LOCUS       sequence-439876-        7108 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac transposon plasmid for doxycycline inducible expression of 
            NFYB.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7108)
  AUTHORS   Pierson Smela M, Adams J, Ma C, Breimann L, Widocki U, Shioda T, 
            Church GM
  TITLE     Induction of Meiosis from Human Pluripotent Stem Cells
  JOURNAL   bioRxiv 2024.05.31.596483
REFERENCE   2  (bases 1 to 7108)
  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: "bioRxiv 
            2024.05.31.596483"
COMMENT     Sequence Label: pPB-cT3G-cERP2-NFYB
FEATURES             Location/Qualifiers
     source          1..7108
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    17..35
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    53..71
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    89..107
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    125..143
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    161..179
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    197..215
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    233..251
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     278..302
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    368..392
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1023..1047)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1049..1075
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1242..1376
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   1648..1682
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1693..1710)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1693..1709)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1702..1724)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1918..1937)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2039..2061
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2100..2118)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2186..2290
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2291..2359,2360..3151)
                     /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(2509..2528)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3322..3910
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3811..3830
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4064..4081
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4287..4308
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4323..4328,4329..4346,4347..4353)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4361..4377
                     /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     4366..4388
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4385..4401
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4385..4401
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4427..4489
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    4665..4746
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5463..5519)
                     /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(5529..6272)
                     /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(6273..6296)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6296..6305
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6325..6536)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        6537..6916
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 atcacctcga gtttactccc tatcagtgat agagaacgta tgaagagttt actccctatc
       61 agtgatagag aacgtatgca gactttactc cctatcagtg atagagaacg tataaggagt
      121 ttactcccta tcagtgatag agaacgtatg accagtttac tccctatcag tgatagagaa
      181 cgtatctaca gtttactccc tatcagtgat agagaacgta tatccagttt actccctatc
      241 agtgatagag aacgtataag cttgcctata taagcagagc tcgtttagtg aaccgtcaga
      301 tcgcctggag acgccatcca cgctgttttg acctccatag aagacaccgg gaccgatcca
      361 gcctaccaca agtttgtaca aaaaagcagg ctgccaccat gacaatggat ggtgacagtt
      421 ctacaacaga tgcttctcaa ctaggaatct ctgcagacta tattggagga agtcattatg
      481 ttatacagcc tcatgatgat actgaggaca gcatgaatga tcatgaagac acaaatggtt
      541 caaaagaaag tttcagagaa caagatatat atcttccaat agcaaacgtg gctaggataa
      601 tgaaaaatgc catacctcaa acgggaaaga ttgcaaaaga tgccaaagaa tgtgttcaag
      661 aatgtgtaag tgagttcatc agttttataa catctgaagc aagtgaaagg tgccatcaag
      721 agaaacggaa aacaatcaat ggagaagata ttctctttgc tatgtctact ttaggctttg
      781 acagttatgt ggaacctctg aaattatacc ttcagaaatt cagagaggct atgaaaggag
      841 aaaagggaat tggtggagca gtcacagcta cagatggact aagtgaagag cttacagagg
      901 aggcatttac taaccagtta ccagctggct taataaccac agacggtcaa caacaaaatg
      961 ttatggttta cacaacatca tatcaacaga tttctggtgt tcagcaaatt cagttttcat
     1021 gaacccagct ttcttgtaca aagtggtata cccatacgat gttccagatt acgcttagta
     1081 atgagaaata ctcagaagat gtcactcacc atagtccgca gtgaaagata aatgatcgac
     1141 tgtaactgcg attgcaagtt ttagagctag aaatagcaag taggccactg agcaagaact
     1201 atgaatgaga tctttagaca tattgcggcc gctttctccg cgatccagac atgataagat
     1261 acattgatga gtttggacaa accaaaacta gaatgcagtg aaaaaaatgc cttatttgtg
     1321 aaatttgtga tgctattgcc ttatttgtaa ccattataag ctgcaataaa caagttatct
     1381 ataacaagaa aatatatata taataagtta tcacgtaagt agaacatgaa ataacaatat
     1441 aattatcgta tgagttaaat cttaaaagtc acgtaaaaga taatcatgcg tcattttgac
     1501 tcacgcggtc gttatagttc aaaatcagtg acacttaccg cattgacaag cacgcctcac
     1561 gggagctcca agcggcgact gagatgtcct aaatgcacag cgacggattc gcgctattta
     1621 gaaagagaga gcaatatttc aagaatgcat gcgtcaattt tacgcagact atctttctag
     1681 ggttaagaat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac
     1741 ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc
     1801 ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg
     1861 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatggtgca ctctcagtac
     1921 aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc
     1981 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccatgtctcc
