LOCUS       sequence-442524-        7045 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac transposon plasmid for doxycycline inducible expression of 
            DMRTB1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7045)
  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 7045)
  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-DMRTB1
FEATURES             Location/Qualifiers
     source          1..7045
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      781..1369
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1270..1289
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1523..1540
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1746..1767
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1782..1787,1788..1805,1806..1812)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1820..1836
                     /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     1825..1847
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1844..1860
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1844..1860
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   1886..1948
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    2124..2205
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(2922..2978)
                     /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(2988..3731)
                     /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(3732..3755)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      3755..3764
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(3784..3995)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        3996..4375
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     protein_bind    4584..4602
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4620..4638
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4656..4674
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4692..4710
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4728..4746
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4764..4782
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4800..4818
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     4845..4869
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    4935..4959
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(5527..5551)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             5553..5579
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    5746..5880
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   6152..6186
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(6197..6214)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6197..6213)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6206..6228)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(6422..6441)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6543..6565
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6604..6622)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6690..6794
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6795..6863,6864..7045,1..610)
                     /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(7013..7032)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
       61 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
      121 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
      181 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
      241 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
      301 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
      361 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
      421 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtggttcac gcggtatcat
      481 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
      541 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
      601 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
      661 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
      721 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
      781 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
      841 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      901 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      961 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     1021 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     1081 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     1141 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     1201 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     1261 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     1321 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     1381 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     1441 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     1501 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggatg agcgcccaat
     1561 acgcaaaccg cctctcaccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
     1621 tcccgactgg aaagcgggca gtgagcgcaa cgcaaaccgc ctctccccgc gcgttggccg
     1681 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac
     1741 gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg
     1801 gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac
     1861 catgattacg ccaaggtcga cttaacccta gaaagataat catattgtga cgtacgttaa
     1921 agataatcat gcgtaaaatt gacgcatgtg ttttatcggt ctgtatatcg aggtttattt
     1981 attaatttga atagatatta agttttatta tatttacact tacatactaa taataaattc
     2041 aacaaacaat ttatttatgt ttatttattt attaaaaaaa aacaaaaact caaaatttct
     2101 tctataaagt aacaaaactt ttagcagtga aaaaaatgct ttatttgtga aatttgtgat
     2161 gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc
     2221 attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaaag caagtaaaac
     2281 ctctacaaat gtggtatggc tgattatgat cctctggaga ttcatgcgcc aggttttctg
