LOCUS       sequence-442525-        7123 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac transposon plasmid for doxycycline inducible expression of 
            HMGB1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7123)
  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 7123)
  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-HMGB1
FEATURES             Location/Qualifiers
     source          1..7123
                     /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    complement(1037..1061)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1063..1089
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1256..1390
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   1662..1696
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1707..1724)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1707..1723)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1716..1738)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1932..1951)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2053..2075
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2114..2132)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2200..2304
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2305..2373,2374..3165)
                     /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(2523..2542)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3336..3924
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3825..3844
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4078..4095
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4301..4322
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4337..4342,4343..4360,4361..4367)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4375..4391
                     /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     4380..4402
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4399..4415
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4399..4415
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4441..4503
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    4679..4760
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5477..5533)
                     /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(5543..6286)
                     /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(6287..6310)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6310..6319
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6339..6550)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        6551..6930
                     /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 aacagttggc atgggcacag gagatcctaa gaagccgaga
      421 ggcaaaatgt catcatatgc attttttgtg caaacttgtc gggaggagca taagaagaag
      481 cacccagatg cttcagtcaa cttctcagag ttttctaaga agtgctcaga gaggtggaag
      541 accatgtctg ctaaagagaa aggaaaattt gaagatatgg caaaggcgga caaggcccgt
      601 tatgaaagag aaatgaaaac ctatatccct cccaaagggg agacaaaaaa gaagttcaag
      661 gatcccaatg cacccaagag gcctccttcg gccttcttcc tcttctgctc tgagtatcgc
      721 ccaaaaatca aaggagaaca tcctggcctg tccattggtg atgttgcgaa gaaactggga
      781 gagatgtgga ataacactgc tgcagatgac aagcagcctt atgaaaagaa ggctgcgaag
      841 ctgaaggaaa aatacgaaaa ggatattgct gcatatcgag ctaaaggaaa gcctgatgca
      901 gcaaaaaagg gagttgtcaa ggctgaaaaa agcaagaaaa agaaggaaga ggaggaagat
      961 gaggaagatg aagaggatga ggaggaggag gaagatgaag aagatgaaga tgaagaagag
     1021 gatgatgatg atgaataccc aactttcttg tacaaagtgg tatacccata cgatgttcca
     1081 gattacgctt agtaatgaga aatactcaga agatgtcact caccatagtc cgcagtgaaa
     1141 gataaatgat cggcagtggt gaacgcagat agttttagag ctagaaatag caagtaggcc
     1201 actgagcaag aactatgaat gagatcttta gacatattgc ggccgctttc tccgcgatcc
     1261 agacatgata agatacattg atgagtttgg acaaaccaaa actagaatgc agtgaaaaaa
     1321 atgccttatt tgtgaaattt gtgatgctat tgccttattt gtaaccatta taagctgcaa
     1381 taaacaagtt atctataaca agaaaatata tatataataa gttatcacgt aagtagaaca
     1441 tgaaataaca atataattat cgtatgagtt aaatcttaaa agtcacgtaa aagataatca
     1501 tgcgtcattt tgactcacgc ggtcgttata gttcaaaatc agtgacactt accgcattga
     1561 caagcacgcc tcacgggagc tccaagcggc gactgagatg tcctaaatgc acagcgacgg
     1621 attcgcgcta tttagaaaga gagagcaata tttcaagaat gcatgcgtca attttacgca
     1681 gactatcttt ctagggttaa gaattcactg gccgtcgttt tacaacgtcg tgactgggaa
     1741 aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt
     1801 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa
     1861 tggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatatgg
     1921 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca
     1981 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     2041 gtgaccatgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacacg
     2101 cgagttcgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
     2161 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     2221 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     2281 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     2341 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     2401 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     2461 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     2521 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     2581 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     2641 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     2701 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     2761 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     2821 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     2881 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     2941 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     3001 tctggagccg gtgagcgtgg ttcacgcggt atcattgcag cactggggcc agatggtaag
