LOCUS       sequence-377024-        6709 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Barcoded piggybac transposon vector with Dox-inducible expression of
            TSC22D3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6709)
  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 6709)
  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-TSC22D3
FEATURES             Location/Qualifiers
     source          1..6709
                     /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(613..637)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             639..665
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    832..966
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   1238..1272
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1283..1300)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1283..1299)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1292..1314)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1508..1527)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1629..1651
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1690..1708)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1776..1880
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1881..1949,1950..2741)
                     /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(2099..2118)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2912..3500
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3401..3420
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3654..3671
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3877..3898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3913..3918,3919..3936,3937..3943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3951..3967
                     /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     3956..3978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3975..3991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3975..3991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4017..4079
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    4255..4336
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5053..5109)
                     /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(5119..5862)
                     /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(5863..5886)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      5886..5895
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(5915..6126)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        6127..6506
                     /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 aaaagtgggc atggatctgg tgaagaatca tctgatgtat gctgtgagag
      421 aggaggtgga gatcctgaag gagcagatcc gagagctggt ggagaagaac tcccagctag
      481 agcgtgagaa caccctgttg aagaccctgg caagcccaga gcagctggag aagttccagt
      541 cctgtctgag ccctgaagag ccagctcccg aatccccaca agtgcccgag gcccctggtg
      601 gttctgcggt ctacccaact ttcttgtaca aagtggtata cccatacgat gttccagatt
      661 acgcttagta atgagaaata ctcagaagat gtcactcacc atagtccgca gtgaaagata
      721 aatgatcaac cattgaaatc cgatgctgtt ttagagctag aaatagcaag taggccactg
      781 agcaagaact atgaatgaga tctttagaca tattgcggcc gctttctccg cgatccagac
      841 atgataagat acattgatga gtttggacaa accaaaacta gaatgcagtg aaaaaaatgc
      901 cttatttgtg aaatttgtga tgctattgcc ttatttgtaa ccattataag ctgcaataaa
      961 caagttatct ataacaagaa aatatatata taataagtta tcacgtaagt agaacatgaa
     1021 ataacaatat aattatcgta tgagttaaat cttaaaagtc acgtaaaaga taatcatgcg
     1081 tcattttgac tcacgcggtc gttatagttc aaaatcagtg acacttaccg cattgacaag
     1141 cacgcctcac gggagctcca agcggcgact gagatgtcct aaatgcacag cgacggattc
     1201 gcgctattta gaaagagaga gcaatatttc aagaatgcat gcgtcaattt tacgcagact
     1261 atctttctag ggttaagaat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc
     1321 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata
     1381 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc
     1441 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatggtgca
     1501 ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac
     1561 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     1621 ccatgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacacgcgag
     1681 ttcgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     1741 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     1801 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     1861 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     1921 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     1981 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     2041 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     2101 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     2161 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     2221 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     2281 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     2341 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     2401 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     2461 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     2521 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     2581 gagccggtga gcgtggttca cgcggtatca ttgcagcact ggggccagat ggtaagccct
