LOCUS       sequence-376598-        7384 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Barcoded piggybac transposon vector with Dox-inducible expression of
            WT1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7384)
  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 7384)
  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-WT1
FEATURES             Location/Qualifiers
     source          1..7384
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     20..44
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    110..134
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             457..468
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     protein_bind    complement(1041..1065)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1067..1093
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1260..1394
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   1666..1700
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1711..1728)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1711..1727)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1720..1742)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1936..1955)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2057..2079
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2118..2136)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2204..2308
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2309..2377,2378..3169)
                     /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(2527..2546)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3340..3928
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3829..3848
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4082..4099
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4305..4326
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4341..4346,4347..4364,4365..4371)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4379..4395
                     /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     4384..4406
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4403..4419
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4403..4419
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4445..4507
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    4683..4764
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5481..5537)
                     /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(5547..6290)
                     /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(6291..6314)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6314..6323
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6343..6554)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        6555..6934
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     protein_bind    7143..7161
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7179..7197
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7215..7233
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7251..7269
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7287..7305
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7323..7341
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7359..7377
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
ORIGIN
        1 agcttgccta tataagcaga gctcgtttag tgaaccgtca gatcgcctgg agacgccatc
       61 cacgctgttt tgacctccat agaagacacc gggaccgatc cagcctacca caagtttgta
      121 caaaaaagtt ggcatggaga agggttacag cacggtcacc ttcgacggga cgcccagcta
      181 cggtcacacg ccctcgcacc atgcggcgca gttccccaac cactcattca agcatgagga
      241 tcccatgggc cagcagggct cgctgggtga gcagcagtac tcggtgccgc ccccggtcta
      301 tggctgccac acccccaccg acagctgcac cggcagccag gctttgctgc tgaggacgcc
      361 ctacagcagt gacaatttat accaaatgac atcccagctt gaatgcatga cctggaatca
      421 gatgaactta ggagccacct taaagggagt tgctgctggg agctccagct cagtgaaatg
      481 gacagaaggg cagagcaacc acagcacagg gtacgagagc gataaccaca caacgcccat
      541 cctctgcgga gcccaataca gaatgcacac gcacggtgtc ttcagaggca ttcaggatgt
      601 gcggcgtgtg cctggagtag ccccgactct tgtacggtcg gcatctgaga ccagtgagaa
      661 acgccccttc atgtgtgctt acccaggctg caataagaga tattttaagc tgtcccactt
      721 acagatgcac agcaggaagc acactggtga gaaaccatac cagtgtgact tcaaggactg
      781 tgaacgaagg ttttctcgtt cagaccagct caaaagacac caaaggagac atacaggtgt
      841 gaaaccattc cagtgtaaaa cttgtcagcg aaagttctcc cggtccgacc acctgaagac
      901 ccacaccagg actcatacag gtgaaaagcc cttcagctgt cggtggccaa gttgtcagaa
      961 aaagtttgcc cggtcagatg aattagtccg ccatcacaac atgcatcaga gaaacatgac
     1021 caaactccag ctggcgcttt gcccaacttt cttgtacaaa gtggtatacc catacgatgt
     1081 tccagattac gcttagtaat gagaaatact cagaagatgt cactcaccat agtccgcagt
     1141 gaaagataaa tgatctctat gtggagttga aataggtttt agagctagaa atagcaagta
     1201 ggccactgag caagaactat gaatgagatc tttagacata ttgcggccgc tttctccgcg
     1261 atccagacat gataagatac attgatgagt ttggacaaac caaaactaga atgcagtgaa
     1321 aaaaatgcct tatttgtgaa atttgtgatg ctattgcctt atttgtaacc attataagct
     1381 gcaataaaca agttatctat aacaagaaaa tatatatata ataagttatc acgtaagtag
     1441 aacatgaaat aacaatataa ttatcgtatg agttaaatct taaaagtcac gtaaaagata
     1501 atcatgcgtc attttgactc acgcggtcgt tatagttcaa aatcagtgac acttaccgca
     1561 ttgacaagca cgcctcacgg gagctccaag cggcgactga gatgtcctaa atgcacagcg
     1621 acggattcgc gctatttaga aagagagagc aatatttcaa gaatgcatgc gtcaatttta
     1681 cgcagactat ctttctaggg ttaagaattc actggccgtc gttttacaac gtcgtgactg
     1741 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg
     1801 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg
     1861 cgaatggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
     1921 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc
     1981 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     2041 agctgtgacc atgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     2101 cacgcgagtt cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
