LOCUS       sequence-437996-        6656 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac transposon plasmid for doxycycline inducible expression of 
            UBB.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6656)
  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 6656)
  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-UBB
FEATURES             Location/Qualifiers
     source          1..6656
                     /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"
     CDS             419..646
                     /codon_start=1
                     /product="human ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     CDS             419..646
                     /codon_start=1
                     /gene="Homo sapiens UBB"
                     /product="human ubiquitin B"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     CDS             647..874
                     /codon_start=1
                     /product="human ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     CDS             647..874
                     /codon_start=1
                     /gene="Homo sapiens UBB"
                     /product="human ubiquitin B"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     CDS             875..1102
                     /codon_start=1
                     /product="human ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     protein_bind    complement(1110..1134)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1136..1162
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1329..1463
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   1735..1769
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1780..1797)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1780..1796)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1789..1811)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2005..2024)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2126..2148
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2187..2205)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2273..2377
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2378..2446,2447..3238)
                     /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(2596..2615)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3409..3997
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3898..3917
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4151..4168
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4374..4395
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4410..4415,4416..4433,4434..4440)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4448..4464
                     /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     4453..4475
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4472..4488
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4472..4488
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4514..4576
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    4752..4833
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5550..5606)
                     /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(5616..6359)
                     /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(6360..6383)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6383..6392
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6412..6623)
                     /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 gcagatcttc gtgaaaaccc ttaccggcaa gaccatcacc cttgaggtgg agcccagtga
      481 caccatcgaa aatgtgaagg ccaagatcca ggataaggaa ggcattcccc ccgaccagca
      541 gaggctcatc tttgcaggca agcagctgga agatggccgt actctttctg actacaacat
      601 tcagaaggag tcgaccctgc acctggtcct gcgtctgaga ggtggtatgc agatcttcgt
      661 gaagaccctg accggcaaga ccatcaccct ggaagtggag cccagtgaca ccatcgaaaa
      721 tgtgaaggcc aagatccagg ataaagaagg catccctccc gaccagcaga ggctcatctt
      781 tgcaggcaag cagctggaag atggccgcac tctttctgac tacaacatcc agaaggagtc
      841 gaccctgcac ctggtcctgc gtctgagagg tggtatgcag atcttcgtga agaccctgac
      901 cggcaagacc atcactctgg aggtggagcc cagtgacacc atcgaaaatg tgaaggccaa
      961 gatccaagat aaagaaggca tcccccccga ccagcagagg ctcatctttg caggcaagca
     1021 gctggaagat ggccgcactc tttctgacta caacatccag aaagagtcga ccctgcacct
     1081 ggtcctgcgc ctgaggggtg gctgttaata cccagctttc ttgtacaaag tggtataccc
     1141 atacgatgtt ccagattacg cttagtaatg agaaatactc agaagatgtc actcaccata
     1201 gtccgcagtg aaagataaat gatctgaatc acagaagaat actcgtttta gagctagaaa
     1261 tagcaagtag gccactgagc aagaactatg aatgagatct ttagacatat tgcggccgct
     1321 ttctccgcga tccagacatg ataagataca ttgatgagtt tggacaaacc aaaactagaa
     1381 tgcagtgaaa aaaatgcctt atttgtgaaa tttgtgatgc tattgcctta tttgtaacca
     1441 ttataagctg caataaacaa gttatctata acaagaaaat atatatataa taagttatca
     1501 cgtaagtaga acatgaaata acaatataat tatcgtatga gttaaatctt aaaagtcacg
     1561 taaaagataa tcatgcgtca ttttgactca cgcggtcgtt atagttcaaa atcagtgaca
     1621 cttaccgcat tgacaagcac gcctcacggg agctccaagc ggcgactgag atgtcctaaa
     1681 tgcacagcga cggattcgcg ctatttagaa agagagagca atatttcaag aatgcatgcg
     1741 tcaattttac gcagactatc tttctagggt taagaattca ctggccgtcg ttttacaacg
     1801 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt
     1861 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
     1921 cctgaatggc gaatggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     1981 acaccgcata tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc
     2041 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     2101 ttacagacaa gctgtgacca tgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     2161 tcaccgaaac acgcgagttc gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     2221 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     2281 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     2341 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     2401 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     2461 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     2521 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     2581 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     2641 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     2701 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     2761 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     2821 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     2881 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     2941 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     3001 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     3061 tattgctgat aaatctggag ccggtgagcg tggttcacgc ggtatcattg cagcactggg
     3121 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     3181 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     3241 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     3301 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     3361 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     3421 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     3481 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     3541 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     3601 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     3661 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     3721 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     3781 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     3841 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     3901 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     3961 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     4021 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     4081 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     4141 gaccgagcgc agcgagtcag tgagcgagga agcggawgag cgcccaatac gcaaaccgcc
     4201 tctcmccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
     4261 agcgggcagt gagcgcaacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
     4321 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg
     4381 agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg
     4441 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc
     4501 aaggtcgact taaccctaga aagataatca tattgtgacg tacgttaaag ataatcatgc
     4561 gtaaaattga cgcatgtgtt ttatcggtct gtatatcgag gtttatttat taatttgaat
     4621 agatattaag ttttattata tttacactta catactaata ataaattcaa caaacaattt
     4681 atttatgttt atttatttat taaaaaaaaa caaaaactca aaatttcttc tataaagtaa
     4741 caaaactttt agcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta
     4801 tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg
     4861 tttcaggttc agggggaggt gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt
     4921 ggtatggctg attatgatcc tctggagatt catgcgccag gttttctggt catgcaccac
     4981 gttcgtgggc cctcaggaac ctccacgtca gcggtaactg tgaagcccag tcgctcgtaa
     5041 aaaggaagat tccggggagc actggtttcg aggaacgccg gcacgccagc ccgttccgcg
     5101 gcctcaacac caggaagaac gacagcgctt cccaaacctt taccttggtg atctgggctg
     5161 acaccgacag tggcaagaaa ccacgcaggc tcctttggtc tatgaggagc cagcaagccc
     5221 tccatttgct gttgtgccgc caacctactc cctgacagct cggccatccg cggacctatt
     5281 tccgcgaaca ctgcgccggc ttcgacgctt tccggagtag tccagaccgc aactgcggca
     5341 ccatcgtccg caacccaaac tttaccaatg tccagaccca cgcgcgtaag aaacagctct
     5401 tgcaactcag ttaccctctc tatgtgtctg tccggatcta cggtatggcg ggtcgctgga
     5461 taatctgcaa atgctgcggc aagtgttcgg acagcccgtg gtacatcgtc acgggtagcg
     5521 agcctaacag tgggcttata ttccgtcatg gggccggggt tctcctccac gtcgccggcc
     5581 tgcttcagca ggctgaagtt ggtggcgccg ctgcccccgg ggagcatgtc aaggtcaaaa
     5641 tcgtcaagag cgtcagcagg cagcatatca aggtcaaagt cgtcaagggc atcggctggg
     5701 agcatgtcta agtcaaaatc gtcaagggcg tcggtcggcc cgccgctttc gcactttagc
     5761 tgtttctcca ggccacatat gattagttcc aggccgaaaa ggaaggcagg ttcggctccc
     5821 tgccggtcga acagctcaat tgcttgtttc agaagtgggg gcatagaatc ggtggtaggt
     5881 gtctctcttt cctcttttgc tacttgatgc tcctgttcct ccaatacgca gcccagtgta
     5941 aagtggccca cggcggacag agcgtacagt gcgttctcca gggagaagcc ttgctgacac
     6001 aggaacgcga gctgattttc cagggtttcg tactgtttct ctgttgggcg ggtgccgaga
     6061 tgcactttag ccccgtcgcg atgtgagagg agagcacagc ggtatgactt ggcgttgttc
     6121 cgcagaaagt cttgccatga ctcgccttcc agggggcagg agtgggtatg atgcctgtcc
     6181 agcatctcga ttggcagggc atcgagcagg gcccgcttgt tcttcacgtg ccagtacagg
     6241 gtaggctgct caactcccag cttttgagcg agtttccttg tcgtcaggcc ttcgataccg
     6301 actccattga gtaattccag agcagagttt atgactttgc tcttgtccag tctagacatc
     6361 ttatcgtcat cgtctttgta atccatggtg gcggatcccg cgtcacgaca cctgtgttct
     6421 ggcggcaaac ccgttgcgaa aaagaacgtt cacggcgact actgcactta tatacggttc
     6481 tcccccaccc tcgggaaaaa ggcggagcca gtacacgaca tcactttccc agtttacccc
     6541 gcgccacctt ctctaggcac cggttcaatt gccgacccct ccccccaact tctcggggac
     6601 tgtgggcgat gtgcgctctg cccactgacg ggcaccggag ccactcgagt ggaatt
//