     3061 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     3121 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     3181 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     3241 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     3301 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     3361 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     3421 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     3481 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     3541 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     3601 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     3661 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     3721 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     3781 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     3841 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     3901 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     3961 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     4021 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     4081 gagtcagtga gcgaggaagc ggatgagcgc ccaatacgca aaccgcctct caccgcgcgt
     4141 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
     4201 cgcaacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     4261 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc
     4321 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga
     4381 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag gtcgacttaa
     4441 ccctagaaag ataatcatat tgtgacgtac gttaaagata atcatgcgta aaattgacgc
     4501 atgtgtttta tcggtctgta tatcgaggtt tatttattaa tttgaataga tattaagttt
     4561 tattatattt acacttacat actaataata aattcaacaa acaatttatt tatgtttatt
     4621 tatttattaa aaaaaaacaa aaactcaaaa tttcttctat aaagtaacaa aacttttagc
     4681 agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta
     4741 taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg
     4801 gggaggtgtg ggaggttttt taaagcaagt aaaacctcta caaatgtggt atggctgatt
     4861 atgatcctct ggagattcat gcgccaggtt ttctggtcat gcaccacgtt cgtgggccct
     4921 caggaacctc cacgtcagcg gtaactgtga agcccagtcg ctcgtaaaaa ggaagattcc
     4981 ggggagcact ggtttcgagg aacgccggca cgccagcccg ttccgcggcc tcaacaccag
     5041 gaagaacgac agcgcttccc aaacctttac cttggtgatc tgggctgaca ccgacagtgg
     5101 caagaaacca cgcaggctcc tttggtctat gaggagccag caagccctcc atttgctgtt
     5161 gtgccgccaa cctactccct gacagctcgg ccatccgcgg acctatttcc gcgaacactg
     5221 cgccggcttc gacgctttcc ggagtagtcc agaccgcaac tgcggcacca tcgtccgcaa
     5281 cccaaacttt accaatgtcc agacccacgc gcgtaagaaa cagctcttgc aactcagtta
     5341 ccctctctat gtgtctgtcc ggatctacgg tatggcgggt cgctggataa tctgcaaatg
     5401 ctgcggcaag tgttcggaca gcccgtggta catcgtcacg ggtagcgagc ctaacagtgg
     5461 gcttatattc cgtcatgggg ccggggttct cctccacgtc gccggcctgc ttcagcaggc
     5521 tgaagttggt ggcgccgctg cccccgggga gcatgtcaag gtcaaaatcg tcaagagcgt
     5581 cagcaggcag catatcaagg tcaaagtcgt caagggcatc ggctgggagc atgtctaagt
     5641 caaaatcgtc aagggcgtcg gtcggcccgc cgctttcgca ctttagctgt ttctccaggc
     5701 cacatatgat tagttccagg ccgaaaagga aggcaggttc ggctccctgc cggtcgaaca
     5761 gctcaattgc ttgtttcaga agtgggggca tagaatcggt ggtaggtgtc tctctttcct
     5821 cttttgctac ttgatgctcc tgttcctcca atacgcagcc cagtgtaaag tggcccacgg
     5881 cggacagagc gtacagtgcg ttctccaggg agaagccttg ctgacacagg aacgcgagct
     5941 gattttccag ggtttcgtac tgtttctctg ttgggcgggt gccgagatgc actttagccc
     6001 cgtcacgatg tgagaggaga gcacagcggt atgacttggc gttgttccgc agaaagtctt
     6061 gccatgactc gccttccagg gggcaggagt gggtatgatg cctgtccagc atctcgattg
     6121 gcagggcatc gagcagggcc cgcttgttct tcacgtgcca gtacagggta ggctgctcaa
     6181 ctcccagctt ttgagcgagt ttccttgtcg tcaggccttc gataccgact ccattgagta
     6241 attccagagc agagtttatg actttgctct tgtccagtct agacatctta tcgtcatcgt
     6301 ctttgtaatc catggtggcg gatcccgcgt cacgacacct gtgttctggc ggcaaacccg
     6361 ttgcgaaaaa gaacgttcac ggcgactact gcacttatat acggttctcc cccaccctcg
     6421 ggaaaaaggc ggagccagta cacgacatca ctttcccagt ttaccccgcg ccaccttctc
     6481 taggcaccgg ttcaattgcc gacccctccc cccaacttct cgggcactgt gggccatgtg
     6541 cgctctgccc catggtaata gccatgacta atacgtagat gtactgccaa gtaggaaagt
     6601 gccataaggt catgtactgg gcataatgcc aggcgggcca tttaccgtca ttgacgtcaa
     6661 tagggggcgt acttggcata tgatacactt gatgtactgc caagtgggca gtttaccgta
     6721 aatactccac ccattgacgt caatggaaag tgcctattgg cgttactatg ggaacatacg
     6781 tcattattga cgtcaatggg cgggggtcgt tgggcggtca gccaggcggg ccatttaccg
     6841 taagttatgt aacgcggaac tccatatatg ggctatgaac taatgacccc gtaattgatt
     6901 actattaata actagtcaat aatcaatgtc ataagctcta atcgcataat cgagctaaga
     6961 gcgattaata tgcttagcta gcttagctaa gctagatttc tctattctcc cacaatatat
     7021 agcagctatt agcttagcag tccgaatacg atcgtccacg atccataagc gtaacacatt
     7081 tgagtagcga ctgacgggca ccggagccac tcgagtggaa tta
//
