LOCUS       sequence-442402-        7843 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac transposon plasmid for doxycycline inducible expression of 
            RARG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7843)
  AUTHORS   Pierson Smela M, Adams J, Ma C, Breimann L, Widocki U, Shioda T, 
            Church GM
  TITLE     Induction of Meiosis from Human Pluripotent Stem Cells
  JOURNAL   bioRxiv 2024.05.31.596483
REFERENCE   2  (bases 1 to 7843)
  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 
            2024.05.31.596483"
COMMENT     Sequence Label: pPB-cT3G-cERP2-RARG
FEATURES             Location/Qualifiers
     source          1..7843
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(95..114)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      908..1496
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1397..1416
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1650..1667
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1873..1894
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1909..1914,1915..1932,1933..1939)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1947..1963
                     /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     1952..1974
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1971..1987
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1971..1987
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   2013..2075
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    2251..2332
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(3049..3105)
                     /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(3115..3858)
                     /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(3859..3882)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      3882..3891
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(3911..4122)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        4123..4502
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     protein_bind    4711..4729
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4747..4765
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4783..4801
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4819..4837
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4855..4873
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4891..4909
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4927..4945
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     4972..4996
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    5062..5086
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(6452..6476)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             6478..6504
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    6671..6805
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   7077..7111
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(7122..7139)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7122..7138)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7131..7153)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(7347..7366)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7468..7490
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7529..7547)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7615..7719
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7720..7788,7789..7843,1..737)
                     /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"
ORIGIN
        1 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
       61 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
      121 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
      181 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
      241 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
      301 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
      361 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
      421 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
      481 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
      541 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt ggttcacgcg
      601 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
      661 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
      721 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
