LOCUS       sequence-442481-        7795 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac transposon plasmid for doxycycline inducible expression of 
            PAX6b.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7795)
  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 7795)
  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-PAX6b
FEATURES             Location/Qualifiers
     source          1..7795
                     /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(1710..1734)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1736..1762
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1929..2063
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   2335..2369
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2380..2397)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2380..2396)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2389..2411)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2605..2624)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2726..2748
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2787..2805)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2873..2977
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2978..3046,3047..3838)
                     /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(3196..3215)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4009..4597
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4498..4517
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4751..4768
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4974..4995
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5010..5015,5016..5033,5034..5040)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5048..5064
                     /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     5053..5075
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5072..5088
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5072..5088
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   5114..5176
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    5352..5433
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(6150..6206)
                     /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(6216..6959)
                     /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(6960..6983)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6983..6992
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(7012..7223)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        7224..7603
                     /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 cagaacagtc acagcggagt
      421 gaatcagctc ggtggtgtct ttgtcaacgg gcggccactg ccggactcca cccggcagaa
      481 gattgtagag ctagctcaca gcggggcccg gccgtgcgac atttcccgaa ttctgcagac
      541 ccatgcagat gcaaaagtcc aagtgctgga caatcaaaac gtgtccaacg gatgtgtgag
      601 taaaattctg ggcaggtatt acgagactgg ctccatcaga cccagggcaa tcggtggtag
      661 taaaccgaga gtagcgactc cagaagttgt aagcaaaata gcccagtata agcgggagtg
      721 cccgtccatc tttgcttggg aaatccgaga cagattactg tccgaggggg tctgtaccaa
      781 cgataacata ccaagcgtgt catcaataaa cagagttctt cgcaacctgg ctagcgaaaa
      841 gcaacagatg ggcgcagacg gcatgtatga taaactaagg atgttgaacg ggcagaccgg
      901 aagctggggc acccgccctg gttggtatcc ggggacttcg gtgccagggc aacctacgca
      961 agatggctgc cagcaacagg aaggaggggg agagaatacc aactccatca gttccaacgg
     1021 agaagattca gatgaggctc aaatgcgact tcagctgaag cggaagctgc aaagaaatag
     1081 aacatccttt acccaagagc aaattgaggc cctggagaaa gagtttgaga gaacccatta
     1141 tccagatgtg tttgcccgag aaagactagc agccaaaata gatctacctg aagcaagaat
     1201 acaggtatgg ttttctaatc gaagggccaa atggagaaga gaagaaaaac tgaggaatca
     1261 gagaagacag gccagcaaca cacctagtca tattcctatc agcagtagtt tcagcaccag
     1321 tgtctaccaa ccaattccac aacccaccac accggtttcc tccttcacat ctggctccat
     1381 gttgggccga acagacacag ccctcacaaa cacctacagc gctctgccgc ctatgcccag
     1441 cttcaccatg gcaaataacc tgcctatgca acccccagtc cccagccaga cctcctcata
     1501 ctcctgcatg ctgcccacca gcccttcggt gaatgggcgg agttatgata cctacacccc
     1561 cccacatatg cagacacaca tgaacagtca gccaatgggc acctcgggca ccacttcaac
     1621 aggactcatt tcccctggtg tgtcagttcc agttcaagtt cccggaagtg aacctgatat
     1681 gtctcaatac tggccaagat tacagtaaga cccagctttc ttgtacaaag tggtataccc
     1741 atacgatgtt ccagattacg cttagtaatg agaaatactc agaagatgtc actcaccata
     1801 gtccgcagtg aaagataaat gatcattgaa ggacttttca gttagtttta gagctagaaa
     1861 tagcaagtag gccactgagc aagaactatg aatgagatct ttagacatat tgcggccgct
     1921 ttctccgcga tccagacatg ataagataca ttgatgagtt tggacaaacc aaaactagaa
     1981 tgcagtgaaa aaaatgcctt atttgtgaaa tttgtgatgc tattgcctta tttgtaacca
     2041 ttataagctg caataaacaa gttatctata acaagaaaat atatatataa taagttatca
     2101 cgtaagtaga acatgaaata acaatataat tatcgtatga gttaaatctt aaaagtcacg
     2161 taaaagataa tcatgcgtca ttttgactca cgcggtcgtt atagttcaaa atcagtgaca
     2221 cttaccgcat tgacaagcac gcctcacggg agctccaagc ggcgactgag atgtcctaaa
     2281 tgcacagcga cggattcgcg ctatttagaa agagagagca atatttcaag aatgcatgcg
     2341 tcaattttac gcagactatc tttctagggt taagaattca ctggccgtcg ttttacaacg
     2401 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt
     2461 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
     2521 cctgaatggc gaatggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     2581 acaccgcata tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc
     2641 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     2701 ttacagacaa gctgtgacca tgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     2761 tcaccgaaac acgcgagttc gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     2821 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     2881 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     2941 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     3001 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     3061 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     3121 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     3181 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     3241 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     3301 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     3361 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     3421 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     3481 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     3541 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     3601 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     3661 tattgctgat aaatctggag ccggtgagcg tggttcacgc ggtatcattg cagcactggg
     3721 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     3781 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     3841 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     3901 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     3961 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     4021 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     4081 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     4141 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     4201 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     4261 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     4321 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     4381 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     4441 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     4501 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     4561 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     4621 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     4681 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     4741 gaccgagcgc agcgagtcag tgagcgagga agcggatgag cgcccaatac gcaaaccgcc
     4801 tctcaccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
     4861 agcgggcagt gagcgcaacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
     4921 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg
     4981 agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg
     5041 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc
     5101 aaggtcgact taaccctaga aagataatca tattgtgacg tacgttaaag ataatcatgc
     5161 gtaaaattga cgcatgtgtt ttatcggtct gtatatcgag gtttatttat taatttgaat
     5221 agatattaag ttttattata tttacactta catactaata ataaattcaa caaacaattt
     5281 atttatgttt atttatttat taaaaaaaaa caaaaactca aaatttcttc tataaagtaa
     5341 caaaactttt agcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta
     5401 tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg
     5461 tttcaggttc agggggaggt gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt
     5521 ggtatggctg attatgatcc tctggagatt catgcgccag gttttctggt catgcaccac
     5581 gttcgtgggc cctcaggaac ctccacgtca gcggtaactg tgaagcccag tcgctcgtaa
     5641 aaaggaagat tccggggagc actggtttcg aggaacgccg gcacgccagc ccgttccgcg
     5701 gcctcaacac caggaagaac gacagcgctt cccaaacctt taccttggtg atctgggctg
     5761 acaccgacag tggcaagaaa ccacgcaggc tcctttggtc tatgaggagc cagcaagccc
     5821 tccatttgct gttgtgccgc caacctactc cctgacagct cggccatccg cggacctatt
     5881 tccgcgaaca ctgcgccggc ttcgacgctt tccggagtag tccagaccgc aactgcggca
     5941 ccatcgtccg caacccaaac tttaccaatg tccagaccca cgcgcgtaag aaacagctct
     6001 tgcaactcag ttaccctctc tatgtgtctg tccggatcta cggtatggcg ggtcgctgga
     6061 taatctgcaa atgctgcggc aagtgttcgg acagcccgtg gtacatcgtc acgggtagcg
     6121 agcctaacag tgggcttata ttccgtcatg gggccggggt tctcctccac gtcgccggcc
     6181 tgcttcagca ggctgaagtt ggtggcgccg ctgcccccgg ggagcatgtc aaggtcaaaa
     6241 tcgtcaagag cgtcagcagg cagcatatca aggtcaaagt cgtcaagggc atcggctggg
     6301 agcatgtcta agtcaaaatc gtcaagggcg tcggtcggcc cgccgctttc gcactttagc
     6361 tgtttctcca ggccacatat gattagttcc aggccgaaaa ggaaggcagg ttcggctccc
     6421 tgccggtcga acagctcaat tgcttgtttc agaagtgggg gcatagaatc ggtggtaggt
     6481 gtctctcttt cctcttttgc tacttgatgc tcctgttcct ccaatacgca gcccagtgta
     6541 aagtggccca cggcggacag agcgtacagt gcgttctcca gggagaagcc ttgctgacac
     6601 aggaacgcga gctgattttc cagggtttcg tactgtttct ctgttgggcg ggtgccgaga
     6661 tgcactttag ccccgtcacg atgtgagagg agagcacagc ggtatgactt ggcgttgttc
     6721 cgcagaaagt cttgccatga ctcgccttcc agggggcagg agtgggtatg atgcctgtcc
     6781 agcatctcga ttggcagggc atcgagcagg gcccgcttgt tcttcacgtg ccagtacagg
     6841 gtaggctgct caactcccag cttttgagcg agtttccttg tcgtcaggcc ttcgataccg
     6901 actccattga gtaattccag agcagagttt atgactttgc tcttgtccag tctagacatc
     6961 ttatcgtcat cgtctttgta atccatggtg gcggatcccg cgtcacgaca cctgtgttct
     7021 ggcggcaaac ccgttgcgaa aaagaacgtt cacggcgact actgcactta tatacggttc
     7081 tcccccaccc tcgggaaaaa ggcggagcca gtacacgaca tcactttccc agtttacccc
     7141 gcgccacctt ctctaggcac cggttcaatt gccgacccct ccccccaact tctcgggcac
     7201 tgtgggccat gtgcgctctg ccccatggta atagccatga ctaatacgta gatgtactgc
     7261 caagtaggaa agtgccataa ggtcatgtac tgggcataat gccaggcggg ccatttaccg
     7321 tcattgacgt caataggggg cgtacttggc atatgataca cttgatgtac tgccaagtgg
     7381 gcagtttacc gtaaatactc cacccattga cgtcaatgga aagtgcctat tggcgttact
     7441 atgggaacat acgtcattat tgacgtcaat gggcgggggt cgttgggcgg tcagccaggc
     7501 gggccattta ccgtaagtta tgtaacgcgg aactccatat atgggctatg aactaatgac
     7561 cccgtaattg attactatta ataactagtc aataatcaat gtcataagct ctaatcgcat
     7621 aatcgagcta agagcgatta atatgcttag ctagcttagc taagctagat ttctctattc
     7681 tcccacaata tatagcagct attagcttag cagtccgaat acgatcgtcc acgatccata
     7741 agcgtaacac atttgagtag cgactgacgg gcaccggagc cactcgagtg gaatt
//
