LOCUS       sequence-442469-        8400 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac transposon plasmid for doxycycline inducible expression of 
            MEIOSIN.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8400)
  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 8400)
  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-MEIOSIN
FEATURES             Location/Qualifiers
     source          1..8400
                     /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(2316..2340)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2342..2368
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    2534..2668
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   2940..2974
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2985..3002)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2985..3001)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2994..3016)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3210..3229)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3331..3353
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3392..3410)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3478..3582
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3583..3651,3652..4443)
                     /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(3801..3820)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4614..5202
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5103..5122
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5356..5373
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5579..5600
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5615..5620,5621..5638,5639..5645)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5653..5669
                     /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     5658..5680
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5677..5693
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5677..5693
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   5719..5781
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    5957..6038
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(6755..6811)
                     /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(6821..7564)
                     /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(7565..7588)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      7588..7597
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(7617..7828)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        7829..8208
                     /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 gtttggttcc agcagatact
      421 tgggttcctc tgaacagccc agagctaatt cactgggtcc cagtgacagg accttggttt
      481 tgtgctccct agtggaaggg gaagataagg tcaacccctc cgaaccacat ggactgagaa
      541 tggaggagaa atggttgctc aaaggaaaac tgaggaatca gaggaaccaa aacaagctcc
      601 tgtccccaaa caagaagcag aggaaaaacc acactagcaa gctgcaagag ttggcactgc
      661 tgctgcccat agccctgaag acggggacca agaagctcac aaagaaggag attctggtgc
      721 atgtcctgca gtacattcag tacctccaga gaaacatcga tgccgccaag gccttgttca
      781 aatgccacat caccactggg gaaggtggac ttgcggggct gggtcagaaa ccagcctggg
      841 gcccagccag gcggaggaga cactctaccc cctccagctc cccaagctct cagaagtcct
      901 gtctccaggg ggcgtgccag aagcctcgga agaagaagct gacccaggca tcagaaagcc
      961 agactcggac cccgaagcct cgccgctctc tggccctgaa caagcctgag aagctggtgg
     1021 ccccatcccc agaccagaaa ggaagtggca caggggggac cactacccct ccaaggtgcc
     1081 ctgactcctg cggtcacccg aggcctgcat catcctcacc tccaggtgac agaaaaggag
     1141 gacagtccca gctgacgctg ttggatctgg ccgaggacac catccactgt gacatctcaa
     1201 gctgctggtg ccagggcagt gtccaggatg acgcaccttt ccctgcgctc ctggctcagg
     1261 aagatgtggc gaggatccat tttctcaaca agacccagcc ccatcccagg cagaagctgg
     1321 tgttctatga ttccagcgag gatgtggaca aagggtccct agacgctgac ccttggctcc
     1381 ctgcctggac cccagagaac agcccccaag ggagcccact gttcctgggg cctccccaga
     1441 ttgatgtctg gagtggaaca ggccacccaa gtgagatcct cgggctcagc cctagccttt
     1501 tcagctcccc agggaaactg ctgccagacg agatcttgga ggatgacatg gagtacctga
