LOCUS       sequence-377932-        7734 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Barcoded piggybac transposon vector with Dox-inducible expression of
            NR2F2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7734)
  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 7734)
  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-NR2F2
FEATURES             Location/Qualifiers
     source          1..7734
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     21..45
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    111..135
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      140..149
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1023..1040
                     /codon_start=1
                     /product="N-myristoylation signal from Src kinase (Pellman 
                     et al., 1985; Kaplan et al., 1988)"
                     /label=myr
                     /translation="GSSKSK"
     protein_bind    complement(1392..1416)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1418..1444
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1611..1745
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   2017..2051
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2062..2079)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2062..2078)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2071..2093)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2287..2306)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2408..2430
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2469..2487)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2555..2659
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2660..2728,2729..3520)
                     /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(2878..2897)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3691..4279
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4180..4199
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4433..4450
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4656..4677
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4692..4697,4698..4715,4716..4722)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4730..4746
                     /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     4735..4757
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4754..4770
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4754..4770
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4796..4858
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    5034..5115
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5832..5888)
                     /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(5898..6641)
                     /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(6642..6665)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6665..6674
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6694..6905)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        6906..7285
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     protein_bind    7494..7512
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7530..7548
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7566..7584
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7602..7620
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7638..7656
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7674..7692
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7710..7728
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
ORIGIN
        1 aagcttgcct atataagcag agctcgttta gtgaaccgtc agatcgcctg gagacgccat
       61 ccacgctgtt ttgacctcca tagaagacac cgggaccgat ccagcctacc acaagtttgt
      121 acaaaaaagc aggctctccg ccaccatggc aatggtagtc agcacgtggc gcgaccccca
      181 ggacgaggtg cccggctcac agggcagcca ggcctcgcag gcgccgcccg tgcccggccc
      241 gccgcccggc gccccgcaca cgccacagac gcccggccaa gggggcccag ccagcacgcc
      301 agcccagacg gcggccggtg gccagggcgg ccctggcggc ccgggtagcg acaagcagca
      361 gcagcagcaa cacatcgagt gcgtggtgtg cggagacaag tcgagcggca agcactacgg
      421 ccagttcacg tgcgagggct gcaagagctt cttcaagcgc agcgtgcgga ggaacctgag
      481 ctacacgtgc cgcgccaacc ggaactgtcc catcgaccag caccatcgca accagtgcca
      541 gtactgccgc ctcaaaaagt gcctcaaagt gggcatgaga cgggaagcgg tgcagagggg
