LOCUS       sequence-437997-        6986 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac transposon plasmid for doxycycline inducible expression of 
            RAD51.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6986)
  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 6986)
  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-RAD51
FEATURES             Location/Qualifiers
     source          1..6986
                     /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    386..410
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      413..422
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(1440..1464)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1466..1492
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1659..1793
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   2065..2099
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2110..2127)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2110..2126)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2119..2141)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2335..2354)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2456..2478
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2517..2535)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2603..2707
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2708..2776,2777..3568)
                     /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(2926..2945)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3739..4327
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4228..4247
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4481..4498
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4704..4725
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4740..4745,4746..4763,4764..4770)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4778..4794
                     /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     4783..4805
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4802..4818
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4802..4818
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4844..4906
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    5082..5163
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5880..5936)
                     /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(5946..6689)
                     /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(6690..6713)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6713..6722
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6742..6953)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
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 gcctccgcgg tggcggcatc ggtacacaag tttgtacaaa aaagcaggct tagccaccat
      421 ggcgatgcag atgcagttgg aagcgaatgc agatactagt gtcgaggaag agtcatttgg
      481 cccgcaaccc atctcgcgtt tagagcaatg tggcatcaat gcaaacgatg tgaaaaaatt
      541 agaggaagct ggattccaca cggtcgaagc ggtcgcatac gcaccgaaaa aagagctgat
      601 caacatcaaa ggcatcagcg aggcgaaagc cgataagatt cttgcagagg cggcgaaatt
      661 agttcccatg ggatttacga cggcgactga gttccatcaa cgtcgttccg agatcattca
      721 aatcacgacc ggaagcaagg agttggataa actgcttcaa ggaggcatcg aaactggctc
      781 gatcactgaa atgtttggag agtttcgtac tggcaaaacc cagatctgcc acacattggc
      841 cgtcacttgc cagcttccga tcgatcgtgg tgggggcgag gggaaggcca tgtacattga
      901 tactgagggg acgtttcgtc ccgaacgcct tcttgcggtg gcagagcgtt acggcttgtc
      961 agggtctgat gttcttgaca atgttgccta tgctcgcgca tttaataccg accaccagac
     1021 gcagcttctg taccaagcaa gcgcgatgat ggtagagtct cgttacgctt tattgattgt
     1081 tgactcagca acagcgttgt accgcacaga ttattctggg cgcggcgaat tgtcggcacg
     1141 ccaaatgcat ttagcccgct ttttgcgtat gttgctgcgc cttgcagacg aatttggggt
     1201 ggctgtggtg atcaccaacc aagttgtagc acaggtggac ggtgccgcta tgtttgccgc
     1261 tgatccgaaa aaacccatcg gagggaatat tatcgcacat gcatcgacaa ctcgcttata
     1321 tctgcgtaag ggccgtggag aaacacgcat ctgcaaaatc tatgactccc catgcctgcc
     1381 ggaggcggaa gccatgttcg caattaacgc agatggcgta ggggacgcca aagattgata
     1441 cccagctttc ttgtacaaag tggtataccc atacgatgtt ccagattacg cttagtaatg
     1501 agaaatactc agaagatgtc actcaccata gtccgcagtg aaagataaat gatcaagacc
     1561 atctgactca aacagtttta gagctagaaa tagcaagtag gccactgagc aagaactatg
     1621 aatgagatct ttagacatat tgcggccgct ttctccgcga tccagacatg ataagataca
     1681 ttgatgagtt tggacaaacc aaaactagaa tgcagtgaaa aaaatgcctt atttgtgaaa
     1741 tttgtgatgc tattgcctta tttgtaacca ttataagctg caataaacaa gttatctata
     1801 acaagaaaat atatatataa taagttatca cgtaagtaga acatgaaata acaatataat
     1861 tatcgtatga gttaaatctt aaaagtcacg taaaagataa tcatgcgtca ttttgactca
     1921 cgcggtcgtt atagttcaaa atcagtgaca cttaccgcat tgacaagcac gcctcacggg
     1981 agctccaagc ggcgactgag atgtcctaaa tgcacagcga cggattcgcg ctatttagaa
     2041 agagagagca atatttcaag aatgcatgcg tcaattttac gcagactatc tttctagggt
     2101 taagaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
     2161 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
     2221 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc tgatgcggta
     2281 ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc tcagtacaat
     2341 ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc
     2401 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgacca tgtctccggg
