LOCUS       sequence-377939-        6697 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Barcoded piggybac transposon vector with Dox-inducible expression of
            HOPX.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6697)
  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 6697)
  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-HOPX
FEATURES             Location/Qualifiers
     source          1..6697
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    17..35
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    53..71
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    89..107
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    125..143
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    161..179
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    197..215
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     242..266
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    332..356
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(576..600)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             602..628
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    795..929
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   1201..1235
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1246..1263)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1246..1262)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1255..1277)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1471..1490)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1592..1614
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1653..1671)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1739..1843
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1844..1912,1913..2704)
                     /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(2062..2081)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2875..3463
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3364..3383
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3617..3634
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3840..3861
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3876..3881,3882..3899,3900..3906)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3914..3930
                     /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     3919..3941
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3938..3954
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3938..3954
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   3980..4042
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    4218..4299
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5016..5072)
                     /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(5082..5825)
                     /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(5826..5849)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      5849..5858
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(5878..6089)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        6090..6469
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     protein_bind    6678..6696
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
ORIGIN
        1 cgtatgaaga gtttactccc tatcagtgat agagaacgta tgcagacttt actccctatc
       61 agtgatagag aacgtataag gagtttactc cctatcagtg atagagaacg tatgaccagt
      121 ttactcccta tcagtgatag agaacgtatc tacagtttac tccctatcag tgatagagaa
      181 cgtatatcca gtttactccc tatcagtgat agagaacgta taagcttgcc tatataagca
      241 gagctcgttt agtgaaccgt cagatcgcct ggagacgcca tccacgctgt tttgacctcc
      301 atagaagaca ccgggaccga tccagcctac cacaagtttg tacaaaaaag ttggcatgtc
      361 ggcggagacc gcgagcggcc ccacagagga ccaggtggaa atcctggagt acaacttcaa
      421 caaggtcgac aagcacccgg attccaccac gctgtgcctc atcgcggccg aggcaggcct
      481 ttccgaggag gagacccaga aatggtttaa gcagcgcctg gcaaagtggc ggcgctcaga
      541 aggcctgccc tcagagtgca gatccgtcat agacaaccca actttcttgt acaaagtggt
      601 atacccatac gatgttccag attacgctta gtaatgagaa atactcagaa gatgtcactc
      661 accatagtcc gcagtgaaag ataaatgatc gttcttctcc ttttgaaaat gttttagagc
      721 tagaaatagc aagtaggcca ctgagcaaga actatgaatg agatctttag acatattgcg
      781 gccgctttct ccgcgatcca gacatgataa gatacattga tgagtttgga caaaccaaaa
      841 ctagaatgca gtgaaaaaaa tgccttattt gtgaaatttg tgatgctatt gccttatttg
      901 taaccattat aagctgcaat aaacaagtta tctataacaa gaaaatatat atataataag
      961 ttatcacgta agtagaacat gaaataacaa tataattatc gtatgagtta aatcttaaaa
     1021 gtcacgtaaa agataatcat gcgtcatttt gactcacgcg gtcgttatag ttcaaaatca
     1081 gtgacactta ccgcattgac aagcacgcct cacgggagct ccaagcggcg actgagatgt
     1141 cctaaatgca cagcgacgga ttcgcgctat ttagaaagag agagcaatat ttcaagaatg
     1201 catgcgtcaa ttttacgcag actatctttc tagggttaag aattcactgg ccgtcgtttt
     1261 acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc
     1321 ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt
     1381 gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     1441 tatttcacac cgcatatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     1501 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     1561 atccgcttac agacaagctg tgaccatgtc tccgggagct gcatgtgtca gaggttttca
     1621 ccgtcatcac cgaaacacgc gagttcgaaa gggcctcgtg atacgcctat ttttataggt
     1681 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
