LOCUS       sequence-377023-        7672 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Barcoded piggybac transposon vector with Dox-inducible expression of
            LHX9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7672)
  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 7672)
  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-LHX9
FEATURES             Location/Qualifiers
     source          1..7672
                     /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(1576..1600)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1602..1628
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1795..1929
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   2201..2235
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2246..2263)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2246..2262)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2255..2277)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2471..2490)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2592..2614
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2653..2671)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2739..2843
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2844..2912,2913..3704)
                     /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(3062..3081)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3875..4463
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4364..4383
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4617..4634
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4840..4861
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4876..4881,4882..4899,4900..4906)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4914..4930
                     /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     4919..4941
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4938..4954
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4938..4954
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4980..5042
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    5218..5299
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(6016..6072)
                     /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(6082..6825)
                     /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(6826..6849)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6849..6858
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6878..7089)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        7090..7469
                     /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 aaaagttggc accatggaaa tagtggggtg ccgagcagaa gacaactcgt
      421 gtcctttccg ccccccagcc atgctctttc acgggatctc cggaggccac atccaaggca
      481 tcatggagga gatggagcgc agatccaaga ctgaggcccg tctggccaaa ggcgcccagc
      541 tcaacggccg cgacgcgggc atgcccccgc tcagcccgga gaagcccgcc ctgtgcgccg
      601 gctgcggggg caagatctcg gacaggtact atctgctggc tgtggacaaa cagtggcatc
      661 tgagatgcct gaagtgctgt gaatgtaagc tggccctcga gtccgagctc acctgctttg
      721 ccaaggacgg tagcatttac tgcaaggagg attactacag aaggttctct gtgcagagat
      781 gtgcccgctg ccaccttggc atttccgcct cggagatggt catgcgcgcc cgagactctg
      841 tctaccacct gagctgcttc acctgctcca cttgcaacaa gactctgacc acgggcgacc
      901 atttcggcat gaaggacagc ctggtgtact gccgcgccca cttcgagacc ctcttgcaag
      961 gagagtatcc accgcagctg agctacacgg agctggcggc caagagcggc ggcctggccc
     1021 tgccttactt caacggtacg ggcaccgtgc agaaagggcg gccccggaag cggaagagcc
     1081 cagcgctggg agtggacatc gtcaattaca actcaggttg taatgagaat gaggcagacc
     1141 acttggaccg ggaccagcag ccttatccac cctcgcagaa gaccaagcgc atgcgaacct
     1201 ctttcaagca tcaccagctc cggaccatga aatcctactt tgccatcaac cacaacccgg
     1261 atgccaagga cctcaagcag cttgcccaga aaacaggtct gaccaaaaga gttttgcagg
     1321 tttggttcca aaacgcacga gccaaattca gaaggaacct tttgcggcag gagaatgggg
     1381 gtgttgataa agctgacggc acgtcgcttc cggccccgcc ctcagcagac agcggagctc
     1441 tcactccacc cggcactgcg accactttaa cagacctgac caatcccact atcactgtag
     1501 tgacatccgt gacctctaac atggacagcc acgaatccgg aagcccctca caaactacct
     1561 taacaaacct tttcttgcca actttcttgt acaaagtggt atacccatac gatgttccag
     1621 attacgctta gtaatgagaa atactcagaa gatgtcactc accatagtcc gcagtgaaag
     1681 ataaatgatc attcggaaga tggcaatgac gttttagagc tagaaatagc aagtaggcca
     1741 ctgagcaaga actatgaatg agatctttag acatattgcg gccgctttct ccgcgatcca
     1801 gacatgataa gatacattga tgagtttgga caaaccaaaa ctagaatgca gtgaaaaaaa
     1861 tgccttattt gtgaaatttg tgatgctatt gccttatttg taaccattat aagctgcaat
     1921 aaacaagtta tctataacaa gaaaatatat atataataag ttatcacgta agtagaacat
     1981 gaaataacaa tataattatc gtatgagtta aatcttaaaa gtcacgtaaa agataatcat
     2041 gcgtcatttt gactcacgcg gtcgttatag ttcaaaatca gtgacactta ccgcattgac
     2101 aagcacgcct cacgggagct ccaagcggcg actgagatgt cctaaatgca cagcgacgga
     2161 ttcgcgctat ttagaaagag agagcaatat ttcaagaatg catgcgtcaa ttttacgcag
     2221 actatctttc tagggttaag aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa
     2281 accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta
     2341 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat
     2401 ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt
     2461 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     2521 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     2581 tgaccatgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacacgc
     2641 gagttcgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     2701 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     2761 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     2821 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     2881 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     2941 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     3001 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     3061 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     3121 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     3181 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     3241 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     3301 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     3361 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     3421 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     3481 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     3541 ctggagccgg tgagcgtggt tcacgcggta tcattgcagc actggggcca gatggtaagc
     3601 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     3661 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     3721 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     3781 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     3841 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     3901 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     3961 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     4021 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     4081 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     4141 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     4201 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     4261 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     4321 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     4381 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     4441 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     4501 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     4561 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     4621 agtcagtgag cgaggaagcg gatgagcgcc caatacgcaa accgcctctc accgcgcgtt
     4681 ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc
     4741 gcaacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
     4801 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca
     4861 ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag
     4921 cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagg tcgacttaac
     4981 cctagaaaga taatcatatt gtgacgtacg ttaaagataa tcatgcgtaa aattgacgca
     5041 tgtgttttat cggtctgtat atcgaggttt atttattaat ttgaatagat attaagtttt
     5101 attatattta cacttacata ctaataataa attcaacaaa caatttattt atgtttattt
     5161 atttattaaa aaaaaacaaa aactcaaaat ttcttctata aagtaacaaa acttttagca
     5221 gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat
     5281 aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg
     5341 ggaggtgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta tggctgatta
     5401 tgatcctctg gagattcatg cgccaggttt tctggtcatg caccacgttc gtgggccctc
     5461 aggaacctcc acgtcagcgg taactgtgaa gcccagtcgc tcgtaaaaag gaagattccg
     5521 gggagcactg gtttcgagga acgccggcac gccagcccgt tccgcggcct caacaccagg
     5581 aagaacgaca gcgcttccca aacctttacc ttggtgatct gggctgacac cgacagtggc
     5641 aagaaaccac gcaggctcct ttggtctatg aggagccagc aagccctcca tttgctgttg
     5701 tgccgccaac ctactccctg acagctcggc catccgcgga cctatttccg cgaacactgc
     5761 gccggcttcg acgctttccg gagtagtcca gaccgcaact gcggcaccat cgtccgcaac
     5821 ccaaacttta ccaatgtcca gacccacgcg cgtaagaaac agctcttgca actcagttac
     5881 cctctctatg tgtctgtccg gatctacggt atggcgggtc gctggataat ctgcaaatgc
     5941 tgcggcaagt gttcggacag cccgtggtac atcgtcacgg gtagcgagcc taacagtggg
     6001 cttatattcc gtcatggggc cggggttctc ctccacgtcg ccggcctgct tcagcaggct
     6061 gaagttggtg gcgccgctgc ccccggggag catgtcaagg tcaaaatcgt caagagcgtc
     6121 agcaggcagc atatcaaggt caaagtcgtc aagggcatcg gctgggagca tgtctaagtc
     6181 aaaatcgtca agggcgtcgg tcggcccgcc gctttcgcac tttagctgtt tctccaggcc
     6241 acatatgatt agttccaggc cgaaaaggaa ggcaggttcg gctccctgcc ggtcgaacag
     6301 ctcaattgct tgtttcagaa gtgggggcat agaatcggtg gtaggtgtct ctctttcctc
     6361 ttttgctact tgatgctcct gttcctccaa tacgcagccc agtgtaaagt ggcccacggc
     6421 ggacagagcg tacagtgcgt tctccaggga gaagccttgc tgacacagga acgcgagctg
     6481 attttccagg gtttcgtact gtttctctgt tgggcgggtg ccgagatgca ctttagcccc
     6541 gtcacgatgt gagaggagag cacagcggta tgacttggcg ttgttccgca gaaagtcttg
     6601 ccatgactcg ccttccaggg ggcaggagtg ggtatgatgc ctgtccagca tctcgattgg
     6661 cagggcatcg agcagggccc gcttgttctt cacgtgccag tacagggtag gctgctcaac
     6721 tcccagcttt tgagcgagtt tccttgtcgt caggccttcg ataccgactc cattgagtaa
     6781 ttccagagca gagtttatga ctttgctctt gtccagtcta gacatcttat cgtcatcgtc
     6841 tttgtaatcc atggtggcgg atcccgcgtc acgacacctg tgttctggcg gcaaacccgt
     6901 tgcgaaaaag aacgttcacg gcgactactg cacttatata cggttctccc ccaccctcgg
     6961 gaaaaaggcg gagccagtac acgacatcac tttcccagtt taccccgcgc caccttctct
     7021 aggcaccggt tcaattgccg acccctcccc ccaacttctc gggcactgtg ggccatgtgc
     7081 gctctgcccc atggtaatag ccatgactaa tacgtagatg tactgccaag taggaaagtg
     7141 ccataaggtc atgtactggg cataatgcca ggcgggccat ttaccgtcat tgacgtcaat
     7201 agggggcgta cttggcatat gatacacttg atgtactgcc aagtgggcag tttaccgtaa
     7261 atactccacc cattgacgtc aatggaaagt gcctattggc gttactatgg gaacatacgt
     7321 cattattgac gtcaatgggc gggggtcgtt gggcggtcag ccaggcgggc catttaccgt
     7381 aagttatgta acgcggaact ccatatatgg gctatgaact aatgaccccg taattgatta
     7441 ctattaataa ctagtcaata atcaatgtca taagctctaa tcgcataatc gagctaagag
     7501 cgattaatat gcttagctag cttagctaag ctagatttct ctattctccc acaatatata
     7561 gcagctatta gcttagcagt ccgaatacga tcgtccacga tccataagcg taacacattt
     7621 gagtagcgac tgacgggcac cggagccact cgagtggaat tatcacctcg ag
//
