LOCUS       sequence-377204-        7850 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Barcoded piggybac transposon vector with Dox-inducible expression of
            TOX2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7850)
  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 7850)
  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-TOX2
FEATURES             Location/Qualifiers
     source          1..7850
                     /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(1528..1552)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1554..1580
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1727..1861
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   2133..2167
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2178..2195)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2178..2194)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2187..2209)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2403..2422)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2524..2546
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2585..2603)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2671..2775
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2776..2844,2845..3636)
                     /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(2994..3013)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3807..4395
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4296..4315
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4549..4566
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4772..4793
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4808..4813,4814..4831,4832..4838)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4846..4862
                     /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     4851..4873
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4870..4886
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4870..4886
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4912..4974
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    5150..5231
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5948..6004)
                     /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(6014..6757)
                     /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(6758..6781)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6781..6790
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6810..7021)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        7022..7401
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     protein_bind    7610..7628
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7646..7664
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7682..7700
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7718..7736
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7754..7772
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7790..7808
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7826..7844
                     /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 tggcatgagt gacggaaacc cagagctcct gtcaaccagc cagacctaca
      181 acggccagag cgagaacaac gaagactatg agatcccccc gataacacct cccaacctcc
      241 cggagccatc cctcctgcac ctgggggacc acgaagccag ctaccactcg ctgtgccacg
      301 gcctcacccc caacggtctg ctccctgcct actcctatca ggccatggac ctcccagcca
      361 tcatggtgtc caacatgcta gcacaggaca gccacctgct gtcgggccag ctgcccacga
      421 tccaggagat ggtccactcg gaagtggctg cctatgactc gggccggccc gggcccctgc
      481 tgggtcgccc ggcaatgctg gccagccaca tgagtgccct cagccagtcc cagctcatct
      541 cgcagatggg catccggagc agcatcgccc acagctcccc atcaccgccg gggagcaagt
      601 cagcgacccc ctctccctcc agctccactc aggaagagga gtcggaagtg catttcaaga
      661 tctcgggaga aaagagacct tcagccgacc caggaaaaaa ggccaagaac ccgaagaaga
      721 agaaaaagaa ggaccccaat gagccgcaga agcctgtgtc ggcctacgca ctcttcttca
      781 gagacactca ggccgccatc aagggtcaga accccagtgc cactttcggt gacgtgtcca
      841 aaatcgtggc ctccatgtgg gacagcctgg gagaggaaca gaagcaggcc tacaagagga
      901 agacagaagc agcaaagaag gaatatctga aggccctggc agcctaccgg gctagcctcg
      961 tctccaagag ctccccagat caaggtgaga ccaagagcac tcaggcaaac ccaccagcca
     1021 aaatgctccc acccaagcag cccatgtatg ccatgccagg cctggcctcc ttcctgacgc
     1081 cgtcggacct gcaggccttc cgcagtgggg cctcccctgc cagcctcgcc cggacgctgg
     1141 gctccaagtc tctgctgcca ggcctcagtg cgtccccgcc gccgccaccc tccttcccgc
     1201 tcagccccac actgcaccag cagctgtcac tgccccctca cgcccagggc gccctcctca
     1261 gtccacctgt tagcatgtcc ccagcccccc agccccctgt cctgcccacc cccatggcac
     1321 tccaggtgca gctggcgatg agcccctcac ctccagggcc acaggacttc ccgcacatct
     1381 ctgagttccc cagcagctcg ggatcctgct cacctggccc atccaacccc accagcagcg
     1441 gggactggga cagcagctac cccagtgggg agtgtggcat cagcacctgc agcctgctcc
     1501 ccagggacaa atcgctctac ctcacctacc caactttctt gtacaaagtg gtatacccat
     1561 acgatgttcc agattacgct tagtaatgag aaatactcag aagatgtcac tcaccatagt
     1621 cckcagtgaa wsmwaamtrw tckyywtrga gctagaaata gcaagtaggc cactgagcaa
     1681 gaactatgaa tgagatcttt agacatattg cggccgcttt ctccgcgatc cagacatgat
     1741 aagatacatt gatgagtttg gacaaaccaa aactagaatg cagtgaaaaa aatgccttat
     1801 ttgtgaaatt tgtgatgcta ttgccttatt tgtaaccatt ataagctgca ataaacaagt
     1861 tatctataac aagaaaatat atatataata agttatcacg taagtagaac atgaaataac
     1921 aatataatta tcgtatgagt taaatcttaa aagtcacgta aaagataatc atgcgtcatt
     1981 ttgactcacg cggtcgttat agttcaaaat cagtgacact taccgcattg acaagcacgc
     2041 ctcacgggag ctccaagcgg cgactgagat gtcctaaatg cacagcgacg gattcgcgct
     2101 atttagaaag agagagcaat atttcaagaa tgcatgcgtc aattttacgc agactatctt
     2161 tctagggtta agaattcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc
     2221 gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa
     2281 gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggcgcctg
     2341 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatatg gtgcactctc
     2401 agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct
     2461 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccatg
     2521 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacac gcgagttcga
