LOCUS       sequence-439420-        7627 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac transposon plasmid for doxycycline inducible expression of 
            NELFE.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7627)
  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 7627)
  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-NELFE
FEATURES             Location/Qualifiers
     source          1..7627
                     /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    368..392
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1542..1566)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1568..1594
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1761..1895
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   2167..2201
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2212..2229)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2212..2228)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2221..2243)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2437..2456)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2558..2580
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2619..2637)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2705..2809
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2810..2878,2879..3670)
                     /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(3028..3047)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3841..4429
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4330..4349
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4583..4600
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4806..4827
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4842..4847,4848..4865,4866..4872)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4880..4896
                     /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     4885..4907
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4904..4920
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4904..4920
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4946..5008
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    5184..5265
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5982..6038)
                     /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(6048..6791)
                     /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(6792..6815)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6815..6824
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6844..7055)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        7056..7435
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
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 gcctaccaca agtttgtaca aaaaagcagg ctgccaccat gctggtcatt ccacctggtt
      421 tatctgaaga ggaagaagct ctccaaaaga agtttaataa attaaagaag aagaagaaag
      481 ccctgctcgc cctcaagaaa cagtcatctt cttcaactac ttcccagggc ggcgttaaga
      541 ggtctttaag cgaacaacca gtaatggata ccgctacggc cactgaacaa gcgaaacaac
      601 ttgtaaaatc cggtgctatt tcagcaataa aagcggaaac taagaatagt ggttttaaaa
      661 gaagcaggac actggaagga aaactgaaag atccagaaaa ggggcccgta cctacctttc
      721 aaccttttca acgctctatt agcgccgacg acgatttgca ggaaagcagc cgccggccac
      781 aacgaaagtc actttacgaa agtttcgtca gttccagcga ccgcttacgg gagctggggc
      841 cggatggcga ggaagctgaa ggacctggcg ctggcgacgg accacccaga tcattcgatt
      901 ggggttacga ggaaaggtcc ggtgctcata gctctgcaag tccaccgcgt tcaaggtccc
      961 gtgatcggtc tcacgaaaga aatagggaca gggaccgcga tcgcgaacga gaccgcgata
     1021 gggacagaga tcgggatcgg gaacgtgatc gcgaccgtga ccgcgacagg gacagggata
     1081 gagagaggga tcgggaccga gaacgagacc gggataggga tcgcgaagga ccgtttcggc
     1141 ggtccgacag ttttccggag agaagggcgc cccgaaaggg aaacaccttg tacgtctacg
     1201 gggaagatat gacccctact ctgcttcgag gagcgttttc ccccttcggg aatattatcg
     1261 atttaagcat ggatccgcca cgcaattgcg cttttgtgac atacgagaaa atggaatctg
     1321 ctgaccaagc tgtggccgaa cttaatggca cacaagtaga aagcgtgcaa ctgaaggtga
     1381 atattgcaag aaagcaacct atgttggacg cggccacagg gaaaagcgtg tggggatctt
     1441 tggccgtgca aaattcccca aaagggtgtc atcgtgataa acgtacacaa atcgtttata
     1501 gcgacgatgt ttataaagag aatctggtcg acggtttcta aacccagctt tcttgtacaa
     1561 agtggtatac ccatacgatg ttccagatta cgcttagtaa tgagaaatac tcagaagatg
     1621 tcactcacca tagtccgcag tgaaagataa atgatctgag caatatacaa aagacagttt
     1681 tagagctaga aatagcaagt aggccactga gcaagaacta tgaatgagat ctttagacat
     1741 attgcggccg ctttctccgc gatccagaca tgataagata cattgatgag tttggacaaa
     1801 ccaaaactag aatgcagtga aaaaaatgcc ttatttgtga aatttgtgat gctattgcct
     1861 tatttgtaac cattataagc tgcaataaac aagttatcta taacaagaaa atatatatat
     1921 aataagttat cacgtaagta gaacatgaaa taacaatata attatcgtat gagttaaatc
     1981 ttaaaagtca cgtaaaagat aatcatgcgt cattttgact cacgcggtcg ttatagttca
     2041 aaatcagtga cacttaccgc attgacaagc acgcctcacg ggagctccaa gcggcgactg
     2101 agatgtccta aatgcacagc gacggattcg cgctatttag aaagagagag caatatttca
     2161 agaatgcatg cgtcaatttt acgcagacta tctttctagg gttaagaatt cactggccgt
     2221 cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc
     2281 acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca
     2341 acagttgcgc agcctgaatg gcgaatggcg cctgatgcgg tattttctcc ttacgcatct
     2401 gtgcggtatt tcacaccgca tatggtgcac tctcagtaca atctgctctg atgccgcata
     2461 gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     2521 cccggcatcc gcttacagac aagctgtgac catgtctccg ggagctgcat gtgtcagagg
     2581 ttttcaccgt catcaccgaa acacgcgagt tcgaaagggc ctcgtgatac gcctattttt
     2641 ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa
     2701 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     2761 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     2821 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     2881 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     2941 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     3001 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     3061 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     3121 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     3181 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     3241 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     3301 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     3361 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     3421 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     3481 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtggttcac gcggtatcat
     3541 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     3601 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     3661 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     3721 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     3781 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     3841 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     3901 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     3961 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     4021 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     4081 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     4141 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     4201 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     4261 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     4321 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     4381 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     4441 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     4501 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     4561 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggatg agcgcccaat
     4621 acgcaaaccg cctctcaccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
     4681 tcccgactgg aaagcgggca gtgagcgcaa cgcaaaccgc ctctccccgc gcgttggccg
     4741 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac
     4801 gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg
     4861 gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac
     4921 catgattacg ccaaggtcga cttaacccta gaaagataat catattgtga cgtacgttaa
     4981 agataatcat gcgtaaaatt gacgcatgtg ttttatcggt ctgtatatcg aggtttattt
     5041 attaatttga atagatatta agttttatta tatttacact tacatactaa taataaattc
     5101 aacaaacaat ttatttatgt ttatttattt attaaaaaaa aacaaaaact caaaatttct
     5161 tctataaagt aacaaaactt ttagcagtga aaaaaatgct ttatttgtga aatttgtgat
     5221 gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc
     5281 attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaaag caagtaaaac
     5341 ctctacaaat gtggtatggc tgattatgat cctctggaga ttcatgcgcc aggttttctg
     5401 gtcatgcacc acgttcgtgg gccctcagga acctccacgt cagcggtaac tgtgaagccc
     5461 agtcgctcgt aaaaaggaag attccgggga gcactggttt cgaggaacgc cggcacgcca
     5521 gcccgttccg cggcctcaac accaggaaga acgacagcgc ttcccaaacc tttaccttgg
     5581 tgatctgggc tgacaccgac agtggcaaga aaccacgcag gctcctttgg tctatgagga
     5641 gccagcaagc cctccatttg ctgttgtgcc gccaacctac tccctgacag ctcggccatc
     5701 cgcggaccta tttccgcgaa cactgcgccg gcttcgacgc tttccggagt agtccagacc
     5761 gcaactgcgg caccatcgtc cgcaacccaa actttaccaa tgtccagacc cacgcgcgta
     5821 agaaacagct cttgcaactc agttaccctc tctatgtgtc tgtccggatc tacggtatgg
     5881 cgggtcgctg gataatctgc aaatgctgcg gcaagtgttc ggacagcccg tggtacatcg
     5941 tcacgggtag cgagcctaac agtgggctta tattccgtca tggggccggg gttctcctcc
     6001 acgtcgccgg cctgcttcag caggctgaag ttggtggcgc cgctgccccc ggggagcatg
     6061 tcaaggtcaa aatcgtcaag agcgtcagca ggcagcatat caaggtcaaa gtcgtcaagg
     6121 gcatcggctg ggagcatgtc taagtcaaaa tcgtcaaggg cgtcggtcgg cccgccgctt
     6181 tcgcacttta gctgtttctc caggccacat atgattagtt ccaggccgaa aaggaaggca
     6241 ggttcggctc cctgccggtc gaacagctca attgcttgtt tcagaagtgg gggcatagaa
     6301 tcggtggtag gtgtctctct ttcctctttt gctacttgat gctcctgttc ctccaatacg
     6361 cagcccagtg taaagtggcc cacggcggac agagcgtaca gtgcgttctc cagggagaag
     6421 ccttgctgac acaggaacgc gagctgattt tccagggttt cgtactgttt ctctgttggg
     6481 cgggtgccga gatgcacttt agccccgtca cgatgtgaga ggagagcaca gcggtatgac
     6541 ttggcgttgt tccgcagaaa gtcttgccat gactcgcctt ccagggggca ggagtgggta
     6601 tgatgcctgt ccagcatctc gattggcagg gcatcgagca gggcccgctt gttcttcacg
     6661 tgccagtaca gggtaggctg ctcaactccc agcttttgag cgagtttcct tgtcgtcagg
     6721 ccttcgatac cgactccatt gagtaattcc agagcagagt ttatgacttt gctcttgtcc
     6781 agtctagaca tcttatcgtc atcgtctttg taatccatgg tggcggatcc cgcgtcacga
     6841 cacctgtgtt ctggcggcaa acccgttgcg aaaaagaacg ttcacggcga ctactgcact
     6901 tatatacggt tctcccccac cctcgggaaa aaggcggagc cagtacacga catcactttc
     6961 ccagtttacc ccgcgccacc ttctctaggc accggttcaa ttgccgaccc ctccccccaa
     7021 cttctcgggc actgtgggcc atgtgcgctc tgccccatgg taatagccat gactaatacg
     7081 tagatgtact gccaagtagg aaagtgccat aaggtcatgt actgggcata atgccaggcg
     7141 ggccatttac cgtcattgac gtcaataggg ggcgtacttg gcatatgata cacttgatgt
     7201 actgccaagt gggcagttta ccgtaaatac tccacccatt gacgtcaatg gaaagtgcct
     7261 attggcgtta ctatgggaac atacgtcatt attgacgtca atgggcgggg gtcgttgggc
     7321 ggtcagccag gcgggccatt taccgtaagt tatgtaacgc ggaactccat atatgggcta
     7381 tgaactaatg accccgtaat tgattactat taataactag tcaataatca atgtcataag
     7441 ctctaatcgc ataatcgagc taagagcgat taatatgctt agctagctta gctaagctag
     7501 atttctctat tctcccacaa tatatagcag ctattagctt agcagtccga atacgatcgt
     7561 ccacgatcca taagcgtaac acatttgagt agcgactgac gggcaccgga gccactcgag
     7621 tggaatt
//