     2461 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac acgcgagttc gaaagggcct
     2521 cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg
     2581 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     2641 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     2701 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     2761 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     2821 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     2881 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     2941 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     3001 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     3061 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     3121 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     3181 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     3241 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     3301 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     3361 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     3421 tggttcacgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     3481 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     3541 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     3601 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     3661 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     3721 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     3781 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     3841 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     3901 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     3961 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     4021 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     4081 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     4141 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     4201 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     4261 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     4321 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     4381 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     4441 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     4501 agcggatgag cgcccaatac gcaaaccgcc tctcaccgcg cgttggccga ttcattaatg
     4561 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caaaccgcct
     4621 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
     4681 gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct
     4741 ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac
     4801 acaggaaaca gctatgacca tgattacgcc aaggtcgact taaccctaga aagataatca
     4861 tattgtgacg tacgttaaag ataatcatgc gtaaaattga cgcatgtgtt ttatcggtct
     4921 gtatatcgag gtttatttat taatttgaat agatattaag ttttattata tttacactta
     4981 catactaata ataaattcaa caaacaattt atttatgttt atttatttat taaaaaaaaa
     5041 caaaaactca aaatttcttc tataaagtaa caaaactttt agcagtgaaa aaaatgcttt
     5101 atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa
     5161 gttaacaaca acaattgcat tcattttatg tttcaggttc agggggaggt gtgggaggtt
     5221 ttttaaagca agtaaaacct ctacaaatgt ggtatggctg attatgatcc tctggagatt
     5281 catgcgccag gttttctggt catgcaccac gttcgtgggc cctcaggaac ctccacgtca
     5341 gcggtaactg tgaagcccag tcgctcgtaa aaaggaagat tccggggagc actggtttcg
     5401 aggaacgccg gcacgccagc ccgttccgcg gcctcaacac caggaagaac gacagcgctt
     5461 cccaaacctt taccttggtg atctgggctg acaccgacag tggcaagaaa ccacgcaggc
     5521 tcctttggtc tatgaggagc cagcaagccc tccatttgct gttgtgccgc caacctactc
     5581 cctgacagct cggccatccg cggacctatt tccgcgaaca ctgcgccggc ttcgacgctt
     5641 tccggagtag tccagaccgc aactgcggca ccatcgtccg caacccaaac tttaccaatg
     5701 tccagaccca cgcgcgtaag aaacagctct tgcaactcag ttaccctctc tatgtgtctg
     5761 tccggatcta cggtatggcg ggtcgctgga taatctgcaa atgctgcggc aagtgttcgg
     5821 acagcccgtg gtacatcgtc acgggtagcg agcctaacag tgggcttata ttccgtcatg
     5881 gggccggggt tctcctccac gtcgccggcc tgcttcagca ggctgaagtt ggtggcgccg
     5941 ctgcccccgg ggagcatgtc aaggtcaaaa tcgtcaagag cgtcagcagg cagcatatca
     6001 aggtcaaagt cgtcaagggc atcggctggg agcatgtcta agtcaaaatc gtcaagggcg
     6061 tcggtcggcc cgccgctttc gcactttagc tgtttctcca ggccacatat gattagttcc
     6121 aggccgaaaa ggaaggcagg ttcggctccc tgccggtcga acagctcaat tgcttgtttc
     6181 agaagtgggg gcatagaatc ggtggtaggt gtctctcttt cctcttttgc tacttgatgc
     6241 tcctgttcct ccaatacgca gcccagtgta aagtggccca cggcggacag agcgtacagt
     6301 gcgttctcca gggagaagcc ttgctgacac aggaacgcga gctgattttc cagggtttcg
     6361 tactgtttct ctgttgggcg ggtgccgaga tgcactttag ccccgtcgcg atgtgagagg
     6421 agagcacagc ggtatgactt ggcgttgttc cgcagaaagt cttgccatga ctcgccttcc
     6481 agggggcagg agtgggtatg atgcctgtcc agcatctcga ttggcagggc atcgagcagg
     6541 gcccgcttgt tcttcacgtg ccagtacagg gtaggctgct caactcccag cttttgagcg
     6601 agtttccttg tcgtcaggcc ttcgataccg actccattga gtaattccag agcagagttt
     6661 atgactttgc tcttgtccag tctagacatc ttatcgtcat cgtctttgta atccatggtg
     6721 gcggatcccg cgtcacgaca cctgtgttct ggcggcaaac ccgttgcgaa aaagaacgtt
     6781 cacggcgact actgcactta tatacggttc tcccccaccc tcgggaaaaa ggcggagcca
     6841 gtacacgaca tcactttccc agtttacccc gcgccacctt ctctaggcac cggttcaatt
     6901 gccgacccct ccccccaact tctcggggac tgtgggcgat gtgcgctctg cccactgacg
     6961 ggcaccggag ccactcgagt ggaatt
//
