LOCUS       sequence-442452-        8326 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac transposon plasmid for doxycycline inducible expression of 
            RFX5.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8326)
  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 8326)
  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-RFX5
FEATURES             Location/Qualifiers
     source          1..8326
                     /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(2240..2264)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2266..2292
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    2459..2593
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   2865..2899
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2910..2927)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2910..2926)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2919..2941)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3135..3154)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3256..3278
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3317..3335)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3403..3507
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3508..3576,3577..4368)
                     /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(3726..3745)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4539..5127
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5028..5047
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5281..5298
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5504..5525
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5540..5545,5546..5563,5564..5570)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5578..5594
                     /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     5583..5605
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5602..5618
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5602..5618
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   5644..5706
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    5882..5963
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(6680..6736)
                     /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(6746..7489)
                     /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(7490..7513)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      7513..7522
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(7542..7753)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        7754..8133
                     /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 aaaagatggc atggcagaag atgagcctga tgctaagagc
      421 cccaagactg ggggaagggc ccccccaggt ggtgctgagg ctggggaacc taccaccctt
      481 cttcagaggc tccgaggtac catttccaag gccgtgcaga acaaagtaga ggggatcctg
      541 caagatgtac agaaattttc tgacaatgac aagctgtatc tctaccttca gctcccctca
      601 ggacccacca ctggagacaa aagctcagag ccaagtacac tgagcaatga ggagtacatg
      661 tatgcctata ggtggatccg caaccacctg gaagagcaca ctgacacctg tctgccaaag
      721 caaagtgttt atgatgccta tcggaagtac tgtgagagtc ttgcctgttg ccgcccactc
      781 agcacagcca actttggcaa gatcatcaga gagatcttcc ctgacatcaa agctcgaagg
      841 cttggtggcc ggggccagtc caaatattgc tacagtggca taaggaggaa gaccttggtg
      901 tctatgccac ccctgcctgg acttgaccta aagggttctg agagtccaga aatgggccca
      961 gaagtaaccc cagcacctcg agatgaactg gtggaggcag cgtgtgccct gacctgtgac
     1021 tgggcagagc ggatcctgaa acggtccttc agttccatcg ttgaggtcgc ccgcttcctg
     1081 ctacagcagc atctcatctc tgcccgatct gcacatgccc atgtgcttaa ggccatgggg
     1141 cttgctgaag aggacgaaca tgcacctcgg gaacggtcat ctaaaccaaa gaatggttta
     1201 gagaacccag agggtggagc ccacaagaag ccagagagac tggcccagcc tcctaaggat
     1261 ctggaagccc gaactggggc cggtcctctc gcacgtggag agcggaagaa gagtgtagtt
     1321 gagagctcgg ccccaggagc caataacctg caggttaatg ccctagtggc tcggctgcct
     1381 ctgctccttc cccgggcccc tcgctcacta attccgccaa tcccagtctc tccacctatt
     1441 ctggccccca ggctttcttc aggtgccctg aaagtggcta cactgcctct gtctagtagg
     1501 gccggggcac ccccagcagc tgtgcccatc attaacatga tcttaccaac tgttcctgct
