LOCUS       sequence-442527-        7124 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac transposon plasmid for doxycycline inducible expression of 
            RNF138.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7124)
  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 7124)
  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-RNF138
FEATURES             Location/Qualifiers
     source          1..7124
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      362..950
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     851..870
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1104..1121
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1238..1259
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1274..1279,1280..1297,1298..1304)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1312..1328
                     /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     1317..1339
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1336..1352
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1336..1352
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   1378..1440
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    1616..1697
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(2414..2470)
                     /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(2480..3223)
                     /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(3224..3247)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      3247..3256
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(3276..3487)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        3488..3867
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     protein_bind    4076..4094
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4112..4130
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4148..4166
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4184..4202
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4220..4238
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4256..4274
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4292..4310
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     4337..4361
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    4427..4451
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(5187..5211)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             5213..5239
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    5406..5540
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   5812..5846
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5857..5874)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5857..5873)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5866..5888)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(6082..6101)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6203..6225
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6264..6282)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6350..6454
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6455..6523,6524..7124,1..191)
                     /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(6673..6692)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 cggctggctg gtttattgct gataaatctg gagccggtga gcgtggttca cgcggtatca
       61 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
      121 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
      181 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
      241 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
      301 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
      361 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
      421 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
      481 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
      541 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
      601 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
      661 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
      721 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
      781 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
      841 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
      901 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
      961 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     1021 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     1081 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggat gagcgcccaa
     1141 tacgcaaacc gcctctcacc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
     1201 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt
     1261 aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg
     1321 gataacaatt tcacacagga aacagctatg accatgatta cgccaaggtc gacttaaccc
     1381 tagaaagata atcatattgt gacgtacgtt aaagataatc atgcgtaaaa ttgacgcatg
     1441 tgttttatcg gtctgtatat cgaggtttat ttattaattt gaatagatat taagttttat
     1501 tatatttaca cttacatact aataataaat tcaacaaaca atttatttat gtttatttat
     1561 ttattaaaaa aaaacaaaaa ctcaaaattt cttctataaa gtaacaaaac ttttagcagt
     1621 gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa
     1681 gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg
     1741 aggtgtggga ggttttttaa agcaagtaaa acctctacaa atgtggtatg gctgattatg
     1801 atcctctgga gattcatgcg ccaggttttc tggtcatgca ccacgttcgt gggccctcag
     1861 gaacctccac gtcagcggta actgtgaagc ccagtcgctc gtaaaaagga agattccggg
     1921 gagcactggt ttcgaggaac gccggcacgc cagcccgttc cgcggcctca acaccaggaa
     1981 gaacgacagc gcttcccaaa cctttacctt ggtgatctgg gctgacaccg acagtggcaa
     2041 gaaaccacgc aggctccttt ggtctatgag gagccagcaa gccctccatt tgctgttgtg
     2101 ccgccaacct actccctgac agctcggcca tccgcggacc tatttccgcg aacactgcgc
     2161 cggcttcgac gctttccgga gtagtccaga ccgcaactgc ggcaccatcg tccgcaaccc
     2221 aaactttacc aatgtccaga cccacgcgcg taagaaacag ctcttgcaac tcagttaccc
     2281 tctctatgtg tctgtccgga tctacggtat ggcgggtcgc tggataatct gcaaatgctg
