LOCUS       sequence-442479-        7753 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac transposon plasmid for doxycycline inducible expression of 
            PAX6a.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7753)
  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 7753)
  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-PAX6a
FEATURES             Location/Qualifiers
     source          1..7753
                     /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(1668..1692)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1694..1720
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1887..2021
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   2293..2327
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2338..2355)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2338..2354)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2347..2369)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2563..2582)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2684..2706
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2745..2763)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2831..2935
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2936..3004,3005..3796)
                     /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(3154..3173)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3967..4555
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4456..4475
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4709..4726
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4932..4953
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4968..4973,4974..4991,4992..4998)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5006..5022
                     /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     5011..5033
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5030..5046
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5030..5046
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   5072..5134
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    5310..5391
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(6108..6164)
                     /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(6174..6917)
                     /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(6918..6941)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6941..6950
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6970..7181)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        7182..7561
                     /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 ctgcaccatg cagaacagtc acagcggagt
      421 gaatcagctc ggtggtgtct ttgtcaacgg gcggccactg ccggactcca cccggcagaa
      481 gattgtagag ctagctcaca gcggggcccg gccgtgcgac atttcccgaa ttctgcaggt
      541 gtccaacgga tgtgtgagta aaattctggg caggtattac gagactggct ccatcagacc
      601 cagggcaatc ggtggtagta aaccgagagt agcgactcca gaagttgtaa gcaaaatagc
      661 ccagtataag cgggagtgcc cgtccatctt tgcttgggaa atccgagaca gattactgtc
      721 cgagggggtc tgtaccaacg ataacatacc aagcgtgtca tcaataaaca gagttcttcg
      781 caacctggct agcgaaaagc aacagatggg cgcagacggc atgtatgata aactaaggat
      841 gttgaacggg cagaccggaa gctggggcac ccgccctggt tggtatccgg ggacttcggt
      901 gccagggcaa cctacgcaag atggctgcca gcaacaggaa ggagggggag agaataccaa
      961 ctccatcagt tccaacggag aagattcaga tgaggctcaa atgcgacttc agctgaagcg
     1021 gaagctgcaa agaaatagaa catcctttac ccaagagcaa attgaggccc tggagaaaga
     1081 gtttgagaga acccattatc cagatgtgtt tgcccgagaa agactagcag ccaaaataga
     1141 tctacctgaa gcaagaatac aggtatggtt ttctaatcga agggccaaat ggagaagaga
     1201 agaaaaactg aggaatcaga gaagacaggc cagcaacaca cctagtcata ttcctatcag
     1261 cagtagtttc agcaccagtg tctaccaacc aattccacaa cccaccacac cggtttcctc
     1321 cttcacatct ggctccatgt tgggccgaac agacacagcc ctcacaaaca cctacagcgc
     1381 tctgccgcct atgcccagct tcaccatggc aaataacctg cctatgcaac ccccagtccc
     1441 cagccagacc tcctcatact cctgcatgct gcccaccagc ccttcggtga atgggcggag
     1501 ttatgatacc tacacccccc cacatatgca gacacacatg aacagtcagc caatgggcac
     1561 ctcgggcacc acttcaacag gactcatttc ccctggtgtg tcagttccag ttcaagttcc
     1621 cggaagtgaa cctgatatgt ctcaatactg gccaagatta cagtaagacc cagctttctt
     1681 gtacaaagtg gtatacccat acgatgttcc agattacgct tagtaatgag aaatactcag
     1741 aagatgtcac tcaccatagt ccgcagtgaa agataaatga tccttttcaa ataagacgtc
     1801 tagttttaga gctagaaata gcaagtaggc cactgagcaa gaactatgaa tgagatcttt
     1861 agacatattg cggccgcttt ctccgcgatc cagacatgat aagatacatt gatgagtttg
     1921 gacaaaccaa aactagaatg cagtgaaaaa aatgccttat ttgtgaaatt tgtgatgcta
     1981 ttgccttatt tgtaaccatt ataagctgca ataaacaagt tatctataac aagaaaatat
     2041 atatataata agttatcacg taagtagaac atgaaataac aatataatta tcgtatgagt
     2101 taaatcttaa aagtcacgta aaagataatc atgcgtcatt ttgactcacg cggtcgttat
     2161 agttcaaaat cagtgacact taccgcattg acaagcacgc ctcacgggag ctccaagcgg
     2221 cgactgagat gtcctaaatg cacagcgacg gattcgcgct atttagaaag agagagcaat
     2281 atttcaagaa tgcatgcgtc aattttacgc agactatctt tctagggtta agaattcact
     2341 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
     2401 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
     2461 ttcccaacag ttgcgcagcc tgaatggcga atggcgcctg atgcggtatt ttctccttac
     2521 gcatctgtgc ggtatttcac accgcatatg gtgcactctc agtacaatct gctctgatgc
     2581 cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     2641 tctgctcccg gcatccgctt acagacaagc tgtgaccatg tctccgggag ctgcatgtgt
     2701 cagaggtttt caccgtcatc accgaaacac gcgagttcga aagggcctcg tgatacgcct
