LOCUS       sequence-437882-        7705 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac transposon plasmid for doxycycline inducible expression of 
            ESX1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7705)
  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 7705)
  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-ESX1
FEATURES             Location/Qualifiers
     source          1..7705
                     /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"
     regulatory      393..402
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(1620..1644)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1646..1672
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1839..1973
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   2245..2279
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2290..2307)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2290..2306)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2299..2321)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2515..2534)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2636..2658
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2697..2715)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2783..2887
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2888..2956,2957..3748)
                     /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(3106..3125)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3919..4507
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4408..4427
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4661..4678
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4884..4905
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4920..4925,4926..4943,4944..4950)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4958..4974
                     /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     4963..4985
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4982..4998
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4982..4998
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   5024..5086
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    5262..5343
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(6060..6116)
                     /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(6126..6869)
                     /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(6870..6893)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6893..6902
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6922..7133)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        7134..7513
                     /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 ggagtctctt cgcgggtaca
      421 cccacagtga tattggctac cgcagcctgg cagtcggcga ggacatcgag gaagtgaatg
      481 atgagaaact taccgtgacc tcgctgatgg caaggggagg agaggacgag gagaatacac
      541 ggtccaaacc tgagtacgga acagaagcgg aaaacaacgt tggcacggaa gggtccgtcc
      601 cctcggacga ccaagaccgt gagggtggcg gcggccacga gccggagcaa cagcaggagg
      661 agccgcccct gaccaagccg gagcaacagc aggaggagcc gcccctgctc gagctgaagc
      721 aagagcagga ggagccgccc cagacgaccg tggaggggcc acagccggcg gaggggccac
      781 aaaccgctga gggaccacag cccccagaga ggaaacgccg ccgccgcacc gcgttcacgc
      841 agtttcagct gcaggagcta gagaactttt tcgatgaatc tcaatatccc gacgttgtgg
      901 cgcgagagag acttgcagca cgcctgaatt tgactgaaga cagagtgcag gtttggtttc
      961 agaacagaag agccaagtgg aaacgaaatc agagggtgct aatgttgaga aacactgcta
     1021 ctgctgacct ggcccaccct ttggacatgt tcttgggtgg ggcctattat gctgctcctg
     1081 ctctggatcc tgctttgtgt gttcatctgg tgccacaact acctagacca cctgtgctgc
     1141 ctgtgccacc tatgccaccc aggccaccca tggtccctat gccacccagg ccacccatag
     1201 cacctatgcc acccatggcg cctgtgccac ccggctcacg catggcgcct gtgccacccg
     1261 ggccacgcat ggcgcctgtg ccaccctggc cgcccatggc gcctgtgcca ccctggccgc
     1321 ccatggcgcc tgtgccaacc gggccgccca tggcgcctgt gccacccggg ccgcccatgg
     1381 cgcgtgtgcc acccgggccg cccatggcgc gtgtgccacc cgggccgccc atggcgcctc
     1441 tgccacccgg gccgcccatg gcgcctctgc cacccgggcc gcccatggcg cctctgccac
     1501 ccgggccgcc catggcgcct ctgccaccca ggtcacatgt gcctcacact ggcctggctc
     1561 ctgtacacat cacatgggcc cctgtcatca acagttatta tgcatgtccc tttttctaaa
     1621 cccagctttc ttgtacaaag tggtataccc atacgatgtt ccagattacg cttagtaatg
     1681 agaaatactc agaagatgtc actcaccata gtccgcagtg aaagataaat gatcgaacca
     1741 gaaacaaaat aaaagtttta gagctagaaa tagcaagtag gccactgagc aagaactatg
     1801 aatgagatct ttagacatat tgcggccgct ttctccgcga tccagacatg ataagataca
     1861 ttgatgagtt tggacaaacc aaaactagaa tgcagtgaaa aaaatgcctt atttgtgaaa
     1921 tttgtgatgc tattgcctta tttgtaacca ttataagctg caataaacaa gttatctata
     1981 acaagaaaat atatatataa taagttatca cgtaagtaga acatgaaata acaatataat
     2041 tatcgtatga gttaaatctt aaaagtcacg taaaagataa tcatgcgtca ttttgactca
     2101 cgcggtcgtt atagttcaaa atcagtgaca cttaccgcat tgacaagcac gcctcacggg
     2161 agctccaagc ggcgactgag atgtcctaaa tgcacagcga cggattcgcg ctatttagaa
     2221 agagagagca atatttcaag aatgcatgcg tcaattttac gcagactatc tttctagggt
     2281 taagaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
     2341 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
     2401 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc tgatgcggta
     2461 ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc tcagtacaat
     2521 ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc
     2581 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgacca tgtctccggg
     2641 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac acgcgagttc gaaagggcct
     2701 cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg
