LOCUS       sequence-442467-        7147 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac transposon plasmid for doxycycline inducible expression of 
            SAP30.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7147)
  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 7147)
  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-SAP30
FEATURES             Location/Qualifiers
     source          1..7147
                     /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"
     protein_bind    complement(1062..1086)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1088..1114
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1281..1415
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   1687..1721
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1732..1749)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1732..1748)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1741..1763)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1957..1976)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2078..2100
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2139..2157)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2225..2329
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2330..2398,2399..3190)
                     /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(2548..2567)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3361..3949
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3850..3869
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4103..4120
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4326..4347
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4362..4367,4368..4385,4386..4392)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4400..4416
                     /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     4405..4427
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4424..4440
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4424..4440
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4466..4528
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    4704..4785
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5502..5558)
                     /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(5568..6311)
                     /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(6312..6335)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6335..6344
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6364..6575)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        6576..6955
                     /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 gaatggtttt acacccgatg
      421 aaatgtctag aggcggtgac gctgctgcag ctgtcgctgc tgtcgtggcc gctgctgctg
      481 cagcagctag tgccggaaat ggaacaggtg ctggtacggg cgcagaagtc cctggagccg
      541 gagccgtttc tgctgcaggc ccacctggcg ctgcaggccc tggacctggt cagctctgtt
      601 gtctcaggga agacggagaa aggtgtgggc gagccgctgg aaatgcgtct tttagtaaac
      661 gtattcagaa atccatttca caaaagaaag ttaaaattga attagacaaa tccgctcgcc
      721 acctgtatat ctgcgactac cacaagaatc tcatccaatc cgtgaggaat aggcgcaaac
      781 gtaagggctc tgacgacgac ggtggggact cccccgtcca ggacatcgac actcccgaag
      841 tggacctgta tcagctccag gtcaacacgc tgcgccgata taagaggcat tttaaactgc
      901 ccactaggcc gggcctcaac aaggcccagt tggtcgaaat tgtggggtgt catttccgga
      961 gtatcccggt taacgagaag gatacactga cgtactttat atatagcgtt aagaacgata
     1021 agaataagtc tgacctgaaa gtggactccg gcgtccatta aacccagctt tcttgtacaa
     1081 agtggtatac ccatacgatg ttccagatta cgcttagtaa tgagaaatac tcagaagatg
     1141 tcactcacca tagtccgcag tgaaagataa atgatctaaa tgtgtgggta aaagttgttt
     1201 tagagctaga aatagcaagt aggccactga gcaagaacta tgaatgagat ctttagacat
     1261 attgcggccg ctttctccgc gatccagaca tgataagata cattgatgag tttggacaaa
     1321 ccaaaactag aatgcagtga aaaaaatgcc ttatttgtga aatttgtgat gctattgcct
     1381 tatttgtaac cattataagc tgcaataaac aagttatcta taacaagaaa atatatatat
     1441 aataagttat cacgtaagta gaacatgaaa taacaatata attatcgtat gagttaaatc
     1501 ttaaaagtca cgtaaaagat aatcatgcgt cattttgact cacgcggtcg ttatagttca
     1561 aaatcagtga cacttaccgc attgacaagc acgcctcacg ggagctccaa gcggcgactg
     1621 agatgtccta aatgcacagc gacggattcg cgctatttag aaagagagag caatatttca
     1681 agaatgcatg cgtcaatttt acgcagacta tctttctagg gttaagaatt cactggccgt
     1741 cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc
     1801 acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca
     1861 acagttgcgc agcctgaatg gcgaatggcg cctgatgcgg tattttctcc ttacgcatct
     1921 gtgcggtatt tcacaccgca tatggtgcac tctcagtaca atctgctctg atgccgcata
     1981 gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     2041 cccggcatcc gcttacagac aagctgtgac catgtctccg ggagctgcat gtgtcagagg
     2101 ttttcaccgt catcaccgaa acacgcgagt tcgaaagggc ctcgtgatac gcctattttt
     2161 ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa
     2221 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     2281 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     2341 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     2401 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     2461 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     2521 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     2581 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     2641 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     2701 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     2761 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     2821 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     2881 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     2941 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     3001 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtggttcac gcggtatcat
