LOCUS       sequence-439854-        7300 bp    DNA     circular SYN 15-APR-2025
DEFINITION  PiggyBac transposon plasmid for doxycycline inducible expression of 
            HHEX.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7300)
  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 7300)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-HHEX
FEATURES             Location/Qualifiers
     source          1..7300
                     /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(1214..1238)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1240..1266
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1433..1567
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   1839..1873
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1884..1901)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1884..1900)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1893..1915)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2109..2128)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2230..2252
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2291..2309)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2377..2481
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2482..2550,2551..3342)
                     /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(2700..2719)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3513..4101
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4002..4021
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4255..4272
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4478..4499
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4514..4519,4520..4537,4538..4544)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4552..4568
                     /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     4557..4579
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4576..4592
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4576..4592
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4618..4680
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    4856..4937
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5654..5710)
                     /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(5720..6463)
                     /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(6464..6487)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6487..6496
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6516..6727)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        6728..7107
                     /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 ttagccacca tgcagtaccc gcaccccggg
      421 ccggcggcgg gcgccgtggg ggtgccgctg tacgcgccca cgccgctgct gcaacccgca
      481 cacccgacgc ccttttacat cgaggacatc ctgggccgcg ggcccgccgc gcccacgccc
      541 gcccccacgc tgccgtcccc caactcctcc ttcaccagcc tcgtgtcccc ctaccggacc
      601 ccggtgtacg agcccacgcc gatccatcca gccttctcgc accactccgc cgccgcgctg
      661 gccgctgcct acggacccgg cggcttcggg ggccctctgt accccttccc gcggacggtg
      721 aacgactaca cgcacgccct gctccgccac gaccccctgg gcaaacctct actctggagc
      781 cccttcttgc agaggcctct gcataaaagg aaaggcggcc aggtgagatt ctccaacgac
      841 cagaccatcg agctggagaa gaaattcgag acgcagaaat atctctctcc gcccgagagg
      901 aagcgtctgg ccaagatgct gcagctcagc gagagacagg tcaaaacctg gtttcagaat
      961 cgacgcgcta aatggaggag actaaaacag gagaaccctc aaagcaataa aaaagaagaa
     1021 ctggaaagtt tggacagttc ctgtgatcag aggcaagatt tgcccagtga acagaataaa
     1081 ggtgcttctt tggatagctc tcaatgttcg ccctcccctg cctcccagga agaccttgaa
     1141 tcagagattt cagaggattc tgatcaggaa gtggacattg agggcgataa aagctatttt
     1201 aatgctggat gatacccagc tttcttgtac aaagtggtat acccatacga tgttccagat
     1261 tacgcttagt aatgagaaat actcagaaga tgtcactcac catagtccgc agtgaaagat
     1321 aaatgatcga atgccgtttc gttgcattgt tttagagcta gaaatagcaa gtaggccact
     1381 gagcaagaac tatgaatgag atctttagac atattgcggc cgctttctcc gcgatccaga
     1441 catgataaga tacattgatg agtttggaca aaccaaaact agaatgcagt gaaaaaaatg
     1501 ccttatttgt gaaatttgtg atgctattgc cttatttgta accattataa gctgcaataa
     1561 acaagttatc tataacaaga aaatatatat ataataagtt atcacgtaag tagaacatga
     1621 aataacaata taattatcgt atgagttaaa tcttaaaagt cacgtaaaag ataatcatgc
     1681 gtcattttga ctcacgcggt cgttatagtt caaaatcagt gacacttacc gcattgacaa
     1741 gcacgcctca cgggagctcc aagcggcgac tgagatgtcc taaatgcaca gcgacggatt
     1801 cgcgctattt agaaagagag agcaatattt caagaatgca tgcgtcaatt ttacgcagac
     1861 tatctttcta gggttaagaa ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac
     1921 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat
     1981 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
     2041 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catatggtgc
     2101 actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca
     2161 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     2221 accatgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacacgcga
     2281 gttcgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt
     2341 tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     2401 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     2461 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     2521 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     2581 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     2641 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     2701 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     2761 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     2821 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     2881 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     2941 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     3001 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     3061 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     3121 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     3181 ggagccggtg agcgtggttc acgcggtatc attgcagcac tggggccaga tggtaagccc
     3241 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     3301 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     3361 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     3421 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     3481 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     3541 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     3601 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     3661 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     3721 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     3781 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     3841 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     3901 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     3961 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     4021 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     4081 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     4141 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     4201 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     4261 tcagtgagcg aggaagcgga tgagcgccca atacgcaaac cgcctctcac cgcgcgttgg
     4321 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
     4381 aacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
     4441 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt
     4501 aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg
     4561 gataacaatt tcacacagga aacagctatg accatgatta cgccaaggtc gacttaaccc
     4621 tagaaagata atcatattgt gacgtacgtt aaagataatc atgcgtaaaa ttgacgcatg
     4681 tgttttatcg gtctgtatat cgaggtttat ttattaattt gaatagatat taagttttat
     4741 tatatttaca cttacatact aataataaat tcaacaaaca atttatttat gtttatttat
     4801 ttattaaaaa aaaacaaaaa ctcaaaattt cttctataaa gtaacaaaac ttttagcagt
     4861 gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa
     4921 gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg
     4981 aggtgtggga ggttttttaa agcaagtaaa acctctacaa atgtggtatg gctgattatg
     5041 atcctctgga gattcatgcg ccaggttttc tggtcatgca ccacgttcgt gggccctcag
     5101 gaacctccac gtcagcggta actgtgaagc ccagtcgctc gtaaaaagga agattccggg
     5161 gagcactggt ttcgaggaac gccggcacgc cagcccgttc cgcggcctca acaccaggaa
     5221 gaacgacagc gcttcccaaa cctttacctt ggtgatctgg gctgacaccg acagtggcaa
     5281 gaaaccacgc aggctccttt ggtctatgag gagccagcaa gccctccatt tgctgttgtg
     5341 ccgccaacct actccctgac agctcggcca tccgcggacc tatttccgcg aacactgcgc
     5401 cggcttcgac gctttccgga gtagtccaga ccgcaactgc ggcaccatcg tccgcaaccc
     5461 aaactttacc aatgtccaga cccacgcgcg taagaaacag ctcttgcaac tcagttaccc
     5521 tctctatgtg tctgtccgga tctacggtat ggcgggtcgc tggataatct gcaaatgctg
     5581 cggcaagtgt tcggacagcc cgtggtacat cgtcacgggt agcgagccta acagtgggct
     5641 tatattccgt catggggccg gggttctcct ccacgtcgcc ggcctgcttc agcaggctga
     5701 agttggtggc gccgctgccc ccggggagca tgtcaaggtc aaaatcgtca agagcgtcag
     5761 caggcagcat atcaaggtca aagtcgtcaa gggcatcggc tgggagcatg tctaagtcaa
     5821 aatcgtcaag ggcgtcggtc ggcccgccgc tttcgcactt tagctgtttc tccaggccac
     5881 atatgattag ttccaggccg aaaaggaagg caggttcggc tccctgccgg tcgaacagct
     5941 caattgcttg tttcagaagt gggggcatag aatcggtggt aggtgtctct ctttcctctt
     6001 ttgctacttg atgctcctgt tcctccaata cgcagcccag tgtaaagtgg cccacggcgg
     6061 acagagcgta cagtgcgttc tccagggaga agccttgctg acacaggaac gcgagctgat
     6121 tttccagggt ttcgtactgt ttctctgttg ggcgggtgcc gagatgcact ttagccccgt
     6181 cacgatgtga gaggagagca cagcggtatg acttggcgtt gttccgcaga aagtcttgcc
     6241 atgactcgcc ttccaggggg caggagtggg tatgatgcct gtccagcatc tcgattggca
     6301 gggcatcgag cagggcccgc ttgttcttca cgtgccagta cagggtaggc tgctcaactc
     6361 ccagcttttg agcgagtttc cttgtcgtca ggccttcgat accgactcca ttgagtaatt
     6421 ccagagcaga gtttatgact ttgctcttgt ccagtctaga catcttatcg tcatcgtctt
     6481 tgtaatccat ggtggcggat cccgcgtcac gacacctgtg ttctggcggc aaacccgttg
     6541 cgaaaaagaa cgttcacggc gactactgca cttatatacg gttctccccc accctcggga
     6601 aaaaggcgga gccagtacac gacatcactt tcccagttta ccccgcgcca ccttctctag
     6661 gcaccggttc aattgccgac ccctcccccc aacttctcgg gcactgtggg ccatgtgcgc
     6721 tctgccccat ggtaatagcc atgactaata cgtagatgta ctgccaagta ggaaagtgcc
     6781 ataaggtcat gtactgggca taatgccagg cgggccattt accgtcattg acgtcaatag
     6841 ggggcgtact tggcatatga tacacttgat gtactgccaa gtgggcagtt taccgtaaat
     6901 actccaccca ttgacgtcaa tggaaagtgc ctattggcgt tactatggga acatacgtca
     6961 ttattgacgt caatgggcgg gggtcgttgg gcggtcagcc aggcgggcca tttaccgtaa
     7021 gttatgtaac gcggaactcc atatatgggc tatgaactaa tgaccccgta attgattact
     7081 attaataact agtcaataat caatgtcata agctctaatc gcataatcga gctaagagcg
     7141 attaatatgc ttagctagct tagctaagct agatttctct attctcccac aatatatagc
     7201 agctattagc ttagcagtcc gaatacgatc gtccacgatc cataagcgta acacatttga
     7261 gtagcgactg acgggcaccg gagccactcg agtggaatta
//
