LOCUS       sequence-442409-        7909 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac transposon plasmid for doxycycline inducible expression of 
            ZCWPW1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7909)
  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 7909)
  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-ZCWPW1
FEATURES             Location/Qualifiers
     source          1..7909
                     /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(1823..1847)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1849..1875
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    2042..2176
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   2448..2482
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2493..2510)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2493..2509)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2502..2524)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2718..2737)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2839..2861
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2900..2918)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2986..3090
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3091..3159,3160..3951)
                     /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(3309..3328)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4122..4710
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4611..4630
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4864..4881
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5087..5108
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5123..5128,5129..5146,5147..5153)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5161..5177
                     /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     5166..5188
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5185..5201
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5185..5201
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   5227..5289
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    5465..5546
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(6263..6319)
                     /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(6329..7072)
                     /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(7073..7096)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      7096..7105
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(7125..7336)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        7337..7716
                     /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 atgggatctg gccttgattt tgcagagact
      421 tcttgtgccc agcccgtagt atctacccaa tcagacaagg agccaggaat tactgcttct
      481 gctgctgata ctgataatgc taatggagag gaggtaccac atactcaaga gatttcagtg
      541 tcttgggaag gtgaagctgc ccctgagata aggacatcta agttaggcca gccagatcct
      601 gcaccctcta agaagaaatc caatagactc accttaagca aaagaaagaa ggaagctcaa
      661 gatgagaagg tggagaaaac tcaaggtgga catgagcaca gacaggaaga ccgactaaag
      721 aaaacagttc aggatcattc tcagatcagg gaccagcaaa aaggagagat aagtggtttt
      781 ggtcaatgtc tggtctgggt ccagtgttcc ttcccaaact gtgggaaatg gaggcggctg
      841 tgtgggaaca ttgacccctc agttctccca gataattggt cctgtgatca gaacacagat
      901 gtgcagtata atcgctgtga tattcctgag gagacctgga cagggcttga gagtgatgtg
      961 gcctatgcct cctacatccc aggatccatc atctgggcca agcaatacgg ttacccctgg
     1021 tggccaggca tgatagaatc tgatcctgac ttaggggaat attttctttt tacttcccat
     1081 cttgattccc tgccgtctaa gtaccatgtg acgttttttg gagaaacagt ttctcgtgca
     1141 tggatcccag tcaacatgct aaagaacttc caggagctgt ccctggagct atcagtcatg
     1201 aaaaagcgca gaaatgactg cagccagaaa ctgggggtgg ccctgatgat ggctcaagag
     1261 gcagaacaga tcagcattca ggaacgggtt aacttgtttg gtttctggag ccgattcaac
     1321 ggatctaaca gtaatgggga aagaaaagac ttacagctct ctggtttgaa cagcccagga
     1381 tcctgcttag agaaaaagga gaaagaggaa gagttggaaa aggaggaagg agagaaaaca
     1441 gacccaattt tgcccattcg taagcgagtc aaaatacaga cccaaaaaac caagccaaga
     1501 ggccctaaga aaaaatttaa agctccccag agcaaggcct tggcagccag cttttcagag
     1561 ggaaaagaag ttagaacagt gccaaagaac ctgggcctat cagcgtgtaa gggggcctgc
     1621 ccctcatctg cgaaagaaga gcccagacac cgggaacccc tgacccagga ggctggaagt
     1681 gtcccccttg aggacgaagc ctccagtgac ctggacctgg agcaactcat ggaagatgtt
     1741 gggagagagc tggggcagag cggggagctg cagcacagca acagtgatgg cgaggacttc
     1801 cccgtggcgc tgtttgggaa gtacccaact ttcttgtaca aagtggtata cccatacgat
     1861 gttccagatt acgcttagta atgagaaata ctcagaagat gtcactcacc atagtccgca
     1921 gtgaaagata aatgatcagc aggtttatct gatgcaggtt ttagagctag aaatagcaag
     1981 taggccactg agcaagaact atgaatgaga tctttagaca tattgcggcc gctttctccg
     2041 cgatccagac atgataagat acattgatga gtttggacaa accaaaacta gaatgcagtg
     2101 aaaaaaatgc cttatttgtg aaatttgtga tgctattgcc ttatttgtaa ccattataag
     2161 ctgcaataaa caagttatct ataacaagaa aatatatata taataagtta tcacgtaagt
     2221 agaacatgaa ataacaatat aattatcgta tgagttaaat cttaaaagtc acgtaaaaga
     2281 taatcatgcg tcattttgac tcacgcggtc gttatagttc aaaatcagtg acacttaccg
     2341 cattgacaag cacgcctcac gggagctcca agcggcgact gagatgtcct aaatgcacag
     2401 cgacggattc gcgctattta gaaagagaga gcaatatttc aagaatgcat gcgtcaattt
     2461 tacgcagact atctttctag ggttaagaat tcactggccg tcgttttaca acgtcgtgac
     2521 tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc
     2581 tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat
     2641 ggcgaatggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     2701 atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac
     2761 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     2821 caagctgtga ccatgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     2881 aacacgcgag ttcgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     2941 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     3001 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     3061 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     3121 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     3181 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     3241 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     3301 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     3361 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     3421 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     3481 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     3541 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     3601 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     3661 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     3721 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     3781 gataaatctg gagccggtga gcgtggttca cgcggtatca ttgcagcact ggggccagat
     3841 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     3901 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     3961 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     4021 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     4081 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     4141 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     4201 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     4261 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     4321 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     4381 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     4441 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     4501 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     4561 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     4621 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     4681 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     4741 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     4801 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     4861 cgcagcgagt cagtgagcga ggaagcggat gagcgcccaa tacgcaaacc gcctctcacc
     4921 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     4981 agtgagcgca acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
     5041 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
     5101 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
     5161 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaaggtcg
     5221 acttaaccct agaaagataa tcatattgtg acgtacgtta aagataatca tgcgtaaaat
     5281 tgacgcatgt gttttatcgg tctgtatatc gaggtttatt tattaatttg aatagatatt
     5341 aagttttatt atatttacac ttacatacta ataataaatt caacaaacaa tttatttatg
     5401 tttatttatt tattaaaaaa aaacaaaaac tcaaaatttc ttctataaag taacaaaact
     5461 tttagcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa
     5521 ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg
     5581 ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtatgg
     5641 ctgattatga tcctctggag attcatgcgc caggttttct ggtcatgcac cacgttcgtg
     5701 ggccctcagg aacctccacg tcagcggtaa ctgtgaagcc cagtcgctcg taaaaaggaa
     5761 gattccgggg agcactggtt tcgaggaacg ccggcacgcc agcccgttcc gcggcctcaa
     5821 caccaggaag aacgacagcg cttcccaaac ctttaccttg gtgatctggg ctgacaccga
     5881 cagtggcaag aaaccacgca ggctcctttg gtctatgagg agccagcaag ccctccattt
     5941 gctgttgtgc cgccaaccta ctccctgaca gctcggccat ccgcggacct atttccgcga
     6001 acactgcgcc ggcttcgacg ctttccggag tagtccagac cgcaactgcg gcaccatcgt
     6061 ccgcaaccca aactttacca atgtccagac ccacgcgcgt aagaaacagc tcttgcaact
     6121 cagttaccct ctctatgtgt ctgtccggat ctacggtatg gcgggtcgct ggataatctg
     6181 caaatgctgc ggcaagtgtt cggacagccc gtggtacatc gtcacgggta gcgagcctaa
     6241 cagtgggctt atattccgtc atggggccgg ggttctcctc cacgtcgccg gcctgcttca
     6301 gcaggctgaa gttggtggcg ccgctgcccc cggggagcat gtcaaggtca aaatcgtcaa
     6361 gagcgtcagc aggcagcata tcaaggtcaa agtcgtcaag ggcatcggct gggagcatgt
     6421 ctaagtcaaa atcgtcaagg gcgtcggtcg gcccgccgct ttcgcacttt agctgtttct
     6481 ccaggccaca tatgattagt tccaggccga aaaggaaggc aggttcggct ccctgccggt
     6541 cgaacagctc aattgcttgt ttcagaagtg ggggcataga atcggtggta ggtgtctctc
     6601 tttcctcttt tgctacttga tgctcctgtt cctccaatac gcagcccagt gtaaagtggc
     6661 ccacggcgga cagagcgtac agtgcgttct ccagggagaa gccttgctga cacaggaacg
     6721 cgagctgatt ttccagggtt tcgtactgtt tctctgttgg gcgggtgccg agatgcactt
     6781 tagccccgtc acgatgtgag aggagagcac agcggtatga cttggcgttg ttccgcagaa
     6841 agtcttgcca tgactcgcct tccagggggc aggagtgggt atgatgcctg tccagcatct
     6901 cgattggcag ggcatcgagc agggcccgct tgttcttcac gtgccagtac agggtaggct
     6961 gctcaactcc cagcttttga gcgagtttcc ttgtcgtcag gccttcgata ccgactccat
     7021 tgagtaattc cagagcagag tttatgactt tgctcttgtc cagtctagac atcttatcgt
     7081 catcgtcttt gtaatccatg gtggcggatc ccgcgtcacg acacctgtgt tctggcggca
     7141 aacccgttgc gaaaaagaac gttcacggcg actactgcac ttatatacgg ttctccccca
     7201 ccctcgggaa aaaggcggag ccagtacacg acatcacttt cccagtttac cccgcgccac
     7261 cttctctagg caccggttca attgccgacc cctcccccca acttctcggg cactgtgggc
     7321 catgtgcgct ctgccccatg gtaatagcca tgactaatac gtagatgtac tgccaagtag
     7381 gaaagtgcca taaggtcatg tactgggcat aatgccaggc gggccattta ccgtcattga
     7441 cgtcaatagg gggcgtactt ggcatatgat acacttgatg tactgccaag tgggcagttt
     7501 accgtaaata ctccacccat tgacgtcaat ggaaagtgcc tattggcgtt actatgggaa
     7561 catacgtcat tattgacgtc aatgggcggg ggtcgttggg cggtcagcca ggcgggccat
     7621 ttaccgtaag ttatgtaacg cggaactcca tatatgggct atgaactaat gaccccgtaa
     7681 ttgattacta ttaataacta gtcaataatc aatgtcataa gctctaatcg cataatcgag
     7741 ctaagagcga ttaatatgct tagctagctt agctaagcta gatttctcta ttctcccaca
     7801 atatatagca gctattagct tagcagtccg aatacgatcg tccacgatcc ataagcgtaa
     7861 cacatttgag tagcgactga cgggcaccgg agccactcga gtggaatta
//