     1561 cccaagcggc tttctttgaa gaagtgtgct tagatctgga gtcttcacct tcagcctaca
     1621 cgcaggaggc tccacaggaa aaggacacag cctccaaggc ccccaaagac cctcctgagt
     1681 cccacagcct gcaccggtcc tcagtctcgc tggaccactg ctacctctcg ctgagcggga
     1741 acagcaaggc gccatcatca tcttctagct ctagctcaag cagttcatca tcagaggaca
     1801 gcgactcgga gcccctgtgg aagcagcgag aggatatgca ggccaaccct gtgggcacgc
     1861 cgggctccag cgaagaggac gaagacacca catggacccc cacccggctg gcctcacccc
     1921 tgctggcagc tgagaaaaag gccacgaagg gccaagtagc cagggcccct gtgaagccca
     1981 aggagaagaa gaaaggcccc tgcccacccc agatgaagaa gaagtgtgtc aacggcttca
     2041 tcatgttctg caggatgaac cggaagcagt acatccgatc ctgccctgga accgcttcca
     2101 cagctgccac caaggagctg gcccagctgt ggcgggtgat gacccagcag gagcggaggc
     2161 catactgcac caaggctcgc aggttcagcc gccagcacaa ccgcatcgtg aagcaggacg
     2221 gctccagcag cgaggctgag gactgggaga cgccaaagcc cttctaccag ctgctggccg
     2281 agaaggcctt gccgctgccc ccgcacctcc agtgaaccca gctttcttgt acaaagtggt
     2341 atacccatac gatgttccag attacgctta gtaatgagaa atactcagaa gatgtcactc
     2401 accatagtcc gcagtgaaag ataaatgatc cacactcatt gaaacttgag ttttagagct
     2461 agaaatagca agtaggccac tgagcaagaa ctatgaatga gatctttaga catattgcgg
     2521 ccgctttctc cgcgatccag acatgataag atacattgat gagtttggac aaaccaaaac
     2581 tagaatgcag tgaaaaaaat gccttatttg tgaaatttgt gatgctattg ccttatttgt
     2641 aaccattata agctgcaata aacaagttat ctataacaag aaaatatata tataataagt
     2701 tatcacgtaa gtagaacatg aaataacaat ataattatcg tatgagttaa atcttaaaag
     2761 tcacgtaaaa gataatcatg cgtcattttg actcacgcgg tcgttatagt tcaaaatcag
     2821 tgacacttac cgcattgaca agcacgcctc acgggagctc caagcggcga ctgagatgtc
     2881 ctaaatgcac agcgacggat tcgcgctatt tagaaagaga gagcaatatt tcaagaatgc
     2941 atgcgtcaat tttacgcaga ctatctttct agggttaaga attcactggc cgtcgtttta
     3001 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc
     3061 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg
     3121 cgcagcctga atggcgaatg gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     3181 atttcacacc gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     3241 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     3301 tccgcttaca gacaagctgt gaccatgtct ccgggagctg catgtgtcag aggttttcac
     3361 cgtcatcacc gaaacacgcg agttcgaaag ggcctcgtga tacgcctatt tttataggtt
     3421 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
     3481 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     3541 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     3601 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     3661 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     3721 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     3781 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     3841 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     3901 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     3961 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     4021 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     4081 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     4141 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     4201 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     4261 tggtttattg ctgataaatc tggagccggt gagcgtggtt cacgcggtat cattgcagca
     4321 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     4381 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     4441 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     4501 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     4561 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     4621 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     4681 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     4741 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     4801 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     4861 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     4921 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     4981 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     5041 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     5101 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     5161 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     5221 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     5281 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     5341 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg atgagcgccc aatacgcaaa
