LOCUS       sequence-442405-        7078 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac transposon plasmid for doxycycline inducible expression of 
            HMGB3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7078)
  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 7078)
  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-HMGB3
FEATURES             Location/Qualifiers
     source          1..7078
                     /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(992..1016)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1018..1044
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1211..1345
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   1617..1651
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1662..1679)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1662..1678)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1671..1693)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1887..1906)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2008..2030
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2069..2087)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2155..2259
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2260..2328,2329..3120)
                     /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(2478..2497)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3291..3879
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3780..3799
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4033..4050
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4256..4277
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4292..4297,4298..4315,4316..4322)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4330..4346
                     /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     4335..4357
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4354..4370
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4354..4370
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4396..4458
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    4634..4715
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5432..5488)
                     /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(5498..6241)
                     /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(6242..6265)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6265..6274
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6294..6505)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        6506..6885
                     /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 atggctaaag gtgaccccaa gaaaccaaag
      421 ggcaagatgt ccgcttatgc cttctttgtg cagacatgca gagaagaaca taagaagaaa
      481 aacccagagg tccctgtcaa ttttgcggaa ttttccaaga agtgctctga gaggtggaag
      541 acgatgtccg ggaaagagaa atctaaattt gatgaaatgg caaaggcaga taaagtgcgc
      601 tatgatcggg aaatgaagga ttatggacca gctaagggag gcaagaagaa gaaggatcct
      661 aatgctccca aaaggccacc gtctggattc ttcctgttct gttcagaatt ccgccccaag
      721 atcaaatcca caaaccccgg catctctatt ggagacgtgg caaaaaagct gggtgagatg
      781 tggaataatt taaatgacag tgaaaagcag ccttacatca ctaaggcggc aaagctgaag
      841 gagaagtatg agaaggatgt tgctgactat aagtcgaaag gaaagtttga tggtgcaaag
      901 ggtcctgcta aagttgcccg gaaaaaggtg gaagaggaag atgaagaaga ggaggaggaa
      961 gaagaggagg aggaggagga ggaggatgaa tacccaactt tcttgtacaa agtggtatac
     1021 ccatacgatg ttccagatta cgcttagtaa tgagaaatac tcagaagatg tcactcacca
     1081 tagtccgcag tgaaagataa atgatcttta tcgtttgtac aatgaagttt tagagctaga
     1141 aatagcaagt aggccactga gcaagaacta tgaatgagat ctttagacat attgcggccg
     1201 ctttctccgc gatccagaca tgataagata cattgatgag tttggacaaa ccaaaactag
     1261 aatgcagtga aaaaaatgcc ttatttgtga aatttgtgat gctattgcct tatttgtaac
     1321 cattataagc tgcaataaac aagttatcta taacaagaaa atatatatat aataagttat
     1381 cacgtaagta gaacatgaaa taacaatata attatcgtat gagttaaatc ttaaaagtca
     1441 cgtaaaagat aatcatgcgt cattttgact cacgcggtcg ttatagttca aaatcagtga
     1501 cacttaccgc attgacaagc acgcctcacg ggagctccaa gcggcgactg agatgtccta
     1561 aatgcacagc gacggattcg cgctatttag aaagagagag caatatttca agaatgcatg
     1621 cgtcaatttt acgcagacta tctttctagg gttaagaatt cactggccgt cgttttacaa
     1681 cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct
     1741 ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
     1801 agcctgaatg gcgaatggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     1861 tcacaccgca tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     1921 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     1981 gcttacagac aagctgtgac catgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     2041 catcaccgaa acacgcgagt tcgaaagggc ctcgtgatac gcctattttt ataggttaat
     2101 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     2161 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     2221 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     2281 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     2341 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     2401 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     2461 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     2521 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     2581 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     2641 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     2701 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     2761 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     2821 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     2881 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     2941 tttattgctg ataaatctgg agccggtgag cgtggttcac gcggtatcat tgcagcactg