     3061 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     3121 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     3181 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     3241 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     3301 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     3361 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     3421 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     3481 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     3541 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     3601 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     3661 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     3721 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     3781 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     3841 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     3901 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     3961 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     4021 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     4081 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggatg agcgcccaat
     4141 acgcaaaccg cctctcaccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
     4201 tcccgactgg aaagcgggca gtgagcgcaa cgcaaaccgc ctctccccgc gcgttggccg
     4261 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac
     4321 gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg
     4381 gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac
     4441 catgattacg ccaaggtcga cttaacccta gaaagataat catattgtga cgtacgttaa
     4501 agataatcat gcgtaaaatt gacgcatgtg ttttatcggt ctgtatatcg aggtttattt
     4561 attaatttga atagatatta agttttatta tatttacact tacatactaa taataaattc
     4621 aacaaacaat ttatttatgt ttatttattt attaaaaaaa aacaaaaact caaaatttct
     4681 tctataaagt aacaaaactt ttagcagtga aaaaaatgct ttatttgtga aatttgtgat
     4741 gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc
     4801 attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaaag caagtaaaac
     4861 ctctacaaat gtggtatggc tgattatgat cctctggaga ttcatgcgcc aggttttctg
     4921 gtcatgcacc acgttcgtgg gccctcagga acctccacgt cagcggtaac tgtgaagccc
     4981 agtcgctcgt aaaaaggaag attccgggga gcactggttt cgaggaacgc cggcacgcca
     5041 gcccgttccg cggcctcaac accaggaaga acgacagcgc ttcccaaacc tttaccttgg
     5101 tgatctgggc tgacaccgac agtggcaaga aaccacgcag gctcctttgg tctatgagga
     5161 gccagcaagc cctccatttg ctgttgtgcc gccaacctac tccctgacag ctcggccatc
     5221 cgcggaccta tttccgcgaa cactgcgccg gcttcgacgc tttccggagt agtccagacc
     5281 gcaactgcgg caccatcgtc cgcaacccaa actttaccaa tgtccagacc cacgcgcgta
     5341 agaaacagct cttgcaactc agttaccctc tctatgtgtc tgtccggatc tacggtatgg
     5401 cgggtcgctg gataatctgc aaatgctgcg gcaagtgttc ggacagcccg tggtacatcg
     5461 tcacgggtag cgagcctaac agtgggctta tattccgtca tggggccggg gttctcctcc
     5521 acgtcgccgg cctgcttcag caggctgaag ttggtggcgc cgctgccccc ggggagcatg
     5581 tcaaggtcaa aatcgtcaag agcgtcagca ggcagcatat caaggtcaaa gtcgtcaagg
     5641 gcatcggctg ggagcatgtc taagtcaaaa tcgtcaaggg cgtcggtcgg cccgccgctt
     5701 tcgcacttta gctgtttctc caggccacat atgattagtt ccaggccgaa aaggaaggca
     5761 ggttcggctc cctgccggtc gaacagctca attgcttgtt tcagaagtgg gggcatagaa
     5821 tcggtggtag gtgtctctct ttcctctttt gctacttgat gctcctgttc ctccaatacg
     5881 cagcccagtg taaagtggcc cacggcggac agagcgtaca gtgcgttctc cagggagaag
     5941 ccttgctgac acaggaacgc gagctgattt tccagggttt cgtactgttt ctctgttggg
     6001 cgggtgccga gatgcacttt agccccgtca cgatgtgaga ggagagcaca gcggtatgac
     6061 ttggcgttgt tccgcagaaa gtcttgccat gactcgcctt ccagggggca ggagtgggta
     6121 tgatgcctgt ccagcatctc gattggcagg gcatcgagca gggcccgctt gttcttcacg
     6181 tgccagtaca gggtaggctg ctcaactccc agcttttgag cgagtttcct tgtcgtcagg
     6241 ccttcgatac cgactccatt gagtaattcc agagcagagt ttatgacttt gctcttgtcc
     6301 agtctagaca tcttatcgtc atcgtctttg taatccatgg tggcggatcc cgcgtcacga
     6361 cacctgtgtt ctggcggcaa acccgttgcg aaaaagaacg ttcacggcga ctactgcact
     6421 tatatacggt tctcccccac cctcgggaaa aaggcggagc cagtacacga catcactttc
     6481 ccagtttacc ccgcgccacc ttctctaggc accggttcaa ttgccgaccc ctccccccaa
     6541 cttctcgggc actgtgggcc atgtgcgctc tgccccatgg taatagccat gactaatacg
     6601 tagatgtact gccaagtagg aaagtgccat aaggtcatgt actgggcata atgccaggcg
     6661 ggccatttac cgtcattgac gtcaataggg ggcgtacttg gcatatgata cacttgatgt
     6721 actgccaagt gggcagttta ccgtaaatac tccacccatt gacgtcaatg gaaagtgcct
     6781 attggcgtta ctatgggaac atacgtcatt attgacgtca atgggcgggg gtcgttgggc
     6841 ggtcagccag gcgggccatt taccgtaagt tatgtaacgc ggaactccat atatgggcta
     6901 tgaactaatg accccgtaat tgattactat taataactag tcaataatca atgtcataag
     6961 ctctaatcgc ataatcgagc taagagcgat taatatgctt agctagctta gctaagctag
     7021 atttctctat tctcccacaa tatatagcag ctattagctt agcagtccga atacgatcgt
     7081 ccacgatcca taagcgtaac acatttgagt agcgactgac gggcaccgga gccactcgag
     7141 tggaatt
//
