LOCUS       sequence-442533-        7105 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac transposon plasmid for doxycycline inducible expression of 
            HMGB2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7105)
  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 7105)
  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-HMGB2
FEATURES             Location/Qualifiers
     source          1..7105
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      783..1371
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1272..1291
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1525..1542
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1748..1769
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1784..1789,1790..1807,1808..1814)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1822..1838
                     /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     1827..1849
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1846..1862
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1846..1862
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   1888..1950
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    2126..2207
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(2924..2980)
                     /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(2990..3733)
                     /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(3734..3757)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      3757..3766
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(3786..3997)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        3998..4377
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     protein_bind    4586..4604
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4622..4640
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4658..4676
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4694..4712
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4730..4748
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4766..4784
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4802..4820
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     4847..4871
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    4937..4961
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(5589..5613)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             5615..5641
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    5808..5942
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   6214..6248
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(6259..6276)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6259..6275)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6268..6290)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(6484..6503)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6605..6627
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6666..6684)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6752..6856
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6857..6925,6926..7105,1..612)
                     /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(7075..7094)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
       61 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
      121 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
      181 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
      241 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
      301 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
      361 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
      421 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtggttc acgcggtatc
      481 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
      541 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
      601 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
      661 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
      721 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
      781 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
      841 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
      901 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
      961 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     1021 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     1081 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     1141 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     1201 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     1261 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     1321 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     1381 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     1441 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     1501 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga tgagcgccca
     1561 atacgcaaac cgcctctcac cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
     1621 tttcccgact ggaaagcggg cagtgagcgc aacgcaaacc gcctctcccc gcgcgttggc
     1681 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     1741 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
     1801 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
     1861 accatgatta cgccaaggtc gacttaaccc tagaaagata atcatattgt gacgtacgtt
     1921 aaagataatc atgcgtaaaa ttgacgcatg tgttttatcg gtctgtatat cgaggtttat
     1981 ttattaattt gaatagatat taagttttat tatatttaca cttacatact aataataaat
     2041 tcaacaaaca atttatttat gtttatttat ttattaaaaa aaaacaaaaa ctcaaaattt
     2101 cttctataaa gtaacaaaac ttttagcagt gaaaaaaatg ctttatttgt gaaatttgtg
     2161 atgctattgc tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt
     2221 gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa agcaagtaaa
     2281 acctctacaa atgtggtatg gctgattatg atcctctgga gattcatgcg ccaggttttc
     2341 tggtcatgca ccacgttcgt gggccctcag gaacctccac gtcagcggta actgtgaagc