     2641 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     2701 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     2761 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     2821 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     2881 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     2941 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     3001 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     3061 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     3121 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     3181 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     3241 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     3301 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     3361 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     3421 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     3481 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     3541 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     3601 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     3661 cagtgagcga ggaagcggat gagcgcccaa tacgcaaacc gcctctcacc gcgcgttggc
     3721 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     3781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
     3841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
     3901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
     3961 ataacaattt cacacaggaa acagctatga ccatgattac gccaaggtcg acttaaccct
     4021 agaaagataa tcatattgtg acgtacgtta aagataatca tgcgtaaaat tgacgcatgt
     4081 gttttatcgg tctgtatatc gaggtttatt tattaatttg aatagatatt aagttttatt
     4141 atatttacac ttacatacta ataataaatt caacaaacaa tttatttatg tttatttatt
     4201 tattaaaaaa aaacaaaaac tcaaaatttc ttctataaag taacaaaact tttagcagtg
     4261 aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag
     4321 ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga
     4381 ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtatgg ctgattatga
     4441 tcctctggag attcatgcgc caggttttct ggtcatgcac cacgttcgtg ggccctcagg
     4501 aacctccacg tcagcggtaa ctgtgaagcc cagtcgctcg taaaaaggaa gattccgggg
     4561 agcactggtt tcgaggaacg ccggcacgcc agcccgttcc gcggcctcaa caccaggaag
     4621 aacgacagcg cttcccaaac ctttaccttg gtgatctggg ctgacaccga cagtggcaag
     4681 aaaccacgca ggctcctttg gtctatgagg agccagcaag ccctccattt gctgttgtgc
     4741 cgccaaccta ctccctgaca gctcggccat ccgcggacct atttccgcga acactgcgcc
     4801 ggcttcgacg ctttccggag tagtccagac cgcaactgcg gcaccatcgt ccgcaaccca
     4861 aactttacca atgtccagac ccacgcgcgt aagaaacagc tcttgcaact cagttaccct
     4921 ctctatgtgt ctgtccggat ctacggtatg gcgggtcgct ggataatctg caaatgctgc
     4981 ggcaagtgtt cggacagccc gtggtacatc gtcacgggta gcgagcctaa cagtgggctt
     5041 atattccgtc atggggccgg ggttctcctc cacgtcgccg gcctgcttca gcaggctgaa
     5101 gttggtggcg ccgctgcccc cggggagcat gtcaaggtca aaatcgtcaa gagcgtcagc
     5161 aggcagcata tcaaggtcaa agtcgtcaag ggcatcggct gggagcatgt ctaagtcaaa
     5221 atcgtcaagg gcgtcggtcg gcccgccgct ttcgcacttt agctgtttct ccaggccaca
     5281 tatgattagt tccaggccga aaaggaaggc aggttcggct ccctgccggt cgaacagctc
     5341 aattgcttgt ttcagaagtg ggggcataga atcggtggta ggtgtctctc tttcctcttt
     5401 tgctacttga tgctcctgtt cctccaatac gcagcccagt gtaaagtggc ccacggcgga
     5461 cagagcgtac agtgcgttct ccagggagaa gccttgctga cacaggaacg cgagctgatt
     5521 ttccagggtt tcgtactgtt tctctgttgg gcgggtgccg agatgcactt tagccccgtc
     5581 acgatgtgag aggagagcac agcggtatga cttggcgttg ttccgcagaa agtcttgcca
     5641 tgactcgcct tccagggggc aggagtgggt atgatgcctg tccagcatct cgattggcag
     5701 ggcatcgagc agggcccgct tgttcttcac gtgccagtac agggtaggct gctcaactcc
     5761 cagcttttga gcgagtttcc ttgtcgtcag gccttcgata ccgactccat tgagtaattc
     5821 cagagcagag tttatgactt tgctcttgtc cagtctagac atcttatcgt catcgtcttt
     5881 gtaatccatg gtggcggatc ccgcgtcacg acacctgtgt tctggcggca aacccgttgc
     5941 gaaaaagaac gttcacggcg actactgcac ttatatacgg ttctccccca ccctcgggaa
     6001 aaaggcggag ccagtacacg acatcacttt cccagtttac cccgcgccac cttctctagg
     6061 caccggttca attgccgacc cctcccccca acttctcggg cactgtgggc catgtgcgct
     6121 ctgccccatg gtaatagcca tgactaatac gtagatgtac tgccaagtag gaaagtgcca
     6181 taaggtcatg tactgggcat aatgccaggc gggccattta ccgtcattga cgtcaatagg
     6241 gggcgtactt ggcatatgat acacttgatg tactgccaag tgggcagttt accgtaaata
     6301 ctccacccat tgacgtcaat ggaaagtgcc tattggcgtt actatgggaa catacgtcat
     6361 tattgacgtc aatgggcggg ggtcgttggg cggtcagcca ggcgggccat ttaccgtaag
     6421 ttatgtaacg cggaactcca tatatgggct atgaactaat gaccccgtaa ttgattacta
     6481 ttaataacta gtcaataatc aatgtcataa gctctaatcg cataatcgag ctaagagcga
     6541 ttaatatgct tagctagctt agctaagcta gatttctcta ttctcccaca atatatagca
     6601 gctattagct tagcagtccg aatacgatcg tccacgatcc ataagcgtaa cacatttgag
     6661 tagcgactga cgggcaccgg agccactcga gtggaattat cacctcgag
//
