaagccacag cagttcccca actgttttga
     1861 attctagtgg cagaatggat gaatctgttt ggcgaccata ttgcccaact ttcttgtaca
     1921 aagtggtata cccatacgat gttccagatt acgcttagta atgagaaata ctcagaagat
     1981 gtcactcacc atagtccgca gtgaaagata aatgatcaga ctggcctgca gtgctcagtt
     2041 ttagagctag aaatagcaag taggccactg agcaagaact atgaatgaga tctttagaca
     2101 tattgcggcc gctttctccg cgatccagac atgataagat acattgatga gtttggacaa
     2161 accaaaacta gaatgcagtg aaaaaaatgc cttatttgtg aaatttgtga tgctattgcc
     2221 ttatttgtaa ccattataag ctgcaataaa caagttatct ataacaagaa aatatatata
     2281 taataagtta tcacgtaagt agaacatgaa ataacaatat aattatcgta tgagttaaat
     2341 cttaaaagtc acgtaaaaga taatcatgcg tcattttgac tcacgcggtc gttatagttc
     2401 aaaatcagtg acacttaccg cattgacaag cacgcctcac gggagctcca agcggcgact
     2461 gagatgtcct aaatgcacag cgacggattc gcgctattta gaaagagaga gcaatatttc
     2521 aagaatgcat gcgtcaattt tacgcagact atctttctag ggttaagaat tcactggccg
     2581 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag
     2641 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
     2701 aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg gtattttctc cttacgcatc
     2761 tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct gatgccgcat
     2821 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     2881 tcccggcatc cgcttacaga caagctgtga ccatgtctcc gggagctgca tgtgtcagag
     2941 gttttcaccg tcatcaccga aacacgcgag ttcgaaaggg cctcgtgata cgcctatttt
     3001 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa
     3061 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     3121 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     3181 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     3241 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     3301 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     3361 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     3421 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     3481 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     3541 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     3601 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     3661 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     3721 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     3781 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     3841 cggctggctg gtttattgct gataaatctg gagccggtga gcgtggttca cgcggtatca
     3901 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     3961 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     4021 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     4081 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     4141 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     4201 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     4261 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     4321 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     4381 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     4441 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     4501 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     4561 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     4621 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     4681 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     4741 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     4801 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     4861 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     4921 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggat gagcgcccaa
     4981 tacgcaaacc gcctctcacc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
     5041 ttcccgactg gaaagcgggc agtgagcgca acgcaaaccg cctctccccg cgcgttggcc
     5101 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa
     5161 cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc
