LOCUS       sequence-377943-        7270 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Barcoded piggybac transposon vector with Dox-inducible expression of
            EMX2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7270)
  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 7270)
  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-EMX2
FEATURES             Location/Qualifiers
     source          1..7270
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        225..604
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        605..816
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      836..845
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             845..868
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             869..1612
                     /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             1622..1678
                     /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"
     polyA_signal    2395..2476
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   2652..2714
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2740..2756)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2740..2756)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2753..2775)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2764..2780
                     /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)."
     promoter        complement(join(2788..2794,2795..2812,2813..2818))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2833..2854
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3060..3077)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3231..3819)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3311..3330)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3990..4781,4782..4850))
                     /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     4613..4632
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4851..4955)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5023..5041
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5080..5102)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5204..5223
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5417..5439
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5431..5448
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5432..5448
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   5459..5493
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    5765..5899
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(6066..6092)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    6094..6118
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(6875..6899)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(6965..6989)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    7016..7034
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7052..7070
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7088..7106
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7124..7142
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7160..7178
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7196..7214
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7232..7250
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    join(7268..7270,1..16)
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
ORIGIN
        1 ctatcactga tagggagtaa actcgaggtg ataattccac tcgagtggct ccggtgcccg
       61 tcagtcgcta ctcaaatgtg ttacgcttat ggatcgtgga cgatcgtatt cggactgcta
      121 agctaatagc tgctatatat tgtgggagaa tagagaaatc tagcttagct aagctagcta
      181 agcatattaa tcgctcttag ctcgattatg cgattagagc ttatgacatt gattattgac
      241 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
      301 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      361 gacgtcaata atgacgtatg ttcccatagt aacgccaata ggcactttcc attgacgtca
      421 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      481 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      541 catgacctta tggcactttc ctacttggca gtacatctac gtattagtca tggctattac
      601 catggggcag agcgcacatg gcccacagtg cccgagaagt tggggggagg ggtcggcaat
      661 tgaaccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg
      721 ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac
      781 gttctttttc gcaacgggtt tgccgccaga acacaggtgt cgtgacgcgg gatccgccac
      841 catggattac aaagacgatg acgataagat gtctagactg gacaagagca aagtcataaa
      901 ctctgctctg gaattactca atggagtcgg tatcgaaggc ctgacgacaa ggaaactcgc
      961 tcaaaagctg ggagttgagc agcctaccct gtactggcac gtgaagaaca agcgggccct
     1021 gctcgatgcc ctgccaatcg agatgctgga caggcatcat acccactcct gccccctgga
     1081 aggcgagtca tggcaagact ttctgcggaa caacgccaag tcataccgct gtgctctcct
     1141 ctcacatcgt gacggggcta aagtgcatct cggcacccgc ccaacagaga aacagtacga
     1201 aaccctggaa aatcagctcg cgttcctgtg tcagcaaggc ttctccctgg agaacgcact
     1261 gtacgctctg tccgccgtgg gccactttac actgggctgc gtattggagg aacaggagca
     1321 tcaagtagca aaagaggaaa gagagacacc taccaccgat tctatgcccc cacttctgaa
     1381 acaagcaatt gagctgttcg accggcaggg agccgaacct gccttccttt tcggcctgga
     1441 actaatcata tgtggcctgg agaaacagct aaagtgcgaa agcggcgggc cgaccgacgc
     1501 ccttgacgat tttgacttag acatgctccc agccgatgcc cttgacgact ttgaccttga
     1561 tatgctgcct gctgacgctc ttgacgattt tgaccttgac atgctccccg ggggcagcgg
     1621 cgccaccaac ttcagcctgc tgaagcaggc cggcgacgtg gaggagaacc ccggccccat
     1681 gacggaatat aagcccactg ttaggctcgc tacccgtgac gatgtaccac gggctgtccg
     1741 aacacttgcc gcagcatttg cagattatcc agcgacccgc cataccgtag atccggacag
     1801 acacatagag agggtaactg agttgcaaga gctgtttctt acgcgcgtgg gtctggacat
     1861 tggtaaagtt tgggttgcgg acgatggtgc cgcagttgcg gtctggacta ctccggaaag
     1921 cgtcgaagcc ggcgcagtgt tcgcggaaat aggtccgcgg atggccgagc tgtcagggag
     1981 taggttggcg gcacaacagc aaatggaggg cttgctggct cctcatagac caaaggagcc
     2041 tgcgtggttt cttgccactg tcggtgtcag cccagatcac caaggtaaag gtttgggaag
     2101 cgctgtcgtt cttcctggtg ttgaggccgc ggaacgggct ggcgtgccgg cgttcctcga