     1561 ttgcctggac ctggacctgg gcctgggcga gctccacctg ggggactcac tcagccccgg
     1621 ggcacagaga acagagaggt aggcataggt ggtgaccaag gaccacatga caagggtgtc
     1681 aagaggacag ctgaagtacc tgtgagtgag gccagtgggc aggctccacc agctaaagca
     1741 gcaaagcagg atatagagga tacagcaagt gatgccaaaa ggaaacgggg gcgccctcga
     1801 aaaaagtcag gtggaagtgg ggaaaggaat tctacccctc tcaagtcagc agctgccatg
     1861 gaatctgccc agtcctcaag gttaccatgg gagacatggg gctcaggagg ggaaggcaac
     1921 tcagctggag gggcagagag gccagggcca atgggagagg ctgaaaaggg ggcagtactt
     1981 gcccagggtc agggagatgg tactgtttcc aaaggaggaa ggggccccgg ttcccagcat
     2041 accaaagaag cagaagataa aattcccttg gtcccctcaa aagtgagtgt catcaagggc
     2101 agcagaagcc aaaaggaggc ttttcctttg gcaaagggag aggtagacac tgcaccacag
     2161 ggtaataaag acttaaagga gcatgtgctt caaagttcct tatcccagga gcataaagac
     2221 ccaaaagcaa cacccccatg cccaactttc ttgtacaaag tggtataccc atacgatgtt
     2281 ccagattacg cttagtaatg agaaatactc agaagatgtc actcaccata gtccgcagtg
     2341 aaagataaat gatcaatgac tttgagccaa cagagtttta gagctagaaa tagcaagtag
     2401 gccactgagc aagaactatg aatgagatct ttagacatat tgcggccgct ttctccgcga
     2461 tccagacatg ataagataca ttgatgagtt tggacaaacc aaaactagaa tgcagtgaaa
     2521 aaaatgcctt atttgtgaaa tttgtgatgc tattgcctta tttgtaacca ttataagctg
     2581 caataaacaa gttatctata acaagaaaat atatatataa taagttatca cgtaagtaga
     2641 acatgaaata acaatataat tatcgtatga gttaaatctt aaaagtcacg taaaagataa
     2701 tcatgcgtca ttttgactca cgcggtcgtt atagttcaaa atcagtgaca cttaccgcat
     2761 tgacaagcac gcctcacggg agctccaagc ggcgactgag atgtcctaaa tgcacagcga
     2821 cggattcgcg ctatttagaa agagagagca atatttcaag aatgcatgcg tcaattttac
     2881 gcagactatc tttctagggt taagaattca ctggccgtcg ttttacaacg tcgtgactgg
     2941 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg
     3001 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc
     3061 gaatggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     3121 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg
     3181 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     3241 gctgtgacca tgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     3301 acgcgagttc gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     3361 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     3421 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     3481 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     3541 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     3601 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     3661 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     3721 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     3781 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     3841 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     3901 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     3961 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     4021 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     4081 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     4141 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     4201 aaatctggag ccggtgagcg tggttcacgc ggtatcattg cagcactggg gccagatggt
     4261 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     4321 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     4381 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     4441 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     4501 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     4561 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     4621 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     4681 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     4741 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     4801 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     4861 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     4921 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     4981 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     5041 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     5101 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     5161 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     5221 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     5281 agcgagtcag tgagcgagga agcggatgag cgcccaatac gcaaaccgcc tctcaccgcg