      781 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
      841 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
      901 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
      961 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     1021 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     1081 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     1141 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     1201 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     1261 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     1321 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     1381 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     1441 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     1501 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     1561 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     1621 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggatgagc
     1681 gcccaatacg caaaccgcct ctcaccgcgc gttggccgat tcattaatgc agctggcacg
     1741 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aaaccgcctc tccccgcgcg
     1801 ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga
     1861 gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat
     1921 gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag
     1981 ctatgaccat gattacgcca aggtcgactt aaccctagaa agataatcat attgtgacgt
     2041 acgttaaaga taatcatgcg taaaattgac gcatgtgttt tatcggtctg tatatcgagg
     2101 tttatttatt aatttgaata gatattaagt tttattatat ttacacttac atactaataa
     2161 taaattcaac aaacaattta tttatgttta tttatttatt aaaaaaaaac aaaaactcaa
     2221 aatttcttct ataaagtaac aaaactttta gcagtgaaaa aaatgcttta tttgtgaaat
     2281 ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa
     2341 caattgcatt cattttatgt ttcaggttca gggggaggtg tgggaggttt tttaaagcaa
     2401 gtaaaacctc tacaaatgtg gtatggctga ttatgatcct ctggagattc atgcgccagg
     2461 ttttctggtc atgcaccacg ttcgtgggcc ctcaggaacc tccacgtcag cggtaactgt
     2521 gaagcccagt cgctcgtaaa aaggaagatt ccggggagca ctggtttcga ggaacgccgg
     2581 cacgccagcc cgttccgcgg cctcaacacc aggaagaacg acagcgcttc ccaaaccttt
     2641 accttggtga tctgggctga caccgacagt ggcaagaaac cacgcaggct cctttggtct
     2701 atgaggagcc agcaagccct ccatttgctg ttgtgccgcc aacctactcc ctgacagctc
     2761 ggccatccgc ggacctattt ccgcgaacac tgcgccggct tcgacgcttt ccggagtagt
     2821 ccagaccgca actgcggcac catcgtccgc aacccaaact ttaccaatgt ccagacccac
     2881 gcgcgtaaga aacagctctt gcaactcagt taccctctct atgtgtctgt ccggatctac
     2941 ggtatggcgg gtcgctggat aatctgcaaa tgctgcggca agtgttcgga cagcccgtgg
     3001 tacatcgtca cgggtagcga gcctaacagt gggcttatat tccgtcatgg ggccggggtt
     3061 ctcctccacg tcgccggcct gcttcagcag gctgaagttg gtggcgccgc tgcccccggg
     3121 gagcatgtca aggtcaaaat cgtcaagagc gtcagcaggc agcatatcaa ggtcaaagtc
     3181 gtcaagggca tcggctggga gcatgtctaa gtcaaaatcg tcaagggcgt cggtcggccc
     3241 gccgctttcg cactttagct gtttctccag gccacatatg attagttcca ggccgaaaag
     3301 gaaggcaggt tcggctccct gccggtcgaa cagctcaatt gcttgtttca gaagtggggg
     3361 catagaatcg gtggtaggtg tctctctttc ctcttttgct acttgatgct cctgttcctc
     3421 caatacgcag cccagtgtaa agtggcccac ggcggacaga gcgtacagtg cgttctccag
     3481 ggagaagcct tgctgacaca ggaacgcgag ctgattttcc agggtttcgt actgtttctc
     3541 tgttgggcgg gtgccgagat gcactttagc cccgtcacga tgtgagagga gagcacagcg
     3601 gtatgacttg gcgttgttcc gcagaaagtc ttgccatgac tcgccttcca gggggcagga
     3661 gtgggtatga tgcctgtcca gcatctcgat tggcagggca tcgagcaggg cccgcttgtt
     3721 cttcacgtgc cagtacaggg taggctgctc aactcccagc ttttgagcga gtttccttgt
     3781 cgtcaggcct tcgataccga ctccattgag taattccaga gcagagttta tgactttgct
     3841 cttgtccagt ctagacatct tatcgtcatc gtctttgtaa tccatggtgg cggatcccgc
     3901 gtcacgacac ctgtgttctg gcggcaaacc cgttgcgaaa aagaacgttc acggcgacta
     3961 ctgcacttat atacggttct cccccaccct cgggaaaaag gcggagccag tacacgacat
     4021 cactttccca gtttaccccg cgccaccttc tctaggcacc ggttcaattg ccgacccctc
     4081 cccccaactt ctcgggcact gtgggccatg tgcgctctgc cccatggtaa tagccatgac
     4141 taatacgtag atgtactgcc aagtaggaaa gtgccataag gtcatgtact gggcataatg
     4201 ccaggcgggc catttaccgt cattgacgtc aatagggggc gtacttggca tatgatacac
     4261 ttgatgtact gccaagtggg cagtttaccg taaatactcc acccattgac gtcaatggaa
     4321 agtgcctatt ggcgttacta tgggaacata cgtcattatt gacgtcaatg ggcgggggtc