     1741 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     1801 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     1861 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     1921 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     1981 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     2041 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     2101 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     2161 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     2221 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     2281 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     2341 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     2401 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     2461 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     2521 ctggtttatt gctgataaat ctggagccgg tgagcgtggt tcacgcggta tcattgcagc
     2581 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     2641 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     2701 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     2761 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     2821 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     2881 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     2941 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     3001 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     3061 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     3121 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     3181 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     3241 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     3301 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     3361 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     3421 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     3481 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     3541 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     3601 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gatgagcgcc caatacgcaa
     3661 accgcctctc accgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga
     3721 ctggaaagcg ggcagtgagc gcaacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
     3781 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
     3841 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt
     3901 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat
     3961 tacgccaagg tcgacttaac cctagaaaga taatcatatt gtgacgtacg ttaaagataa
     4021 tcatgcgtaa aattgacgca tgtgttttat cggtctgtat atcgaggttt atttattaat
     4081 ttgaatagat attaagtttt attatattta cacttacata ctaataataa attcaacaaa
     4141 caatttattt atgtttattt atttattaaa aaaaaacaaa aactcaaaat ttcttctata
     4201 aagtaacaaa acttttagca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt
     4261 gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat
     4321 tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta aaacctctac
     4381 aaatgtggta tggctgatta tgatcctctg gagattcatg cgccaggttt tctggtcatg
     4441 caccacgttc gtgggccctc aggaacctcc acgtcagcgg taactgtgaa gcccagtcgc
     4501 tcgtaaaaag gaagattccg gggagcactg gtttcgagga acgccggcac gccagcccgt
     4561 tccgcggcct caacaccagg aagaacgaca gcgcttccca aacctttacc ttggtgatct
     4621 gggctgacac cgacagtggc aagaaaccac gcaggctcct ttggtctatg aggagccagc
     4681 aagccctcca tttgctgttg tgccgccaac ctactccctg acagctcggc catccgcgga
     4741 cctatttccg cgaacactgc gccggcttcg acgctttccg gagtagtcca gaccgcaact
     4801 gcggcaccat cgtccgcaac ccaaacttta ccaatgtcca gacccacgcg cgtaagaaac
     4861 agctcttgca actcagttac cctctctatg tgtctgtccg gatctacggt atggcgggtc
     4921 gctggataat ctgcaaatgc tgcggcaagt gttcggacag cccgtggtac atcgtcacgg
     4981 gtagcgagcc taacagtggg cttatattcc gtcatggggc cggggttctc ctccacgtcg
     5041 ccggcctgct tcagcaggct gaagttggtg gcgccgctgc ccccggggag catgtcaagg
     5101 tcaaaatcgt caagagcgtc agcaggcagc atatcaaggt caaagtcgtc aagggcatcg
     5161 gctgggagca tgtctaagtc aaaatcgtca agggcgtcgg tcggcccgcc gctttcgcac
     5221 tttagctgtt tctccaggcc acatatgatt agttccaggc cgaaaaggaa ggcaggttcg
     5281 gctccctgcc ggtcgaacag ctcaattgct tgtttcagaa gtgggggcat agaatcggtg
     5341 gtaggtgtct ctctttcctc ttttgctact tgatgctcct gttcctccaa tacgcagccc
     5401 agtgtaaagt ggcccacggc ggacagagcg tacagtgcgt tctccaggga gaagccttgc
     5461 tgacacagga acgcgagctg attttccagg gtttcgtact gtttctctgt tgggcgggtg
     5521 ccgagatgca ctttagcccc gtcacgatgt gagaggagag cacagcggta tgacttggcg
     5581 ttgttccgca gaaagtcttg ccatgactcg ccttccaggg ggcaggagtg ggtatgatgc
     5641 ctgtccagca tctcgattgg cagggcatcg agcagggccc gcttgttctt cacgtgccag
     5701 tacagggtag gctgctcaac tcccagcttt tgagcgagtt tccttgtcgt caggccttcg
     5761 ataccgactc cattgagtaa ttccagagca gagtttatga ctttgctctt gtccagtcta
     5821 gacatcttat cgtcatcgtc tttgtaatcc atggtggcgg atcccgcgtc acgacacctg
     5881 tgttctggcg gcaaacccgt tgcgaaaaag aacgttcacg gcgactactg cacttatata
     5941 cggttctccc ccaccctcgg gaaaaaggcg gagccagtac acgacatcac tttcccagtt
     6001 taccccgcgc caccttctct aggcaccggt tcaattgccg acccctcccc ccaacttctc
     6061 gggcactgtg ggccatgtgc gctctgcccc atggtaatag ccatgactaa tacgtagatg
     6121 tactgccaag taggaaagtg ccataaggtc atgtactggg cataatgcca ggcgggccat
     6181 ttaccgtcat tgacgtcaat agggggcgta cttggcatat gatacacttg atgtactgcc
     6241 aagtgggcag tttaccgtaa atactccacc cattgacgtc aatggaaagt gcctattggc
     6301 gttactatgg gaacatacgt cattattgac gtcaatgggc gggggtcgtt gggcggtcag
     6361 ccaggcgggc catttaccgt aagttatgta acgcggaact ccatatatgg gctatgaact
     6421 aatgaccccg taattgatta ctattaataa ctagtcaata atcaatgtca taagctctaa
     6481 tcgcataatc gagctaagag cgattaatat gcttagctag cttagctaag ctagatttct
     6541 ctattctccc acaatatata gcagctatta gcttagcagt ccgaatacga tcgtccacga
     6601 tccataagcg taacacattt gagtagcgac tgacgggcac cggagccact cgagtggaat
     6661 tatcacctcg agtttactcc ctatcagtga tagagaa
//