     5221 ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga
     5281 ccatgattac gccaaggtcg acttaaccct agaaagataa tcatattgtg acgtacgtta
     5341 aagataatca tgcgtaaaat tgacgcatgt gttttatcgg tctgtatatc gaggtttatt
     5401 tattaatttg aatagatatt aagttttatt atatttacac ttacatacta ataataaatt
     5461 caacaaacaa tttatttatg tttatttatt tattaaaaaa aaacaaaaac tcaaaatttc
     5521 ttctataaag taacaaaact tttagcagtg aaaaaaatgc tttatttgtg aaatttgtga
     5581 tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg
     5641 cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa
     5701 cctctacaaa tgtggtatgg ctgattatga tcctctggag attcatgcgc caggttttct
     5761 ggtcatgcac cacgttcgtg ggccctcagg aacctccacg tcagcggtaa ctgtgaagcc
     5821 cagtcgctcg taaaaaggaa gattccgggg agcactggtt tcgaggaacg ccggcacgcc
     5881 agcccgttcc gcggcctcaa caccaggaag aacgacagcg cttcccaaac ctttaccttg
     5941 gtgatctggg ctgacaccga cagtggcaag aaaccacgca ggctcctttg gtctatgagg
     6001 agccagcaag ccctccattt gctgttgtgc cgccaaccta ctccctgaca gctcggccat
     6061 ccgcggacct atttccgcga acactgcgcc ggcttcgacg ctttccggag tagtccagac
     6121 cgcaactgcg gcaccatcgt ccgcaaccca aactttacca atgtccagac ccacgcgcgt
     6181 aagaaacagc tcttgcaact cagttaccct ctctatgtgt ctgtccggat ctacggtatg
     6241 gcgggtcgct ggataatctg caaatgctgc ggcaagtgtt cggacagccc gtggtacatc
     6301 gtcacgggta gcgagcctaa cagtgggctt atattccgtc atggggccgg ggttctcctc
     6361 cacgtcgccg gcctgcttca gcaggctgaa gttggtggcg ccgctgcccc cggggagcat
     6421 gtcaaggtca aaatcgtcaa gagcgtcagc aggcagcata tcaaggtcaa agtcgtcaag
     6481 ggcatcggct gggagcatgt ctaagtcaaa atcgtcaagg gcgtcggtcg gcccgccgct
     6541 ttcgcacttt agctgtttct ccaggccaca tatgattagt tccaggccga aaaggaaggc
     6601 aggttcggct ccctgccggt cgaacagctc aattgcttgt ttcagaagtg ggggcataga
     6661 atcggtggta ggtgtctctc tttcctcttt tgctacttga tgctcctgtt cctccaatac
     6721 gcagcccagt gtaaagtggc ccacggcgga cagagcgtac agtgcgttct ccagggagaa
     6781 gccttgctga cacaggaacg cgagctgatt ttccagggtt tcgtactgtt tctctgttgg
     6841 gcgggtgccg agatgcactt tagccccgtc acgatgtgag aggagagcac agcggtatga
     6901 cttggcgttg ttccgcagaa agtcttgcca tgactcgcct tccagggggc aggagtgggt
     6961 atgatgcctg tccagcatct cgattggcag ggcatcgagc agggcccgct tgttcttcac
     7021 gtgccagtac agggtaggct gctcaactcc cagcttttga gcgagtttcc ttgtcgtcag
     7081 gccttcgata ccgactccat tgagtaattc cagagcagag tttatgactt tgctcttgtc
     7141 cagtctagac atcttatcgt catcgtcttt gtaatccatg gtggcggatc ccgcgtcacg
     7201 acacctgtgt tctggcggca aacccgttgc gaaaaagaac gttcacggcg actactgcac
     7261 ttatatacgg ttctccccca ccctcgggaa aaaggcggag ccagtacacg acatcacttt
     7321 cccagtttac cccgcgccac cttctctagg caccggttca attgccgacc cctcccccca
     7381 acttctcggg cactgtgggc catgtgcgct ctgccccatg gtaatagcca tgactaatac
     7441 gtagatgtac tgccaagtag gaaagtgcca taaggtcatg tactgggcat aatgccaggc
     7501 gggccattta ccgtcattga cgtcaatagg gggcgtactt ggcatatgat acacttgatg
     7561 tactgccaag tgggcagttt accgtaaata ctccacccat tgacgtcaat ggaaagtgcc
     7621 tattggcgtt actatgggaa catacgtcat tattgacgtc aatgggcggg ggtcgttggg
     7681 cggtcagcca ggcgggccat ttaccgtaag ttatgtaacg cggaactcca tatatgggct
     7741 atgaactaat gaccccgtaa ttgattacta ttaataacta gtcaataatc aatgtcataa
     7801 gctctaatcg cataatcgag ctaagagcga ttaatatgct tagctagctt agctaagcta
     7861 gatttctcta ttctcccaca atatatagca gctattagct tagcagtccg aatacgatcg
     7921 tccacgatcc ataagcgtaa cacatttgag tagcgactga cgggcaccgg agccactcga
     7981 gtggaattat cacctcgagt ttactcccta tcagtgatag agaacgtatg aagagtttac
     8041 tccctatcag tgatagagaa cgtatgcaga ctttactccc tatcagtgat agagaacgta
     8101 taaggagttt actccctatc agtgatagag aacgtatgac cagtttactc cctatcagtg
     8161 atagagaacg tatctacagt ttactcccta tcagtgatag agaacgtata tccagtttac
     8221 tccctatcag tgatagagaa cgtat
//
