LOCUS       sequence-439859-        7756 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac transposon plasmid for doxycycline inducible expression of 
            SOX17.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7756)
  AUTHORS   Pierson Smela M, Kramme CC, Fortuna PRJ, Wolf B, Srikar Kavirayuni 
            V, Adams J, Ma C, Velychko S, Widocki U, Goel S, Chen T, Vincoff S, 
            Dong E, Kohman RE, Kobayashi M, Shioda T, Church GM, Chatterjee P
  TITLE     Rapid Human Oogonia-like Cell Specification via Combinatorial 
            Transcription Factor-Directed Differentiation
  JOURNAL   bioRxiv 2022.07.11.499564
REFERENCE   2  (bases 1 to 7756)
  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 
            2022.07.11.499564"
COMMENT     Sequence Label: pPB-cT3G-cERP2-SOX17
FEATURES             Location/Qualifiers
     source          1..7756
                     /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"
     CDS             1357..1374
                     /codon_start=1
                     /product="HQHQHQ metal affinity tag"
                     /label=HQ tag
                     /note="The HQ tag binds to nickel and other metal ions much
                     like the 6xHis tag, but may allow elution at lower 
                     concentrations of imidazole (Godat et al., 2008)."
                     /translation="HQHQHQ"
     CDS             1363..1380
                     /codon_start=1
                     /product="HQHQHQ metal affinity tag"
                     /label=HQ tag
                     /note="The HQ tag binds to nickel and other metal ions much
                     like the 6xHis tag, but may allow elution at lower 
                     concentrations of imidazole (Godat et al., 2008)."
                     /translation="HQHQHQ"
     protein_bind    complement(1670..1694)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1696..1722
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1889..2023
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   2295..2329
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2340..2357)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2340..2356)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2349..2371)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2565..2584)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2686..2708
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2747..2765)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2833..2937
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2938..3006,3007..3798)
                     /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(3156..3175)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3969..4557
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4458..4477
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4711..4728
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4934..4955
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4970..4975,4976..4993,4994..5000)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5008..5024
                     /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     5013..5035
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5032..5048
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5032..5048
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   5074..5136
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    5312..5393
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(6110..6166)
                     /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(6176..6919)
                     /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(6920..6943)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6943..6952
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6972..7183)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        7184..7563
                     /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 aaaagcaggc ttggaaggag ttcgaaccat gagcagcccg
      421 gatgcgggat acgccagtga cgaccagagc cagacccaga gcgcgctgcc cgcggtgatg
      481 gccgggctgg gcccctgccc ctgggccgag tcgctgagcc ccatcgggga catgaaggtg
      541 aagggcgagg cgccggcgaa cagcggagca ccggccgggg ccgcgggccg agccaagggc
      601 gagtcccgta tccggcggcc gatgaacgct ttcatggtgt gggctaagga cgagcgcaag
