LOCUS       sequence-439887-        7372 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac transposon plasmid for doxycycline inducible expression of 
            BOLL.
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-BOLL
FEATURES             Location/Qualifiers
     source          1..7372
                     /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"
     regulatory      392..401
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(1286..1310)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1312..1338
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1505..1639
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   1911..1945
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1956..1973)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1956..1972)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1965..1987)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2181..2200)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2302..2324
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2363..2381)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2449..2553
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2554..2622,2623..3414)
                     /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(2772..2791)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3585..4173
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4074..4093
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4327..4344
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4550..4571
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4586..4591,4592..4609,4610..4616)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4624..4640
                     /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     4629..4651
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4648..4664
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4648..4664
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4690..4752
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    4928..5009
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5726..5782)
                     /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(5792..6535)
                     /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(6536..6559)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6559..6568
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6588..6799)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        6800..7179
                     /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 aaaagcaggc tgccaccatg gaaaccgagt ccgggccgca
      421 aacatcaaac cagatgcaaa cagattcatt atctccatcc cctaatcctg tgtcacctgt
      481 gcctttgaat aacccaacaa gtgccccaag atatggaaca gtgatcccta atcgcatctt
      541 tgtaggagga attgatttta agacaaacga aagtgattta agaaaatttt tttcccagta
      601 tgggtctgtg aaagaagtga agattgtaaa tgacagagct ggagtatcca aagggtatgg
      661 tttcgtcact tttgaaacac aagaagatgc acaaaaaatt ttacaagagg ctgaaaaact
      721 taattataag gataagaagc tgaacattgg tccagcaata agaaaacaac aagtagggat
      781 ccctcgttct agtataatgc cagcagctgg aacaatgtat ctaacaactt caactggata
      841 tccttatact taccataatg gtgttgctta ttttcatact ccagaggtaa cttcggtccc
      901 accgccttgg ccttcacgtt ctgtatgtag ctcccctgtg atggtagctc agcccattta
      961 tcagcaacct gcatatcact accaggccac cacacagtat ttaccaggac agtggcagtg
     1021 gagtgttcct cagccttctg cctcttctgc tccattctta tacctgcaac cttctgaggt
     1081 tatttatcaa ccagtggaaa ttgcacagga tggtggatgt gttcctcctc cactgtctct
     1141 gatggaaact tcagttccag agccttattc tgatcatgga gttcaagcaa catatcacca
     1201 ggtttatgct ccaagtgcca tcactatgcc tgcgcctgtg atgcagcctg agccaattaa
     1261 aacagtgtgg agcattcatt attaaaccca gctttcttgt acaaagtggt atacccatac
     1321 gatgttccag attacgctta gtaatgagaa atactcagaa gatgtcactc accatagtcc
     1381 gcagtgaaag ataaatgatc atggggctcg gagcaggaca gttttagagc tagaaatagc
     1441 aagtaggcca ctgagcaaga actatgaatg agatctttag acatattgcg gccgctttct
     1501 ccgcgatcca gacatgataa gatacattga tgagtttgga caaaccaaaa ctagaatgca
     1561 gtgaaaaaaa tgccttattt gtgaaatttg tgatgctatt gccttatttg taaccattat
     1621 aagctgcaat aaacaagtta tctataacaa gaaaatatat atataataag ttatcacgta
     1681 agtagaacat gaaataacaa tataattatc gtatgagtta aatcttaaaa gtcacgtaaa
     1741 agataatcat gcgtcatttt gactcacgcg gtcgttatag ttcaaaatca gtgacactta
     1801 ccgcattgac aagcacgcct cacgggagct ccaagcggcg actgagatgt cctaaatgca
     1861 cagcgacgga ttcgcgctat ttagaaagag agagcaatat ttcaagaatg catgcgtcaa
     1921 ttttacgcag actatctttc tagggttaag aattcactgg ccgtcgtttt acaacgtcgt
     1981 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
     2041 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
     2101 aatggcgaat ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     2161 cgcatatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
     2221 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     2281 agacaagctg tgaccatgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     2341 cgaaacacgc gagttcgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
