LOCUS       sequence-382106-        7060 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Barcoded piggybac transposon vector with Dox-inducible expression of
            TCF21.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7060)
  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 7060)
  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-TCF21
FEATURES             Location/Qualifiers
     source          1..7060
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    21..39
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    57..75
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    93..111
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    129..147
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    165..183
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    201..219
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    237..255
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     282..306
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    372..396
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(943..967)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             969..995
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1162..1296
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   1568..1602
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1613..1630)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1613..1629)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1622..1644)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1838..1857)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1959..1981
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2020..2038)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2106..2210
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2211..2279,2280..3071)
                     /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(2429..2448)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3242..3830
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3731..3750
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3984..4001
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4207..4228
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4243..4248,4249..4266,4267..4273)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4281..4297
                     /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     4286..4308
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4305..4321
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4305..4321
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4347..4409
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    4585..4666
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5383..5439)
                     /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(5449..6192)
                     /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(6193..6216)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6216..6225
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6245..6456)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        6457..6836
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     protein_bind    join(7045..7060,1..3)
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
ORIGIN
        1 agaacgtatg aagagtttac tccctatcag tgatagagaa cgtatgcaga stttactccc
       61 tatcagtgat agagaacgta trmrgasttt actccctatc agtgatagag aacgtataag
      121 gagtttactc cctatcagtg atagagaacg tatgaccagt ttactcccta tcagtgatag
      181 agaacgtatc tacagtttac tccctatcag tgatagagaa cgtatatcca gtttactccc
      241 tatcagtgat agagaacgta taagcttgcc tatataagca gagctcgttt agtgaaccgt
      301 cagatcgcct ggagacgcca tccacgctgt tttgacctcc atagaagaca ccgggaccga
      361 tccagcctac cacaagtttg tacaaaaaag caggctccac catgtccacc ggctccctca
      421 gcgatgtgga ggaccttcaa gaggtggaga tgttggaatg tgacgggttg aaaatggatt
      481 cgaacaagga atttgtgact tccaacgaga gcaccgagga gagctccaac tgcgagaatg
      541 ggtctcccca gaagggccgc ggcggcctgg gcaagaggag gaaggcgccc accaagaaga
      601 gccccctgag cggggtcagc caggagggga agcaggtcca gcgcaacgcc gccaacgcgc
      661 gagagcgggc ccgcatgcga gtgctgagca aggccttctc cagactcaag accaccctgc
      721 cctgggtgcc ccccgacacc aagctctcca agctggacac gctcaggctg gcgtccagct
      781 acatcgccca cttgaggcag atcctggcta acgacaaata cgagaacggg tacattcacc
      841 cggtcaacct gacgtggccc tttatggtgg ccgggaaacc cgagagtgac ctgaaagaag
      901 tggtgaccgc gagccgctta tgtggaacca ccgcgtcctt ggacccagct ttcttgtaca
      961 aagtggtata cccatacgat gttccagatt acgcttagta atgagaaata ctcagaagat
     1021 gtcactcacc atagtccgca gtgaaagata aatgatcctg gaggctctca aattagagtt
     1081 ttagagctag aaatagcaag taggccactg agcaagaact atgaatgaga tctttagaca
     1141 tattgcggcc gctttctccg cgatccagac atgataagat acattgatga gtttggacaa
     1201 accaaaacta gaatgcagtg aaaaaaatgc cttatttgtg aaatttgtga tgctattgcc
     1261 ttatttgtaa ccattataag ctgcaataaa caagttatct ataacaagaa aatatatata
     1321 taataagtta tcacgtaagt agaacatgaa ataacaatat aattatcgta tgagttaaat
     1381 cttaaaagtc acgtaaaaga taatcatgcg tcattttgac tcacgcggtc gttatagttc
     1441 aaaatcagtg acacttaccg cattgacaag cacgcctcac gggagctcca agcggcgact
     1501 gagatgtcct aaatgcacag cgacggattc gcgctattta gaaagagaga gcaatatttc
     1561 aagaatgcat gcgtcaattt tacgcagact atctttctag ggttaagaat tcactggccg
     1621 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag
     1681 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
     1741 aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg gtattttctc cttacgcatc
     1801 tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct gatgccgcat
     1861 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     1921 tcccggcatc cgcttacaga caagctgtga ccatgtctcc gggagctgca tgtgtcagag
     1981 gttttcaccg tcatcaccga aacacgcgag ttcgaaaggg cctcgtgata cgcctatttt
     2041 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa
     2101 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     2161 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     2221 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     2281 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     2341 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     2401 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     2461 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     2521 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     2581 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     2641 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     2701 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     2761 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     2821 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     2881 cggctggctg gtttattgct gataaatctg gagccggtga gcgtggttca cgcggtatca
     2941 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     3001 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     3061 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     3121 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     3181 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     3241 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     3301 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     3361 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     3421 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     3481 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     3541 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     3601 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     3661 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     3721 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     3781 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     3841 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     3901 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     3961 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggat gagcgcccaa
     4021 tacgcaaacc gcctctcacc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
     4081 ttcccgactg gaaagcgggc agtgagcgca acgcaaaccg cctctccccg cgcgttggcc
     4141 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa
     4201 cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc
     4261 ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga
     4321 ccatgattac gccaaggtcg acttaaccct agaaagataa tcatattgtg acgtacgtta
     4381 aagataatca tgcgtaaaat tgacgcatgt gttttatcgg tctgtatatc gaggtttatt
     4441 tattaatttg aatagatatt aagttttatt atatttacac ttacatacta ataataaatt
     4501 caacaaacaa tttatttatg tttatttatt tattaaaaaa aaacaaaaac tcaaaatttc
     4561 ttctataaag taacaaaact tttagcagtg aaaaaaatgc tttatttgtg aaatttgtga
     4621 tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg
     4681 cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa
     4741 cctctacaaa tgtggtatgg ctgattatga tcctctggag attcatgcgc caggttttct
     4801 ggtcatgcac cacgttcgtg ggccctcagg aacctccacg tcagcggtaa ctgtgaagcc
     4861 cagtcgctcg taaaaaggaa gattccgggg agcactggtt tcgaggaacg ccggcacgcc
     4921 agcccgttcc gcggcctcaa caccaggaag aacgacagcg cttcccaaac ctttaccttg
     4981 gtgatctggg ctgacaccga cagtggcaag aaaccacgca ggctcctttg gtctatgagg
     5041 agccagcaag ccctccattt gctgttgtgc cgccaaccta ctccctgaca gctcggccat
     5101 ccgcggacct atttccgcga acactgcgcc ggcttcgacg ctttccggag tagtccagac
     5161 cgcaactgcg gcaccatcgt ccgcaaccca aactttacca atgtccagac ccacgcgcgt
     5221 aagaaacagc tcttgcaact cagttaccct ctctatgtgt ctgtccggat ctacggtatg
     5281 gcgggtcgct ggataatctg caaatgctgc ggcaagtgtt cggacagccc gtggtacatc
     5341 gtcacgggta gcgagcctaa cagtgggctt atattccgtc atggggccgg ggttctcctc
     5401 cacgtcgccg gcctgcttca gcaggctgaa gttggtggcg ccgctgcccc cggggagcat
     5461 gtcaaggtca aaatcgtcaa gagcgtcagc aggcagcata tcaaggtcaa agtcgtcaag
     5521 ggcatcggct gggagcatgt ctaagtcaaa atcgtcaagg gcgtcggtcg gcccgccgct
     5581 ttcgcacttt agctgtttct ccaggccaca tatgattagt tccaggccga aaaggaaggc
     5641 aggttcggct ccctgccggt cgaacagctc aattgcttgt ttcagaagtg ggggcataga
     5701 atcggtggta ggtgtctctc tttcctcttt tgctacttga tgctcctgtt cctccaatac
     5761 gcagcccagt gtaaagtggc ccacggcgga cagagcgtac agtgcgttct ccagggagaa
     5821 gccttgctga cacaggaacg cgagctgatt ttccagggtt tcgtactgtt tctctgttgg
     5881 gcgggtgccg agatgcactt tagccccgtc acgatgtgag aggagagcac agcggtatga
     5941 cttggcgttg ttccgcagaa agtcttgcca tgactcgcct tccagggggc aggagtgggt
     6001 atgatgcctg tccagcatct cgattggcag ggcatcgagc agggcccgct tgttcttcac
     6061 gtgccagtac agggtaggct gctcaactcc cagcttttga gcgagtttcc ttgtcgtcag
     6121 gccttcgata ccgactccat tgagtaattc cagagcagag tttatgactt tgctcttgtc
     6181 cagtctagac atcttatcgt catcgtcttt gtaatccatg gtggcggatc ccgcgtcacg
     6241 acacctgtgt tctggcggca aacccgttgc gaaaaagaac gttcacggcg actactgcac
     6301 ttatatacgg ttctccccca ccctcgggaa aaaggcggag ccagtacacg acatcacttt
     6361 cccagtttac cccgcgccac cttctctagg caccggttca attgccgacc cctcccccca
     6421 acttctcggg cactgtgggc catgtgcgct ctgccccatg gtaatagcca tgactaatac
     6481 gtagatgtac tgccaagtag gaaagtgcca taaggtcatg tactgggcat aatgccaggc
     6541 gggccattta ccgtcattga cgtcaatagg gggcgtactt ggcatatgat acacttgatg
     6601 tactgccaag tgggcagttt accgtaaata ctccacccat tgacgtcaat ggaaagtgcc
     6661 tattggcgtt actatgggaa catacgtcat tattgacgtc aatgggcggg ggtcgttggg
     6721 cggtcagcca ggcgggccat ttaccgtaag ttatgtaacg cggaactcca tatatgggct
     6781 atgaactaat gaccccgtaa ttgattacta ttaataacta gtcaataatc aatgtcataa
     6841 gctctaatcg cataatcgag ctaagagcga ttaatatgct tagctagctt agctaagcta
     6901 gatttctcta ttctcccaca atatatagca gctattagct tagcagtccg aatacgatcg
     6961 tccacgatcc ataagcgtaa cacatttgag tagcgactga cgggcaccgg agccactcga
     7021 gtggaattat cacctcgagt ttactcccta tcagtgatag
//