     4381 gttgggcggt cagccaggcg ggccatttac cgtaagttat gtaacgcgga actccatata
     4441 tgggctatga actaatgacc ccgtaattga ttactattaa taactagtca ataatcaatg
     4501 tcataagctc taatcgcata atcgagctaa gagcgattaa tatgcttagc tagcttagct
     4561 aagctagatt tctctattct cccacaatat atagcagcta ttagcttagc agtccgaata
     4621 cgatcgtcca cgatccataa gcgtaacaca tttgagtagc gactgacggg caccggagcc
     4681 actcgagtgg aattatcacc tcgagtttac tccctatcag tgatagagaa cgtatgaaga
     4741 gtttactccc tatcagtgat agagaacgta tgcagacttt actccctatc agtgatagag
     4801 aacgtataag gagtttactc cctatcagtg atagagaacg tatgaccagt ttactcccta
     4861 tcagtgatag agaacgtatc tacagtttac tccctatcag tgatagagaa cgtatatcca
     4921 gtttactccc tatcagtgat agagaacgta taagcttgcc tatataagca gagctcgttt
     4981 agtgaaccgt cagatcgcct ggagacgcca tccacgctgt tttgacctcc atagaagaca
     5041 ccgggaccga tccagcctac cacaagtttg tacaaaaaag ttggcaccat ggccaccaat
     5101 aaggagcgac tctttgcggc tggtgccctg gggcctggat ctggctaccc aggggcaggt
     5161 ttccccttcg ccttcccagg ggcactcagg gggtctccgc ctttcgagat gctgagccct
     5221 agcttccggg gcctgggcca gcctgacctc cccaaggaga tggcctctct gtcggtggag
     5281 acacagagca ccagctcaga ggagatggtg cccagctcgc cctcgccccc tccgcctcct
     5341 cgggtctaca agccatgctt cgtgtgcaat gacaagtcct ctggctacca ctatggggtc
     5401 agctcttgtg aaggctgcaa gggcttcttt cgccgaagca tccagaagaa catggtgtac
     5461 acgtgtcacc gcgacaaaaa ctgtatcatc aacaaggtga ccaggaatcg ctgccagtac
     5521 tgccggctac agaagtgctt cgaagtgggc atgtccaagg aagctgtgcg aaatgaccgg
     5581 aacaagaaga agaaagaggt gaaggaagaa gggtcacctg acagctatga gctgagccct
     5641 cagttagaag agctcatcac caaggtcagc aaagcccatc aggagacttt cccctcgctc
     5701 tgccagctgg gcaagtatac cacgaactcc agtgcagacc accgcgtgca gctggatctg
     5761 gggctgtggg acaagttcag tgagctggct accaagtgca tcatcaagat cgtggagttt
     5821 gccaagcggt tgcctggctt tacagggctc agcattgctg accagatcac tctgctcaaa
     5881 gctgcctgcc tagatatcct gatgctgcgt atctgcacaa ggtacacccc agagcaggac
     5941 accatgacct tctccgacgg gctgaccctg aaccggaccc agatgcacaa tgccggcttc
     6001 gggcccctca cagaccttgt ctttgccttt gctgggcagc tcctgcccct ggagatggat
     6061 gacaccgaga cagggctgct cagcgccatc tgcctcatct gcggagaccg catggacctg
     6121 gaggagcccg aaaaagtgga caagctgcag gagccactgc tggaagccct gaggctgtac
     6181 gcccggcgcc ggcggcccag ccagccctac atgttcccaa ggatgctaat gaaaatcacc
     6241 gacctccggg gcatcagcac taagggagct gaaagggcca ttactctgaa gatggagatt
     6301 ccaggcccga tgcctccctt aatccgagag atgctggaga accctgaaat gtttgaggat
     6361 gactcctcgc agcctggtcc ccaccccaat gcctctagcg aggatgaggt tcctgggggc
     6421 cagggcaaag ggggcctgaa gtccccagcc ttgccaactt tcttgtacaa agtggtatac
     6481 ccatacgatg ttccagatta cgcttagtaa tgagaaatac tcagaagatg tcactcacca
     6541 tagtccgcag tgaaagataa atgatctaag gaagtgagcc agatctgttt tagagctaga
     6601 aatagcaagt aggccactga gcaagaacta tgaatgagat ctttagacat attgcggccg
     6661 ctttctccgc gatccagaca tgataagata cattgatgag tttggacaaa ccaaaactag
     6721 aatgcagtga aaaaaatgcc ttatttgtga aatttgtgat gctattgcct tatttgtaac
     6781 cattataagc tgcaataaac aagttatcta taacaagaaa atatatatat aataagttat
     6841 cacgtaagta gaacatgaaa taacaatata attatcgtat gagttaaatc ttaaaagtca
     6901 cgtaaaagat aatcatgcgt cattttgact cacgcggtcg ttatagttca aaatcagtga
     6961 cacttaccgc attgacaagc acgcctcacg ggagctccaa gcggcgactg agatgtccta
     7021 aatgcacagc gacggattcg cgctatttag aaagagagag caatatttca agaatgcatg
     7081 cgtcaatttt acgcagacta tctttctagg gttaagaatt cactggccgt cgttttacaa
     7141 cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct
     7201 ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
     7261 agcctgaatg gcgaatggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     7321 tcacaccgca tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     7381 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     7441 gcttacagac aagctgtgac catgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     7501 catcaccgaa acacgcgagt tcgaaagggc ctcgtgatac gcctattttt ataggttaat
     7561 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     7621 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     7681 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     7741 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     7801 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gag
//