     2401 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     2461 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     2521 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     2581 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     2641 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     2701 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     2761 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     2821 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     2881 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     2941 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     3001 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     3061 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     3121 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     3181 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     3241 gctgataaat ctggagccgg tgagcgtggt tcacgcggta tcattgcagc actggggcca
     3301 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     3361 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     3421 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     3481 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     3541 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     3601 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     3661 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     3721 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     3781 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     3841 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     3901 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     3961 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     4021 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     4081 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     4141 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     4201 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     4261 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     4321 gagcgcagcg agtcagtgag cgaggaagcg gatgagcgcc caatacgcaa accgcctctc
     4381 accgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
     4441 ggcagtgagc gcaacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     4501 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt
     4561 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg
     4621 gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagg
     4681 tcgacttaac cctagaaaga taatcatatt gtgacgtacg ttaaagataa tcatgcgtaa
     4741 aattgacgca tgtgttttat cggtctgtat atcgaggttt atttattaat ttgaatagat
     4801 attaagtttt attatattta cacttacata ctaataataa attcaacaaa caatttattt
     4861 atgtttattt atttattaaa aaaaaacaaa aactcaaaat ttcttctata aagtaacaaa
     4921 acttttagca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg
     4981 taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc
     5041 aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta
     5101 tggctgatta tgatcctctg gagattcatg cgccaggttt tctggtcatg caccacgttc
     5161 gtgggccctc aggaacctcc acgtcagcgg taactgtgaa gcccagtcgc tcgtaaaaag
     5221 gaagattccg gggagcactg gtttcgagga acgccggcac gccagcccgt tccgcggcct
     5281 caacaccagg aagaacgaca gcgcttccca aacctttacc ttggtgatct gggctgacac
     5341 cgacagtggc aagaaaccac gcaggctcct ttggtctatg aggagccagc aagccctcca
     5401 tttgctgttg tgccgccaac ctactccctg acagctcggc catccgcgga cctatttccg
     5461 cgaacactgc gccggcttcg acgctttccg gagtagtcca gaccgcaact gcggcaccat
     5521 cgtccgcaac ccaaacttta ccaatgtcca gacccacgcg cgtaagaaac agctcttgca
     5581 actcagttac cctctctatg tgtctgtccg gatctacggt atggcgggtc gctggataat
     5641 ctgcaaatgc tgcggcaagt gttcggacag cccgtggtac atcgtcacgg gtagcgagcc
     5701 taacagtggg cttatattcc gtcatggggc cggggttctc ctccacgtcg ccggcctgct
     5761 tcagcaggct gaagttggtg gcgccgctgc ccccggggag catgtcaagg tcaaaatcgt
     5821 caagagcgtc agcaggcagc atatcaaggt caaagtcgtc aagggcatcg gctgggagca
     5881 tgtctaagtc aaaatcgtca agggcgtcgg tcggcccgcc gctttcgcac tttagctgtt
     5941 tctccaggcc acatatgatt agttccaggc cgaaaaggaa ggcaggttcg gctccctgcc
     6001 ggtcgaacag ctcaattgct tgtttcagaa gtgggggcat agaatcggtg gtaggtgtct
     6061 ctctttcctc ttttgctact tgatgctcct gttcctccaa tacgcagccc agtgtaaagt
     6121 ggcccacggc ggacagagcg tacagtgcgt tctccaggga gaagccttgc tgacacagga
     6181 acgcgagctg attttccagg gtttcgtact gtttctctgt tgggcgggtg ccgagatgca
     6241 ctttagcccc gtcacgatgt gagaggagag cacagcggta tgacttggcg ttgttccgca
     6301 gaaagtcttg ccatgactcg ccttccaggg ggcaggagtg ggtatgatgc ctgtccagca
     6361 tctcgattgg cagggcatcg agcagggccc gcttgttctt cacgtgccag tacagggtag
     6421 gctgctcaac tcccagcttt tgagcgagtt tccttgtcgt caggccttcg ataccgactc
     6481 cattgagtaa ttccagagca gagtttatga ctttgctctt gtccagtcta gacatcttat
     6541 cgtcatcgtc tttgtaatcc atggtggcgg atcccgcgtc acgacacctg tgttctggcg
     6601 gcaaacccgt tgcgaaaaag aacgttcacg gcgactactg cacttatata cggttctccc
     6661 ccaccctcgg gaaaaaggcg gagccagtac acgacatcac tttcccagtt taccccgcgc
     6721 caccttctct aggcaccggt tcaattgccg acccctcccc ccaacttctc gggcactgtg
     6781 ggccatgtgc gctctgcccc atggtaatag ccatgactaa tacgtagatg tactgccaag
     6841 taggaaagtg ccataaggtc atgtactggg cataatgcca ggcgggccat ttaccgtcat
     6901 tgacgtcaat agggggcgta cttggcatat gatacacttg atgtactgcc aagtgggcag
     6961 tttaccgtaa atactccacc cattgacgtc aatggaaagt gcctattggc gttactatgg
     7021 gaacatacgt cattattgac gtcaatgggc gggggtcgtt gggcggtcag ccaggcgggc
     7081 catttaccgt aagttatgta acgcggaact ccatatatgg gctatgaact aatgaccccg
     7141 taattgatta ctattaataa ctagtcaata atcaatgtca taagctctaa tcgcataatc
     7201 gagctaagag cgattaatat gcttagctag cttagctaag ctagatttct ctattctccc
     7261 acaatatata gcagctatta gcttagcagt ccgaatacga tcgtccacga tccataagcg
     7321 taacacattt gagtagcgac tgacgggcac cggagccact cgagtggaat ta
//
