LOCUS       sequence-437875-        7372 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac transposon plasmid for doxycycline inducible expression of 
            DAZL.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7372)
  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 7372)
  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-DAZL
FEATURES             Location/Qualifiers
     source          1..7372
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    634..715
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   891..953
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(979..995)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(979..995)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(992..1014)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1003..1019
                     /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)."
     promoter        complement(join(1027..1033,1034..1051,1052..1057))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1072..1093
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1299..1316)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1470..2058)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1550..1569)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2229..3020,3021..3089))
                     /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     2852..2871
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3090..3194)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3262..3280
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3319..3341)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3443..3462
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3656..3678
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3670..3687
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3671..3687
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   3698..3732
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    4004..4138
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(4305..4331)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    4333..4357
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(5252..5276)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(5342..5366)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    5393..5411
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5429..5447
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5465..5483
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5501..5519
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5537..5555
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5573..5591
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5609..5627
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     enhancer        5836..6215
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6216..6427
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      6447..6456
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6456..6479
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6480..7223
                     /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             7233..7289
                     /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"
ORIGIN
        1 agattatcca gcgacccgcc ataccgtaga tccggacaga cacatagaga gggtaactga
       61 gttgcaagag ctgtttctta cgcgcgtggg tctggacatt ggtaaagttt gggttgcgga
      121 cgatggtgcc gcagttgcgg tctggactac tccggaaagc gtcgaagccg gcgcagtgtt
      181 cgcggaaata ggtccgcgga tggccgagct gtcagggagt aggttggcgg cacaacagca
      241 aatggagggc ttgctggctc ctcatagacc aaaggagcct gcgtggtttc ttgccactgt
      301 cggtgtcagc ccagatcacc aaggtaaagg tttgggaagc gctgtcgttc ttcctggtgt
      361 tgaggccgcg gaacgggctg gcgtgccggc gttcctcgaa accagtgctc cccggaatct
      421 tcctttttac gagcgactgg gcttcacagt taccgctgac gtggaggttc ctgagggccc
      481 acgaacgtgg tgcatgacca gaaaacctgg cgcatgaatc tccagaggat cataatcagc
      541 cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac
      601 ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt
      661 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgctaaaa
      721 gttttgttac tttatagaag aaattttgag tttttgtttt tttttaataa ataaataaac
      781 ataaataaat tgtttgttga atttattatt agtatgtaag tgtaaatata ataaaactta
      841 atatctattc aaattaataa ataaacctcg atatacagac cgataaaaca catgcgtcaa
      901 ttttacgcat gattatcttt aacgtacgtc acaatatgat tatctttcta gggttaagtc
      961 gaccttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     1021 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     1081 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     1141 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgtt gcgctcactg
     1201 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     1261 gtgagaggcg gtttgcgtat tgggcgctca tccgcttcct cgctcactga ctcgctgcgc
     1321 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     1381 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     1441 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     1501 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     1561 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     1621 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     1681 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     1741 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     1801 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     1861 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     1921 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     1981 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     2041 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     2101 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     2161 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     2221 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     2281 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     2341 tctggcccca gtgctgcaat gataccgcgt gaaccacgct caccggctcc agatttatca