     3001 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     3061 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     3121 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     3181 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     3241 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     3301 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     3361 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     3421 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     3481 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     3541 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     3601 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     3661 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     3721 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     3781 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     3841 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     3901 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     3961 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     4021 acgaccgagc gcagcgagtc agtgagcgag gaagcggatg agcgcccaat acgcaaaccg
     4081 cctctcaccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
     4141 aaagcgggca gtgagcgcaa cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     4201 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     4261 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
     4321 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg
     4381 ccaaggtcga cttaacccta gaaagataat catattgtga cgtacgttaa agataatcat
     4441 gcgtaaaatt gacgcatgtg ttttatcggt ctgtatatcg aggtttattt attaatttga
     4501 atagatatta agttttatta tatttacact tacatactaa taataaattc aacaaacaat
     4561 ttatttatgt ttatttattt attaaaaaaa aacaaaaact caaaatttct tctataaagt
     4621 aacaaaactt ttagcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt
     4681 tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta
     4741 tgtttcaggt tcagggggag gtgtgggagg ttttttaaag caagtaaaac ctctacaaat
     4801 gtggtatggc tgattatgat cctctggaga ttcatgcgcc aggttttctg gtcatgcacc
     4861 acgttcgtgg gccctcagga acctccacgt cagcggtaac tgtgaagccc agtcgctcgt
     4921 aaaaaggaag attccgggga gcactggttt cgaggaacgc cggcacgcca gcccgttccg
     4981 cggcctcaac accaggaaga acgacagcgc ttcccaaacc tttaccttgg tgatctgggc
     5041 tgacaccgac agtggcaaga aaccacgcag gctcctttgg tctatgagga gccagcaagc
     5101 cctccatttg ctgttgtgcc gccaacctac tccctgacag ctcggccatc cgcggaccta
     5161 tttccgcgaa cactgcgccg gcttcgacgc tttccggagt agtccagacc gcaactgcgg
     5221 caccatcgtc cgcaacccaa actttaccaa tgtccagacc cacgcgcgta agaaacagct
     5281 cttgcaactc agttaccctc tctatgtgtc tgtccggatc tacggtatgg cgggtcgctg
     5341 gataatctgc aaatgctgcg gcaagtgttc ggacagcccg tggtacatcg tcacgggtag
     5401 cgagcctaac agtgggctta tattccgtca tggggccggg gttctcctcc acgtcgccgg
     5461 cctgcttcag caggctgaag ttggtggcgc cgctgccccc ggggagcatg tcaaggtcaa
     5521 aatcgtcaag agcgtcagca ggcagcatat caaggtcaaa gtcgtcaagg gcatcggctg
     5581 ggagcatgtc taagtcaaaa tcgtcaaggg cgtcggtcgg cccgccgctt tcgcacttta
     5641 gctgtttctc caggccacat atgattagtt ccaggccgaa aaggaaggca ggttcggctc
     5701 cctgccggtc gaacagctca attgcttgtt tcagaagtgg gggcatagaa tcggtggtag
     5761 gtgtctctct ttcctctttt gctacttgat gctcctgttc ctccaatacg cagcccagtg
     5821 taaagtggcc cacggcggac agagcgtaca gtgcgttctc cagggagaag ccttgctgac
     5881 acaggaacgc gagctgattt tccagggttt cgtactgttt ctctgttggg cgggtgccga
     5941 gatgcacttt agccccgtca cgatgtgaga ggagagcaca gcggtatgac ttggcgttgt
     6001 tccgcagaaa gtcttgccat gactcgcctt ccagggggca ggagtgggta tgatgcctgt
     6061 ccagcatctc gattggcagg gcatcgagca gggcccgctt gttcttcacg tgccagtaca
     6121 gggtaggctg ctcaactccc agcttttgag cgagtttcct tgtcgtcagg ccttcgatac
     6181 cgactccatt gagtaattcc agagcagagt ttatgacttt gctcttgtcc agtctagaca
     6241 tcttatcgtc atcgtctttg taatccatgg tggcggatcc cgcgtcacga cacctgtgtt
     6301 ctggcggcaa acccgttgcg aaaaagaacg ttcacggcga ctactgcact tatatacggt
     6361 tctcccccac cctcgggaaa aaggcggagc cagtacacga catcactttc ccagtttacc
     6421 ccgcgccacc ttctctaggc accggttcaa ttgccgaccc ctccccccaa cttctcgggc
     6481 actgtgggcc atgtgcgctc tgccccatgg taatagccat gactaatacg tagatgtact
     6541 gccaagtagg aaagtgccat aaggtcatgt actgggcata atgccaggcg ggccatttac
     6601 cgtcattgac gtcaataggg ggcgtacttg gcatatgata cacttgatgt actgccaagt
     6661 gggcagttta ccgtaaatac tccacccatt gacgtcaatg gaaagtgcct attggcgtta
     6721 ctatgggaac atacgtcatt attgacgtca atgggcgggg gtcgttgggc ggtcagccag
     6781 gcgggccatt taccgtaagt tatgtaacgc ggaactccat atatgggcta tgaactaatg
     6841 accccgtaat tgattactat taataactag tcaataatca atgtcataag ctctaatcgc
     6901 ataatcgagc taagagcgat taatatgctt agctagctta gctaagctag atttctctat
     6961 tctcccacaa tatatagcag ctattagctt agcagtccga atacgatcgt ccacgatcca
     7021 taagcgtaac acatttgagt agcgactgac gggcaccgga gccactcgag tggaatta
//
