LOCUS       sequence-439860-        7147 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac transposon plasmid for doxycycline inducible expression of 
            FIGLA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7147)
  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 7147)
  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-FIGLA
FEATURES             Location/Qualifiers
     source          1..7147
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1..17
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   43..105
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    281..362
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(1079..1135)
                     /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(1145..1888)
                     /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(1889..1912)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      1912..1921
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(1941..2152)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        2153..2532
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     protein_bind    2741..2759
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2777..2795
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2813..2831
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2849..2867
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2885..2903
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2921..2939
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2957..2975
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     3002..3026
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    3092..3116
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      3119..3128
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(3786..3810)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3812..3838
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    4005..4139
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   4411..4445
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(4456..4473)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4456..4472)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4465..4487)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(4681..4700)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4802..4824
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4863..4881)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4949..5053
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5054..5122,5123..5914)
                     /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(5272..5291)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6085..6673
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6574..6593
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6827..6844
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7050..7071
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7086..7091,7092..7109,7110..7116)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7124..7140
                     /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     join(7129..7147,1..4)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
ORIGIN
        1 caggaaacag ctatgaccat gattacgcca aggtcgactt aaccctagaa agataatcat
       61 attgtgacgt acgttaaaga taatcatgcg taaaattgac gcatgtgttt tatcggtctg
      121 tatatcgagg tttatttatt aatttgaata gatattaagt tttattatat ttacacttac
      181 atactaataa taaattcaac aaacaattta tttatgttta tttatttatt aaaaaaaaac
      241 aaaaactcaa aatttcttct ataaagtaac aaaactttta gcagtgaaaa aaatgcttta
      301 tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag
      361 ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggaggtg tgggaggttt
      421 tttaaagcaa gtaaaacctc tacaaatgtg gtatggctga ttatgatcct ctggagattc
      481 atgcgccagg ttttctggtc atgcaccacg ttcgtgggcc ctcaggaacc tccacgtcag
      541 cggtaactgt gaagcccagt cgctcgtaaa aaggaagatt ccggggagca ctggtttcga
      601 ggaacgccgg cacgccagcc cgttccgcgg cctcaacacc aggaagaacg acagcgcttc
      661 ccaaaccttt accttggtga tctgggctga caccgacagt ggcaagaaac cacgcaggct
      721 cctttggtct atgaggagcc agcaagccct ccatttgctg ttgtgccgcc aacctactcc
      781 ctgacagctc ggccatccgc ggacctattt ccgcgaacac tgcgccggct tcgacgcttt
      841 ccggagtagt ccagaccgca actgcggcac catcgtccgc aacccaaact ttaccaatgt
      901 ccagacccac gcgcgtaaga aacagctctt gcaactcagt taccctctct atgtgtctgt
      961 ccggatctac ggtatggcgg gtcgctggat aatctgcaaa tgctgcggca agtgttcgga
     1021 cagcccgtgg tacatcgtca cgggtagcga gcctaacagt gggcttatat tccgtcatgg
     1081 ggccggggtt ctcctccacg tcgccggcct gcttcagcag gctgaagttg gtggcgccgc
     1141 tgcccccggg gagcatgtca aggtcaaaat cgtcaagagc gtcagcaggc agcatatcaa
     1201 ggtcaaagtc gtcaagggca tcggctggga gcatgtctaa gtcaaaatcg tcaagggcgt
     1261 cggtcggccc gccgctttcg cactttagct gtttctccag gccacatatg attagttcca
     1321 ggccgaaaag gaaggcaggt tcggctccct gccggtcgaa cagctcaatt gcttgtttca
     1381 gaagtggggg catagaatcg gtggtaggtg tctctctttc ctcttttgct acttgatgct
     1441 cctgttcctc caatacgcag cccagtgtaa agtggcccac ggcggacaga gcgtacagtg
     1501 cgttctccag ggagaagcct tgctgacaca ggaacgcgag ctgattttcc agggtttcgt
     1561 actgtttctc tgttgggcgg gtgccgagat gcactttagc cccgtcacga tgtgagagga
     1621 gagcacagcg gtatgacttg gcgttgttcc gcagaaagtc ttgccatgac tcgccttcca
     1681 gggggcagga gtgggtatga tgcctgtcca gcatctcgat tggcagggca tcgagcaggg
     1741 cccgcttgtt cttcacgtgc cagtacaggg taggctgctc aactcccagc ttttgagcga
     1801 gtttccttgt cgtcaggcct tcgataccga ctccattgag taattccaga gcagagttta
     1861 tgactttgct cttgtccagt ctagacatct tatcgtcatc gtctttgtaa tccatggtgg
     1921 cggatcccgc gtcacgacac ctgtgttctg gcggcaaacc cgttgcgaaa aagaacgttc
     1981 acggcgacta ctgcacttat atacggttct cccccaccct cgggaaaaag gcggagccag
     2041 tacacgacat cactttccca gtttaccccg cgccaccttc tctaggcacc ggttcaattg
