LOCUS       sequence-377909-        7459 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Barcoded piggybac transposon vector with Dox-inducible expression of
            YBX1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7459)
  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 7459)
  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-YBX1
FEATURES             Location/Qualifiers
     source          1..7459
                     /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(1363..1387)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1389..1415
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1582..1716
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   1988..2022
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2033..2050)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2033..2049)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2042..2064)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2258..2277)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2379..2401
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2440..2458)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2526..2630
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2631..2699,2700..3491)
                     /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(2849..2868)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3662..4250
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4151..4170
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4404..4421
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4627..4648
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4663..4668,4669..4686,4687..4693)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4701..4717
                     /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     4706..4728
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4725..4741
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4725..4741
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4767..4829
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    5005..5086
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5803..5859)
                     /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(5869..6612)
                     /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(6613..6636)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6636..6645
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6665..6876)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        6877..7256
                     /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 gcagcgaggc cgagacccag cagccgcccg
      421 ccgccccccc cgccgccccc gccctcagcg ccgccgacac caagcccggc actacgggca
      481 gcggcgcagg gagcggtggc ccgggcggcc tcacatcggc ggcgcctgcc ggcggggaca
      541 agaaggtcat cgcaacgaag gttttgggaa cagtaaaatg gttcaatgta aggaacggat
      601 atggtttcat caacaggaat gacaccaagg aagatgtatt tgtacaccag actgccataa
      661 agaagaataa ccccaggaag taccttcgca gtgtaggaga tggagagact gtggagtttg
      721 atgttgttga aggagaaaag ggtgcggagg cagcaaatgt tacaggtcct ggtggtgttc
      781 cagttcaagg cagtaaatat gcagcagacc gtaaccatta tagacgctat ccacgtcgta
      841 ggggtcctcc acgcaattac cagcaaaatt accagaatag tgagagtggg gaaaagaacg
      901 agggatcgga gagtgctccc gaaggccagg cccaacaacg ccggccctac cgcaggcgaa
      961 ggttcccacc ttactacatg cggagaccct atgggcgtcg accacagtat tccaaccctc
     1021 ctgtgcaggg agaagtgatg gagggtgctg acaaccaggg tgcaggagaa caaggtagac
     1081 cagtgaggca gaatatgtat cggggatata gaccacgatt ccgcaggggc cctcctcgcc
     1141 aaagacagcc tagagaggac ggcaatgaag aagataaaga aaatcaagga gatgagaccc
     1201 aaggtcagca gccacctcaa cgtcggtacc gccgcaactt caattaccga cgcagacgcc
     1261 cagaaaaccc taaaccacaa gatggcaaag agacaaaagc agccgatcca ccagctgaga
     1321 attcgtccgc tcccgaggct gagcagggcg gggctgagtt ggacccagct ttcttgtaca
     1381 aagtggtata cccatacgat gttccagatt acgcttagta atgagaaata ctcagaagat
     1441 gtcactcacc atagtccgca gtgaaagata aatgatcatc ttcatcttcg tcttctagtt
     1501 ttagagctag aaatagcaag taggccactg agcaagaact atgaatgaga tctttagaca
     1561 tattgcggcc gctttctccg cgatccagac atgataagat acattgatga gtttggacaa
     1621 accaaaacta gaatgcagtg aaaaaaatgc cttatttgtg aaatttgtga tgctattgcc
     1681 ttatttgtaa ccattataag ctgcaataaa caagttatct ataacaagaa aatatatata
     1741 taataagtta tcacgtaagt agaacatgaa ataacaatat aattatcgta tgagttaaat
     1801 cttaaaagtc acgtaaaaga taatcatgcg tcattttgac tcacgcggtc gttatagttc
     1861 aaaatcagtg acacttaccg cattgacaag cacgcctcac gggagctcca agcggcgact
     1921 gagatgtcct aaatgcacag cgacggattc gcgctattta gaaagagaga gcaatatttc
     1981 aagaatgcat gcgtcaattt tacgcagact atctttctag ggttaagaat tcactggccg
     2041 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag
     2101 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
     2161 aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg gtattttctc cttacgcatc
     2221 tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct gatgccgcat
     2281 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     2341 tcccggcatc cgcttacaga caagctgtga ccatgtctcc gggagctgca tgtgtcagag
     2401 gttttcaccg tcatcaccga aacacgcgag ttcgaaaggg cctcgtgata cgcctatttt
     2461 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa
     2521 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     2581 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     2641 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     2701 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     2761 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     2821 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     2881 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     2941 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     3001 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     3061 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     3121 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     3181 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     3241 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     3301 cggctggctg gtttattgct gataaatctg gagccggtga gcgtggttca cgcggtatca
     3361 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     3421 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     3481 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     3541 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     3601 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     3661 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     3721 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     3781 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     3841 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     3901 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     3961 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     4021 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     4081 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     4141 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     4201 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     4261 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     4321 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     4381 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggat gagcgcccaa
     4441 tacgcaaacc gcctctcacc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
     4501 ttcccgactg gaaagcgggc agtgagcgca acgcaaaccg cctctccccg cgcgttggcc
     4561 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa
     4621 cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc
     4681 ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga
     4741 ccatgattac gccaaggtcg acttaaccct agaaagataa tcatattgtg acgtacgtta
     4801 aagataatca tgcgtaaaat tgacgcatgt gttttatcgg tctgtatatc gaggtttatt
     4861 tattaatttg aatagatatt aagttttatt atatttacac ttacatacta ataataaatt
     4921 caacaaacaa tttatttatg tttatttatt tattaaaaaa aaacaaaaac tcaaaatttc
     4981 ttctataaag taacaaaact tttagcagtg aaaaaaatgc tttatttgtg aaatttgtga
     5041 tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg
     5101 cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa
     5161 cctctacaaa tgtggtatgg ctgattatga tcctctggag attcatgcgc caggttttct
     5221 ggtcatgcac cacgttcgtg ggccctcagg aacctccacg tcagcggtaa ctgtgaagcc
     5281 cagtcgctcg taaaaaggaa gattccgggg agcactggtt tcgaggaacg ccggcacgcc
     5341 agcccgttcc gcggcctcaa caccaggaag aacgacagcg cttcccaaac ctttaccttg
     5401 gtgatctggg ctgacaccga cagtggcaag aaaccacgca ggctcctttg gtctatgagg
     5461 agccagcaag ccctccattt gctgttgtgc cgccaaccta ctccctgaca gctcggccat
     5521 ccgcggacct atttccgcga acactgcgcc ggcttcgacg ctttccggag tagtccagac
     5581 cgcaactgcg gcaccatcgt ccgcaaccca aactttacca atgtccagac ccacgcgcgt
     5641 aagaaacagc tcttgcaact cagttaccct ctctatgtgt ctgtccggat ctacggtatg
     5701 gcgggtcgct ggataatctg caaatgctgc ggcaagtgtt cggacagccc gtggtacatc
     5761 gtcacgggta gcgagcctaa cagtgggctt atattccgtc atggggccgg ggttctcctc
     5821 cacgtcgccg gcctgcttca gcaggctgaa gttggtggcg ccgctgcccc cggggagcat
     5881 gtcaaggtca aaatcgtcaa gagcgtcagc aggcagcata tcaaggtcaa agtcgtcaag
     5941 ggcatcggct gggagcatgt ctaagtcaaa atcgtcaagg gcgtcggtcg gcccgccgct
     6001 ttcgcacttt agctgtttct ccaggccaca tatgattagt tccaggccga aaaggaaggc
     6061 aggttcggct ccctgccggt cgaacagctc aattgcttgt ttcagaagtg ggggcataga
     6121 atcggtggta ggtgtctctc tttcctcttt tgctacttga tgctcctgtt cctccaatac
     6181 gcagcccagt gtaaagtggc ccacggcgga cagagcgtac agtgcgttct ccagggagaa
     6241 gccttgctga cacaggaacg cgagctgatt ttccagggtt tcgtactgtt tctctgttgg
     6301 gcgggtgccg agatgcactt tagccccgtc acgatgtgag aggagagcac agcggtatga
     6361 cttggcgttg ttccgcagaa agtcttgcca tgactcgcct tccagggggc aggagtgggt
     6421 atgatgcctg tccagcatct cgattggcag ggcatcgagc agggcccgct tgttcttcac
     6481 gtgccagtac agggtaggct gctcaactcc cagcttttga gcgagtttcc ttgtcgtcag
     6541 gccttcgata ccgactccat tgagtaattc cagagcagag tttatgactt tgctcttgtc
     6601 cagtctagac atcttatcgt catcgtcttt gtaatccatg gtggcggatc ccgcgtcacg
     6661 acacctgtgt tctggcggca aacccgttgc gaaaaagaac gttcacggcg actactgcac
     6721 ttatatacgg ttctccccca ccctcgggaa aaaggcggag ccagtacacg acatcacttt
     6781 cccagtttac cccgcgccac cttctctagg caccggttca attgccgacc cctcccccca
     6841 acttctcggg cactgtgggc catgtgcgct ctgccccatg gtaatagcca tgactaatac
     6901 gtagatgtac tgccaagtag gaaagtgcca taaggtcatg tactgggcat aatgccaggc
     6961 gggccattta ccgtcattga cgtcaatagg gggcgtactt ggcatatgat acacttgatg
     7021 tactgccaag tgggcagttt accgtaaata ctccacccat tgacgtcaat ggaaagtgcc
     7081 tattggcgtt actatgggaa catacgtcat tattgacgtc aatgggcggg ggtcgttggg
     7141 cggtcagcca ggcgggccat ttaccgtaag ttatgtaacg cggaactcca tatatgggct
     7201 atgaactaat gaccccgtaa ttgattacta ttaataacta gtcaataatc aatgtcataa
     7261 gctctaatcg cataatcgag ctaagagcga ttaatatgct tagctagctt agctaagcta
     7321 gatttctcta ttctcccaca atatatagca gctattagct tagcagtccg aatacgatcg
     7381 tccacgatcc ataagcgtaa cacatttgag tagcgactga cgggcaccgg agccactcga
     7441 gtggaattat cacctcgag
//
