LOCUS       sequence-442400-        7486 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac transposon plasmid for doxycycline inducible expression of 
            BCL2, with hygromycin resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7486)
  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 7486)
  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-cERH-BCL2
FEATURES             Location/Qualifiers
     source          1..7486
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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(1128..1152)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1154..1180
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1194..1328
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   1600..1634
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1645..1662)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1645..1661)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1654..1676)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1870..1889)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1991..2013
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2052..2070)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2138..2242
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2243..2311,2312..3103)
                     /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(2461..2480)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3274..3862
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3763..3782
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4016..4033
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4239..4260
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4275..4280,4281..4298,4299..4305)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4313..4329
                     /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     4318..4340
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4337..4353
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4337..4353
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4379..4441
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    4617..4698
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(4815..5840)
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVHRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(5841..5897)
                     /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(5907..6650)
                     /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
                     DSMPPLLKQAIELFDRQGAEPAFLFGL*LIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     regulatory      6674..6683
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6703..6914)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        6915..7294
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 atcacctcga gtttactcca 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 aaaaagttgg cccccgcggc ggcggcacca tggcgcacgc
      421 tgggagaaca gggtacgata accgggagat agtgatgaag tacatccatt ataagctgtc
      481 gcagaggggc tacgagtggg atgcgggaga tgtgggcgcc gcgcccccgg gggccgcccc
      541 cgcaccgggc atcttctcct cccagcccgg gcacacgccc catccagccg catcccggga
      601 cccggtcgcc aggacctcgc cgctgcagac cccggctgcc cccggcgccg ccgcggggcc
      661 tgcgctcagc ccggtgccac ctgtggtcca cctgaccctc cgccaggccg gcgacgactt
      721 ctcccgccgc taccgccgcg acttcgccga gatgtccagc cagctgcacc tgacgccctt
      781 caccgcgcgg ggacgctttg ccacggtggt ggaggagctc ttcagggacg gggtgaactg
      841 ggggaggatt gtggccttct ttgagttcgg tggggtcatg tgtgtggaga gcgtcaacag
      901 ggagatgtcg cccctggtgg acaacatcgc cctgtggatg actgagtacc tgaaccggca
      961 cctgcacacc tggatccagg ataacggagg ctgggatgcc tttgtggaac tgtacggccc
     1021 cagcatgcgg cctctgtttg atttctcctg gctgtctctg aagactctgc tcagtttggc
     1081 cctggtggga gcttgcatca ccctgggtgc ctatctgggc cacaagttgc caactttctt
     1141 gtacaaagtg gtatacccat acgatgttcc agattacgct taactttctc cgcgatccag
     1201 acatgataag atacattgat gagtttggac aaaccaaaac tagaatgcag tgaaaaaaat
     1261 gccttatttg tgaaatttgt gatgctattg ccttatttgt aaccattata agctgcaata
     1321 aacaagttat ctataacaag aaaatatata tataataagt tatcacgtaa gtagaacatg
     1381 aaataacaat ataattatcg tatgagttaa atcttaaaag tcacgtaaaa gataatcatg
     1441 cgtcattttg actcacgcgg tcgttatagt tcaaaatcag tgacacttac cgcattgaca
     1501 agcacgcctc acgggagctc caagcggcga ctgagatgtc ctaaatgcac agcgacggat
     1561 tcgcgctatt tagaaagaga gagcaatatt tcaagaatgc atgcgtcaat tttacgcaga
     1621 ctatctttct agggttaaga attcactggc cgtcgtttta caacgtcgtg actgggaaaa
     1681 ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa
     1741 tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg
     1801 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatggtg
     1861 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac
     1921 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     1981 gaccatgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacacgcg
     2041 agttcgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
     2101 ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     2161 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     2221 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     2281 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     2341 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     2401 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     2461 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     2521 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     2581 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     2641 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     2701 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     2761 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     2821 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     2881 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     2941 tggagccggt gagcgtggtt cacgcggtat cattgcagca ctggggccag atggtaagcc
