LOCUS       sequence-442529-        7033 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac transposon plasmid for doxycycline inducible expression of 
            HESX1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7033)
  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 7033)
  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-HESX1
FEATURES             Location/Qualifiers
     source          1..7033
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    16..34
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    52..70
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    88..106
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    124..142
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    160..178
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    196..214
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    232..250
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     277..301
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    367..391
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(947..971)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             973..999
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1166..1300
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   1572..1606
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1617..1634)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1617..1633)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1626..1648)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1842..1861)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1963..1985
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2024..2042)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2110..2214
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2215..2283,2284..3075)
                     /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(2433..2452)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3246..3834
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3735..3754
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3988..4005
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4211..4232
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4247..4252,4253..4270,4271..4277)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4285..4301
                     /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     4290..4312
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4309..4325
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4309..4325
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4351..4413
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    4589..4670
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5387..5443)
                     /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(5453..6196)
                     /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(6197..6220)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6220..6229
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6249..6460)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        6461..6840
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 tcacctcgag tttactccct atcagtgata gagaacgtat gaagagttta ctccctatca
       61 gtgatagaga acgtatgcag actttactcc ctatcagtga tagagaacgt ataaggagtt
      121 tactccctat cagtgataga gaacgtatga ccagtttact ccctatcagt gatagagaac
      181 gtatctacag tttactccct atcagtgata gagaacgtat atccagttta ctccctatca
      241 gtgatagaga acgtataagc ttgcctatat aagcagagct cgtttagtga accgtcagat
      301 cgcctggaga cgccatccac gctgttttga cctccataga agacaccggg accgatccag
      361 cctaccacaa gtttgtacaa aaaagttggc atgtctccca gccttcagga aggcgctcag
      421 ctcggggaaa acaaaccctc aacttgctcc ttttcaattg agagaatctt aggactggac
      481 cagaagaaag actgtgttcc attaatgaaa ccccacaggc cctgggcaga cacctgcagc
      541 tcatcaggga aagatggtaa cttatgtcta catgtcccaa atcctcccag tgggatttca
      601 ttccctagcg tggtggatca cccaatgcca gaagaaagag cttcgaaata tgaaaattac
      661 ttttcagcct cagaaagact gtctttgaaa agagagttga gttggtatag aggccgaaga
      721 ccaagaactg cttttactca aaaccagatt gaagtgttag aaaatgtctt tagagtaaac
      781 tgctatcctg gtatcgatat tagagaagac ttagctcaaa aattgaatct agaggaagac
      841 agaatccaga tttggtttca aaatcggcgt gcaaaactga aaaggtccca tagagaatca
      901 cagtttctaa tggcgaaaaa aaatttcaac acaaatctgc tggaataccc aactttcttg
      961 tacaaagtgg tatacccata cgatgttcca gattacgctt agtaatgaga aatactcaga
     1021 agatgtcact caccatagtc cgcagtgaaa gataaatgat ccgaacagta cacatcacgt
     1081 cgttttagag ctagaaatag caagtaggcc actgagcaag aactatgaat gagatcttta
     1141 gacatattgc ggccgctttc tccgcgatcc agacatgata agatacattg atgagtttgg
     1201 acaaaccaaa actagaatgc agtgaaaaaa atgccttatt tgtgaaattt gtgatgctat
     1261 tgccttattt gtaaccatta taagctgcaa taaacaagtt atctataaca agaaaatata
     1321 tatataataa gttatcacgt aagtagaaca tgaaataaca atataattat cgtatgagtt
     1381 aaatcttaaa agtcacgtaa aagataatca tgcgtcattt tgactcacgc ggtcgttata
     1441 gttcaaaatc agtgacactt accgcattga caagcacgcc tcacgggagc tccaagcggc
     1501 gactgagatg tcctaaatgc acagcgacgg attcgcgcta tttagaaaga gagagcaata
     1561 tttcaagaat gcatgcgtca attttacgca gactatcttt ctagggttaa gaattcactg
     1621 gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt
     1681 gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct
     1741 tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt tctccttacg
     1801 catctgtgcg gtatttcaca ccgcatatgg tgcactctca gtacaatctg ctctgatgcc
     1861 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     1921 ctgctcccgg catccgctta cagacaagct gtgaccatgt ctccgggagc tgcatgtgtc
     1981 agaggttttc accgtcatca ccgaaacacg cgagttcgaa agggcctcgt gatacgccta
