LOCUS       sequence-376599-        7762 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Barcoded piggybac transposon vector with Dox-inducible expression of
            ELK1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7762)
  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 7762)
  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-ELK1
FEATURES             Location/Qualifiers
     source          1..7762
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     22..46
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    112..136
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1421..1445)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1447..1473
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1640..1774
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   2046..2080
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2091..2108)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2091..2107)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2100..2122)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2316..2335)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2437..2459
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2498..2516)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2584..2688
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2689..2757,2758..3549)
                     /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(2907..2926)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3720..4308
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4209..4228
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4462..4479
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4685..4706
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4721..4726,4727..4744,4745..4751)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4759..4775
                     /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     4764..4786
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4783..4799
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4783..4799
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4825..4887
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    5063..5144
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5861..5917)
                     /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(5927..6670)
                     /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(6671..6694)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6694..6703
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6723..6934)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        6935..7314
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     protein_bind    7523..7541
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7559..7577
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7595..7613
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7631..7649
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7667..7685
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7703..7721
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7739..7757
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
ORIGIN
        1 taagcttgcc tatataagca gagctcgttt agtgaaccgt cagatcgcct ggagacgcca
       61 tccacgctgt tttgacctcc atagaagaca ccgggaccga tccagcctac cacaagtttg
      121 tacaaaaaag ttggcatgga cccatctgtg acgctgtggc agtttctgct gcagctgctg
      181 agagagcaag gcaatggcca catcatctcc tggacttcac gggatggtgg tgaattcaag
      241 ctggtggatg cagaggaggt ggcccggctg tgggggctac gcaagaacaa gaccaacatg
      301 aattacgaca agctcagccg ggccttgcgg tactactatg acaagaacat catccgcaag
      361 gtgagcggcc agaagttcgt ctacaagttt gtgtcctacc ctgaggtcgc agggtgctcc
      421 actgaggact gcccgcccca gccagaggtg tctgttacct ccaccatgcc aaatgtggcc
      481 cctgctgcta tacatgccgc cccaggggac actgtctctg gaaagccagg cacacccaag
      541 ggtgcaggaa tggcaggccc aggcggtttg gcacgcagca gccggaacga gtacatgcgc
      601 tcgggcctct attccacctt caccatccag tctctgcagc cgcagccacc ccctcatcct
      661 cggcctgctg tggtgctccc cagtgcagct cctgcagggg cagcagcgcc cccctcgggg
      721 agcaggagca ccagtccaag ccccttggag gcctgtctgg aggctgaaga ggccggcttg
      781 cctctgcagg tcatcctgac cccgcccgag gccccaaacc tgaaatcgga agagcttaat
