LOCUS       sequence-442478-        7660 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac transposon plasmid for doxycycline inducible expression of 
            PAX5.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7660)
  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 7660)
  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-PAX5
FEATURES             Location/Qualifiers
     source          1..7660
                     /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(1575..1599)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1601..1627
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1794..1928
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   2200..2234
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2245..2262)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2245..2261)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2254..2276)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2470..2489)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2591..2613
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2652..2670)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2738..2842
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2843..2911,2912..3703)
                     /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(3061..3080)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3874..4462
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4363..4382
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4616..4633
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4839..4860
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4875..4880,4881..4898,4899..4905)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4913..4929
                     /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     4918..4940
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4937..4953
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4937..4953
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4979..5041
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    5217..5298
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(6015..6071)
                     /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(6081..6824)
                     /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(6825..6848)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6848..6857
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6877..7088)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        7089..7468
                     /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 ctgcaccatg gatttagaga aaaattatcc
      421 gactcctcgg accagcagga caggacatgg aggagtgaat cagcttgggg gggtttttgt
      481 gaatggacgg ccactcccgg atgtagtccg ccagaggata gtggaacttg ctcatcaagg
      541 tgtcaggccc tgcgacatct ccaggcagct tcgggtcagc catggttgtg tcagcaaaat
      601 tcttggcagg tattatgaga caggaagcat caagcctggg gtaattggag gatccaaacc
      661 aaaggtcgcc acacccaaag tggtggaaaa aatcgctgaa tataaacgcc aaaatcccac
      721 catgtttgcc tgggagatca gggaccggct gctggcagag cgggtgtgtg acaatgacac
      781 cgtgcctagc gtcagttcca tcaacaggat catccggaca aaagtacagc agccacccaa
      841 ccaaccagtc ccagcttcca gtcacagcat agtgtccact ggctccgtga cgcaggtgtc
      901 ctcggtgagc acggattcgg ccggctcgtc gtactccatc agcggcatcc tgggcatcac
      961 gtcccccagc gccgacacca acaagcgcaa gagagacgaa ggtattcagg agtctccggt
     1021 gccgaacggc cactcgcttc cgggcagaga cttcctccgg aagcagatgc ggggagactt
     1081 gttcacacag cagcagctgg aggtgctgga ccgcgtgttt gagaggcagc actactcaga
     1141 catcttcacc accacagagc ccatcaagcc cgagcagacc acagagtatt cagccatggc
     1201 ctcgctggct ggtgggctgg acgacatgaa ggccaatctg gccagcccca cccctgctga
     1261 catcgggagc agtgtgccag gcccgcagtc ctaccccatt gtgacaggcc gtgacttggc
     1321 gagcacgacc ctccccgggt accctccaca cgtccccccc gctggacagg gcagctactc
     1381 agcaccgacg ctgacaggga tggtgcctgg gagtgagttt tccgggagtc cctacagcca
     1441 ccctcagtat tcctcgtaca acgactcctg gaggttcccc aacccggggc tgcttggctc
     1501 cccctactat tatagcgctg ccgcccgagg agccgcccca cctgcagccg ccactgccta
     1561 tgaccgtcac tgagacccag ctttcttgta caaagtggta tacccatacg atgttccaga
     1621 ttacgcttag taatgagaaa tactcagaag atgtcactca ccatagtccg cagtgaaaga
     1681 taaatgatcg ctgcctctca gatttctgag ttttagagct agaaatagca agtaggccac
     1741 tgagcaagaa ctatgaatga gatctttaga catattgcgg ccgctttctc cgcgatccag
     1801 acatgataag atacattgat gagtttggac aaaccaaaac tagaatgcag tgaaaaaaat
     1861 gccttatttg tgaaatttgt gatgctattg ccttatttgt aaccattata agctgcaata
     1921 aacaagttat ctataacaag aaaatatata tataataagt tatcacgtaa gtagaacatg
     1981 aaataacaat ataattatcg tatgagttaa atcttaaaag tcacgtaaaa gataatcatg
     2041 cgtcattttg actcacgcgg tcgttatagt tcaaaatcag tgacacttac cgcattgaca
     2101 agcacgcctc acgggagctc caagcggcga ctgagatgtc ctaaatgcac agcgacggat
     2161 tcgcgctatt tagaaagaga gagcaatatt tcaagaatgc atgcgtcaat tttacgcaga
     2221 ctatctttct agggttaaga attcactggc cgtcgtttta caacgtcgtg actgggaaaa
     2281 ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa
     2341 tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg
     2401 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatggtg
     2461 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac
     2521 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     2581 gaccatgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacacgcg
     2641 agttcgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