     2341 gtcatgcacc acgttcgtgg gccctcagga acctccacgt cagcggtaac tgtgaagccc
     2401 agtcgctcgt aaaaaggaag attccgggga gcactggttt cgaggaacgc cggcacgcca
     2461 gcccgttccg cggcctcaac accaggaaga acgacagcgc ttcccaaacc tttaccttgg
     2521 tgatctgggc tgacaccgac agtggcaaga aaccacgcag gctcctttgg tctatgagga
     2581 gccagcaagc cctccatttg ctgttgtgcc gccaacctac tccctgacag ctcggccatc
     2641 cgcggaccta tttccgcgaa cactgcgccg gcttcgacgc tttccggagt agtccagacc
     2701 gcaactgcgg caccatcgtc cgcaacccaa actttaccaa tgtccagacc cacgcgcgta
     2761 agaaacagct cttgcaactc agttaccctc tctatgtgtc tgtccggatc tacggtatgg
     2821 cgggtcgctg gataatctgc aaatgctgcg gcaagtgttc ggacagcccg tggtacatcg
     2881 tcacgggtag cgagcctaac agtgggctta tattccgtca tggggccggg gttctcctcc
     2941 acgtcgccgg cctgcttcag caggctgaag ttggtggcgc cgctgccccc ggggagcatg
     3001 tcaaggtcaa aatcgtcaag agcgtcagca ggcagcatat caaggtcaaa gtcgtcaagg
     3061 gcatcggctg ggagcatgtc taagtcaaaa tcgtcaaggg cgtcggtcgg cccgccgctt
     3121 tcgcacttta gctgtttctc caggccacat atgattagtt ccaggccgaa aaggaaggca
     3181 ggttcggctc cctgccggtc gaacagctca attgcttgtt tcagaagtgg gggcatagaa
     3241 tcggtggtag gtgtctctct ttcctctttt gctacttgat gctcctgttc ctccaatacg
     3301 cagcccagtg taaagtggcc cacggcggac agagcgtaca gtgcgttctc cagggagaag
     3361 ccttgctgac acaggaacgc gagctgattt tccagggttt cgtactgttt ctctgttggg
     3421 cgggtgccga gatgcacttt agccccgtca cgatgtgaga ggagagcaca gcggtatgac
     3481 ttggcgttgt tccgcagaaa gtcttgccat gactcgcctt ccagggggca ggagtgggta
     3541 tgatgcctgt ccagcatctc gattggcagg gcatcgagca gggcccgctt gttcttcacg
     3601 tgccagtaca gggtaggctg ctcaactccc agcttttgag cgagtttcct tgtcgtcagg
     3661 ccttcgatac cgactccatt gagtaattcc agagcagagt ttatgacttt gctcttgtcc
     3721 agtctagaca tcttatcgtc atcgtctttg taatccatgg tggcggatcc cgcgtcacga
     3781 cacctgtgtt ctggcggcaa acccgttgcg aaaaagaacg ttcacggcga ctactgcact
     3841 tatatacggt tctcccccac cctcgggaaa aaggcggagc cagtacacga catcactttc
     3901 ccagtttacc ccgcgccacc ttctctaggc accggttcaa ttgccgaccc ctccccccaa
     3961 cttctcgggc actgtgggcc atgtgcgctc tgccccatgg taatagccat gactaatacg
     4021 tagatgtact gccaagtagg aaagtgccat aaggtcatgt actgggcata atgccaggcg
     4081 ggccatttac cgtcattgac gtcaataggg ggcgtacttg gcatatgata cacttgatgt
     4141 actgccaagt gggcagttta ccgtaaatac tccacccatt gacgtcaatg gaaagtgcct
     4201 attggcgtta ctatgggaac atacgtcatt attgacgtca atgggcgggg gtcgttgggc
     4261 ggtcagccag gcgggccatt taccgtaagt tatgtaacgc ggaactccat atatgggcta
     4321 tgaactaatg accccgtaat tgattactat taataactag tcaataatca atgtcataag
     4381 ctctaatcgc ataatcgagc taagagcgat taatatgctt agctagctta gctaagctag
     4441 atttctctat tctcccacaa tatatagcag ctattagctt agcagtccga atacgatcgt
     4501 ccacgatcca taagcgtaac acatttgagt agcgactgac gggcaccgga gccactcgag
     4561 tggaattatc acctcgagtt tactccctat cagtgataga gaacgtatga agagtttact
     4621 ccctatcagt gatagagaac gtatgcagac tttactccct atcagtgata gagaacgtat
     4681 aaggagttta ctccctatca gtgatagaga acgtatgacc agtttactcc ctatcagtga
     4741 tagagaacgt atctacagtt tactccctat cagtgataga gaacgtatat ccagtttact
     4801 ccctatcagt gatagagaac gtataagctt gcctatataa gcagagctcg tttagtgaac
     4861 cgtcagatcg cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgggac
     4921 cgatccagcc taccacaagt ttgtacaaaa aagttggcat gcccagcctt gcgggacccc
     4981 cttttggggc ggaggccgca ggcagtggct accctggccc cctagacctg cgcaggccga
     5041 tgcggaccgt gcccggccca ctgttcaccg actttgtgcg ccctctgaac atcaacccgg
     5101 accgtgcact gggccctgag taccctggtg gctccagcat gcacccctac tgcccgttcc
     5161 cgctgggcta cctggacgcc cctcctggcg tccccctgca gcagggcttc cggcatgtgt
     5221 cccgcagcca gtaccaaggc ggaggcttgg tgtcagaacc aggaggagac ttccagccaa
     5281 gctactacct gccgccgccg ccgccgccac tgccgcccct tccaccgctt ccaccgcagc
     5341 cccagttcct cccgccaggc tacctctctg cgctccactt cctccccccg ccaccgccac
     5401 caccacctcc atcatctttc tcactgaccg tcctgtttga tactgacaag gagaacactg
     5461 atgaccagga tgcagaggta ctgtcgggtg agcccagcca gccatcgtct caggagcagt
     5521 ccgactaccc aactttcttg tacaaagtgg tatacccata cgatgttcca gattacgctt
     5581 agtaatgaga aatactcaga agatgtcact caccatagtc cgcagtgaaa gataaatgat
     5641 ccaagtttga cagaccttcg agttttagag ctagaaatag caagtaggcc actgagcaag
     5701 aactatgaat gagatcttta gacatattgc ggccgctttc tccgcgatcc agacatgata
     5761 agatacattg atgagtttgg acaaaccaaa actagaatgc agtgaaaaaa atgccttatt
     5821 tgtgaaattt gtgatgctat tgccttattt gtaaccatta taagctgcaa taaacaagtt
     5881 atctataaca agaaaatata tatataataa gttatcacgt aagtagaaca tgaaataaca
     5941 atataattat cgtatgagtt aaatcttaaa agtcacgtaa aagataatca tgcgtcattt
     6001 tgactcacgc ggtcgttata gttcaaaatc agtgacactt accgcattga caagcacgcc
     6061 tcacgggagc tccaagcggc gactgagatg tcctaaatgc acagcgacgg attcgcgcta
     6121 tttagaaaga gagagcaata tttcaagaat gcatgcgtca attttacgca gactatcttt
     6181 ctagggttaa gaattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg
     6241 ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag
     6301 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga
     6361 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatatgg tgcactctca
     6421 gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg
     6481 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccatgt
     6541 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacacg cgagttcgaa
     6601 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     6661 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     6721 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     6781 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     6841 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     6901 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     6961 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     7021 gcgcggtatt atcccgtatt gacgc
//