      841 gtggagccgg gtttgggccg ggctttgccc ccagaagtga aagtagaagg gcccaaggaa
      901 gagttggaag ttgcggggga gagagggttt gtgccagaaa ccaccaaggc cgagccagaa
      961 gtccctccac aggagggcgt gccagcccgg ctgcccgcgg ttgttatgga caccgcaggg
     1021 caggcgggcg gccatgcggc ttccagccct gagatctccc agccgcagaa gggccggaag
     1081 ccccgggacc tagagcttcc actcagcccg agcctgctag gtgggccggg acccgaacgg
     1141 accccaggat cgggaagtgg ctccggcctc caggctccgg ggccggcgct gaccccatcc
     1201 ctgcttccta cgcatacatt gaccccggtg ctgctgacac ccagctcgct gcctcctagc
     1261 attcacttct ggagcaccct gagtcccatt gcgccccgta gcccggccaa gctctccttc
     1321 cagtttccat ccagtggcag cgcccaggtg cacatccctt ctatcagcgt ggatggcctc
     1381 tcgacccccg tggtgctctc cccagggccc cagaagccat gcccaacttt cttgtacaaa
     1441 gtggtatacc catacgatgt tccagattac gcttagtaat gagaaatact cagaagatgt
     1501 cactcaccat agtccgcagt gaaagataaa tgatcgcagg aagcgttgtg ttgcagtttt
     1561 agagctagaa atagcaagta ggccactgag caagaactat gaatgagatc tttagacata
     1621 ttgcggccgc tttctccgcg atccagacat gataagatac attgatgagt ttggacaaac
     1681 caaaactaga atgcagtgaa aaaaatgcct tatttgtgaa atttgtgatg ctattgcctt
     1741 atttgtaacc attataagct gcaataaaca agttatctat aacaagaaaa tatatatata
     1801 ataagttatc acgtaagtag aacatgaaat aacaatataa ttatcgtatg agttaaatct
     1861 taaaagtcac gtaaaagata atcatgcgtc attttgactc acgcggtcgt tatagttcaa
     1921 aatcagtgac acttaccgca ttgacaagca cgcctcacgg gagctccaag cggcgactga
     1981 gatgtcctaa atgcacagcg acggattcgc gctatttaga aagagagagc aatatttcaa
     2041 gaatgcatgc gtcaatttta cgcagactat ctttctaggg ttaagaattc actggccgtc
     2101 gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca
     2161 catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa
     2221 cagttgcgca gcctgaatgg cgaatggcgc ctgatgcggt attttctcct tacgcatctg
     2281 tgcggtattt cacaccgcat atggtgcact ctcagtacaa tctgctctga tgccgcatag
     2341 ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     2401 ccggcatccg cttacagaca agctgtgacc atgtctccgg gagctgcatg tgtcagaggt
     2461 tttcaccgtc atcaccgaaa cacgcgagtt cgaaagggcc tcgtgatacg cctattttta
     2521 taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat
     2581 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     2641 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     2701 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     2761 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     2821 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     2881 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     2941 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     3001 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     3061 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     3121 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     3181 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     3241 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     3301 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     3361 gctggctggt ttattgctga taaatctgga gccggtgagc gtggttcacg cggtatcatt
     3421 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     3481 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     3541 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     3601 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     3661 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     3721 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     3781 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     3841 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     3901 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     3961 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     4021 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     4081 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     4141 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     4201 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     4261 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     4321 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     4381 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     4441 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggatga gcgcccaata