     2161 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     2221 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     2281 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     2341 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     2401 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     2461 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     2521 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     2581 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     2641 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     2701 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     2761 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     2821 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     2881 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     2941 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     3001 taaatctgga gccggtgagc gtggttcacg cggtatcatt gcagcactgg ggccagatgg
     3061 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     3121 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     3181 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     3241 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     3301 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     3361 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     3421 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     3481 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     3541 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     3601 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     3661 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     3721 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     3781 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     3841 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     3901 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     3961 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     4021 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     4081 cagcgagtca gtgagcgagg aagcggatga gcgcccaata cgcaaaccgc ctctcaccgc
     4141 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     4201 tgagcgcaac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
     4261 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc
     4321 actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt
     4381 gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc caaggtcgac
     4441 ttaaccctag aaagataatc atattgtgac gtacgttaaa gataatcatg cgtaaaattg
     4501 acgcatgtgt tttatcggtc tgtatatcga ggtttattta ttaatttgaa tagatattaa
     4561 gttttattat atttacactt acatactaat aataaattca acaaacaatt tatttatgtt
     4621 tatttattta ttaaaaaaaa acaaaaactc aaaatttctt ctataaagta acaaaacttt
     4681 tagcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc
     4741 attataagct gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt
     4801 cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtatggct
     4861 gattatgatc ctctggagat tcatgcgcca ggttttctgg tcatgcacca cgttcgtggg
     4921 ccctcaggaa cctccacgtc agcggtaact gtgaagccca gtcgctcgta aaaaggaaga
     4981 ttccggggag cactggtttc gaggaacgcc ggcacgccag cccgttccgc ggcctcaaca
     5041 ccaggaagaa cgacagcgct tcccaaacct ttaccttggt gatctgggct gacaccgaca
     5101 gtggcaagaa accacgcagg ctcctttggt ctatgaggag ccagcaagcc ctccatttgc
     5161 tgttgtgccg ccaacctact ccctgacagc tcggccatcc gcggacctat ttccgcgaac
     5221 actgcgccgg cttcgacgct ttccggagta gtccagaccg caactgcggc accatcgtcc
     5281 gcaacccaaa ctttaccaat gtccagaccc acgcgcgtaa gaaacagctc ttgcaactca
     5341 gttaccctct ctatgtgtct gtccggatct acggtatggc gggtcgctgg ataatctgca
     5401 aatgctgcgg caagtgttcg gacagcccgt ggtacatcgt cacgggtagc gagcctaaca
     5461 gtgggcttat attccgtcat ggggccgggg ttctcctcca cgtcgccggc ctgcttcagc
     5521 aggctgaagt tggtggcgcc gctgcccccg gggagcatgt caaggtcaaa atcgtcaaga
     5581 gcgtcagcag gcagcatatc aaggtcaaag tcgtcaaggg catcggctgg gagcatgtct
     5641 aagtcaaaat cgtcaagggc gtcggtcggc ccgccgcttt cgcactttag ctgtttctcc
     5701 aggccacata tgattagttc caggccgaaa aggaaggcag gttcggctcc ctgccggtcg
     5761 aacagctcaa ttgcttgttt cagaagtggg ggcatagaat cggtggtagg tgtctctctt
     5821 tcctcttttg ctacttgatg ctcctgttcc tccaatacgc agcccagtgt aaagtggccc
     5881 acggcggaca gagcgtacag tgcgttctcc agggagaagc cttgctgaca caggaacgcg
     5941 agctgatttt ccagggtttc gtactgtttc tctgttgggc gggtgccgag atgcacttta
     6001 gccccgtcac gatgtgagag gagagcacag cggtatgact tggcgttgtt ccgcagaaag
     6061 tcttgccatg actcgccttc cagggggcag gagtgggtat gatgcctgtc cagcatctcg
     6121 attggcaggg catcgagcag ggcccgcttg ttcttcacgt gccagtacag ggtaggctgc
     6181 tcaactccca gcttttgagc gagtttcctt gtcgtcaggc cttcgatacc gactccattg
     6241 agtaattcca gagcagagtt tatgactttg ctcttgtcca gtctagacat cttatcgtca
     6301 tcgtctttgt aatccatggt ggcggatccc gcgtcacgac acctgtgttc tggcggcaaa
     6361 cccgttgcga aaaagaacgt tcacggcgac tactgcactt atatacggtt ctcccccacc
     6421 ctcgggaaaa aggcggagcc agtacacgac atcactttcc cagtttaccc cgcgccacct
     6481 tctctaggca ccggttcaat tgccgacccc tccccccaac ttctcgggca ctgtgggcca
     6541 tgtgcgctct gccccatggt aatagccatg actaatacgt agatgtactg ccaagtagga
     6601 aagtgccata aggtcatgta ctgggcataa tgccaggcgg gccatttacc gtcattgacg
     6661 tcaatagggg gcgtacttgg catatgatac acttgatgta ctgccaagtg ggcagtttac
     6721 cgtaaatact ccacccattg acgtcaatgg aaagtgccta ttggcgttac tatgggaaca
     6781 tacgtcatta ttgacgtcaa tgggcggggg tcgttgggcg gtcagccagg cgggccattt
     6841 accgtaagtt atgtaacgcg gaactccata tatgggctat gaactaatga ccccgtaatt
     6901 gattactatt aataactagt caataatcaa tgtcataagc tctaatcgca taatcgagct
     6961 aagagcgatt aatatgctta gctagcttag ctaagctaga tttctctatt ctcccacaat
     7021 atatagcagc tattagctta gcagtccgaa tacgatcgtc cacgatccat aagcgtaaca
     7081 catttgagta gcgactgacg ggcaccggag ccactcgagt ggaattatca cctcgagttt
     7141 actccctatc agtgatagag aacgtatgaa gagtttactc cctatcagtg atagagaacg
     7201 tatgcagact ttactcccta tcagtgatag agaacgtata aggagtttac tccctatcag
     7261 tgatagagaa cgtatgacca gtttactccc tatcagtgat agagaacgta tctacagttt
     7321 actccctatc agtgatagag aacgtatatc cagtttactc cctatcagtg atagagaacg
     7381 tata
//
