LOCUS       sequence-442526-        7126 bp    DNA     circular SYN 15-APR-2025
DEFINITION  PiggyBac transposon plasmid for doxycycline inducible expression of 
            RAN.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7126)
  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 7126)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-RAN
FEATURES             Location/Qualifiers
     source          1..7126
                     /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(1040..1064)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1066..1092
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1259..1393
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   1665..1699
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1710..1727)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1710..1726)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1719..1741)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1935..1954)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2056..2078
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2117..2135)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2203..2307
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2308..2376,2377..3168)
                     /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(2526..2545)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3339..3927
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3828..3847
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4081..4098
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4304..4325
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4340..4345,4346..4363,4364..4370)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4378..4394
                     /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     4383..4405
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4402..4418
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4402..4418
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4444..4506
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    4682..4763
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5480..5536)
                     /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(5546..6289)
                     /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(6290..6313)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6313..6322
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6342..6553)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        6554..6933
                     /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 aaaagttggc atggctgcgc agggagagcc ccaggtccag
      421 ttcaaacttg tattggttgg tgatggtggt actggaaaaa cgaccttcgt gaaacgtcat
      481 ttgactggtg aatttgagaa gaagtatgta gccaccttgg gtgttgaggt tcatccccta
      541 gtgttccaca ccaacagagg acctattaag ttcaatgtat gggacacagc cggccaggag
      601 aaattcggtg gactgagaga tggctattat atccaagccc agtgtgccat cataatgttt
      661 gatgtaacat cgagagttac ttacaagaat gtgcctaact ggcatagaga tctggtacga
      721 gtgtgtgaaa acatccccat tgtgttgtgt ggcaacaaag tggatattaa ggacaggaaa
      781 gtgaaggcga aatccattgt cttccaccga aagaagaatc ttcagtacta cgacatttct
      841 gccaaaagta actacaactt tgaaaagccc ttcctctggc ttgctaggaa gctcattgga
      901 gaccctaact tggaatttgt tgccatgcct gctctcgccc caccagaagt tgtcatggac
      961 ccagctttgg cagcacagta tgagcacgac ttagaggttg ctcagacaac tgctctcccg
     1021 gatgaggatg atgacctgtg cccaactttc ttgtacaaag tggtataccc atacgatgtt
     1081 ccagattacg cttagtaatg agaaatactc agaagatgtc actcaccata gtccgcagtg
     1141 aaagataaat gatcgaacgg tcaacaacat ttaagtttta gagctagaaa tagcaagtag
     1201 gccactgagc aagaactatg aatgagatct ttagacatat tgcggccgct ttctccgcga
     1261 tccagacatg ataagataca ttgatgagtt tggacaaacc aaaactagaa tgcagtgaaa
     1321 aaaatgcctt atttgtgaaa tttgtgatgc tattgcctta tttgtaacca ttataagctg
     1381 caataaacaa gttatctata acaagaaaat atatatataa taagttatca cgtaagtaga
     1441 acatgaaata acaatataat tatcgtatga gttaaatctt aaaagtcacg taaaagataa
     1501 tcatgcgtca ttttgactca cgcggtcgtt atagttcaaa atcagtgaca cttaccgcat
     1561 tgacaagcac gcctcacggg agctccaagc ggcgactgag atgtcctaaa tgcacagcga
     1621 cggattcgcg ctatttagaa agagagagca atatttcaag aatgcatgcg tcaattttac
     1681 gcagactatc tttctagggt taagaattca ctggccgtcg ttttacaacg tcgtgactgg
     1741 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg
     1801 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc
     1861 gaatggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     1921 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg
     1981 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     2041 gctgtgacca tgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     2101 acgcgagttc gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     2161 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     2221 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     2281 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     2341 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     2401 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     2461 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     2521 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     2581 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     2641 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     2701 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     2761 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     2821 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     2881 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     2941 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     3001 aaatctggag ccggtgagcg tggttcacgc ggtatcattg cagcactggg gccagatggt