     2161 aaccagtgct ccccggaatc ttccttttta cgagcgactg ggcttcacag ttaccgctga
     2221 cgtggaggtt cctgagggcc cacgaacgtg gtgcatgacc agaaaacctg gcgcatgaat
     2281 ctccagagga tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac
     2341 ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg
     2401 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     2461 gcattttttt cactgctaaa agttttgtta ctttatagaa gaaattttga gtttttgttt
     2521 ttttttaata aataaataaa cataaataaa ttgtttgttg aatttattat tagtatgtaa
     2581 gtgtaaatat aataaaactt aatatctatt caaattaata aataaacctc gatatacaga
     2641 ccgataaaac acatgcgtca attttacgca tgattatctt taacgtacgt cacaatatga
     2701 ttatctttct agggttaagt cgaccttggc gtaatcatgg tcatagctgt ttcctgtgtg
     2761 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     2821 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     2881 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     2941 cggtttgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     3001 taatgaatcg gccaacgcgc ggtgagaggc ggtttgcgta ttgggcgctc atccgcttcc
     3061 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     3121 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     3181 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     3241 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     3301 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     3361 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     3421 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     3481 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     3541 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     3601 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     3661 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     3721 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     3781 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     3841 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     3901 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     3961 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     4021 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     4081 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg tgaaccacgc
     4141 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     4201 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     4261 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     4321 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     4381 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     4441 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     4501 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     4561 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     4621 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     4681 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     4741 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     4801 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     4861 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     4921 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     4981 gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg
     5041 ccctttcgaa ctcgcgtgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc
     5101 ggagacatgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc
     5161 gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact atgcggcatc agagcagatt
     5221 gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac
     5281 cgcatcaggc gccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg
     5341 gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg
     5401 gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga attcttaacc
     5461 ctagaaagat agtctgcgta aaattgacgc atgcattctt gaaatattgc tctctctttc
     5521 taaatagcgc gaatccgtcg ctgtgcattt aggacatctc agtcgccgct tggagctccc
     5581 gtgaggcgtg cttgtcaatg cggtaagtgt cactgatttt gaactataac gaccgcgtga
     5641 gtcaaaatga cgcatgatta tcttttacgt gacttttaag atttaactca tacgataatt
     5701 atattgttat ttcatgttct acttacgtga taacttatta tatatatatt ttcttgttat
     5761 agataacttg tttattgcag cttataatgg ttacaaataa ggcaatagca tcacaaattt
     5821 cacaaataag gcattttttt cactgcattc tagttttggt ttgtccaaac tcatcaatgt
     5881 atcttatcat gtctggatcg cggagaaagc ggccgcaata tgtctaaaga tctcattcat
     5941 agttcttgct cagtggccta cttgctattt ctagctctaa aactgaaagc tcaatacggc
     6001 aaggatcatt tatctttcac tgcggactat ggtgagtgac atcttctgag tatttctcat
     6061 tactaagcgt aatctggaac atcgtatggg tataccactt tgtacaagaa agttgggtaa
     6121 tcatctgagg tcacgtctat ttcctccgga ctcgcctgct tggtggcgat tctccaccgg
     6181 ttaatatggt gcgtcccttt tttcttttgt tgcgaatctg agccttcttc ctccagcttc
     6241 tgccttttga actttgttct tcggttctga aaccatactt ttacctgagt ttccgtgagg
     6301 ctgaggctgt gtgccagctg cttcctttcg gcgcccacca cgtagtgatt cttctcaaag
     6361 gcgtgttcca gccttagaag ctgggacggg gagaaggcgg ttcggatccg cttgggcttt
     6421 cgggccagcg cgttgtgcaa aaggaaactc tcggggctag tgtcgttccc ttggaaacga
     6481 tgacccagat atcggtagcg gtggatgagc caggggtaga aggtagacgg atcccgctgc
     6541 tgtgaggcga ataggggatg tggcgagtgt gatgagggta ggggatgagc ggcaagagca
     6601 tgaggcggag gcactgggtg tactggtact gcggggtttg gaggatgcga aaccgcttcg
     6661 gcgaacacta aatccgggtt tgagtatacg cccctaccgg cggctgcagc ggctgctgag
     6721 tggaagccgt tgaggaacgg atttatggga ctggagttag cgtagctgag tgctgctgga
     6781 cgaatggggt cctcggagcg cgatgctggc aggggactgt cttttgccac cagcgactct
     6841 atagtgaagc agcgcttggg tgctggctgg aacatgccaa cttttttgta caaacttgtg
     6901 gtaggctgga tcggtcccgg tgtcttctat ggaggtcaaa acagcgtgga tggcgtctcc
     6961 aggcgatctg acggttcact aaacgagctc tgcttatata ggcaagctta tacgttctct
     7021 atcactgata gggagtaaac tggatatacg ttctctatca ctgataggga gtaaactgta
     7081 gatacgttct ctatcactga tagggagtaa actggtcata cgttctctat cactgatagg
     7141 gagtaaactc cttatacgtt ctctatcact gatagggagt aaastcykya tacgttctct
     7201 atcactgata gggagtaaas tctgcatacg ttctctatca ctgataggga gtaaactctt
     7261 catacgttct
//