     4501 cgcaaaccgc ctctcaccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     4561 cccgactgga aagcgggcag tgagcgcaac gcaaaccgcc tctccccgcg cgttggccga
     4621 ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg
     4681 caattaatgt gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg
     4741 ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc
     4801 atgattacgc caaggtcgac ttaaccctag aaagataatc atattgtgac gtacgttaaa
     4861 gataatcatg cgtaaaattg acgcatgtgt tttatcggtc tgtatatcga ggtttattta
     4921 ttaatttgaa tagatattaa gttttattat atttacactt acatactaat aataaattca
     4981 acaaacaatt tatttatgtt tatttattta ttaaaaaaaa acaaaaactc aaaatttctt
     5041 ctataaagta acaaaacttt tagcagtgaa aaaaatgctt tatttgtgaa atttgtgatg
     5101 ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca
     5161 ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaaagc aagtaaaacc
     5221 tctacaaatg tggtatggct gattatgatc ctctggagat tcatgcgcca ggttttctgg
     5281 tcatgcacca cgttcgtggg ccctcaggaa cctccacgtc agcggtaact gtgaagccca
     5341 gtcgctcgta aaaaggaaga ttccggggag cactggtttc gaggaacgcc ggcacgccag
     5401 cccgttccgc ggcctcaaca ccaggaagaa cgacagcgct tcccaaacct ttaccttggt
     5461 gatctgggct gacaccgaca gtggcaagaa accacgcagg ctcctttggt ctatgaggag
     5521 ccagcaagcc ctccatttgc tgttgtgccg ccaacctact ccctgacagc tcggccatcc
     5581 gcggacctat ttccgcgaac actgcgccgg cttcgacgct ttccggagta gtccagaccg
     5641 caactgcggc accatcgtcc gcaacccaaa ctttaccaat gtccagaccc acgcgcgtaa
     5701 gaaacagctc ttgcaactca gttaccctct ctatgtgtct gtccggatct acggtatggc
     5761 gggtcgctgg ataatctgca aatgctgcgg caagtgttcg gacagcccgt ggtacatcgt
     5821 cacgggtagc gagcctaaca gtgggcttat attccgtcat ggggccgggg ttctcctcca
     5881 cgtcgccggc ctgcttcagc aggctgaagt tggtggcgcc gctgcccccg gggagcatgt
     5941 caaggtcaaa atcgtcaaga gcgtcagcag gcagcatatc aaggtcaaag tcgtcaaggg
     6001 catcggctgg gagcatgtct aagtcaaaat cgtcaagggc gtcggtcggc ccgccgcttt
     6061 cgcactttag ctgtttctcc aggccacata tgattagttc caggccgaaa aggaaggcag
     6121 gttcggctcc ctgccggtcg aacagctcaa ttgcttgttt cagaagtggg ggcatagaat
     6181 cggtggtagg tgtctctctt tcctcttttg ctacttgatg ctcctgttcc tccaatacgc
     6241 agcccagtgt aaagtggccc acggcggaca gagcgtacag tgcgttctcc agggagaagc
     6301 cttgctgaca caggaacgcg agctgatttt ccagggtttc gtactgtttc tctgttgggc
     6361 gggtgccgag atgcacttta gccccgtcac gatgtgagag gagagcacag cggtatgact
     6421 tggcgttgtt ccgcagaaag tcttgccatg actcgccttc cagggggcag gagtgggtat
     6481 gatgcctgtc cagcatctcg attggcaggg catcgagcag ggcccgcttg ttcttcacgt
     6541 gccagtacag ggtaggctgc tcaactccca gcttttgagc gagtttcctt gtcgtcaggc
     6601 cttcgatacc gactccattg agtaattcca gagcagagtt tatgactttg ctcttgtcca
     6661 gtctagacat cttatcgtca tcgtctttgt aatccatggt ggcggatccc gcgtcacgac
     6721 acctgtgttc tggcggcaaa cccgttgcga aaaagaacgt tcacggcgac tactgcactt
     6781 atatacggtt ctcccccacc ctcgggaaaa aggcggagcc agtacacgac atcactttcc
     6841 cagtttaccc cgcgccacct tctctaggca ccggttcaat tgccgacccc tccccccaac
     6901 ttctcgggca ctgtgggcca tgtgcgctct gccccatggt aatagccatg actaatacgt
     6961 agatgtactg ccaagtagga aagtgccata aggtcatgta ctgggcataa tgccaggcgg
     7021 gccatttacc gtcattgacg tcaatagggg gcgtacttgg catatgatac acttgatgta
     7081 ctgccaagtg ggcagtttac cgtaaatact ccacccattg acgtcaatgg aaagtgccta
     7141 ttggcgttac tatgggaaca tacgtcatta ttgacgtcaa tgggcggggg tcgttgggcg
     7201 gtcagccagg cgggccattt accgtaagtt atgtaacgcg gaactccata tatgggctat
     7261 gaactaatga ccccgtaatt gattactatt aataactagt caataatcaa tgtcataagc
     7321 tctaatcgca taatcgagct aagagcgatt aatatgctta gctagcttag ctaagctaga
     7381 tttctctatt ctcccacaat atatagcagc tattagctta gcagtccgaa tacgatcgtc
     7441 cacgatccat aagcgtaaca catttgagta gcgactgacg ggcaccggag ccactcgagt
     7501 ggaattatca cctcgagttt actccctatc agtgatagag aacgtatgaa gagtttactc
     7561 cctatcagtg atagagaacg tatgcagact ttactcccta tcagtgatag agaacgtata
     7621 aggagtttac tccctatcag tgatagagaa cgtatgacca gtttactccc tatcagtgat
     7681 agagaacgta tctacagttt actccctatc agtgatagag aacgtatatc cagtttactc
     7741 cctatcagtg atagagaacg ta
//