     5341 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
     5401 gagcgcaacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
     5461 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca
     5521 ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg
     5581 tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc aaggtcgact
     5641 taaccctaga aagataatca tattgtgacg tacgttaaag ataatcatgc gtaaaattga
     5701 cgcatgtgtt ttatcggtct gtatatcgag gtttatttat taatttgaat agatattaag
     5761 ttttattata tttacactta catactaata ataaattcaa caaacaattt atttatgttt
     5821 atttatttat taaaaaaaaa caaaaactca aaatttcttc tataaagtaa caaaactttt
     5881 agcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca
     5941 ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc
     6001 agggggaggt gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt ggtatggctg
     6061 attatgatcc tctggagatt catgcgccag gttttctggt catgcaccac gttcgtgggc
     6121 cctcaggaac ctccacgtca gcggtaactg tgaagcccag tcgctcgtaa aaaggaagat
     6181 tccggggagc actggtttcg aggaacgccg gcacgccagc ccgttccgcg gcctcaacac
     6241 caggaagaac gacagcgctt cccaaacctt taccttggtg atctgggctg acaccgacag
     6301 tggcaagaaa ccacgcaggc tcctttggtc tatgaggagc cagcaagccc tccatttgct
     6361 gttgtgccgc caacctactc cctgacagct cggccatccg cggacctatt tccgcgaaca
     6421 ctgcgccggc ttcgacgctt tccggagtag tccagaccgc aactgcggca ccatcgtccg
     6481 caacccaaac tttaccaatg tccagaccca cgcgcgtaag aaacagctct tgcaactcag
     6541 ttaccctctc tatgtgtctg tccggatcta cggtatggcg ggtcgctgga taatctgcaa
     6601 atgctgcggc aagtgttcgg acagcccgtg gtacatcgtc acgggtagcg agcctaacag
     6661 tgggcttata ttccgtcatg gggccggggt tctcctccac gtcgccggcc tgcttcagca
     6721 ggctgaagtt ggtggcgccg ctgcccccgg ggagcatgtc aaggtcaaaa tcgtcaagag
     6781 cgtcagcagg cagcatatca aggtcaaagt cgtcaagggc atcggctggg agcatgtcta
     6841 agtcaaaatc gtcaagggcg tcggtcggcc cgccgctttc gcactttagc tgtttctcca
     6901 ggccacatat gattagttcc aggccgaaaa ggaaggcagg ttcggctccc tgccggtcga
     6961 acagctcaat tgcttgtttc agaagtgggg gcatagaatc ggtggtaggt gtctctcttt
     7021 cctcttttgc tacttgatgc tcctgttcct ccaatacgca gcccagtgta aagtggccca
     7081 cggcggacag agcgtacagt gcgttctcca gggagaagcc ttgctgacac aggaacgcga
     7141 gctgattttc cagggtttcg tactgtttct ctgttgggcg ggtgccgaga tgcactttag
     7201 ccccgtcacg atgtgagagg agagcacagc ggtatgactt ggcgttgttc cgcagaaagt
     7261 cttgccatga ctcgccttcc agggggcagg agtgggtatg atgcctgtcc agcatctcga
     7321 ttggcagggc atcgagcagg gcccgcttgt tcttcacgtg ccagtacagg gtaggctgct
     7381 caactcccag cttttgagcg agtttccttg tcgtcaggcc ttcgataccg actccattga
     7441 gtaattccag agcagagttt atgactttgc tcttgtccag tctagacatc ttatcgtcat
     7501 cgtctttgta atccatggtg gcggatcccg cgtcacgaca cctgtgttct ggcggcaaac
     7561 ccgttgcgaa aaagaacgtt cacggcgact actgcactta tatacggttc tcccccaccc
     7621 tcgggaaaaa ggcggagcca gtacacgaca tcactttccc agtttacccc gcgccacctt
     7681 ctctaggcac cggttcaatt gccgacccct ccccccaact tctcgggcac tgtgggccat
     7741 gtgcgctctg ccccatggta atagccatga ctaatacgta gatgtactgc caagtaggaa
     7801 agtgccataa ggtcatgtac tgggcataat gccaggcggg ccatttaccg tcattgacgt
     7861 caataggggg cgtacttggc atatgataca cttgatgtac tgccaagtgg gcagtttacc
     7921 gtaaatactc cacccattga cgtcaatgga aagtgcctat tggcgttact atgggaacat
     7981 acgtcattat tgacgtcaat gggcgggggt cgttgggcgg tcagccaggc gggccattta
     8041 ccgtaagtta tgtaacgcgg aactccatat atgggctatg aactaatgac cccgtaattg
     8101 attactatta ataactagtc aataatcaat gtcataagct ctaatcgcat aatcgagcta
     8161 agagcgatta atatgcttag ctagcttagc taagctagat ttctctattc tcccacaata
     8221 tatagcagct attagcttag cagtccgaat acgatcgtcc acgatccata agcgtaacac
     8281 atttgagtag cgactgacgg gcaccggagc cactcgagtg gaatta
//