     2761 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     2821 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     2881 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     2941 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     3001 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     3061 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     3121 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     3181 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     3241 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     3301 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     3361 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     3421 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     3481 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     3541 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     3601 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg gttcacgcgg
     3661 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     3721 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     3781 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     3841 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     3901 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     3961 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     4021 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     4081 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     4141 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     4201 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     4261 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     4321 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     4381 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     4441 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     4501 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     4561 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     4621 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     4681 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggatgagcg
     4741 cccaatacgc aaaccgcctc tcaccgcgcg ttggccgatt cattaatgca gctggcacga
     4801 caggtttccc gactggaaag cgggcagtga gcgcaacgca aaccgcctct ccccgcgcgt
     4861 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
     4921 cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg
     4981 cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc
     5041 tatgaccatg attacgccaa ggtcgactta accctagaaa gataatcata ttgtgacgta
     5101 cgttaaagat aatcatgcgt aaaattgacg catgtgtttt atcggtctgt atatcgaggt
     5161 ttatttatta atttgaatag atattaagtt ttattatatt tacacttaca tactaataat
     5221 aaattcaaca aacaatttat ttatgtttat ttatttatta aaaaaaaaca aaaactcaaa
     5281 atttcttcta taaagtaaca aaacttttag cagtgaaaaa aatgctttat ttgtgaaatt
     5341 tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac
     5401 aattgcattc attttatgtt tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag
     5461 taaaacctct acaaatgtgg tatggctgat tatgatcctc tggagattca tgcgccaggt
     5521 tttctggtca tgcaccacgt tcgtgggccc tcaggaacct ccacgtcagc ggtaactgtg
     5581 aagcccagtc gctcgtaaaa aggaagattc cggggagcac tggtttcgag gaacgccggc
     5641 acgccagccc gttccgcggc ctcaacacca ggaagaacga cagcgcttcc caaaccttta
     5701 ccttggtgat ctgggctgac accgacagtg gcaagaaacc acgcaggctc ctttggtcta
     5761 tgaggagcca gcaagccctc catttgctgt tgtgccgcca acctactccc tgacagctcg
     5821 gccatccgcg gacctatttc cgcgaacact gcgccggctt cgacgctttc cggagtagtc
     5881 cagaccgcaa ctgcggcacc atcgtccgca acccaaactt taccaatgtc cagacccacg
     5941 cgcgtaagaa acagctcttg caactcagtt accctctcta tgtgtctgtc cggatctacg
     6001 gtatggcggg tcgctggata atctgcaaat gctgcggcaa gtgttcggac agcccgtggt
     6061 acatcgtcac gggtagcgag cctaacagtg ggcttatatt ccgtcatggg gccggggttc
     6121 tcctccacgt cgccggcctg cttcagcagg ctgaagttgg tggcgccgct gcccccgggg
     6181 agcatgtcaa ggtcaaaatc gtcaagagcg tcagcaggca gcatatcaag gtcaaagtcg
     6241 tcaagggcat cggctgggag catgtctaag tcaaaatcgt caagggcgtc ggtcggcccg
     6301 ccgctttcgc actttagctg tttctccagg ccacatatga ttagttccag gccgaaaagg
     6361 aaggcaggtt cggctccctg ccggtcgaac agctcaattg cttgtttcag aagtgggggc
     6421 atagaatcgg tggtaggtgt ctctctttcc tcttttgcta cttgatgctc ctgttcctcc
     6481 aatacgcagc ccagtgtaaa gtggcccacg gcggacagag cgtacagtgc gttctccagg
     6541 gagaagcctt gctgacacag gaacgcgagc tgattttcca gggtttcgta ctgtttctct
     6601 gttgggcggg tgccgagatg cactttagcc ccgtcacgat gtgagaggag agcacagcgg
     6661 tatgacttgg cgttgttccg cagaaagtct tgccatgact cgccttccag ggggcaggag
     6721 tgggtatgat gcctgtccag catctcgatt ggcagggcat cgagcagggc ccgcttgttc
     6781 ttcacgtgcc agtacagggt aggctgctca actcccagct tttgagcgag tttccttgtc
     6841 gtcaggcctt cgataccgac tccattgagt aattccagag cagagtttat gactttgctc
     6901 ttgtccagtc tagacatctt atcgtcatcg tctttgtaat ccatggtggc ggatcccgcg
     6961 tcacgacacc tgtgttctgg cggcaaaccc gttgcgaaaa agaacgttca cggcgactac
     7021 tgcacttata tacggttctc ccccaccctc gggaaaaagg cggagccagt acacgacatc
     7081 actttcccag tttaccccgc gccaccttct ctaggcaccg gttcaattgc cgacccctcc
     7141 ccccaacttc tcgggcactg tgggccatgt gcgctctgcc ccatggtaat agccatgact
     7201 aatacgtaga tgtactgcca agtaggaaag tgccataagg tcatgtactg ggcataatgc
     7261 caggcgggcc atttaccgtc attgacgtca atagggggcg tacttggcat atgatacact
     7321 tgatgtactg ccaagtgggc agtttaccgt aaatactcca cccattgacg tcaatggaaa
     7381 gtgcctattg gcgttactat gggaacatac gtcattattg acgtcaatgg gcgggggtcg
     7441 ttgggcggtc agccaggcgg gccatttacc gtaagttatg taacgcggaa ctccatatat
     7501 gggctatgaa ctaatgaccc cgtaattgat tactattaat aactagtcaa taatcaatgt
     7561 cataagctct aatcgcataa tcgagctaag agcgattaat atgcttagct agcttagcta
     7621 agctagattt ctctattctc ccacaatata tagcagctat tagcttagca gtccgaatac
     7681 gatcgtccac gatccataag cgtaacacat ttgagtagcg actgacgggc accggagcca
     7741 ctcgagtgga att
//