     3061 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     3121 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     3181 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     3241 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     3301 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     3361 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     3421 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     3481 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     3541 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     3601 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     3661 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     3721 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     3781 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     3841 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     3901 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     3961 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     4021 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     4081 agcgagtcag tgagcgagga agcggatgag cgcccaatac gcaaaccgcc tctcaccgcg
     4141 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
     4201 gagcgcaacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
     4261 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca
     4321 ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg
     4381 tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc aaggtcgact
     4441 taaccctaga aagataatca tattgtgacg tacgttaaag ataatcatgc gtaaaattga
     4501 cgcatgtgtt ttatcggtct gtatatcgag gtttatttat taatttgaat agatattaag
     4561 ttttattata tttacactta catactaata ataaattcaa caaacaattt atttatgttt
     4621 atttatttat taaaaaaaaa caaaaactca aaatttcttc tataaagtaa caaaactttt
     4681 agcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca
     4741 ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc
     4801 agggggaggt gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt ggtatggctg
     4861 attatgatcc tctggagatt catgcgccag gttttctggt catgcaccac gttcgtgggc
     4921 cctcaggaac ctccacgtca gcggtaactg tgaagcccag tcgctcgtaa aaaggaagat
     4981 tccggggagc actggtttcg aggaacgccg gcacgccagc ccgttccgcg gcctcaacac
     5041 caggaagaac gacagcgctt cccaaacctt taccttggtg atctgggctg acaccgacag
     5101 tggcaagaaa ccacgcaggc tcctttggtc tatgaggagc cagcaagccc tccatttgct
     5161 gttgtgccgc caacctactc cctgacagct cggccatccg cggacctatt tccgcgaaca
     5221 ctgcgccggc ttcgacgctt tccggagtag tccagaccgc aactgcggca ccatcgtccg
     5281 caacccaaac tttaccaatg tccagaccca cgcgcgtaag aaacagctct tgcaactcag
     5341 ttaccctctc tatgtgtctg tccggatcta cggtatggcg ggtcgctgga taatctgcaa
     5401 atgctgcggc aagtgttcgg acagcccgtg gtacatcgtc acgggtagcg agcctaacag
     5461 tgggcttata ttccgtcatg gggccggggt tctcctccac gtcgccggcc tgcttcagca
     5521 ggctgaagtt ggtggcgccg ctgcccccgg ggagcatgtc aaggtcaaaa tcgtcaagag
     5581 cgtcagcagg cagcatatca aggtcaaagt cgtcaagggc atcggctggg agcatgtcta
     5641 agtcaaaatc gtcaagggcg tcggtcggcc cgccgctttc gcactttagc tgtttctcca
     5701 ggccacatat gattagttcc aggccgaaaa ggaaggcagg ttcggctccc tgccggtcga
     5761 acagctcaat tgcttgtttc agaagtgggg gcatagaatc ggtggtaggt gtctctcttt
     5821 cctcttttgc tacttgatgc tcctgttcct ccaatacgca gcccagtgta aagtggccca
     5881 cggcggacag agcgtacagt gcgttctcca gggagaagcc ttgctgacac aggaacgcga
     5941 gctgattttc cagggtttcg tactgtttct ctgttgggcg ggtgccgaga tgcactttag
     6001 ccccgtcacg atgtgagagg agagcacagc ggtatgactt ggcgttgttc cgcagaaagt
     6061 cttgccatga ctcgccttcc agggggcagg agtgggtatg atgcctgtcc agcatctcga
     6121 ttggcagggc atcgagcagg gcccgcttgt tcttcacgtg ccagtacagg gtaggctgct
     6181 caactcccag cttttgagcg agtttccttg tcgtcaggcc ttcgataccg actccattga
     6241 gtaattccag agcagagttt atgactttgc tcttgtccag tctagacatc ttatcgtcat
     6301 cgtctttgta atccatggtg gcggatcccg cgtcacgaca cctgtgttct ggcggcaaac
     6361 ccgttgcgaa aaagaacgtt cacggcgact actgcactta tatacggttc tcccccaccc
     6421 tcgggaaaaa ggcggagcca gtacacgaca tcactttccc agtttacccc gcgccacctt
     6481 ctctaggcac cggttcaatt gccgacccct ccccccaact tctcgggcac tgtgggccat
     6541 gtgcgctctg ccccatggta atagccatga ctaatacgta gatgtactgc caagtaggaa
     6601 agtgccataa ggtcatgtac tgggcataat gccaggcggg ccatttaccg tcattgacgt
     6661 caataggggg cgtacttggc atatgataca cttgatgtac tgccaagtgg gcagtttacc
     6721 gtaaatactc cacccattga cgtcaatgga aagtgcctat tggcgttact atgggaacat
     6781 acgtcattat tgacgtcaat gggcgggggt cgttgggcgg tcagccaggc gggccattta
     6841 ccgtaagtta tgtaacgcgg aactccatat atgggctatg aactaatgac cccgtaattg
     6901 attactatta ataactagtc aataatcaat gtcataagct ctaatcgcat aatcgagcta
     6961 agagcgatta atatgcttag ctagcttagc taagctagat ttctctattc tcccacaata
     7021 tatagcagct attagcttag cagtccgaat acgatcgtcc acgatccata agcgtaacac
     7081 atttgagtag cgactgacgg gcaccggagc cactcgagtg gaatta
//