     2701 ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     2761 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     2821 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     2881 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     2941 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     3001 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     3061 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     3121 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     3181 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     3241 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     3301 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     3361 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     3421 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     3481 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     3541 tggagccggt gagcgtggtt cacgcggtat cattgcagca ctggggccag atggtaagcc
     3601 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     3661 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     3721 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     3781 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     3841 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     3901 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     3961 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     4021 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     4081 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     4141 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     4201 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     4261 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     4321 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     4381 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     4441 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     4501 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     4561 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     4621 gtcagtgagc gaggaagcgg atgagcgccc aatacgcaaa ccgcctctca ccgcgcgttg
     4681 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg
     4741 caacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
     4801 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat
     4861 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc
     4921 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaaggt cgacttaacc
     4981 ctagaaagat aatcatattg tgacgtacgt taaagataat catgcgtaaa attgacgcat
     5041 gtgttttatc ggtctgtata tcgaggttta tttattaatt tgaatagata ttaagtttta
     5101 ttatatttac acttacatac taataataaa ttcaacaaac aatttattta tgtttattta
     5161 tttattaaaa aaaaacaaaa actcaaaatt tcttctataa agtaacaaaa cttttagcag
     5221 tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata
     5281 agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg
     5341 gaggtgtggg aggtttttta aagcaagtaa aacctctaca aatgtggtat ggctgattat
     5401 gatcctctgg agattcatgc gccaggtttt ctggtcatgc accacgttcg tgggccctca
     5461 ggaacctcca cgtcagcggt aactgtgaag cccagtcgct cgtaaaaagg aagattccgg
     5521 ggagcactgg tttcgaggaa cgccggcacg ccagcccgtt ccgcggcctc aacaccagga
     5581 agaacgacag cgcttcccaa acctttacct tggtgatctg ggctgacacc gacagtggca
     5641 agaaaccacg caggctcctt tggtctatga ggagccagca agccctccat ttgctgttgt
     5701 gccgccaacc tactccctga cagctcggcc atccgcggac ctatttccgc gaacactgcg
     5761 ccggcttcga cgctttccgg agtagtccag accgcaactg cggcaccatc gtccgcaacc
     5821 caaactttac caatgtccag acccacgcgc gtaagaaaca gctcttgcaa ctcagttacc
     5881 ctctctatgt gtctgtccgg atctacggta tggcgggtcg ctggataatc tgcaaatgct
     5941 gcggcaagtg ttcggacagc ccgtggtaca tcgtcacggg tagcgagcct aacagtgggc
     6001 ttatattccg tcatggggcc ggggttctcc tccacgtcgc cggcctgctt cagcaggctg
     6061 aagttggtgg cgccgctgcc cccggggagc atgtcaaggt caaaatcgtc aagagcgtca
     6121 gcaggcagca tatcaaggtc aaagtcgtca agggcatcgg ctgggagcat gtctaagtca
     6181 aaatcgtcaa gggcgtcggt cggcccgccg ctttcgcact ttagctgttt ctccaggcca
     6241 catatgatta gttccaggcc gaaaaggaag gcaggttcgg ctccctgccg gtcgaacagc
     6301 tcaattgctt gtttcagaag tgggggcata gaatcggtgg taggtgtctc tctttcctct
     6361 tttgctactt gatgctcctg ttcctccaat acgcagccca gtgtaaagtg gcccacggcg
     6421 gacagagcgt acagtgcgtt ctccagggag aagccttgct gacacaggaa cgcgagctga
     6481 ttttccaggg tttcgtactg tttctctgtt gggcgggtgc cgagatgcac tttagccccg
     6541 tcacgatgtg agaggagagc acagcggtat gacttggcgt tgttccgcag aaagtcttgc
     6601 catgactcgc cttccagggg gcaggagtgg gtatgatgcc tgtccagcat ctcgattggc
     6661 agggcatcga gcagggcccg cttgttcttc acgtgccagt acagggtagg ctgctcaact
     6721 cccagctttt gagcgagttt ccttgtcgtc aggccttcga taccgactcc attgagtaat
     6781 tccagagcag agtttatgac tttgctcttg tccagtctag acatcttatc gtcatcgtct
     6841 ttgtaatcca tggtggcgga tcccgcgtca cgacacctgt gttctggcgg caaacccgtt
     6901 gcgaaaaaga acgttcacgg cgactactgc acttatatac ggttctcccc caccctcggg
     6961 aaaaaggcgg agccagtaca cgacatcact ttcccagttt accccgcgcc accttctcta
     7021 ggcaccggtt caattgccga cccctccccc caacttctcg ggcactgtgg gccatgtgcg
     7081 ctctgcccca tggtaatagc catgactaat acgtagatgt actgccaagt aggaaagtgc
     7141 cataaggtca tgtactgggc ataatgccag gcgggccatt taccgtcatt gacgtcaata
     7201 gggggcgtac ttggcatatg atacacttga tgtactgcca agtgggcagt ttaccgtaaa
     7261 tactccaccc attgacgtca atggaaagtg cctattggcg ttactatggg aacatacgtc
     7321 attattgacg tcaatgggcg ggggtcgttg ggcggtcagc caggcgggcc atttaccgta
     7381 agttatgtaa cgcggaactc catatatggg ctatgaacta atgaccccgt aattgattac
     7441 tattaataac tagtcaataa tcaatgtcat aagctctaat cgcataatcg agctaagagc
     7501 gattaatatg cttagctagc ttagctaagc tagatttctc tattctccca caatatatag
     7561 cagctattag cttagcagtc cgaatacgat cgtccacgat ccataagcgt aacacatttg
     7621 agtagcgact gacgggcacc ggagccactc gagtggaatt
//
