LOCUS       sequence-376980-        7297 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Barcoded piggybac transposon vector with Dox-inducible expression of
            CEBPD.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7297)
  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 7297)
  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-CEBPD
FEATURES             Location/Qualifiers
     source          1..7297
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    6..24
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    42..60
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    78..96
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    114..132
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    150..168
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    186..204
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    222..240
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     267..291
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    357..381
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1201..1225)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1227..1253
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1420..1554
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   1826..1860
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1871..1888)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1871..1887)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1880..1902)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2096..2115)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2217..2239
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2278..2296)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2364..2468
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2469..2537,2538..3329)
                     /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(2687..2706)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3500..4088
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3989..4008
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4242..4259
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4465..4486
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4501..4506,4507..4524,4525..4531)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4539..4555
                     /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     4544..4566
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4563..4579
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4563..4579
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4605..4667
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    4843..4924
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5641..5697)
                     /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(5707..6450)
                     /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(6451..6474)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6474..6483
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6503..6714)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        6715..7094
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 tttactccct atcagtgata gagaacgtat gaagagttta ctccctatca gtgatagaga
       61 acgtatgcag actttactcc ctatcagtga tagagaacgt ataaggagtt tactccctat
      121 cagtgataga gaacgtatga ccagtttact ccctatcagt gatagagaac gtatctacag
      181 tttactccct atcagtgata gagaacgtat atccagttta ctccctatca gtgatagaga
      241 acgtataagc ttgcctatat aagcagagct cgtttagtga accgtcagat cgcctggaga
      301 cgccatccac gctgttttga cctccataga agacaccggg accgatccag cctaccacaa
      361 gtttgtacaa aaaagcaggc tccaccatga gcgccgcgct cttcagcctg gacggcccgg
      421 cgcgcggcgc gccctggcct gcggagcctg cgcccttcta cgaaccgggc cgggcgggca
      481 agccgggccg cggggccgag ccaggggccc taggcgagcc aggcgccgcc gcccccgcca
      541 tgtacgacga cgagagcgcc atcgacttca gcgcctacat cgactccatg gccgccgtgc
      601 ccaccctgga gctgtgccac gacgagctct tcgccgacct cttcaacagc aatcacaagg
      661 cgggcggcgc ggggcccctg gagcttcttc ccggcggccc cgcgcgcccc ttgggcccgg
      721 gccctgccgc tccccgcctg ctcaagcgcg agcccgactg gggcgacggc gacgcgcccg
      781 gctcgctgtt gcccgcgcag gtggccgcgt gcgcacagac cgtggtgagc ttggcggccg
      841 cagggcagcc caccccgccc acgtcgccgg agccgccgcg cagcagcccc aggcagaccc
      901 ccgcgcccgg ccccgcccgg gagaagagcg ccggcaagag gggcccggac cgcggcagcc
      961 ccgagtaccg gcagcggcgc gagcgcaaca acatcgccgt gcgcaagagc cgcgacaagg
     1021 ccaagcggcg caaccaggag atgcagcaga agttggtgga gctgtcggct gagaacgaga
     1081 agctgcacca gcgcgtggag cagctcacgc gggacctggc cggcctccgg cagttcttca
     1141 agcagctgcc cagcccgccc ttcctgccgg ccgccgggac agcagactgc cggtgaatcc
     1201 acccagcttt cttgtacaaa gtggtatacc catacgatgt tccagattac gcttagtaat
     1261 gagaaatact cagaagatgt cactcaccat agtccgcagt gaaagataaa tgatctgcat
     1321 ttcctttccc tgtttgtttt agagctagaa atagcaagta ggccactgag caagaactat
     1381 gaatgagatc tttagacata ttgcggccgc tttctccgcg atccagacat gataagatac
     1441 attgatgagt ttggacaaac caaaactaga atgcagtgaa aaaaatgcct tatttgtgaa
     1501 atttgtgatg ctattgcctt atttgtaacc attataagct gcaataaaca agttatctat
     1561 aacaagaaaa tatatatata ataagttatc acgtaagtag aacatgaaat aacaatataa
     1621 ttatcgtatg agttaaatct taaaagtcac gtaaaagata atcatgcgtc attttgactc
     1681 acgcggtcgt tatagttcaa aatcagtgac acttaccgca ttgacaagca cgcctcacgg
     1741 gagctccaag cggcgactga gatgtcctaa atgcacagcg acggattcgc gctatttaga
     1801 aagagagagc aatatttcaa gaatgcatgc gtcaatttta cgcagactat ctttctaggg
     1861 ttaagaattc actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc
     1921 aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc
     1981 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc ctgatgcggt
     2041 attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa
     2101 tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc
     2161 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc atgtctccgg