     2401 ccagtcgctc gtaaaaagga agattccggg gagcactggt ttcgaggaac gccggcacgc
     2461 cagcccgttc cgcggcctca acaccaggaa gaacgacagc gcttcccaaa cctttacctt
     2521 ggtgatctgg gctgacaccg acagtggcaa gaaaccacgc aggctccttt ggtctatgag
     2581 gagccagcaa gccctccatt tgctgttgtg ccgccaacct actccctgac agctcggcca
     2641 tccgcggacc tatttccgcg aacactgcgc cggcttcgac gctttccgga gtagtccaga
     2701 ccgcaactgc ggcaccatcg tccgcaaccc aaactttacc aatgtccaga cccacgcgcg
     2761 taagaaacag ctcttgcaac tcagttaccc tctctatgtg tctgtccgga tctacggtat
     2821 ggcgggtcgc tggataatct gcaaatgctg cggcaagtgt tcggacagcc cgtggtacat
     2881 cgtcacgggt agcgagccta acagtgggct tatattccgt catggggccg gggttctcct
     2941 ccacgtcgcc ggcctgcttc agcaggctga agttggtggc gccgctgccc ccggggagca
     3001 tgtcaaggtc aaaatcgtca agagcgtcag caggcagcat atcaaggtca aagtcgtcaa
     3061 gggcatcggc tgggagcatg tctaagtcaa aatcgtcaag ggcgtcggtc ggcccgccgc
     3121 tttcgcactt tagctgtttc tccaggccac atatgattag ttccaggccg aaaaggaagg
     3181 caggttcggc tccctgccgg tcgaacagct caattgcttg tttcagaagt gggggcatag
     3241 aatcggtggt aggtgtctct ctttcctctt ttgctacttg atgctcctgt tcctccaata
     3301 cgcagcccag tgtaaagtgg cccacggcgg acagagcgta cagtgcgttc tccagggaga
     3361 agccttgctg acacaggaac gcgagctgat tttccagggt ttcgtactgt ttctctgttg
     3421 ggcgggtgcc gagatgcact ttagccccgt cacgatgtga gaggagagca cagcggtatg
     3481 acttggcgtt gttccgcaga aagtcttgcc atgactcgcc ttccaggggg caggagtggg
     3541 tatgatgcct gtccagcatc tcgattggca gggcatcgag cagggcccgc ttgttcttca
     3601 cgtgccagta cagggtaggc tgctcaactc ccagcttttg agcgagtttc cttgtcgtca
     3661 ggccttcgat accgactcca ttgagtaatt ccagagcaga gtttatgact ttgctcttgt
     3721 ccagtctaga catcttatcg tcatcgtctt tgtaatccat ggtggcggat cccgcgtcac
     3781 gacacctgtg ttctggcggc aaacccgttg cgaaaaagaa cgttcacggc gactactgca
     3841 cttatatacg gttctccccc accctcggga aaaaggcgga gccagtacac gacatcactt
     3901 tcccagttta ccccgcgcca ccttctctag gcaccggttc aattgccgac ccctcccccc
     3961 aacttctcgg gcactgtggg ccatgtgcgc tctgccccat ggtaatagcc atgactaata
     4021 cgtagatgta ctgccaagta ggaaagtgcc ataaggtcat gtactgggca taatgccagg
     4081 cgggccattt accgtcattg acgtcaatag ggggcgtact tggcatatga tacacttgat
     4141 gtactgccaa gtgggcagtt taccgtaaat actccaccca ttgacgtcaa tggaaagtgc
     4201 ctattggcgt tactatggga acatacgtca ttattgacgt caatgggcgg gggtcgttgg
     4261 gcggtcagcc aggcgggcca tttaccgtaa gttatgtaac gcggaactcc atatatgggc
     4321 tatgaactaa tgaccccgta attgattact attaataact agtcaataat caatgtcata
     4381 agctctaatc gcataatcga gctaagagcg attaatatgc ttagctagct tagctaagct
     4441 agatttctct attctcccac aatatatagc agctattagc ttagcagtcc gaatacgatc
     4501 gtccacgatc cataagcgta acacatttga gtagcgactg acgggcaccg gagccactcg
     4561 agtggaatta tcacctcgag tttactccct atcagtgata gagaacgtat gaagagttta
     4621 ctccctatca gtgatagaga acgtatgcag actttactcc ctatcagtga tagagaacgt
     4681 ataaggagtt tactccctat cagtgataga gaacgtatga ccagtttact ccctatcagt
     4741 gatagagaac gtatctacag tttactccct atcagtgata gagaacgtat atccagttta
     4801 ctccctatca gtgatagaga acgtataagc ttgcctatat aagcagagct cgtttagtga
     4861 accgtcagat cgcctggaga cgccatccac gctgttttga cctccataga agacaccggg
     4921 accgatccag cctaccacaa gtttgtacaa aaaagttggc atgggtaaag gagaccccaa
     4981 caagccgcgg ggcaaaatgt cctcgtacgc cttcttcgtg cagacctgcc gggaagagca
     5041 caagaagaaa cacccggact cttccgtcaa tttcgcggaa ttctccaaga agtgttcgga
     5101 gagatggaag accatgtctg caaaggagaa gtcgaagttt gaagatatgg caaaaagtga
     5161 caaagctcgc tatgacaggg agatgaaaaa ttacgttcct cccaaaggtg ataagaaggg
     5221 gaagaaaaag gaccccaatg ctcctaaaag gccaccatct gccttcttcc tgttttgctc
     5281 tgaacatcgc ccaaagatca aaagtgaaca ccctggccta tccattgggg atactgcaaa
     5341 gaaattgggt gaaatgtggt ctgagcagtc agccaaagat aaacaaccat atgaacagaa
     5401 agcagctaag ctaaaggaga aatatgaaaa ggatattgct gcatatcgtg ccaagggcaa
     5461 aagtgaagca ggaaagaagg gccctggcag gccaacaggc tcaaagaaga agaacgaacc
     5521 agaagatgag gaggaggagg aggaagaaga agatgaagat gaggaggaag aggatgaaga
     5581 tgaagaatac ccaactttct tgtacaaagt ggtataccca tacgatgttc cagattacgc
     5641 ttagtaatga gaaatactca gaagatgtca ctcaccatag tccgcagtga aagataaatg
     5701 atcttgtaag gcacaccgag tctgttttag agctagaaat agcaagtagg ccactgagca
     5761 agaactatga atgagatctt tagacatatt gcggccgctt tctccgcgat ccagacatga
     5821 taagatacat tgatgagttt ggacaaacca aaactagaat gcagtgaaaa aaatgcctta
     5881 tttgtgaaat ttgtgatgct attgccttat ttgtaaccat tataagctgc aataaacaag
     5941 ttatctataa caagaaaata tatatataat aagttatcac gtaagtagaa catgaaataa
     6001 caatataatt atcgtatgag ttaaatctta aaagtcacgt aaaagataat catgcgtcat
     6061 tttgactcac gcggtcgtta tagttcaaaa tcagtgacac ttaccgcatt gacaagcacg
     6121 cctcacggga gctccaagcg gcgactgaga tgtcctaaat gcacagcgac ggattcgcgc
     6181 tatttagaaa gagagagcaa tatttcaaga atgcatgcgt caattttacg cagactatct
     6241 ttctagggtt aagaattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg
     6301 cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga
     6361 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcgcct
     6421 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat ggtgcactct
     6481 cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc
     6541 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccat
     6601 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaaca cgcgagttcg
     6661 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta
     6721 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     6781 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     6841 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     6901 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     6961 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     7021 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     7081 tggcgcggta ttatcccgta ttgac
//