     2341 cggcaagtgt tcggacagcc cgtggtacat cgtcacgggt agcgagccta acagtgggct
     2401 tatattccgt catggggccg gggttctcct ccacgtcgcc ggcctgcttc agcaggctga
     2461 agttggtggc gccgctgccc ccggggagca tgtcaaggtc aaaatcgtca agagcgtcag
     2521 caggcagcat atcaaggtca aagtcgtcaa gggcatcggc tgggagcatg tctaagtcaa
     2581 aatcgtcaag ggcgtcggtc ggcccgccgc tttcgcactt tagctgtttc tccaggccac
     2641 atatgattag ttccaggccg aaaaggaagg caggttcggc tccctgccgg tcgaacagct
     2701 caattgcttg tttcagaagt gggggcatag aatcggtggt aggtgtctct ctttcctctt
     2761 ttgctacttg atgctcctgt tcctccaata cgcagcccag tgtaaagtgg cccacggcgg
     2821 acagagcgta cagtgcgttc tccagggaga agccttgctg acacaggaac gcgagctgat
     2881 tttccagggt ttcgtactgt ttctctgttg ggcgggtgcc gagatgcact ttagccccgt
     2941 cacgatgtga gaggagagca cagcggtatg acttggcgtt gttccgcaga aagtcttgcc
     3001 atgactcgcc ttccaggggg caggagtggg tatgatgcct gtccagcatc tcgattggca
     3061 gggcatcgag cagggcccgc ttgttcttca cgtgccagta cagggtaggc tgctcaactc
     3121 ccagcttttg agcgagtttc cttgtcgtca ggccttcgat accgactcca ttgagtaatt
     3181 ccagagcaga gtttatgact ttgctcttgt ccagtctaga catcttatcg tcatcgtctt
     3241 tgtaatccat ggtggcggat cccgcgtcac gacacctgtg ttctggcggc aaacccgttg
     3301 cgaaaaagaa cgttcacggc gactactgca cttatatacg gttctccccc accctcggga
     3361 aaaaggcgga gccagtacac gacatcactt tcccagttta ccccgcgcca ccttctctag
     3421 gcaccggttc aattgccgac ccctcccccc aacttctcgg gcactgtggg ccatgtgcgc
     3481 tctgccccat ggtaatagcc atgactaata cgtagatgta ctgccaagta ggaaagtgcc
     3541 ataaggtcat gtactgggca taatgccagg cgggccattt accgtcattg acgtcaatag
     3601 ggggcgtact tggcatatga tacacttgat gtactgccaa gtgggcagtt taccgtaaat
     3661 actccaccca ttgacgtcaa tggaaagtgc ctattggcgt tactatggga acatacgtca
     3721 ttattgacgt caatgggcgg gggtcgttgg gcggtcagcc aggcgggcca tttaccgtaa
     3781 gttatgtaac gcggaactcc atatatgggc tatgaactaa tgaccccgta attgattact
     3841 attaataact agtcaataat caatgtcata agctctaatc gcataatcga gctaagagcg
     3901 attaatatgc ttagctagct tagctaagct agatttctct attctcccac aatatatagc
     3961 agctattagc ttagcagtcc gaatacgatc gtccacgatc cataagcgta acacatttga
     4021 gtagcgactg acgggcaccg gagccactcg agtggaatta tcacctcgag tttactccct
     4081 atcagtgata gagaacgtat gaagagttta ctccctatca gtgatagaga acgtatgcag
     4141 actttactcc ctatcagtga tagagaacgt ataaggagtt tactccctat cagtgataga
     4201 gaacgtatga ccagtttact ccctatcagt gatagagaac gtatctacag tttactccct
     4261 atcagtgata gagaacgtat atccagttta ctccctatca gtgatagaga acgtataagc
     4321 ttgcctatat aagcagagct cgtttagtga accgtcagat cgcctggaga cgccatccac
     4381 gctgttttga cctccataga agacaccggg accgatccag cctaccacaa gtttgtacaa
     4441 aaaagttggc atggccgagg acctctctgc ggccacgtcc tacaccgaag atgatttcta
     4501 ctgccccgtc tgtcaggagg tgctcaaaac gcccgtgcgg accacggcct gtcagcacgt
     4561 tttctgtaga aaatgtttcc tgactgcaat gagggaaagc ggagcacatt gtcccctatg
     4621 tcgtggaaat gtgactagaa gagagagagc atgtcctgaa cgggccttag accttgaaaa
     4681 tataatgagg aagttttctg gtagctgcag atgctgtgca aaacagatta aattctatcg
     4741 catgagacat cattacaaat cttgtaagaa gtatcaggat gaatatggtg tttcttctat
     4801 cattccaaac tttcagatct ctcaagattc agtagggaac agcaatagga gtgaaacatc
     4861 cacatctgat aacacagaaa cttaccaaga gaatacaagt tcttctggtc atcctacttt
     4921 taagtgtccc ctgtgtcaag aatcaaattt taccagacag cgtttactgg atcactgtaa
     4981 cagtaatcac ctatttcaga tagttcctgt gacatgtcct atttgtgtgt ctcttccttg
     5041 gggagatcct agccagatta ccagaaattt cgttagtcat ctaaatcaga gacatcaatt
     5101 tgattatgga gaatttgtga atcttcagct agatgaagaa acccaatacc aaactgctgt
     5161 tgaagaatct tttcaagtaa acatctgccc aactttcttg tacaaagtgg tatacccata
     5221 cgatgttcca gattacgctt agtaatgaga aatactcaga agatgtcact caccatagtc
     5281 cgcagtgaaa gataaatgat ctaccgaatg taatgccgca cgttttagag ctagaaatag
     5341 caagtaggcc actgagcaag aactatgaat gagatcttta gacatattgc ggccgctttc
     5401 tccgcgatcc agacatgata agatacattg atgagtttgg acaaaccaaa actagaatgc
     5461 agtgaaaaaa atgccttatt tgtgaaattt gtgatgctat tgccttattt gtaaccatta
     5521 taagctgcaa taaacaagtt atctataaca agaaaatata tatataataa gttatcacgt
     5581 aagtagaaca tgaaataaca atataattat cgtatgagtt aaatcttaaa agtcacgtaa
     5641 aagataatca tgcgtcattt tgactcacgc ggtcgttata gttcaaaatc agtgacactt
     5701 accgcattga caagcacgcc tcacgggagc tccaagcggc gactgagatg tcctaaatgc
     5761 acagcgacgg attcgcgcta tttagaaaga gagagcaata tttcaagaat gcatgcgtca
     5821 attttacgca gactatcttt ctagggttaa gaattcactg gccgtcgttt tacaacgtcg
     5881 tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc
     5941 cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct
     6001 gaatggcgaa tggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     6061 ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
     6121 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     6181 cagacaagct gtgaccatgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     6241 ccgaaacacg cgagttcgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat
     6301 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     6361 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     6421 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     6481 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     6541 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     6601 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     6661 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     6721 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     6781 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     6841 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     6901 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     6961 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     7021 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     7081 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttc
//
