LOCUS       sequence-439874-        7734 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac transposon plasmid for doxycycline inducible expression of 
            TERF1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7734)
  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 7734)
  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-TERF1
FEATURES             Location/Qualifiers
     source          1..7734
                     /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(1649..1673)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1675..1701
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1867..2001
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   2273..2307
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2318..2335)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2318..2334)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2327..2349)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2543..2562)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2664..2686
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2725..2743)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2811..2915
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2916..2984,2985..3776)
                     /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(3134..3153)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3947..4535
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4436..4455
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4689..4706
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4912..4933
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4948..4953,4954..4971,4972..4978)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4986..5002
                     /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     4991..5013
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5010..5026
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5010..5026
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   5052..5114
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    5290..5371
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(6088..6144)
                     /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(6154..6897)
                     /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(6898..6921)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6921..6930
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6950..7161)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        7162..7541
                     /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 aaaagttggc atggcggagg atgtttcctc agcggccccg
      421 agcccgcggg gctgtgcgga tggtagggat gccgacccta ctgaggagca gatggcagaa
      481 acagagagaa acgacgagga gcagttcgaa tgccaggaac tgctcgagtg ccaggtgcag
      541 gtgggggccc ccgaggagga ggaggaggag gaggaggacg cgggcctggt ggccgaggcc
      601 gaggccgtgg ctgccggctg gatgctcgat ttcctctgcc tctctctttg ccgagctttc
      661 cgcgacggcc gctccgagga cttccgcagg acccgcaaca gcgcagaggc tattattcat
      721 ggactatcca gtctaacagc ttgccagttg agaacgatat acatatgtca gtttttgaca
      781 agaattgcag caggaaaaac ccttgatgca cagtttgaaa atgatgaacg aattacaccc
      841 ttggaatcag ccctgatgat ttggggttca attgaaaagg aacatgacaa acttcatgaa
      901 gaaatacaga atttaattaa aattcaggct atagctgttt gtatggaaaa tggcaacttt
      961 aaagaagcag aagaagtctt tgaaagaata tttggtgatc caaattctca tatgcctttc
     1021 aaaagcaaat tgcttatgat aatctctcag aaagatacat ttcattcctt ttttcaacac
     1081 ttcagctaca accacatgat ggagaaaatt aagagttatg tgaattatgt gctaagtgaa
     1141 aaatcatcaa cctttctaat gaaggcagcg gcaaaagtag tagaaagcaa aaggacaaga
     1201 acaataactt ctcaagataa acctagtggt aatgatgttg aaatggaaac tgaagctaat
     1261 ttggatacaa gaaaaaggtc tcacaagaat cttttcttat ctaagttgca acatggaacc
     1321 cagcaacaag accttaataa gaaagaaaga agagtaggaa ctcctcaaag tacaaaaaag
     1381 aaaaaagaaa gcagaagagc cactgaaagc agaatacctg tttcaaagag tcagccggta
     1441 actcctgaaa aacatcgagc tagaaaaaga caggcatggc tttgggaaga agacaagaat
     1501 ttgagatctg gcgtgaggaa atatggagag ggaaactggt ctaaaatact gttgcattat
     1561 aaattcaaca accggacaag tgtcatgtta aaagacagat ggaggaccat gaagaaacta
     1621 aaactgattt cctcagacag cgaagactgc ccaactttct tgtacaaagt ggtataccca
     1681 tacgatgttc cagattacgc ttagtaatga gaaatactca gaagatgtca ctcaccatag
     1741 tccgcagtga aagataaatg atctgacgtt ggtttgatag cggttttaga gctagaaata
     1801 gcaagtaggc cactgagcaa gaactatgaa tgagatcttt agacatattg cggccgcttt
     1861 ctccgcgatc cagacatgat aagatacatt gatgagtttg gacaaaccaa aactagaatg
     1921 cagtgaaaaa aatgccttat ttgtgaaatt tgtgatgcta ttgccttatt tgtaaccatt
     1981 ataagctgca ataaacaagt tatctataac aagaaaatat atatataata agttatcacg
     2041 taagtagaac atgaaataac aatataatta tcgtatgagt taaatcttaa aagtcacgta
     2101 aaagataatc atgcgtcatt ttgactcacg cggtcgttat agttcaaaat cagtgacact
     2161 taccgcattg acaagcacgc ctcacgggag ctccaagcgg cgactgagat gtcctaaatg
     2221 cacagcgacg gattcgcgct atttagaaag agagagcaat atttcaagaa tgcatgcgtc
     2281 aattttacgc agactatctt tctagggtta agaattcact ggccgtcgtt ttacaacgtc
     2341 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg
     2401 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
     2461 tgaatggcga atggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     2521 accgcatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc
     2581 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     2641 acagacaagc tgtgaccatg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     2701 accgaaacac gcgagttcga aagggcctcg tgatacgcct atttttatag gttaatgtca