     2581 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     2641 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     2701 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     2761 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     2821 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     2881 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     2941 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     3001 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     3061 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     3121 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     3181 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     3241 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     3301 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     3361 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     3421 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     3481 ggtgagcgtg gttcacgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     3541 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     3601 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     3661 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     3721 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     3781 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     3841 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     3901 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     3961 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     4021 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     4081 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     4141 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     4201 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     4261 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     4321 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     4381 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     4441 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     4501 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     4561 agcgaggaag cggatgagcg cccaatacgc aaaccgcctc tcaccgcgcg ttggccgatt
     4621 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
     4681 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
     4741 actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac
     4801 cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac
     4861 aatttcacac aggaaacagc tatgaccatg attacgccaa ggtcgactta accctagaaa
     4921 gataatcata ttgtgacgta cgttaaagat aatcatgcgt aaaattgacg catgtgtttt
     4981 atcggtctgt atatcgaggt ttatttatta atttgaatag atattaagtt ttattatatt
     5041 tacacttaca tactaataat aaattcaaca aacaatttat ttatgtttat ttatttatta
     5101 aaaaaaaaca aaaactcaaa atttcttcta taaagtaaca aaacttttag cagtgaaaaa
     5161 aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca
     5221 ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt
     5281 gggaggtttt ttaaagcaag taaaacctct acaaatgtgg tatggctgat tatgatcctc
     5341 tggagattca tgcgccaggt tttctggtca tgcaccacgt tcgtgggccc tcaggaacct
     5401 ccacgtcagc ggtaactgtg aagcccagtc gctcgtaaaa aggaagattc cggggagcac
     5461 tggtttcgag gaacgccggc acgccagccc gttccgcggc ctcaacacca ggaagaacga
     5521 cagcgcttcc caaaccttta ccttggtgat ctgggctgac accgacagtg gcaagaaacc
     5581 acgcaggctc ctttggtcta tgaggagcca gcaagccctc catttgctgt tgtgccgcca
     5641 acctactccc tgacagctcg gccatccgcg gacctatttc cgcgaacact gcgccggctt
     5701 cgacgctttc cggagtagtc cagaccgcaa ctgcggcacc atcgtccgca acccaaactt
     5761 taccaatgtc cagacccacg cgcgtaagaa acagctcttg caactcagtt accctctcta
     5821 tgtgtctgtc cggatctacg gtatggcggg tcgctggata atctgcaaat gctgcggcaa
     5881 gtgttcggac agcccgtggt acatcgtcac gggtagcgag cctaacagtg ggcttatatt
     5941 ccgtcatggg gccggggttc tcctccacgt cgccggcctg cttcagcagg ctgaagttgg
     6001 tggcgccgct gcccccgggg agcatgtcaa ggtcaaaatc gtcaagagcg tcagcaggca
     6061 gcatatcaag gtcaaagtcg tcaagggcat cggctgggag catgtctaag tcaaaatcgt
     6121 caagggcgtc ggtcggcccg ccgctttcgc actttagctg tttctccagg ccacatatga
     6181 ttagttccag gccgaaaagg aaggcaggtt cggctccctg ccggtcgaac agctcaattg
     6241 cttgtttcag aagtgggggc atagaatcgg tggtaggtgt ctctctttcc tcttttgcta
     6301 cttgatgctc ctgttcctcc aatacgcagc ccagtgtaaa gtggcccacg gcggacagag
     6361 cgtacagtgc gttctccagg gagaagcctt gctgacacag gaacgcgagc tgattttcca
     6421 gggtttcgta ctgtttctct gttgggcggg tgccgagatg cactttagcc ccgtcacgat
     6481 gtgagaggag agcacagcgg tatgacttgg cgttgttccg cagaaagtct tgccatgact
     6541 cgccttccag ggggcaggag tgggtatgat gcctgtccag catctcgatt ggcagggcat
     6601 cgagcagggc ccgcttgttc ttcacgtgcc agtacagggt aggctgctca actcccagct
     6661 tttgagcgag tttccttgtc gtcaggcctt cgataccgac tccattgagt aattccagag
     6721 cagagtttat gactttgctc ttgtccagtc tagacatctt atcgtcatcg tctttgtaat
     6781 ccatggtggc ggatcccgcg tcacgacacc tgtgttctgg cggcaaaccc gttgcgaaaa
     6841 agaacgttca cggcgactac tgcacttata tacggttctc ccccaccctc gggaaaaagg
     6901 cggagccagt acacgacatc actttcccag tttaccccgc gccaccttct ctaggcaccg
     6961 gttcaattgc cgacccctcc ccccaacttc tcgggcactg tgggccatgt gcgctctgcc
     7021 ccatggtaat agccatgact aatacgtaga tgtactgcca agtaggaaag tgccataagg
     7081 tcatgtactg ggcataatgc caggcgggcc atttaccgtc attgacgtca atagggggcg
     7141 tacttggcat atgatacact tgatgtactg ccaagtgggc agtttaccgt aaatactcca
     7201 cccattgacg tcaatggaaa gtgcctattg gcgttactat gggaacatac gtcattattg
     7261 acgtcaatgg gcgggggtcg ttgggcggtc agccaggcgg gccatttacc gtaagttatg
     7321 taacgcggaa ctccatatat gggctatgaa ctaatgaccc cgtaattgat tactattaat
     7381 aactagtcaa taatcaatgt cataagctct aatcgcataa tcgagctaag agcgattaat
     7441 atgcttagct agcttagcta agctagattt ctctattctc ccacaatata tagcagctat
     7501 tagcttagca gtccgaatac gatcgtccac gatccataag cgtaacacat ttgagtagcg
     7561 actgacgggc accggagcca ctcgagtgga attatcacct cgagtttact ccctatcagt
     7621 gatagagaac gtatgaagag tttactccct atcagtgata gagaacgtat gcagacttta
     7681 ctccctatca gtgatagaga acgtataagg agtttactcc ctatcagtga tagagaacgt
     7741 atgaccagtt tactccctat cagtgataga gaacgtatct acagtttact ccctatcagt
     7801 gatagagaac gtatatccag tttactccct atcagtgata gagaacgtat
//