     2041 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     2101 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     2161 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     2221 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     2281 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     2341 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     2401 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     2461 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     2521 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     2581 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     2641 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     2701 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     2761 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     2821 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     2881 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg ttcacgcggt
     2941 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     3001 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     3061 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     3121 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     3181 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     3241 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     3301 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     3361 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     3421 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     3481 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     3541 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     3601 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     3661 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     3721 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     3781 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     3841 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     3901 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     3961 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggatgagcgc
     4021 ccaatacgca aaccgcctct caccgcgcgt tggccgattc attaatgcag ctggcacgac
     4081 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa accgcctctc cccgcgcgtt
     4141 ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc
     4201 gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc
     4261 ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct
     4321 atgaccatga ttacgccaag gtcgacttaa ccctagaaag ataatcatat tgtgacgtac
     4381 gttaaagata atcatgcgta aaattgacgc atgtgtttta tcggtctgta tatcgaggtt
     4441 tatttattaa tttgaataga tattaagttt tattatattt acacttacat actaataata
     4501 aattcaacaa acaatttatt tatgtttatt tatttattaa aaaaaaacaa aaactcaaaa
     4561 tttcttctat aaagtaacaa aacttttagc agtgaaaaaa atgctttatt tgtgaaattt
     4621 gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca
     4681 attgcattca ttttatgttt caggttcagg gggaggtgtg ggaggttttt taaagcaagt
     4741 aaaacctcta caaatgtggt atggctgatt atgatcctct ggagattcat gcgccaggtt
     4801 ttctggtcat gcaccacgtt cgtgggccct caggaacctc cacgtcagcg gtaactgtga
     4861 agcccagtcg ctcgtaaaaa ggaagattcc ggggagcact ggtttcgagg aacgccggca
     4921 cgccagcccg ttccgcggcc tcaacaccag gaagaacgac agcgcttccc aaacctttac
     4981 cttggtgatc tgggctgaca ccgacagtgg caagaaacca cgcaggctcc tttggtctat
     5041 gaggagccag caagccctcc atttgctgtt gtgccgccaa cctactccct gacagctcgg
     5101 ccatccgcgg acctatttcc gcgaacactg cgccggcttc gacgctttcc ggagtagtcc
     5161 agaccgcaac tgcggcacca tcgtccgcaa cccaaacttt accaatgtcc agacccacgc
     5221 gcgtaagaaa cagctcttgc aactcagtta ccctctctat gtgtctgtcc ggatctacgg
     5281 tatggcgggt cgctggataa tctgcaaatg ctgcggcaag tgttcggaca gcccgtggta
     5341 catcgtcacg ggtagcgagc ctaacagtgg gcttatattc cgtcatgggg ccggggttct
     5401 cctccacgtc gccggcctgc ttcagcaggc tgaagttggt ggcgccgctg cccccgggga
     5461 gcatgtcaag gtcaaaatcg tcaagagcgt cagcaggcag catatcaagg tcaaagtcgt
     5521 caagggcatc ggctgggagc atgtctaagt caaaatcgtc aagggcgtcg gtcggcccgc
     5581 cgctttcgca ctttagctgt ttctccaggc cacatatgat tagttccagg ccgaaaagga
     5641 aggcaggttc ggctccctgc cggtcgaaca gctcaattgc ttgtttcaga agtgggggca
     5701 tagaatcggt ggtaggtgtc tctctttcct cttttgctac ttgatgctcc tgttcctcca
     5761 atacgcagcc cagtgtaaag tggcccacgg cggacagagc gtacagtgcg ttctccaggg
     5821 agaagccttg ctgacacagg aacgcgagct gattttccag ggtttcgtac tgtttctctg
     5881 ttgggcgggt gccgagatgc actttagccc cgtcacgatg tgagaggaga gcacagcggt
     5941 atgacttggc gttgttccgc agaaagtctt gccatgactc gccttccagg gggcaggagt
     6001 gggtatgatg cctgtccagc atctcgattg gcagggcatc gagcagggcc cgcttgttct
     6061 tcacgtgcca gtacagggta ggctgctcaa ctcccagctt ttgagcgagt ttccttgtcg
     6121 tcaggccttc gataccgact ccattgagta attccagagc agagtttatg actttgctct
     6181 tgtccagtct agacatctta tcgtcatcgt ctttgtaatc catggtggcg gatcccgcgt
     6241 cacgacacct gtgttctggc ggcaaacccg ttgcgaaaaa gaacgttcac ggcgactact
     6301 gcacttatat acggttctcc cccaccctcg ggaaaaaggc ggagccagta cacgacatca
     6361 ctttcccagt ttaccccgcg ccaccttctc taggcaccgg ttcaattgcc gacccctccc
     6421 cccaacttct cgggcactgt gggccatgtg cgctctgccc catggtaata gccatgacta
     6481 atacgtagat gtactgccaa gtaggaaagt gccataaggt catgtactgg gcataatgcc
     6541 aggcgggcca tttaccgtca ttgacgtcaa tagggggcgt acttggcata tgatacactt
     6601 gatgtactgc caagtgggca gtttaccgta aatactccac ccattgacgt caatggaaag
     6661 tgcctattgg cgttactatg ggaacatacg tcattattga cgtcaatggg cgggggtcgt
     6721 tgggcggtca gccaggcggg ccatttaccg taagttatgt aacgcggaac tccatatatg
     6781 ggctatgaac taatgacccc gtaattgatt actattaata actagtcaat aatcaatgtc
     6841 ataagctcta atcgcataat cgagctaaga gcgattaata tgcttagcta gcttagctaa
     6901 gctagatttc tctattctcc cacaatatat agcagctatt agcttagcag tccgaatacg
     6961 atcgtccacg atccataagc gtaacacatt tgagtagcga ctgacgggca ccggagccac
     7021 tcgagtggaa tta
//