     2221 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cacgcgagtt cgaaagggcc
     2281 tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag
     2341 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     2401 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     2461 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     2521 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     2581 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     2641 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     2701 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     2761 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     2821 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     2881 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     2941 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     3001 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     3061 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     3121 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     3181 gtggttcacg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     3241 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     3301 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     3361 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     3421 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     3481 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     3541 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     3601 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     3661 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     3721 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     3781 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     3841 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     3901 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     3961 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     4021 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     4081 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     4141 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     4201 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     4261 aagcggatga gcgcccaata cgcaaaccgc ctctcaccgc gcgttggccg attcattaat
     4321 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaaaccgcc
     4381 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
     4441 agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc
     4501 tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca
     4561 cacaggaaac agctatgacc atgattacgc caaggtcgac ttaaccctag aaagataatc
     4621 atattgtgac gtacgttaaa gataatcatg cgtaaaattg acgcatgtgt tttatcggtc
     4681 tgtatatcga ggtttattta ttaatttgaa tagatattaa gttttattat atttacactt
     4741 acatactaat aataaattca acaaacaatt tatttatgtt tatttattta ttaaaaaaaa
     4801 acaaaaactc aaaatttctt ctataaagta acaaaacttt tagcagtgaa aaaaatgctt
     4861 tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca
     4921 agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg tgtgggaggt
     4981 tttttaaagc aagtaaaacc tctacaaatg tggtatggct gattatgatc ctctggagat
     5041 tcatgcgcca ggttttctgg tcatgcacca cgttcgtggg ccctcaggaa cctccacgtc
     5101 agcggtaact gtgaagccca gtcgctcgta aaaaggaaga ttccggggag cactggtttc
     5161 gaggaacgcc ggcacgccag cccgttccgc ggcctcaaca ccaggaagaa cgacagcgct
     5221 tcccaaacct ttaccttggt gatctgggct gacaccgaca gtggcaagaa accacgcagg
     5281 ctcctttggt ctatgaggag ccagcaagcc ctccatttgc tgttgtgccg ccaacctact
     5341 ccctgacagc tcggccatcc gcggacctat ttccgcgaac actgcgccgg cttcgacgct
     5401 ttccggagta gtccagaccg caactgcggc accatcgtcc gcaacccaaa ctttaccaat
     5461 gtccagaccc acgcgcgtaa gaaacagctc ttgcaactca gttaccctct ctatgtgtct
     5521 gtccggatct acggtatggc gggtcgctgg ataatctgca aatgctgcgg caagtgttcg
     5581 gacagcccgt ggtacatcgt cacgggtagc gagcctaaca gtgggcttat attccgtcat
     5641 ggggccgggg ttctcctcca cgtcgccggc ctgcttcagc aggctgaagt tggtggcgcc
     5701 gctgcccccg gggagcatgt caaggtcaaa atcgtcaaga gcgtcagcag gcagcatatc
     5761 aaggtcaaag tcgtcaaggg catcggctgg gagcatgtct aagtcaaaat cgtcaagggc
     5821 gtcggtcggc ccgccgcttt cgcactttag ctgtttctcc aggccacata tgattagttc
     5881 caggccgaaa aggaaggcag gttcggctcc ctgccggtcg aacagctcaa ttgcttgttt
     5941 cagaagtggg ggcatagaat cggtggtagg tgtctctctt tcctcttttg ctacttgatg
     6001 ctcctgttcc tccaatacgc agcccagtgt aaagtggccc acggcggaca gagcgtacag
     6061 tgcgttctcc agggagaagc cttgctgaca caggaacgcg agctgatttt ccagggtttc
     6121 gtactgtttc tctgttgggc gggtgccgag atgcacttta gccccgtcac gatgtgagag
     6181 gagagcacag cggtatgact tggcgttgtt ccgcagaaag tcttgccatg actcgccttc
     6241 cagggggcag gagtgggtat gatgcctgtc cagcatctcg attggcaggg catcgagcag
     6301 ggcccgcttg ttcttcacgt gccagtacag ggtaggctgc tcaactccca gcttttgagc
     6361 gagtttcctt gtcgtcaggc cttcgatacc gactccattg agtaattcca gagcagagtt
     6421 tatgactttg ctcttgtcca gtctagacat cttatcgtca tcgtctttgt aatccatggt
     6481 ggcggatccc gcgtcacgac acctgtgttc tggcggcaaa cccgttgcga aaaagaacgt
     6541 tcacggcgac tactgcactt atatacggtt ctcccccacc ctcgggaaaa aggcggagcc
     6601 agtacacgac atcactttcc cagtttaccc cgcgccacct tctctaggca ccggttcaat
     6661 tgccgacccc tccccccaac ttctcgggca ctgtgggcca tgtgcgctct gccccatggt
     6721 aatagccatg actaatacgt agatgtactg ccaagtagga aagtgccata aggtcatgta
     6781 ctgggcataa tgccaggcgg gccatttacc gtcattgacg tcaatagggg gcgtacttgg
     6841 catatgatac acttgatgta ctgccaagtg ggcagtttac cgtaaatact ccacccattg
     6901 acgtcaatgg aaagtgccta ttggcgttac tatgggaaca tacgtcatta ttgacgtcaa
     6961 tgggcggggg tcgttgggcg gtcagccagg cgggccattt accgtaagtt atgtaacgcg
     7021 gaactccata tatgggctat gaactaatga ccccgtaatt gattactatt aataactagt
     7081 caataatcaa tgtcataagc tctaatcgca taatcgagct aagagcgatt aatatgctta
     7141 gctagcttag ctaagctaga tttctctatt ctcccacaat atatagcagc tattagctta
     7201 gcagtccgaa tacgatcgtc cacgatccat aagcgtaaca catttgagta gcgactgacg
     7261 ggcaccggag ccactcgagt ggaattatca cctcgag
//