      661 cggctggcgc agcagaatcc agacctgcac aacgccgagt tgagcaagat gctgggcaag
      721 tcgtggaagg cgctgacgct ggcggagaag cggcccttcg tggaggaggc agagcggctg
      781 cgcgtgcagc acatgcagga ccaccccaac tacaagtacc ggccgcggcg gcgcaagcag
      841 gtgaagcggc tgaagcgggt ggagggcggc ttcctgcacg gcctggctga gccgcaggcg
      901 gccgcgctgg gccccgaggg cggccgcgtg gccatggacg gcctgggcct ccagttcccc
      961 gagcagggct tccccgccgg cccgccgctg ctgcctccgc acatgggcgg ccactaccgc
     1021 gactgccaga gtctgggcgc gcctccgctc gacggctacc cgttgcccac gcccgacacg
     1081 tccccgctgg acggcgtgga ccccgacccg gctttcttcg ccgccccgat gcccggggac
     1141 tgcccggcgg ccggcaccta cagctacgcg caggtctcgg actacgctgg ccccccggag
     1201 cctcccgccg gtcccatgca cccccgactc ggcccagagc ccgcgggtcc ctcgattccg
     1261 ggcctcctgg cgccacccag cgcccttcac gtgtactacg gcgcgatggg ctcgcccggg
     1321 gcgggcggcg ggcgcggctt ccagatgcag ccgcaacacc agcaccagca ccagcaccag
     1381 caccaccccc cgggccccgg acagccgtcg ccccctccgg aggcactgcc ctgccgggac
     1441 ggcacggacc ccagtcagcc cgccgagctc ctcggggagg tggaccgcac ggaatttgaa
     1501 cagtatctgc acttcgtgtg caagcctgag atgggcctcc cctaccaggg gcatgactcc
     1561 ggtgtgaatc tccccgacag ccacggggcc atttcctcgg tggtgtccga cgccagctcc
     1621 gcggtatatt actgcaacta tcctgacgtg tagctcgagt gcggccgcaa cccagctttc
     1681 ttgtacaaag tggtataccc atacgatgtt ccagattacg cttagtaatg agaaatactc
     1741 agaagatgtc actcaccata gtccgcagtg aaagataaat gatctctttt ggttcatcta
     1801 ttttgtttta gagctagaaa tagcaagtag gccactgagc aagaactatg aatgagatct
     1861 ttagacatat tgcggccgct ttctccgcga tccagacatg ataagataca ttgatgagtt
     1921 tggacaaacc aaaactagaa tgcagtgaaa aaaatgcctt atttgtgaaa tttgtgatgc
     1981 tattgcctta tttgtaacca ttataagctg caataaacaa gttatctata acaagaaaat
     2041 atatatataa taagttatca cgtaagtaga acatgaaata acaatataat tatcgtatga
     2101 gttaaatctt aaaagtcacg taaaagataa tcatgcgtca ttttgactca cgcggtcgtt
     2161 atagttcaaa atcagtgaca cttaccgcat tgacaagcac gcctcacggg agctccaagc
     2221 ggcgactgag atgtcctaaa tgcacagcga cggattcgcg ctatttagaa agagagagca
     2281 atatttcaag aatgcatgcg tcaattttac gcagactatc tttctagggt taagaattca
     2341 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc
     2401 cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc
     2461 ccttcccaac agttgcgcag cctgaatggc gaatggcgcc tgatgcggta ttttctcctt
     2521 acgcatctgt gcggtatttc acaccgcata tggtgcactc tcagtacaat ctgctctgat
     2581 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     2641 tgtctgctcc cggcatccgc ttacagacaa gctgtgacca tgtctccggg agctgcatgt
     2701 gtcagaggtt ttcaccgtca tcaccgaaac acgcgagttc gaaagggcct cgtgatacgc
     2761 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
     2821 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     2881 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     2941 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     3001 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     3061 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     3121 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     3181 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     3241 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     3301 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     3361 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     3421 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     3481 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     3541 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     3601 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tggttcacgc
     3661 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     3721 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     3781 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     3841 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     3901 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     3961 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     4021 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     4081 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     4141 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     4201 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     4261 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     4321 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     4381 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     4441 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     4501 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     4561 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     4621 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     4681 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggatgag
     4741 cgcccaatac gcaaaccgcc tctcaccgcg cgttggccga ttcattaatg cagctggcac
     4801 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caaaccgcct ctccccgcgc
     4861 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
     4921 agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta
     4981 tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca
     5041 gctatgacca tgattacgcc aaggtcgact taaccctaga aagataatca tattgtgacg
     5101 tacgttaaag ataatcatgc gtaaaattga cgcatgtgtt ttatcggtct gtatatcgag
     5161 gtttatttat taatttgaat agatattaag ttttattata tttacactta catactaata
     5221 ataaattcaa caaacaattt atttatgttt atttatttat taaaaaaaaa caaaaactca
     5281 aaatttcttc tataaagtaa caaaactttt agcagtgaaa aaaatgcttt atttgtgaaa
     5341 tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca
     5401 acaattgcat tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttaaagca
     5461 agtaaaacct ctacaaatgt ggtatggctg attatgatcc tctggagatt catgcgccag
     5521 gttttctggt catgcaccac gttcgtgggc cctcaggaac ctccacgtca gcggtaactg
     5581 tgaagcccag tcgctcgtaa aaaggaagat tccggggagc actggtttcg aggaacgccg
     5641 gcacgccagc ccgttccgcg gcctcaacac caggaagaac gacagcgctt cccaaacctt
     5701 taccttggtg atctgggctg acaccgacag tggcaagaaa ccacgcaggc tcctttggtc
     5761 tatgaggagc cagcaagccc tccatttgct gttgtgccgc caacctactc cctgacagct
     5821 cggccatccg cggacctatt tccgcgaaca ctgcgccggc ttcgacgctt tccggagtag
     5881 tccagaccgc aactgcggca ccatcgtccg caacccaaac tttaccaatg tccagaccca
     5941 cgcgcgtaag aaacagctct tgcaactcag ttaccctctc tatgtgtctg tccggatcta
     6001 cggtatggcg ggtcgctgga taatctgcaa atgctgcggc aagtgttcgg acagcccgtg
     6061 gtacatcgtc acgggtagcg agcctaacag tgggcttata ttccgtcatg gggccggggt
     6121 tctcctccac gtcgccggcc tgcttcagca ggctgaagtt ggtggcgccg ctgcccccgg
     6181 ggagcatgtc aaggtcaaaa tcgtcaagag cgtcagcagg cagcatatca aggtcaaagt
     6241 cgtcaagggc atcggctggg agcatgtcta agtcaaaatc gtcaagggcg tcggtcggcc
     6301 cgccgctttc gcactttagc tgtttctcca ggccacatat gattagttcc aggccgaaaa
     6361 ggaaggcagg ttcggctccc tgccggtcga acagctcaat tgcttgtttc agaagtgggg
     6421 gcatagaatc ggtggtaggt gtctctcttt cctcttttgc tacttgatgc tcctgttcct
     6481 ccaatacgca gcccagtgta aagtggccca cggcggacag agcgtacagt gcgttctcca
     6541 gggagaagcc ttgctgacac aggaacgcga gctgattttc cagggtttcg tactgtttct
     6601 ctgttgggcg ggtgccgaga tgcactttag ccccgtcacg atgtgagagg agagcacagc
     6661 ggtatgactt ggcgttgttc cgcagaaagt cttgccatga ctcgccttcc agggggcagg
     6721 agtgggtatg atgcctgtcc agcatctcga ttggcagggc atcgagcagg gcccgcttgt
     6781 tcttcacgtg ccagtacagg gtaggctgct caactcccag cttttgagcg agtttccttg
     6841 tcgtcaggcc ttcgataccg actccattga gtaattccag agcagagttt atgactttgc
     6901 tcttgtccag tctagacatc ttatcgtcat cgtctttgta atccatggtg gcggatcccg
     6961 cgtcacgaca cctgtgttct ggcggcaaac ccgttgcgaa aaagaacgtt cacggcgact
     7021 actgcactta tatacggttc tcccccaccc tcgggaaaaa ggcggagcca gtacacgaca
     7081 tcactttccc agtttacccc gcgccacctt ctctaggcac cggttcaatt gccgacccct
     7141 ccccccaact tctcgggcac tgtgggccat gtgcgctctg ccccatggta atagccatga
     7201 ctaatacgta gatgtactgc caagtaggaa agtgccataa ggtcatgtac tgggcataat
     7261 gccaggcggg ccatttaccg tcattgacgt caataggggg cgtacttggc atatgataca
     7321 cttgatgtac tgccaagtgg gcagtttacc gtaaatactc cacccattga cgtcaatgga
     7381 aagtgcctat tggcgttact atgggaacat acgtcattat tgacgtcaat gggcgggggt
     7441 cgttgggcgg tcagccaggc gggccattta ccgtaagtta tgtaacgcgg aactccatat
     7501 atgggctatg aactaatgac cccgtaattg attactatta ataactagtc aataatcaat
     7561 gtcataagct ctaatcgcat aatcgagcta agagcgatta atatgcttag ctagcttagc
     7621 taagctagat ttctctattc tcccacaata tatagcagct attagcttag cagtccgaat
     7681 acgatcgtcc acgatccata agcgtaacac atttgagtag cgactgacgg gcaccggagc
     7741 cactcgagtg gaatta
//