      601 caggatgccg ccgacccagc cgacccacgg gcagttcgcg ctgaccaacg gggatcccct
      661 caactgccac tcgtacctgt ccggatatat ttccctgctg ttgcgcgcgg agccctatcc
      721 cacgtcgcgc ttcggcagcc aatgcatgca gcccaacaac atcatgggta tcgagaacat
      781 ttgcgaactg gccgcgagga tgctcttcag cgccgtcgag tgggcccgga acatcccctt
      841 cttccccgac ctgcagatca cggaccaggt ggccctgctt cgcctcacct ggagcgagct
      901 gtttgtgttg aatgcggcgc agtgctccat gcccctccac gtcgccccgc tcctggccgc
      961 cgccggcctg catgcttcgc ccatgtccgc cgaccgggtg gtcgccttta tggaccacat
     1021 acggatcttc caagagcaag tggagaagct caaggcgctg cacgttgact cagccgagta
     1081 cagctgcctc aaggccatag tcctgttcac ctcagatgcc tgtggtctct ctgatgtagc
     1141 ccatgtggaa agcttgcagg aaaagtctca gtgtgctttg gaagaatacg ttaggagcca
     1201 gtaccccaac cagccgacga gattcggaaa gcttttgctt cgcctccctt ccctccgcac
     1261 cgtctcctcc tcagtcatag agcaattgtt tttcgtccgt ttggtaggta aaacccccat
     1321 cgaaaccctc atccgggata tgttactgtc cggcagcagt tttaactggc cgtatatggc
     1381 aattcaataa cacccagctt tcttgtacaa agtggtatac ccatacgatg ttccagatta
     1441 cgcttagtaa tgagaaatac tcagaagatg tcactcacca tagtccgcag tgaaagataa
     1501 atgatcagct ctgtgagaaa ttgtcagttt tagagctaga aatagcaagt aggccactga
     1561 gcaagaacta tgaatgagat ctttagacat attgcggccg ctttctccgc gatccagaca
     1621 tgataagata cattgatgag tttggacaaa ccaaaactag aatgcagtga aaaaaatgcc
     1681 ttatttgtga aatttgtgat gctattgcct tatttgtaac cattataagc tgcaataaac
     1741 aagttatcta taacaagaaa atatatatat aataagttat cacgtaagta gaacatgaaa
     1801 taacaatata attatcgtat gagttaaatc ttaaaagtca cgtaaaagat aatcatgcgt
     1861 cattttgact cacgcggtcg ttatagttca aaatcagtga cacttaccgc attgacaagc
     1921 acgcctcacg ggagctccaa gcggcgactg agatgtccta aatgcacagc gacggattcg
     1981 cgctatttag aaagagagag caatatttca agaatgcatg cgtcaatttt acgcagacta
     2041 tctttctagg gttaagaatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc
     2101 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
     2161 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggcg
     2221 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac
     2281 tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc
     2341 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     2401 catgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acacgcgagt
     2461 tcgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
     2521 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     2581 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     2641 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     2701 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     2761 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     2821 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     2881 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     2941 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     3001 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     3061 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     3121 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     3181 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     3241 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     3301 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     3361 agccggtgag cgtggttcac gcggtatcat tgcagcactg gggccagatg gtaagccctc
     3421 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     3481 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     3541 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     3601 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     3661 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     3721 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     3781 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     3841 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     3901 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     3961 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     4021 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     4081 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     4141 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     4201 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     4261 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     4321 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     4381 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     4441 agtgagcgag gaagcggatg agcgcccaat acgcaaaccg cctctcaccg cgcgttggcc