     3001 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     3061 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     3121 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     3181 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     3241 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     3301 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     3361 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     3421 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     3481 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     3541 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     3601 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     3661 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     3721 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     3781 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     3841 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     3901 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     3961 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     4021 gtcagtgagc gaggaagcgg atgagcgccc aatacgcaaa ccgcctctca ccgcgcgttg
     4081 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg
     4141 caacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
     4201 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat
     4261 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc
     4321 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaaggt cgacttaacc
     4381 ctagaaagat aatcatattg tgacgtacgt taaagataat catgcgtaaa attgacgcat
     4441 gtgttttatc ggtctgtata tcgaggttta tttattaatt tgaatagata ttaagtttta
     4501 ttatatttac acttacatac taataataaa ttcaacaaac aatttattta tgtttattta
     4561 tttattaaaa aaaaacaaaa actcaaaatt tcttctataa agtaacaaaa cttttagcag
     4621 tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata
     4681 agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg
     4741 gaggtgtggg aggtttttta aagcaagtaa aacctctaca aatgtggtat ggctgattat
     4801 gatcctctgg agatctattc ctttgccctc ggacgagtgc tggggcgtcg gtttccacta
     4861 tcggcgagta cttctacaca gccatcggtc cagacggccg cgcttctgcg ggcgatttgt
     4921 gtacgcccga cagtcccggc tccggatcgg acgattgcgt cgcatcgacc ctgcgcccaa
     4981 gctgcatcat cgaaattgcc gtcaaccaag ctctgataga gttggtcaag accaatgcgg
     5041 agcatatacg cccggagccg cggcgatcct gcaagctccg gatgcctccg ctcgaagtag
     5101 cgcgtctgct gctccataca agccaaccac ggcctccaga agaagatgtt ggcgacctcg
     5161 tattgggaat ccccgaacat cgcctcgctc cagtcaatga ccgctgttat gcggccattg
     5221 tccgtcagga cattgttgga gccgaaatcc gcgtgcacga ggtgccggac ttcggggcag
     5281 tcctcggccc aaagcatcag ctcatcgaga gcctgcgcga cggacgcact gacggtgtcg
     5341 tccatcacag tttgccagtg atacacatgg ggatcagcaa tcgcgcatat gaaatcacgc
     5401 cacgtagtgt attgaccgat tccttgcggt ccgaatgggc cgaacccgct cgtctggcta
     5461 agatcggccg cagcgatcgc atccatggcc tccgcgaccg gctgcagaac agcgggcagt
     5521 tcggtttcag gcaggtcttg caacgtgaca ccctgtgcac ggcgggagat gcaataggtc
     5581 aggctctcgc tgaattcccc aatgtcaagc acttccggaa tcgggagcgc ggccgatgca
     5641 aagtgccgat gaacataacg atctttgtag aaaccatcgg cgcagctatt tacccgcagg
     5701 acatatccac gccctcctac atcgaagctg aaagcacgag attcttcgcc ctccgagagc
     5761 tgcatcaggt cggagacgct gtcgaacttt tcgatcagaa acttctcgac agacgtcgcg
     5821 gtgagttcag gctttttcat ggggccgggg ttctcctcca cgtcgccggc ctgcttcagc
     5881 aggctgaagt tggtggcgcc gctgcccccg gggagcatgt caaggtcaaa atcgtcaaga
     5941 gcgtcagcag gcagcatatc aaggtcaaag tcgtcaaggg catcggctgg gagcatgtct
     6001 aagtcaaaat cgtcaagggc gtcggtcggc ccaccgcttt cgcactttag ctgtttctcc
     6061 aggccacata tgattagtta caggccgaaa aggaaggcag gttcggctcc ctgccggtcg
     6121 aacagctcaa ttgcttgttt cagaagtggg ggcatagaat cggtggtagg tgtctctctt
     6181 tcctcttttg ctacttgatg ctcctgttcc tccaatacgc agcccagtgt aaagtggccc
     6241 acggcggaca gagcgtacag tgcgttctcc agggagaagc cttgctgaca caggaacgcg
     6301 agctgatttt ccagggtttc gtactgtttc tctgttgggc gggtgccgag atgcacttta
     6361 gccccgtcac gatgtgagag gagagcacag cggtatgact tggcgttgtt ccgcagaaag
     6421 tcttgccatg actcgccttc cagggggcag gagtgggtat gatgcctgtc cagcatctcg
     6481 attggcaggg catcgagcag ggcccgcttg ttcttcacgt gccagtacag ggtaggctgc
     6541 tcaactccca gcttttgagc gagtttcctt gtcgtcaggc cttcgatacc gactccattg
     6601 agtaattcca gagcagagtt tatgactttg ctcttgtcca gtctagacat cttatcgtca
     6661 tagtctttgt aatccatggt ggcggatccc gcgtcacgac acctgtgttc tggcggcaaa
     6721 cccgttgcga aaaagaacgt tcacggcgac tactgcactt atatacggtt ctcccccacc
     6781 ctcgggaaaa aggcggagcc agtacacgac atcactttcc cagtttaccc cgcgccacct
     6841 tctctaggca ccggttcaat tgccgacccc tccccccaac ttctcgggca ctgtgggcca
     6901 tgtgcgctct gccccatggt aatagccatg actaatacgt agatgtactg ccaagtagga
     6961 aagtgccata aggtcatgta ctgggcataa tgccaggcgg gccatttacc gtcattgacg
     7021 tcaatagggg gcgtacttgg catatgatac acttgatgta ctgccaagtg ggcagtttac
     7081 cgtaaatact ccacccattg acgtcaatgg aaagtgccta ttggcgttac tatgggaaca
     7141 tacgtcatta ttgacgtcaa tgggcggggg tcgttgggcg gtcagccagg cgggccattt
     7201 accgtaagtt atgtaacgcg gaactccata tatgggctat gaactaatga ccccgtaatt
     7261 gattactatt aataactagt caataatcaa tgtcataagc tctaatcgca taatcgagct
     7321 aagagcgatt aatatgctta gctagcttag ctaagctaga tttctctatt ctcccacaat
     7381 atatagcagc tattagctta gcagtccgaa tacgatcgtc cacgatccat aagcgtaaca
     7441 catttgagta gcgactgacg ggcaccggag ccactcgagt ggaatt
//
