LOCUS       sequence-439806-        7303 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac transposon plasmid for doxycycline inducible expression of 
            ZGLP1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7303)
  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 7303)
  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-ZGLP1
FEATURES             Location/Qualifiers
     source          1..7303
                     /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(1218..1242)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1244..1270
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1437..1571
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   1843..1877
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1888..1905)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1888..1904)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1897..1919)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2113..2132)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2234..2256
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2295..2313)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2381..2485
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2486..2554,2555..3346)
                     /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(2704..2723)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3517..4105
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4006..4025
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4259..4276
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4482..4503
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4518..4523,4524..4541,4542..4548)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4556..4572
                     /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     4561..4583
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4580..4596
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4580..4596
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4622..4684
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    4860..4941
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5658..5714)
                     /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(5724..6467)
                     /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(6468..6491)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6491..6500
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6520..6731)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        6732..7111
                     /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 cttagccacc atgactgaac ctcaggtggg
      421 gtgtgtagcc tgtccaaggg tacacaaaga gccggcccaa gttggaaccc cctggccggc
      481 taaacccagg agtcacccaa gaaaacgtga ccccactgcc cttctcccca ggtccctctg
      541 gcctgcatgc caggagtcgg tcaccgccct gtgcttcctc caagagacag tggagaggct
      601 gggtcagtcc cctgcccagg acaccccggt cctggggcct tgctgggacc cgatggctct
      661 ggggactcag ggccgcctgc tgctggacag ggattccaag gacacacaga ccaggatcag
      721 ccaaaagggc cgccgtctgc agcccccggg gactccctcg gccccacccc agagaaggcc
      781 ccggaaacag ctgaacccct gccggggcac cgagagagtg gaccctgggt tcgagggggt
      841 gactctgaag tttcagataa agccggactc cagcctgcag atcatcccca cgtacagcct
      901 gccctgcagt agccgttctc aggaatcccc tgcagatgct gttgggggcc ctgcagccca
      961 cccaggaggc accgaggccc actcagcagg cagcgaggcc ctggagcccc ggcgctgtgc
     1021 ttcctgtcgg acccagagga ccccgctctg gagagacgct gaagatggga cccctctctg
     1081 caacgcctgt gggatcaggt acaagaaata cggcactcgc tgctccagct gctggctggt
     1141 gcccaggaaa aatgtccagc ccaagaggct atgtggcaga tgtggagtgt ccctggaccc
     1201 cattcaggaa ggttaatacc cagctttctt gtacaaagtg gtatacccat acgatgttcc
     1261 agattacgct tagtaatgag aaatactcag aagatgtcac tcaccatagt ccgcagtgaa
     1321 agataaatga tctcaaaaca taagcaagca ttgttttaga gctagaaata gcaagtaggc
     1381 cactgagcaa gaactatgaa tgagatcttt agacatattg cggccgcttt ctccgcgatc
     1441 cagacatgat aagatacatt gatgagtttg gacaaaccaa aactagaatg cagtgaaaaa
     1501 aatgccttat ttgtgaaatt tgtgatgcta ttgccttatt tgtaaccatt ataagctgca
     1561 ataaacaagt tatctataac aagaaaatat atatataata agttatcacg taagtagaac
     1621 atgaaataac aatataatta tcgtatgagt taaatcttaa aagtcacgta aaagataatc
     1681 atgcgtcatt ttgactcacg cggtcgttat agttcaaaat cagtgacact taccgcattg
     1741 acaagcacgc ctcacgggag ctccaagcgg cgactgagat gtcctaaatg cacagcgacg
     1801 gattcgcgct atttagaaag agagagcaat atttcaagaa tgcatgcgtc aattttacgc
     1861 agactatctt tctagggtta agaattcact ggccgtcgtt ttacaacgtc gtgactggga
     1921 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
     1981 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
     2041 atggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatatg
     2101 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc
     2161 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     2221 tgtgaccatg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacac
     2281 gcgagttcga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat
     2341 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     2401 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     2461 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     2521 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     2581 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     2641 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     2701 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     2761 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     2821 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     2881 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     2941 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     3001 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     3061 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     3121 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     3181 atctggagcc ggtgagcgtg gttcacgcgg tatcattgca gcactggggc cagatggtaa
     3241 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     3301 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     3361 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     3421 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     3481 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     3541 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     3601 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     3661 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     3721 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     3781 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     3841 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     3901 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     3961 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     4021 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     4081 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     4141 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     4201 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     4261 cgagtcagtg agcgaggaag cggatgagcg cccaatacgc aaaccgcctc tcaccgcgcg
     4321 ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga
     4381 gcgcaacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
     4441 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact
     4501 cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg
     4561 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa ggtcgactta
     4621 accctagaaa gataatcata ttgtgacgta cgttaaagat aatcatgcgt aaaattgacg
     4681 catgtgtttt atcggtctgt atatcgaggt ttatttatta atttgaatag atattaagtt
     4741 ttattatatt tacacttaca tactaataat aaattcaaca aacaatttat ttatgtttat
     4801 ttatttatta aaaaaaaaca aaaactcaaa atttcttcta taaagtaaca aaacttttag
     4861 cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt
     4921 ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag
     4981 ggggaggtgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg tatggctgat
     5041 tatgatcctc tggagattca tgcgccaggt tttctggtca tgcaccacgt tcgtgggccc
     5101 tcaggaacct ccacgtcagc ggtaactgtg aagcccagtc gctcgtaaaa aggaagattc
     5161 cggggagcac tggtttcgag gaacgccggc acgccagccc gttccgcggc ctcaacacca
     5221 ggaagaacga cagcgcttcc caaaccttta ccttggtgat ctgggctgac accgacagtg
     5281 gcaagaaacc acgcaggctc ctttggtcta tgaggagcca gcaagccctc catttgctgt
     5341 tgtgccgcca acctactccc tgacagctcg gccatccgcg gacctatttc cgcgaacact
     5401 gcgccggctt cgacgctttc cggagtagtc cagaccgcaa ctgcggcacc atcgtccgca
     5461 acccaaactt taccaatgtc cagacccacg cgcgtaagaa acagctcttg caactcagtt
     5521 accctctcta tgtgtctgtc cggatctacg gtatggcggg tcgctggata atctgcaaat
     5581 gctgcggcaa gtgttcggac agcccgtggt acatcgtcac gggtagcgag cctaacagtg
     5641 ggcttatatt ccgtcatggg gccggggttc tcctccacgt cgccggcctg cttcagcagg
     5701 ctgaagttgg tggcgccgct gcccccgggg agcatgtcaa ggtcaaaatc gtcaagagcg
     5761 tcagcaggca gcatatcaag gtcaaagtcg tcaagggcat cggctgggag catgtctaag
     5821 tcaaaatcgt caagggcgtc ggtcggcccg ccgctttcgc actttagctg tttctccagg
     5881 ccacatatga ttagttccag gccgaaaagg aaggcaggtt cggctccctg ccggtcgaac
     5941 agctcaattg cttgtttcag aagtgggggc atagaatcgg tggtaggtgt ctctctttcc
     6001 tcttttgcta cttgatgctc ctgttcctcc aatacgcagc ccagtgtaaa gtggcccacg
     6061 gcggacagag cgtacagtgc gttctccagg gagaagcctt gctgacacag gaacgcgagc
     6121 tgattttcca gggtttcgta ctgtttctct gttgggcggg tgccgagatg cactttagcc
     6181 ccgtcacgat gtgagaggag agcacagcgg tatgacttgg cgttgttccg cagaaagtct
     6241 tgccatgact cgccttccag ggggcaggag tgggtatgat gcctgtccag catctcgatt
     6301 ggcagggcat cgagcagggc ccgcttgttc ttcacgtgcc agtacagggt aggctgctca
     6361 actcccagct tttgagcgag tttccttgtc gtcaggcctt cgataccgac tccattgagt
     6421 aattccagag cagagtttat gactttgctc ttgtccagtc tagacatctt atcgtcatcg
     6481 tctttgtaat ccatggtggc ggatcccgcg tcacgacacc tgtgttctgg cggcaaaccc
     6541 gttgcgaaaa agaacgttca cggcgactac tgcacttata tacggttctc ccccaccctc
     6601 gggaaaaagg cggagccagt acacgacatc actttcccag tttaccccgc gccaccttct
     6661 ctaggcaccg gttcaattgc cgacccctcc ccccaacttc tcgggcactg tgggccatgt
     6721 gcgctctgcc ccatggtaat agccatgact aatacgtaga tgtactgcca agtaggaaag
     6781 tgccataagg tcatgtactg ggcataatgc caggcgggcc atttaccgtc attgacgtca
     6841 atagggggcg tacttggcat atgatacact tgatgtactg ccaagtgggc agtttaccgt
     6901 aaatactcca cccattgacg tcaatggaaa gtgcctattg gcgttactat gggaacatac
     6961 gtcattattg acgtcaatgg gcgggggtcg ttgggcggtc agccaggcgg gccatttacc
     7021 gtaagttatg taacgcggaa ctccatatat gggctatgaa ctaatgaccc cgtaattgat
     7081 tactattaat aactagtcaa taatcaatgt cataagctct aatcgcataa tcgagctaag
     7141 agcgattaat atgcttagct agcttagcta agctagattt ctctattctc ccacaatata
     7201 tagcagctat tagcttagca gtccgaatac gatcgtccac gatccataag cgtaacacat
     7261 ttgagtagcg actgacgggc accggagcca ctcgagtgga att
//