     2041 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacacgcgag ttcgaaaggg
     2101 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc
     2161 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     2221 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     2281 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     2341 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     2401 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     2461 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     2521 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     2581 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     2641 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     2701 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     2761 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     2821 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     2881 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     2941 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     3001 gcgtggttca cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     3061 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     3121 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     3181 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     3241 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     3301 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     3361 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     3421 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     3481 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     3541 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     3601 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     3661 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     3721 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     3781 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     3841 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     3901 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     3961 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     4021 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     4081 ggaagcggat gagcgcccaa tacgcaaacc gcctctcacc gcgcgttggc cgattcatta
     4141 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaaaccg
     4201 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
     4261 aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag
     4321 gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt
     4381 cacacaggaa acagctatga ccatgattac gccaaggtcg acttaaccct agaaagataa
     4441 tcatattgtg acgtacgtta aagataatca tgcgtaaaat tgacgcatgt gttttatcgg
     4501 tctgtatatc gaggtttatt tattaatttg aatagatatt aagttttatt atatttacac
     4561 ttacatacta ataataaatt caacaaacaa tttatttatg tttatttatt tattaaaaaa
     4621 aaacaaaaac tcaaaatttc ttctataaag taacaaaact tttagcagtg aaaaaaatgc
     4681 tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa
     4741 caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag
     4801 gttttttaaa gcaagtaaaa cctctacaaa tgtggtatgg ctgattatga tcctctggag
     4861 attcatgcgc caggttttct ggtcatgcac cacgttcgtg ggccctcagg aacctccacg
     4921 tcagcggtaa ctgtgaagcc cagtcgctcg taaaaaggaa gattccgggg agcactggtt
     4981 tcgaggaacg ccggcacgcc agcccgttcc gcggcctcaa caccaggaag aacgacagcg
     5041 cttcccaaac ctttaccttg gtgatctggg ctgacaccga cagtggcaag aaaccacgca
     5101 ggctcctttg gtctatgagg agccagcaag ccctccattt gctgttgtgc cgccaaccta
     5161 ctccctgaca gctcggccat ccgcggacct atttccgcga acactgcgcc ggcttcgacg
     5221 ctttccggag tagtccagac cgcaactgcg gcaccatcgt ccgcaaccca aactttacca
     5281 atgtccagac ccacgcgcgt aagaaacagc tcttgcaact cagttaccct ctctatgtgt
     5341 ctgtccggat ctacggtatg gcgggtcgct ggataatctg caaatgctgc ggcaagtgtt
     5401 cggacagccc gtggtacatc gtcacgggta gcgagcctaa cagtgggctt atattccgtc
     5461 atggggccgg ggttctcctc cacgtcgccg gcctgcttca gcaggctgaa gttggtggcg
     5521 ccgctgcccc cggggagcat gtcaaggtca aaatcgtcaa gagcgtcagc aggcagcata
     5581 tcaaggtcaa agtcgtcaag ggcatcggct gggagcatgt ctaagtcaaa atcgtcaagg
     5641 gcgtcggtcg gcccgccgct ttcgcacttt agctgtttct ccaggccaca tatgattagt
     5701 tccaggccga aaaggaaggc aggttcggct ccctgccggt cgaacagctc aattgcttgt
     5761 ttcagaagtg ggggcataga atcggtggta ggtgtctctc tttcctcttt tgctacttga
     5821 tgctcctgtt cctccaatac gcagcccagt gtaaagtggc ccacggcgga cagagcgtac
     5881 agtgcgttct ccagggagaa gccttgctga cacaggaacg cgagctgatt ttccagggtt
     5941 tcgtactgtt tctctgttgg gcgggtgccg agatgcactt tagccccgtc acgatgtgag
     6001 aggagagcac agcggtatga cttggcgttg ttccgcagaa agtcttgcca tgactcgcct
     6061 tccagggggc aggagtgggt atgatgcctg tccagcatct cgattggcag ggcatcgagc
     6121 agggcccgct tgttcttcac gtgccagtac agggtaggct gctcaactcc cagcttttga
     6181 gcgagtttcc ttgtcgtcag gccttcgata ccgactccat tgagtaattc cagagcagag
     6241 tttatgactt tgctcttgtc cagtctagac atcttatcgt catcgtcttt gtaatccatg
     6301 gtggcggatc ccgcgtcacg acacctgtgt tctggcggca aacccgttgc gaaaaagaac
     6361 gttcacggcg actactgcac ttatatacgg ttctccccca ccctcgggaa aaaggcggag
     6421 ccagtacacg acatcacttt cccagtttac cccgcgccac cttctctagg caccggttca
     6481 attgccgacc cctcccccca acttctcggg cactgtgggc catgtgcgct ctgccccatg
     6541 gtaatagcca tgactaatac gtagatgtac tgccaagtag gaaagtgcca taaggtcatg
     6601 tactgggcat aatgccaggc gggccattta ccgtcattga cgtcaatagg gggcgtactt
     6661 ggcatatgat acacttgatg tactgccaag tgggcagttt accgtaaata ctccacccat
     6721 tgacgtcaat ggaaagtgcc tattggcgtt actatgggaa catacgtcat tattgacgtc
     6781 aatgggcggg ggtcgttggg cggtcagcca ggcgggccat ttaccgtaag ttatgtaacg
     6841 cggaactcca tatatgggct atgaactaat gaccccgtaa ttgattacta ttaataacta
     6901 gtcaataatc aatgtcataa gctctaatcg cataatcgag ctaagagcga ttaatatgct
     6961 tagctagctt agctaagcta gatttctcta ttctcccaca atatatagca gctattagct
     7021 tagcagtccg aatacgatcg tccacgatcc ataagcgtaa cacatttgag tagcgactga
     7081 cgggcaccgg agccactcga gtggaatt
//
