LOCUS       sequence-439807-        7357 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac transposon plasmid for doxycycline inducible expression of 
            DLX5.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7357)
  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 7357)
  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-DLX5
FEATURES             Location/Qualifiers
     source          1..7357
                     /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(1271..1295)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1297..1323
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1490..1624
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   1896..1930
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1941..1958)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1941..1957)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1950..1972)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2166..2185)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2287..2309
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2348..2366)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2434..2538
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2539..2607,2608..3399)
                     /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(2757..2776)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3570..4158
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4059..4078
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4312..4329
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4535..4556
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4571..4576,4577..4594,4595..4601)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4609..4625
                     /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     4614..4636
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4633..4649
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4633..4649
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4675..4737
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    4913..4994
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5711..5767)
                     /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(5777..6520)
                     /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(6521..6544)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6544..6553
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6573..6784)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        6785..7164
                     /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 tgacaggagt gtttgacaga
      421 agggtcccca gcatccgatc cggcgacttc caagctccgt tccagacgtc cgcagctatg
      481 caccatccgt ctcaggaatc gccaactttg cccgagtctt cagctaccga ttctgactac
      541 tacagcccta cggggggagc cccgcacggc tactgctctc ctacctcggc ttcctatggc
      601 aaagctctca acccctacca gtatcagtat cacggcgtga acggctccgc cgggagctac
      661 ccagccaaag cttatgccga ctatagctac gctagctcct accaccagta cggcggcgcc
      721 tacaaccgcg tcccaagcgc caccaaccag ccagagaaag aagtgaccga gcccgaggtg
      781 agaatggtga atggcaaacc aaagaaagtt cgtaaaccca ggactattta ttccagcttt
      841 cagctggccg cattacagag aaggtttcag aagactcagt acctcgcctt gccggaacgc
      901 gccgagctgg ccgcctcgct gggattgaca caaacacagg tgaaaatctg gtttcagaac
      961 aaaagatcca agatcaagaa gatcatgaaa aacggggaga tgcccccgga gcacagtccc
     1021 agctccagcg acccaatggc gtgtaactcg ccgcagtctc cagcggtgtg ggagccccag
     1081 ggctcgtccc gctcgctcag ccaccaccct catgcccacc ctccgacctc caaccagtcc
     1141 ccagcgtcca gctacctgga gaactctgca tcctggtaca caagtgcagc cagctcaatc
     1201 aattcccacc tgccgccgcc gggctcctta cagcacccgc tggcgctggc ctccgggaca
     1261 ctctattagt acccagcttt cttgtacaaa gtggtatacc catacgatgt tccagattac
     1321 gcttagtaat gagaaatact cagaagatgt cactcaccat agtccgcagt gaaagataaa
     1381 tgatcgtttc atcgttaacg tccaggtttt agagctagaa atagcaagta ggccactgag
     1441 caagaactat gaatgagatc tttagacata ttgcggccgc tttctccgcg atccagacat
     1501 gataagatac attgatgagt ttggacaaac caaaactaga atgcagtgaa aaaaatgcct
     1561 tatttgtgaa atttgtgatg ctattgcctt atttgtaacc attataagct gcaataaaca
     1621 agttatctat aacaagaaaa tatatatata ataagttatc acgtaagtag aacatgaaat
     1681 aacaatataa ttatcgtatg agttaaatct taaaagtcac gtaaaagata atcatgcgtc
     1741 attttgactc acgcggtcgt tatagttcaa aatcagtgac acttaccgca ttgacaagca
     1801 cgcctcacgg gagctccaag cggcgactga gatgtcctaa atgcacagcg acggattcgc
     1861 gctatttaga aagagagagc aatatttcaa gaatgcatgc gtcaatttta cgcagactat
     1921 ctttctaggg ttaagaattc actggccgtc gttttacaac gtcgtgactg ggaaaaccct
     1981 ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc
     2041 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc
     2101 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact
     2161 ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc
     2221 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     2281 atgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cacgcgagtt
     2341 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct
     2401 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     2461 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     2521 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     2581 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     2641 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     2701 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     2761 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     2821 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     2881 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     2941 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     3001 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     3061 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     3121 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     3181 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     3241 gccggtgagc gtggttcacg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     3301 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     3361 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     3421 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     3481 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     3541 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     3601 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     3661 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     3721 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     3781 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     3841 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     3901 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     3961 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     4021 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     4081 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     4141 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     4201 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     4261 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     4321 gtgagcgagg aagcggatga gcgcccaata cgcaaaccgc ctctcaccgc gcgttggccg
     4381 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac
     4441 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
     4501 ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg
     4561 caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat
     4621 aacaatttca cacaggaaac agctatgacc atgattacgc caaggtcgac ttaaccctag
     4681 aaagataatc atattgtgac gtacgttaaa gataatcatg cgtaaaattg acgcatgtgt
     4741 tttatcggtc tgtatatcga ggtttattta ttaatttgaa tagatattaa gttttattat
     4801 atttacactt acatactaat aataaattca acaaacaatt tatttatgtt tatttattta
     4861 ttaaaaaaaa acaaaaactc aaaatttctt ctataaagta acaaaacttt tagcagtgaa
     4921 aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct
     4981 gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg
     5041 tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtatggct gattatgatc
     5101 ctctggagat tcatgcgcca ggttttctgg tcatgcacca cgttcgtggg ccctcaggaa
     5161 cctccacgtc agcggtaact gtgaagccca gtcgctcgta aaaaggaaga ttccggggag
     5221 cactggtttc gaggaacgcc ggcacgccag cccgttccgc ggcctcaaca ccaggaagaa
     5281 cgacagcgct tcccaaacct ttaccttggt gatctgggct gacaccgaca gtggcaagaa
     5341 accacgcagg ctcctttggt ctatgaggag ccagcaagcc ctccatttgc tgttgtgccg
     5401 ccaacctact ccctgacagc tcggccatcc gcggacctat ttccgcgaac actgcgccgg
     5461 cttcgacgct ttccggagta gtccagaccg caactgcggc accatcgtcc gcaacccaaa
     5521 ctttaccaat gtccagaccc acgcgcgtaa gaaacagctc ttgcaactca gttaccctct
     5581 ctatgtgtct gtccggatct acggtatggc gggtcgctgg ataatctgca aatgctgcgg
     5641 caagtgttcg gacagcccgt ggtacatcgt cacgggtagc gagcctaaca gtgggcttat
     5701 attccgtcat ggggccgggg ttctcctcca cgtcgccggc ctgcttcagc aggctgaagt
     5761 tggtggcgcc gctgcccccg gggagcatgt caaggtcaaa atcgtcaaga gcgtcagcag
     5821 gcagcatatc aaggtcaaag tcgtcaaggg catcggctgg gagcatgtct aagtcaaaat
     5881 cgtcaagggc gtcggtcggc ccgccgcttt cgcactttag ctgtttctcc aggccacata
     5941 tgattagttc caggccgaaa aggaaggcag gttcggctcc ctgccggtcg aacagctcaa
     6001 ttgcttgttt cagaagtggg ggcatagaat cggtggtagg tgtctctctt tcctcttttg
     6061 ctacttgatg ctcctgttcc tccaatacgc agcccagtgt aaagtggccc acggcggaca
     6121 gagcgtacag tgcgttctcc agggagaagc cttgctgaca caggaacgcg agctgatttt
     6181 ccagggtttc gtactgtttc tctgttgggc gggtgccgag atgcacttta gccccgtcac
     6241 gatgtgagag gagagcacag cggtatgact tggcgttgtt ccgcagaaag tcttgccatg
     6301 actcgccttc cagggggcag gagtgggtat gatgcctgtc cagcatctcg attggcaggg
     6361 catcgagcag ggcccgcttg ttcttcacgt gccagtacag ggtaggctgc tcaactccca
     6421 gcttttgagc gagtttcctt gtcgtcaggc cttcgatacc gactccattg agtaattcca
     6481 gagcagagtt tatgactttg ctcttgtcca gtctagacat cttatcgtca tcgtctttgt
     6541 aatccatggt ggcggatccc gcgtcacgac acctgtgttc tggcggcaaa cccgttgcga
     6601 aaaagaacgt tcacggcgac tactgcactt atatacggtt ctcccccacc ctcgggaaaa
     6661 aggcggagcc agtacacgac atcactttcc cagtttaccc cgcgccacct tctctaggca
     6721 ccggttcaat tgccgacccc tccccccaac ttctcgggca ctgtgggcca tgtgcgctct
     6781 gccccatggt aatagccatg actaatacgt agatgtactg ccaagtagga aagtgccata
     6841 aggtcatgta ctgggcataa tgccaggcgg gccatttacc gtcattgacg tcaatagggg
     6901 gcgtacttgg catatgatac acttgatgta ctgccaagtg ggcagtttac cgtaaatact
     6961 ccacccattg acgtcaatgg aaagtgccta ttggcgttac tatgggaaca tacgtcatta
     7021 ttgacgtcaa tgggcggggg tcgttgggcg gtcagccagg cgggccattt accgtaagtt
     7081 atgtaacgcg gaactccata tatgggctat gaactaatga ccccgtaatt gattactatt
     7141 aataactagt caataatcaa tgtcataagc tctaatcgca taatcgagct aagagcgatt
     7201 aatatgctta gctagcttag ctaagctaga tttctctatt ctcccacaat atatagcagc
     7261 tattagctta gcagtccgaa tacgatcgtc cacgatccat aagcgtaaca catttgagta
     7321 gcgactgacg ggcaccggag ccactcgagt ggaatta
//
