LOCUS       sequence-437992-        6431 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac transposon plasmid for doxycycline inducible expression of 
            SOD1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6431)
  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 6431)
  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-SOD1
FEATURES             Location/Qualifiers
     source          1..6431
                     /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"
     regulatory      413..422
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(885..909)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             911..937
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1104..1238
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   1510..1544
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1555..1572)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1555..1571)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1564..1586)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1780..1799)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1901..1923
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1962..1980)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2048..2152
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2153..2221,2222..3013)
                     /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(2371..2390)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3184..3772
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3673..3692
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3926..3943
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4149..4170
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4185..4190,4191..4208,4209..4215)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4223..4239
                     /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     4228..4250
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4247..4263
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4247..4263
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4289..4351
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    4527..4608
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5325..5381)
                     /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(5391..6134)
                     /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(6135..6158)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6158..6167
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6187..6398)
                     /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 ggcgacgaag gccgtgtgcg tgctgaaggg cgacggccca gtgcagggca tcatcaattt
      481 cgagcagaag gaaagtaatg gaccagtgaa ggtgtgggga agcattaaag gactgactga
      541 aggcctgcat ggattccatg ttcatgagtt tggagataat acagcaggct gtaccagtgc
      601 aggtcctcac tttaatcctc tatccagaaa acacggtggg ccaaaggatg aagagaggca
      661 tgttggagac ttgggcaatg tgactgctga caaagatggt gtggccgatg tgtctattga
      721 agattctgtg atctcactct caggagacca ttgcatcatt ggccgcacac tggtggtcca
      781 tgaaaaagca gatgacttgg gcaaaggtgg aaatgaagaa agtacaaaga caggaaacgc
      841 tggaagtcgt ttggcttgtg gtgtaattgg gatcgcccaa taatacccag ctttcttgta
      901 caaagtggta tacccatacg atgttccaga ttacgcttag taatgagaaa tactcagaag
      961 atgtcactca ccatagtccg cagtgaaaga taaatgatca ttggcggtgc gctcgccgag
     1021 ttttagagct agaaatagca agtaggccac tgagcaagaa ctatgaatga gatctttaga
     1081 catattgcgg ccgctttctc cgcgatccag acatgataag atacattgat gagtttggac
     1141 aaaccaaaac tagaatgcag tgaaaaaaat gccttatttg tgaaatttgt gatgctattg
     1201 ccttatttgt aaccattata agctgcaata aacaagttat ctataacaag aaaatatata
     1261 tataataagt tatcacgtaa gtagaacatg aaataacaat ataattatcg tatgagttaa
     1321 atcttaaaag tcacgtaaaa gataatcatg cgtcattttg actcacgcgg tcgttatagt
     1381 tcaaaatcag tgacacttac cgcattgaca agcacgcctc acgggagctc caagcggcga
     1441 ctgagatgtc ctaaatgcac agcgacggat tcgcgctatt tagaaagaga gagcaatatt
     1501 tcaagaatgc atgcgtcaat tttacgcaga ctatctttct agggttaaga attcactggc
     1561 cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc
     1621 agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc
     1681 ccaacagttg cgcagcctga atggcgaatg gcgcctgatg cggtattttc tccttacgca
     1741 tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct ctgatgccgc
     1801 atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     1861 gctcccggca tccgcttaca gacaagctgt gaccatgtct ccgggagctg catgtgtcag
     1921 aggttttcac cgtcatcacc gaaacacgcg agttcgaaag ggcctcgtga tacgcctatt
     1981 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg
     2041 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     2101 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     2161 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     2221 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     2281 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     2341 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     2401 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     2461 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     2521 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     2581 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     2641 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     2701 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     2761 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     2821 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtggtt cacgcggtat
     2881 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     2941 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     3001 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     3061 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     3121 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     3181 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     3241 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     3301 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     3361 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     3421 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     3481 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     3541 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     3601 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     3661 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     3721 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     3781 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     3841 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     3901 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg atgagcgccc
     3961 aatacgcaaa ccgcctctca ccgcgcgttg gccgattcat taatgcagct ggcacgacag
     4021 gtttcccgac tggaaagcgg gcagtgagcg caacgcaaac cgcctctccc cgcgcgttgg
     4081 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
     4141 aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt
     4201 ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat
     4261 gaccatgatt acgccaaggt cgacttaacc ctagaaagat aatcatattg tgacgtacgt
     4321 taaagataat catgcgtaaa attgacgcat gtgttttatc ggtctgtata tcgaggttta
     4381 tttattaatt tgaatagata ttaagtttta ttatatttac acttacatac taataataaa
     4441 ttcaacaaac aatttattta tgtttattta tttattaaaa aaaaacaaaa actcaaaatt
     4501 tcttctataa agtaacaaaa cttttagcag tgaaaaaaat gctttatttg tgaaatttgt
     4561 gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat
     4621 tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta aagcaagtaa
     4681 aacctctaca aatgtggtat ggctgattat gatcctctgg agattcatgc gccaggtttt
     4741 ctggtcatgc accacgttcg tgggccctca ggaacctcca cgtcagcggt aactgtgaag
     4801 cccagtcgct cgtaaaaagg aagattccgg ggagcactgg tttcgaggaa cgccggcacg
     4861 ccagcccgtt ccgcggcctc aacaccagga agaacgacag cgcttcccaa acctttacct
     4921 tggtgatctg ggctgacacc gacagtggca agaaaccacg caggctcctt tggtctatga
     4981 ggagccagca agccctccat ttgctgttgt gccgccaacc tactccctga cagctcggcc
     5041 atccgcggac ctatttccgc gaacactgcg ccggcttcga cgctttccgg agtagtccag
     5101 accgcaactg cggcaccatc gtccgcaacc caaactttac caatgtccag acccacgcgc
     5161 gtaagaaaca gctcttgcaa ctcagttacc ctctctatgt gtctgtccgg atctacggta
     5221 tggcgggtcg ctggataatc tgcaaatgct gcggcaagtg ttcggacagc ccgtggtaca
     5281 tcgtcacggg tagcgagcct aacagtgggc ttatattccg tcatggggcc ggggttctcc
     5341 tccacgtcgc cggcctgctt cagcaggctg aagttggtgg cgccgctgcc cccggggagc
     5401 atgtcaaggt caaaatcgtc aagagcgtca gcaggcagca tatcaaggtc aaagtcgtca
     5461 agggcatcgg ctgggagcat gtctaagtca aaatcgtcaa gggcgtcggt cggcccgccg
     5521 ctttcgcact ttagctgttt ctccaggcca catatgatta gttccaggcc gaaaaggaag
     5581 gcaggttcgg ctccctgccg gtcgaacagc tcaattgctt gtttcagaag tgggggcata
     5641 gaatcggtgg taggtgtctc tctttcctct tttgctactt gatgctcctg ttcctccaat
     5701 acgcagccca gtgtaaagtg gcccacggcg gacagagcgt acagtgcgtt ctccagggag
     5761 aagccttgct gacacaggaa cgcgagctga ttttccaggg tttcgtactg tttctctgtt
     5821 gggcgggtgc cgagatgcac tttagccccg tcgcgatgtg agaggagagc acagcggtat
     5881 gacttggcgt tgttccgcag aaagtcttgc catgactcgc cttccagggg gcaggagtgg
     5941 gtatgatgcc tgtccagcat ctcgattggc agggcatcga gcagggcccg cttgttcttc
     6001 acgtgccagt acagggtagg ctgctcaact cccagctttt gagcgagttt ccttgtcgtc
     6061 aggccttcga taccgactcc attgagtaat tccagagcag agtttatgac tttgctcttg
     6121 tccagtctag acatcttatc gtcatcgtct ttgtaatcca tggtggcgga tcccgcgtca
     6181 cgacacctgt gttctggcgg caaacccgtt gcgaaaaaga acgttcacgg cgactactgc
     6241 acttatatac ggttctcccc caccctcggg aaaaaggcgg agccagtaca cgacatcact
     6301 ttcccagttt accccgcgcc accttctcta ggcaccggtt caattgccga cccctccccc
     6361 caacttctcg gggactgtgg gcgatgtgcg ctctgcccac tgacgggcac cggagccact
     6421 cgagtggaat t
//