     2761 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     2821 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     2881 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     2941 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     3001 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     3061 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     3121 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     3181 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     3241 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     3301 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     3361 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     3421 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     3481 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     3541 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     3601 tggttcacgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     3661 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     3721 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     3781 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     3841 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     3901 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     3961 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     4021 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     4081 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     4141 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     4201 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     4261 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     4321 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     4381 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     4441 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     4501 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     4561 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     4621 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     4681 agcggatgag cgcccaatac gcaaaccgcc tctcaccgcg cgttggccga ttcattaatg
     4741 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caaaccgcct
     4801 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
     4861 gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct
     4921 ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac
     4981 acaggaaaca gctatgacca tgattacgcc aaggtcgact taaccctaga aagataatca
     5041 tattgtgacg tacgttaaag ataatcatgc gtaaaattga cgcatgtgtt ttatcggtct
     5101 gtatatcgag gtttatttat taatttgaat agatattaag ttttattata tttacactta
     5161 catactaata ataaattcaa caaacaattt atttatgttt atttatttat taaaaaaaaa
     5221 caaaaactca aaatttcttc tataaagtaa caaaactttt agcagtgaaa aaaatgcttt
     5281 atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa
     5341 gttaacaaca acaattgcat tcattttatg tttcaggttc agggggaggt gtgggaggtt
     5401 ttttaaagca agtaaaacct ctacaaatgt ggtatggctg attatgatcc tctggagatt
     5461 catgcgccag gttttctggt catgcaccac gttcgtgggc cctcaggaac ctccacgtca
     5521 gcggtaactg tgaagcccag tcgctcgtaa aaaggaagat tccggggagc actggtttcg
     5581 aggaacgccg gcacgccagc ccgttccgcg gcctcaacac caggaagaac gacagcgctt
     5641 cccaaacctt taccttggtg atctgggctg acaccgacag tggcaagaaa ccacgcaggc
     5701 tcctttggtc tatgaggagc cagcaagccc tccatttgct gttgtgccgc caacctactc
     5761 cctgacagct cggccatccg cggacctatt tccgcgaaca ctgcgccggc ttcgacgctt
     5821 tccggagtag tccagaccgc aactgcggca ccatcgtccg caacccaaac tttaccaatg
     5881 tccagaccca cgcgcgtaag aaacagctct tgcaactcag ttaccctctc tatgtgtctg
     5941 tccggatcta cggtatggcg ggtcgctgga taatctgcaa atgctgcggc aagtgttcgg
     6001 acagcccgtg gtacatcgtc acgggtagcg agcctaacag tgggcttata ttccgtcatg
     6061 gggccggggt tctcctccac gtcgccggcc tgcttcagca ggctgaagtt ggtggcgccg
     6121 ctgcccccgg ggagcatgtc aaggtcaaaa tcgtcaagag cgtcagcagg cagcatatca
     6181 aggtcaaagt cgtcaagggc atcggctggg agcatgtcta agtcaaaatc gtcaagggcg
     6241 tcggtcggcc cgccgctttc gcactttagc tgtttctcca ggccacatat gattagttcc
     6301 aggccgaaaa ggaaggcagg ttcggctccc tgccggtcga acagctcaat tgcttgtttc
     6361 agaagtgggg gcatagaatc ggtggtaggt gtctctcttt cctcttttgc tacttgatgc
     6421 tcctgttcct ccaatacgca gcccagtgta aagtggccca cggcggacag agcgtacagt
     6481 gcgttctcca gggagaagcc ttgctgacac aggaacgcga gctgattttc cagggtttcg
     6541 tactgtttct ctgttgggcg ggtgccgaga tgcactttag ccccgtcacg atgtgagagg
     6601 agagcacagc ggtatgactt ggcgttgttc cgcagaaagt cttgccatga ctcgccttcc
     6661 agggggcagg agtgggtatg atgcctgtcc agcatctcga ttggcagggc atcgagcagg
     6721 gcccgcttgt tcttcacgtg ccagtacagg gtaggctgct caactcccag cttttgagcg
     6781 agtttccttg tcgtcaggcc ttcgataccg actccattga gtaattccag agcagagttt
     6841 atgactttgc tcttgtccag tctagacatc ttatcgtcat cgtctttgta atccatggtg
     6901 gcggatcccg cgtcacgaca cctgtgttct ggcggcaaac ccgttgcgaa aaagaacgtt
     6961 cacggcgact actgcactta tatacggttc tcccccaccc tcgggaaaaa ggcggagcca
     7021 gtacacgaca tcactttccc agtttacccc gcgccacctt ctctaggcac cggttcaatt
     7081 gccgacccct ccccccaact tctcgggcac tgtgggccat gtgcgctctg ccccatggta
     7141 atagccatga ctaatacgta gatgtactgc caagtaggaa agtgccataa ggtcatgtac
     7201 tgggcataat gccaggcggg ccatttaccg tcattgacgt caataggggg cgtacttggc
     7261 atatgataca cttgatgtac tgccaagtgg gcagtttacc gtaaatactc cacccattga
     7321 cgtcaatgga aagtgcctat tggcgttact atgggaacat acgtcattat tgacgtcaat
     7381 gggcgggggt cgttgggcgg tcagccaggc gggccattta ccgtaagtta tgtaacgcgg
     7441 aactccatat atgggctatg aactaatgac cccgtaattg attactatta ataactagtc
     7501 aataatcaat gtcataagct ctaatcgcat aatcgagcta agagcgatta atatgcttag
     7561 ctagcttagc taagctagat ttctctattc tcccacaata tatagcagct attagcttag
     7621 cagtccgaat acgatcgtcc acgatccata agcgtaacac atttgagtag cgactgacgg
     7681 gcaccggagc cactcgagtg gaatt
//