     2101 ccgacccctc cccccaactt ctcgggcact gtgggccatg tgcgctctgc cccatggtaa
     2161 tagccatgac taatacgtag atgtactgcc aagtaggaaa gtgccataag gtcatgtact
     2221 gggcataatg ccaggcgggc catttaccgt cattgacgtc aatagggggc gtacttggca
     2281 tatgatacac ttgatgtact gccaagtggg cagtttaccg taaatactcc acccattgac
     2341 gtcaatggaa agtgcctatt ggcgttacta tgggaacata cgtcattatt gacgtcaatg
     2401 ggcgggggtc gttgggcggt cagccaggcg ggccatttac cgtaagttat gtaacgcgga
     2461 actccatata tgggctatga actaatgacc ccgtaattga ttactattaa taactagtca
     2521 ataatcaatg tcataagctc taatcgcata atcgagctaa gagcgattaa tatgcttagc
     2581 tagcttagct aagctagatt tctctattct cccacaatat atagcagcta ttagcttagc
     2641 agtccgaata cgatcgtcca cgatccataa gcgtaacaca tttgagtagc gactgacggg
     2701 caccggagcc actcgagtgg aattatcacc tcgagtttac tccctatcag tgatagagaa
     2761 cgtatgaaga gtttactccc tatcagtgat agagaacgta tgcagacttt actccctatc
     2821 agtgatagag aacgtataag gagtttactc cctatcagtg atagagaacg tatgaccagt
     2881 ttactcccta tcagtgatag agaacgtatc tacagtttac tccctatcag tgatagagaa
     2941 cgtatatcca gtttactccc tatcagtgat agagaacgta taagcttgcc tatataagca
     3001 gagctcgttt agtgaaccgt cagatcgcct ggagacgcca tccacgctgt tttgacctcc
     3061 atagaagaca ccgggaccga tccagcctac cacaagtttg tacaaaaaag caggcttagc
     3121 caccatggac cccgcgcccg gcgtcctaga tccacgtgct gcaccaccag cacttctagg
     3181 tactccacaa gcagaagtac tagaagatgt attacgagaa caatttggac cgctgcccca
     3241 gctggccgct gtctgccggc tcaagcggct gccctcgggc ggctactcgt ccactgaaaa
     3301 cctccagttg gtgctggagc ggcggcgtgt ggccaacgcc aaggagcgtg agcggataaa
     3361 aaatctcaac cgtggttttg ccagattgaa ggcacttgtg ccatttcttc cccaaagcag
     3421 gaagcccagc aaagttgata tccttaaagg tgcgactgaa tatatacagg ttctcagtga
     3481 tcttttggaa ggagccaaag actcaaagaa acaagaccca gatgagcaga gctatagtaa
     3541 caacagttct gaatcacata catcctcggc aagacagctg tcaagaaaca tcacccaaca
     3601 tatcagctgt gctttcggct tgaagaatga agaggaaggg ccttgggcag atggtggcag
     3661 tggtgagcca gcacacgctt gtcgccacag tgtgatgtct acgactgaaa ttatctcccc
     3721 aaccagaagt ctggatagat tcccagaagt agaactgctg agtcacagac ttccacaagt
     3781 ataataccca gctttcttgt acaaagtggt atacccatac gatgttccag attacgctta
     3841 gtaatgagaa atactcagaa gatgtcactc accatagtcc gcagtgaaag ataaatgatc
     3901 attgactatg acaccgatga gttttagagc tagaaatagc aagtaggcca ctgagcaaga
     3961 actatgaatg agatctttag acatattgcg gccgctttct ccgcgatcca gacatgataa
     4021 gatacattga tgagtttgga caaaccaaaa ctagaatgca gtgaaaaaaa tgccttattt
     4081 gtgaaatttg tgatgctatt gccttatttg taaccattat aagctgcaat aaacaagtta
     4141 tctataacaa gaaaatatat atataataag ttatcacgta agtagaacat gaaataacaa
     4201 tataattatc gtatgagtta aatcttaaaa gtcacgtaaa agataatcat gcgtcatttt
     4261 gactcacgcg gtcgttatag ttcaaaatca gtgacactta ccgcattgac aagcacgcct
     4321 cacgggagct ccaagcggcg actgagatgt cctaaatgca cagcgacgga ttcgcgctat
     4381 ttagaaagag agagcaatat ttcaagaatg catgcgtcaa ttttacgcag actatctttc
     4441 tagggttaag aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
     4501 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga
     4561 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat
     4621 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag
     4681 tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga
     4741 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccatgtc
     4801 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacacgc gagttcgaaa
     4861 gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac
     4921 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     4981 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     5041 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     5101 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     5161 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     5221 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     5281 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     5341 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     5401 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     5461 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     5521 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     5581 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     5641 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     5701 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     5761 tgagcgtggt tcacgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     5821 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     5881 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     5941 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     6001 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     6061 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     6121 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     6181 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     6241 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     6301 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     6361 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     6421 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     6481 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     6541 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     6601 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     6661 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     6721 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     6781 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     6841 cgaggaagcg gatgagcgcc caatacgcaa accgcctctc accgcgcgtt ggccgattca
     6901 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaaa
     6961 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
     7021 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc
     7081 caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa
     7141 tttcaca
//