     2761 tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     2821 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     2881 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     2941 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     3001 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     3061 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     3121 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     3181 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     3241 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     3301 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     3361 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     3421 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     3481 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     3541 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     3601 ttgctgataa atctggagcc ggtgagcgtg gttcacgcgg tatcattgca gcactggggc
     3661 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     3721 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     3781 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     3841 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     3901 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     3961 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     4021 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     4081 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     4141 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     4201 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     4261 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     4321 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     4381 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     4441 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     4501 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     4561 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     4621 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     4681 ccgagcgcag cgagtcagtg agcgaggaag cggatgagcg cccaatacgc aaaccgcctc
     4741 tcaccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
     4801 cgggcagtga gcgcaacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
     4861 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
     4921 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
     4981 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa
     5041 ggtcgactta accctagaaa gataatcata ttgtgacgta cgttaaagat aatcatgcgt
     5101 aaaattgacg catgtgtttt atcggtctgt atatcgaggt ttatttatta atttgaatag
     5161 atattaagtt ttattatatt tacacttaca tactaataat aaattcaaca aacaatttat
     5221 ttatgtttat ttatttatta aaaaaaaaca aaaactcaaa atttcttcta taaagtaaca
     5281 aaacttttag cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt
     5341 tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt
     5401 tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg
     5461 tatggctgat tatgatcctc tggagattca tgcgccaggt tttctggtca tgcaccacgt
     5521 tcgtgggccc tcaggaacct ccacgtcagc ggtaactgtg aagcccagtc gctcgtaaaa
     5581 aggaagattc cggggagcac tggtttcgag gaacgccggc acgccagccc gttccgcggc
     5641 ctcaacacca ggaagaacga cagcgcttcc caaaccttta ccttggtgat ctgggctgac
     5701 accgacagtg gcaagaaacc acgcaggctc ctttggtcta tgaggagcca gcaagccctc
     5761 catttgctgt tgtgccgcca acctactccc tgacagctcg gccatccgcg gacctatttc
     5821 cgcgaacact gcgccggctt cgacgctttc cggagtagtc cagaccgcaa ctgcggcacc
     5881 atcgtccgca acccaaactt taccaatgtc cagacccacg cgcgtaagaa acagctcttg
     5941 caactcagtt accctctcta tgtgtctgtc cggatctacg gtatggcggg tcgctggata
     6001 atctgcaaat gctgcggcaa gtgttcggac agcccgtggt acatcgtcac gggtagcgag
     6061 cctaacagtg ggcttatatt ccgtcatggg gccggggttc tcctccacgt cgccggcctg
     6121 cttcagcagg ctgaagttgg tggcgccgct gcccccgggg agcatgtcaa ggtcaaaatc
     6181 gtcaagagcg tcagcaggca gcatatcaag gtcaaagtcg tcaagggcat cggctgggag
     6241 catgtctaag tcaaaatcgt caagggcgtc ggtcggcccg ccgctttcgc actttagctg
     6301 tttctccagg ccacatatga ttagttccag gccgaaaagg aaggcaggtt cggctccctg
     6361 ccggtcgaac agctcaattg cttgtttcag aagtgggggc atagaatcgg tggtaggtgt
     6421 ctctctttcc tcttttgcta cttgatgctc ctgttcctcc aatacgcagc ccagtgtaaa
     6481 gtggcccacg gcggacagag cgtacagtgc gttctccagg gagaagcctt gctgacacag
     6541 gaacgcgagc tgattttcca gggtttcgta ctgtttctct gttgggcggg tgccgagatg
     6601 cactttagcc ccgtcacgat gtgagaggag agcacagcgg tatgacttgg cgttgttccg
     6661 cagaaagtct tgccatgact cgccttccag ggggcaggag tgggtatgat gcctgtccag
     6721 catctcgatt ggcagggcat cgagcagggc ccgcttgttc ttcacgtgcc agtacagggt
     6781 aggctgctca actcccagct tttgagcgag tttccttgtc gtcaggcctt cgataccgac
     6841 tccattgagt aattccagag cagagtttat gactttgctc ttgtccagtc tagacatctt
     6901 atcgtcatcg tctttgtaat ccatggtggc ggatcccgcg tcacgacacc tgtgttctgg
     6961 cggcaaaccc gttgcgaaaa agaacgttca cggcgactac tgcacttata tacggttctc
     7021 ccccaccctc gggaaaaagg cggagccagt acacgacatc actttcccag tttaccccgc
     7081 gccaccttct ctaggcaccg gttcaattgc cgacccctcc ccccaacttc tcgggcactg
     7141 tgggccatgt gcgctctgcc ccatggtaat agccatgact aatacgtaga tgtactgcca
     7201 agtaggaaag tgccataagg tcatgtactg ggcataatgc caggcgggcc atttaccgtc
     7261 attgacgtca atagggggcg tacttggcat atgatacact tgatgtactg ccaagtgggc
     7321 agtttaccgt aaatactcca cccattgacg tcaatggaaa gtgcctattg gcgttactat
     7381 gggaacatac gtcattattg acgtcaatgg gcgggggtcg ttgggcggtc agccaggcgg
     7441 gccatttacc gtaagttatg taacgcggaa ctccatatat gggctatgaa ctaatgaccc
     7501 cgtaattgat tactattaat aactagtcaa taatcaatgt cataagctct aatcgcataa
     7561 tcgagctaag agcgattaat atgcttagct agcttagcta agctagattt ctctattctc
     7621 ccacaatata tagcagctat tagcttagca gtccgaatac gatcgtccac gatccataag
     7681 cgtaacacat ttgagtagcg actgacgggc accggagcca ctcgagtgga atta
//