     5401 ccgcctctca ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
     5461 tggaaagcgg gcagtgagcg caacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
     5521 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
     5581 atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta
     5641 tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt
     5701 acgccaaggt cgacttaacc ctagaaagat aatcatattg tgacgtacgt taaagataat
     5761 catgcgtaaa attgacgcat gtgttttatc ggtctgtata tcgaggttta tttattaatt
     5821 tgaatagata ttaagtttta ttatatttac acttacatac taataataaa ttcaacaaac
     5881 aatttattta tgtttattta tttattaaaa aaaaacaaaa actcaaaatt tcttctataa
     5941 agtaacaaaa cttttagcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg
     6001 ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt
     6061 ttatgtttca ggttcagggg gaggtgtggg aggtttttta aagcaagtaa aacctctaca
     6121 aatgtggtat ggctgattat gatcctctgg agattcatgc gccaggtttt ctggtcatgc
     6181 accacgttcg tgggccctca ggaacctcca cgtcagcggt aactgtgaag cccagtcgct
     6241 cgtaaaaagg aagattccgg ggagcactgg tttcgaggaa cgccggcacg ccagcccgtt
     6301 ccgcggcctc aacaccagga agaacgacag cgcttcccaa acctttacct tggtgatctg
     6361 ggctgacacc gacagtggca agaaaccacg caggctcctt tggtctatga ggagccagca
     6421 agccctccat ttgctgttgt gccgccaacc tactccctga cagctcggcc atccgcggac
     6481 ctatttccgc gaacactgcg ccggcttcga cgctttccgg agtagtccag accgcaactg
     6541 cggcaccatc gtccgcaacc caaactttac caatgtccag acccacgcgc gtaagaaaca
     6601 gctcttgcaa ctcagttacc ctctctatgt gtctgtccgg atctacggta tggcgggtcg
     6661 ctggataatc tgcaaatgct gcggcaagtg ttcggacagc ccgtggtaca tcgtcacggg
     6721 tagcgagcct aacagtgggc ttatattccg tcatggggcc ggggttctcc tccacgtcgc
     6781 cggcctgctt cagcaggctg aagttggtgg cgccgctgcc cccggggagc atgtcaaggt
     6841 caaaatcgtc aagagcgtca gcaggcagca tatcaaggtc aaagtcgtca agggcatcgg
     6901 ctgggagcat gtctaagtca aaatcgtcaa gggcgtcggt cggcccgccg ctttcgcact
     6961 ttagctgttt ctccaggcca catatgatta gttccaggcc gaaaaggaag gcaggttcgg
     7021 ctccctgccg gtcgaacagc tcaattgctt gtttcagaag tgggggcata gaatcggtgg
     7081 taggtgtctc tctttcctct tttgctactt gatgctcctg ttcctccaat acgcagccca
     7141 gtgtaaagtg gcccacggcg gacagagcgt acagtgcgtt ctccagggag aagccttgct
     7201 gacacaggaa cgcgagctga ttttccaggg tttcgtactg tttctctgtt gggcgggtgc
     7261 cgagatgcac tttagccccg tcacgatgtg agaggagagc acagcggtat gacttggcgt
     7321 tgttccgcag aaagtcttgc catgactcgc cttccagggg gcaggagtgg gtatgatgcc
     7381 tgtccagcat ctcgattggc agggcatcga gcagggcccg cttgttcttc acgtgccagt
     7441 acagggtagg ctgctcaact cccagctttt gagcgagttt ccttgtcgtc aggccttcga
     7501 taccgactcc attgagtaat tccagagcag agtttatgac tttgctcttg tccagtctag
     7561 acatcttatc gtcatcgtct ttgtaatcca tggtggcgga tcccgcgtca cgacacctgt
     7621 gttctggcgg caaacccgtt gcgaaaaaga acgttcacgg cgactactgc acttatatac
     7681 ggttctcccc caccctcggg aaaaaggcgg agccagtaca cgacatcact ttcccagttt
     7741 accccgcgcc accttctcta ggcaccggtt caattgccga cccctccccc caacttctcg
     7801 ggcactgtgg gccatgtgcg ctctgcccca tggtaatagc catgactaat acgtagatgt
     7861 actgccaagt aggaaagtgc cataaggtca tgtactgggc ataatgccag gcgggccatt
     7921 taccgtcatt gacgtcaata gggggcgtac ttggcatatg atacacttga tgtactgcca
     7981 agtgggcagt ttaccgtaaa tactccaccc attgacgtca atggaaagtg cctattggcg
     8041 ttactatggg aacatacgtc attattgacg tcaatgggcg ggggtcgttg ggcggtcagc
     8101 caggcgggcc atttaccgta agttatgtaa cgcggaactc catatatggg ctatgaacta
     8161 atgaccccgt aattgattac tattaataac tagtcaataa tcaatgtcat aagctctaat
     8221 cgcataatcg agctaagagc gattaatatg cttagctagc ttagctaagc tagatttctc
     8281 tattctccca caatatatag cagctattag cttagcagtc cgaatacgat cgtccacgat
     8341 ccataagcgt aacacatttg agtagcgact gacgggcacc ggagccactc gagtggaatt
//