     2401 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     2461 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     2521 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     2581 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     2641 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     2701 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     2761 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     2821 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     2881 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     2941 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     3001 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     3061 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     3121 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     3181 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt
     3241 atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgaac tcgcgtgttt
     3301 cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacatggt cacagcttgt
     3361 ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg
     3421 tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg
     3481 cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ccattcgcca
     3541 ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag
     3601 ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag
     3661 tcacgacgtt gtaaaacgac ggccagtgaa ttcttaaccc tagaaagata gtctgcgtaa
     3721 aattgacgca tgcattcttg aaatattgct ctctctttct aaatagcgcg aatccgtcgc
     3781 tgtgcattta ggacatctca gtcgccgctt ggagctcccg tgaggcgtgc ttgtcaatgc
     3841 ggtaagtgtc actgattttg aactataacg accgcgtgag tcaaaatgac gcatgattat
     3901 cttttacgtg acttttaaga tttaactcat acgataatta tattgttatt tcatgttcta
     3961 cttacgtgat aacttattat atatatattt tcttgttata gataacttgt ttattgcagc
     4021 ttataatggt tacaaataag gcaatagcat cacaaatttc acaaataagg catttttttc
     4081 actgcattct agttttggtt tgtccaaact catcaatgta tcttatcatg tctggatcgc
     4141 ggagaaagcg gccgcaatat gtctaaagat ctcattcata gttcttgctc agtggcctac
     4201 ttgctatttc tagctctaaa acgtttcgct tacagacaaa ctgatcattt atctttcact
     4261 gcggactatg gtgagtgaca tcttctgagt atttctcatt actaagcgta atctggaaca
     4321 tcgtatgggt ataccacttt gtacaagaaa gctgggttca aacagattta agcattgccc
     4381 gacttcttct aaagtgatgc actcttttat ccttgaagta gtcatcttga gtaacaacag
     4441 agtttctcag tctgttctct ggattaaaca gacaagatac caccgtttgt atgcttcggt
     4501 ccacagattt cttttgtggg ccatttccag agggtggagt agcttcatga actgaacatt
     4561 catttggcac aacttcagct cctggatcaa cttcattaca gtggtagtta acagctgaat
     4621 aagccggagg tacaacatag ctcctttgct ccccaacagg ccactgtggt ggcatctgat
     4681 aattatatac aggcaactga tatccagtga tgacctgaac tggtgaattt gggtaagtag
     4741 gatatgcctg aacatactga gttataggat tcatcgtggt tgtgggctgc atataagttt
     4801 cagtgtttgg attagtccag acattctgaa actgtggtgg aggaggatga ttaaaaacca
     4861 aaggacgtgg ctgcacatga taagcacata aattttgttt cctgattgca gggcccagct
     4921 tcagcttttt accatggaaa tttatctgtg attctactat cttctgcaca tccacgtcat
     4981 taaaaaatga aacaaatcca tagcctttgg acacaccagt tcgatcagtg attatcttca
     5041 cttctttcac tgaaccatat ctagcaaaga agcttctaat ctcagtttca tccatcctaa
     5101 catcaattcc tccaacaaaa acagtgtttg gcatgatttt gccttctggt aaaatatagc
     5161 cttggctggt tgcagctgat gaggactggg tgctggcctc tctggagatg gttgagtttg
     5221 gagtttcagg atttgcagta gacatggtgg cagcctgctt ttttgtacaa acttgtggta
     5281 ggctggatcg gtcccggtgt cttctatgga ggtcaaaaca gcgtggatgg cgtctccagg
     5341 cgatctgacg gttcactaaa cgagctctgc ttatataggc aagcttatac gttctctatc
     5401 actgataggg agtaaactgg atatacgttc tctatcactg atagggagta aactgtagat
     5461 acgttctcta tcactgatag ggagtaaact ggtcatacgt tctctatcac tgatagggag
     5521 taaactcctt atacgttctc tatcactgat agggagtaaa gtctgcatac gttctctatc
     5581 actgataggg agtaaactct tcatacgttc tctatcactg atagggagta aactcgaggt
     5641 gataattcca ctcgagtggc tccggtgccc gtcagtcgct actcaaatgt gttacgctta
     5701 tggatcgtgg acgatcgtat tcggactgct aagctaatag ctgctatata ttgtgggaga
     5761 atagagaaat ctagcttagc taagctagct aagcatatta atcgctctta gctcgattat
     5821 gcgattagag cttatgacat tgattattga ctagttatta atagtaatca attacggggt
     5881 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
     5941 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
     6001 taacgccaat aggcactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
     6061 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg
     6121 gtaaatggcc cgcctggcat tatgcccagt acatgacctt atggcacttt cctacttggc
     6181 agtacatcta cgtattagtc atggctatta ccatggggca gagcgcacat ggcccacagt
     6241 gcccgagaag ttggggggag gggtcggcaa ttgaaccggt gcctagagaa ggtggcgcgg
     6301 ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg gtgggggaga
     6361 accgtatata agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag
     6421 aacacaggtg tcgtgacgcg ggatccgcca ccatggatta caaagacgat gacgataaga
     6481 tgtctagact ggacaagagc aaagtcataa actctgctct ggaattactc aatggagtcg
     6541 gtatcgaagg cctgacgaca aggaaactcg ctcaaaagct gggagttgag cagcctaccc
     6601 tgtactggca cgtgaagaac aagcgggccc tgctcgatgc cctgccaatc gagatgctgg
     6661 acaggcatca tacccactcc tgccccctgg aaggcgagtc atggcaagac tttctgcgga
     6721 acaacgccaa gtcataccgc tgtgctctcc tctcacatcg tgacggggct aaagtgcatc
     6781 tcggcacccg cccaacagag aaacagtacg aaaccctgga aaatcagctc gcgttcctgt
     6841 gtcagcaagg cttctccctg gagaacgcac tgtacgctct gtccgccgtg ggccacttta
     6901 cactgggctg cgtattggag gaacaggagc atcaagtagc aaaagaggaa agagagacac
     6961 ctaccaccga ttctatgccc ccacttctga aacaagcaat tgagctgttc gaccggcagg
     7021 gagccgaacc tgccttcctt ttcggcctgg aactaatcat atgtggcctg gagaaacagc
     7081 taaagtgcga aagcggcggg ccgaccgacg cccttgacga ttttgactta gacatgctcc
     7141 cagccgatgc ccttgacgac tttgaccttg atatgctgcc tgctgacgct cttgacgatt
     7201 ttgaccttga catgctcccc gggggcagcg gcgccaccaa cttcagcctg ctgaagcagg
     7261 ccggcgacgt ggaggagaac cccggcccca tgacggaata taagcccact gttaggctcg
     7321 ctacccgtga cgatgtacca cgggctgtcc gaacacttgc cgcagcattt gc
//