     4501 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa
     4561 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     4621 cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag
     4681 gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga
     4741 taacaatttc acacaggaaa cagctatgac catgattacg ccaaggtcga cttaacccta
     4801 gaaagataat catattgtga cgtacgttaa agataatcat gcgtaaaatt gacgcatgtg
     4861 ttttatcggt ctgtatatcg aggtttattt attaatttga atagatatta agttttatta
     4921 tatttacact tacatactaa taataaattc aacaaacaat ttatttatgt ttatttattt
     4981 attaaaaaaa aacaaaaact caaaatttct tctataaagt aacaaaactt ttagcagtga
     5041 aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc
     5101 tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag
     5161 gtgtgggagg ttttttaaag caagtaaaac ctctacaaat gtggtatggc tgattatgat
     5221 cctctggaga ttcatgcgcc aggttttctg gtcatgcacc acgttcgtgg gccctcagga
     5281 acctccacgt cagcggtaac tgtgaagccc agtcgctcgt aaaaaggaag attccgggga
     5341 gcactggttt cgaggaacgc cggcacgcca gcccgttccg cggcctcaac accaggaaga
     5401 acgacagcgc ttcccaaacc tttaccttgg tgatctgggc tgacaccgac agtggcaaga
     5461 aaccacgcag gctcctttgg tctatgagga gccagcaagc cctccatttg ctgttgtgcc
     5521 gccaacctac tccctgacag ctcggccatc cgcggaccta tttccgcgaa cactgcgccg
     5581 gcttcgacgc tttccggagt agtccagacc gcaactgcgg caccatcgtc cgcaacccaa
     5641 actttaccaa tgtccagacc cacgcgcgta agaaacagct cttgcaactc agttaccctc
     5701 tctatgtgtc tgtccggatc tacggtatgg cgggtcgctg gataatctgc aaatgctgcg
     5761 gcaagtgttc ggacagcccg tggtacatcg tcacgggtag cgagcctaac agtgggctta
     5821 tattccgtca tggggccggg gttctcctcc acgtcgccgg cctgcttcag caggctgaag
     5881 ttggtggcgc cgctgccccc ggggagcatg tcaaggtcaa aatcgtcaag agcgtcagca
     5941 ggcagcatat caaggtcaaa gtcgtcaagg gcatcggctg ggagcatgtc taagtcaaaa
     6001 tcgtcaaggg cgtcggtcgg cccgccgctt tcgcacttta gctgtttctc caggccacat
     6061 atgattagtt ccaggccgaa aaggaaggca ggttcggctc cctgccggtc gaacagctca
     6121 attgcttgtt tcagaagtgg gggcatagaa tcggtggtag gtgtctctct ttcctctttt
     6181 gctacttgat gctcctgttc ctccaatacg cagcccagtg taaagtggcc cacggcggac
     6241 agagcgtaca gtgcgttctc cagggagaag ccttgctgac acaggaacgc gagctgattt
     6301 tccagggttt cgtactgttt ctctgttggg cgggtgccga gatgcacttt agccccgtca
     6361 cgatgtgaga ggagagcaca gcggtatgac ttggcgttgt tccgcagaaa gtcttgccat
     6421 gactcgcctt ccagggggca ggagtgggta tgatgcctgt ccagcatctc gattggcagg
     6481 gcatcgagca gggcccgctt gttcttcacg tgccagtaca gggtaggctg ctcaactccc
     6541 agcttttgag cgagtttcct tgtcgtcagg ccttcgatac cgactccatt gagtaattcc
     6601 agagcagagt ttatgacttt gctcttgtcc agtctagaca tcttatcgtc atcgtctttg
     6661 taatccatgg tggcggatcc cgcgtcacga cacctgtgtt ctggcggcaa acccgttgcg
     6721 aaaaagaacg ttcacggcga ctactgcact tatatacggt tctcccccac cctcgggaaa
     6781 aaggcggagc cagtacacga catcactttc ccagtttacc ccgcgccacc ttctctaggc
     6841 accggttcaa ttgccgaccc ctccccccaa cttctcgggc actgtgggcc atgtgcgctc
     6901 tgccccatgg taatagccat gactaatacg tagatgtact gccaagtagg aaagtgccat
     6961 aaggtcatgt actgggcata atgccaggcg ggccatttac cgtcattgac gtcaataggg
     7021 ggcgtacttg gcatatgata cacttgatgt actgccaagt gggcagttta ccgtaaatac
     7081 tccacccatt gacgtcaatg gaaagtgcct attggcgtta ctatgggaac atacgtcatt
     7141 attgacgtca atgggcgggg gtcgttgggc ggtcagccag gcgggccatt taccgtaagt
     7201 tatgtaacgc ggaactccat atatgggcta tgaactaatg accccgtaat tgattactat
     7261 taataactag tcaataatca atgtcataag ctctaatcgc ataatcgagc taagagcgat
     7321 taatatgctt agctagctta gctaagctag atttctctat tctcccacaa tatatagcag
     7381 ctattagctt agcagtccga atacgatcgt ccacgatcca taagcgtaac acatttgagt
     7441 agcgactgac gggcaccgga gccactcgag tggaattatc acctcgagtt tactccctat
     7501 cagtgataga gaacgtatga agagtttact ccctatcagt gatagagaac gtatgcagac
     7561 tttactccct atcagtgata gagaacgtat aaggagttta ctccctatca gtgatagaga
     7621 acgtatgacc agtttactcc ctatcagtga tagagaacgt atctacagtt tactccctat
     7681 cagtgataga gaacgtatat ccagtttact ccctatcagt gatagagaac gtat
//
