LOCUS       sequence-442407-        8077 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac transposon plasmid for doxycycline inducible expression of 
            RBM46.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8077)
  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 8077)
  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-RBM46
FEATURES             Location/Qualifiers
     source          1..8077
                     /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(1991..2015)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2017..2043
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    2210..2344
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   2616..2650
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2661..2678)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2661..2677)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2670..2692)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2886..2905)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3007..3029
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3068..3086)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3154..3258
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3259..3327,3328..4119)
                     /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(3477..3496)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4290..4878
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4779..4798
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5032..5049
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5255..5276
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5291..5296,5297..5314,5315..5321)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5329..5345
                     /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     5334..5356
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5353..5369
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5353..5369
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   5395..5457
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    5633..5714
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(6431..6487)
                     /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(6497..7240)
                     /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(7241..7264)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      7264..7273
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(7293..7504)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        7505..7884
                     /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 atgaatgaag aaaatataga tggaacaaat
      421 ggatgcagta aagttcgaac tggtattcag aatgaagcag cattacttgc tttgatggaa
      481 aagactggtt acaacatggt tcaggaaaat ggacaaagga aatttggcgg tcctcctcca
      541 ggttgggaag gtccacctcc acctagaggc tgtgaagttt ttgtaggaaa aatacctcgt
      601 gatatgtatg aagatgagtt agttcctgta tttgaaagag ctgggaagat atatgaattt
      661 cgacttatga tggaatttag tggtgaaaat cgaggttatg cttttgtgat gtacactaca
      721 aaagaagaag cccaattagc catcagaatt cttaataatt atgaaattcg accagggaag
      781 tttattggtg tgtgtgtaag cctggataat tgtagattat ttattggagc tattcccaag
      841 gaaaagaaga aagaagaaat tttagatgaa atgaagaaag ttacagaagg agttgtagat
      901 gtcattgttt atccaagtgc aactgataag accaaaaatc gtggttttgc atttgtggaa
      961 tatgaatctc acagagctgc tgctatggca aggaggaaac taattccagg aacattccaa
     1021 ctatggggcc acaccattca ggtagattgg gctgacccag agaaagaggt ggatgaggaa
     1081 accatgcaga gagttaaagt tctttatgta agaaatttaa tgatctcaac tacagaggaa
     1141 acaattaaag cagaattcaa taaatttaag cctggtgcag ttgaacgggt aaagaaactt
     1201 agagattatg cttttgttca ctttttcaac cgagaagatg cagtggctgc catgtctgtt
     1261 atgaatggaa aatgcattga tggagcaagt attgaggtaa cactagctaa accagtaaat
     1321 aaagaaaaca cttggagaca gcatcttaat ggtcagatta gtccaaattc tgaaaatctg
     1381 attgtgtttg ctaacaaaga agagagccac ccaaaaactc taggcaagct gccaactctt
     1441 cctgctcgtc tcaatggtca gcatagccca agtccgcctg aagttgaaag atgcacttac
     1501 cctttttatc ctggaacaaa gcttactcca attagtatgt attctttaaa atccaatcat
     1561 tttaattctg cagtaatgca tttggattat tactgcaaca aaaataactg ggcaccacca
     1621 gaatattatt tatattcaac aacaagtcaa gatgggaaag tactcttggt gtataagata
     1681 gttattcctg ctattgcaaa tggatcccag agttacttca tgccagacaa actctgtact
     1741 acgttagaag atgcaaagga actggcagcc cagtttacat tacttcattt ggactacaat
     1801 ttccatcgca gctcaataaa tagtctttcc cctgttagtg ctaccctctc ttctgggact
     1861 cccagcgtgc ttccttatac ttcaaggcct tattcttatc caggctatcc tttgtcacca
     1921 acaatatcac ttgctaatgg cagccatgtt ggacagcggc tatgtatctc caatcaggcc
     1981 tccttcttct gcccaacttt cttgtacaaa gtggtatacc catacgatgt tccagattac
     2041 gcttagtaat gagaaatact cagaagatgt cactcaccat agtccgcagt gaaagataaa
     2101 tgatctgact aagtattcca ttagcgtttt agagctagaa atagcaagta ggccactgag
     2161 caagaactat gaatgagatc tttagacata ttgcggccgc tttctccgcg atccagacat
     2221 gataagatac attgatgagt ttggacaaac caaaactaga atgcagtgaa aaaaatgcct
     2281 tatttgtgaa atttgtgatg ctattgcctt atttgtaacc attataagct gcaataaaca
     2341 agttatctat aacaagaaaa tatatatata ataagttatc acgtaagtag aacatgaaat
     2401 aacaatataa ttatcgtatg agttaaatct taaaagtcac gtaaaagata atcatgcgtc
     2461 attttgactc acgcggtcgt tatagttcaa aatcagtgac acttaccgca ttgacaagca
     2521 cgcctcacgg gagctccaag cggcgactga gatgtcctaa atgcacagcg acggattcgc
     2581 gctatttaga aagagagagc aatatttcaa gaatgcatgc gtcaatttta cgcagactat
     2641 ctttctaggg ttaagaattc actggccgtc gttttacaac gtcgtgactg ggaaaaccct
     2701 ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc
     2761 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc
     2821 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact
     2881 ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc
     2941 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     3001 atgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cacgcgagtt
     3061 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct
     3121 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     3181 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     3241 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     3301 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     3361 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     3421 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     3481 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     3541 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     3601 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     3661 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     3721 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     3781 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     3841 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     3901 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     3961 gccggtgagc gtggttcacg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     4021 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     4081 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     4141 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     4201 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     4261 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     4321 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     4381 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     4441 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     4501 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     4561 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     4621 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     4681 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     4741 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     4801 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     4861 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     4921 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     4981 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     5041 gtgagcgagg aagcggatga gcgcccaata cgcaaaccgc ctctcaccgc gcgttggccg
     5101 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac
     5161 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
     5221 ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg
     5281 caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat
     5341 aacaatttca cacaggaaac agctatgacc atgattacgc caaggtcgac ttaaccctag
     5401 aaagataatc atattgtgac gtacgttaaa gataatcatg cgtaaaattg acgcatgtgt
     5461 tttatcggtc tgtatatcga ggtttattta ttaatttgaa tagatattaa gttttattat
     5521 atttacactt acatactaat aataaattca acaaacaatt tatttatgtt tatttattta
     5581 ttaaaaaaaa acaaaaactc aaaatttctt ctataaagta acaaaacttt tagcagtgaa
     5641 aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct
     5701 gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg
     5761 tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtatggct gattatgatc
     5821 ctctggagat tcatgcgcca ggttttctgg tcatgcacca cgttcgtggg ccctcaggaa
     5881 cctccacgtc agcggtaact gtgaagccca gtcgctcgta aaaaggaaga ttccggggag
     5941 cactggtttc gaggaacgcc ggcacgccag cccgttccgc ggcctcaaca ccaggaagaa
     6001 cgacagcgct tcccaaacct ttaccttggt gatctgggct gacaccgaca gtggcaagaa
     6061 accacgcagg ctcctttggt ctatgaggag ccagcaagcc ctccatttgc tgttgtgccg
     6121 ccaacctact ccctgacagc tcggccatcc gcggacctat ttccgcgaac actgcgccgg
     6181 cttcgacgct ttccggagta gtccagaccg caactgcggc accatcgtcc gcaacccaaa
     6241 ctttaccaat gtccagaccc acgcgcgtaa gaaacagctc ttgcaactca gttaccctct
     6301 ctatgtgtct gtccggatct acggtatggc gggtcgctgg ataatctgca aatgctgcgg
     6361 caagtgttcg gacagcccgt ggtacatcgt cacgggtagc gagcctaaca gtgggcttat
     6421 attccgtcat ggggccgggg ttctcctcca cgtcgccggc ctgcttcagc aggctgaagt
     6481 tggtggcgcc gctgcccccg gggagcatgt caaggtcaaa atcgtcaaga gcgtcagcag
     6541 gcagcatatc aaggtcaaag tcgtcaaggg catcggctgg gagcatgtct aagtcaaaat
     6601 cgtcaagggc gtcggtcggc ccgccgcttt cgcactttag ctgtttctcc aggccacata
     6661 tgattagttc caggccgaaa aggaaggcag gttcggctcc ctgccggtcg aacagctcaa
     6721 ttgcttgttt cagaagtggg ggcatagaat cggtggtagg tgtctctctt tcctcttttg
     6781 ctacttgatg ctcctgttcc tccaatacgc agcccagtgt aaagtggccc acggcggaca
     6841 gagcgtacag tgcgttctcc agggagaagc cttgctgaca caggaacgcg agctgatttt
     6901 ccagggtttc gtactgtttc tctgttgggc gggtgccgag atgcacttta gccccgtcac
     6961 gatgtgagag gagagcacag cggtatgact tggcgttgtt ccgcagaaag tcttgccatg
     7021 actcgccttc cagggggcag gagtgggtat gatgcctgtc cagcatctcg attggcaggg
     7081 catcgagcag ggcccgcttg ttcttcacgt gccagtacag ggtaggctgc tcaactccca
     7141 gcttttgagc gagtttcctt gtcgtcaggc cttcgatacc gactccattg agtaattcca
     7201 gagcagagtt tatgactttg ctcttgtcca gtctagacat cttatcgtca tcgtctttgt
     7261 aatccatggt ggcggatccc gcgtcacgac acctgtgttc tggcggcaaa cccgttgcga
     7321 aaaagaacgt tcacggcgac tactgcactt atatacggtt ctcccccacc ctcgggaaaa
     7381 aggcggagcc agtacacgac atcactttcc cagtttaccc cgcgccacct tctctaggca
     7441 ccggttcaat tgccgacccc tccccccaac ttctcgggca ctgtgggcca tgtgcgctct
     7501 gccccatggt aatagccatg actaatacgt agatgtactg ccaagtagga aagtgccata
     7561 aggtcatgta ctgggcataa tgccaggcgg gccatttacc gtcattgacg tcaatagggg
     7621 gcgtacttgg catatgatac acttgatgta ctgccaagtg ggcagtttac cgtaaatact
     7681 ccacccattg acgtcaatgg aaagtgccta ttggcgttac tatgggaaca tacgtcatta
     7741 ttgacgtcaa tgggcggggg tcgttgggcg gtcagccagg cgggccattt accgtaagtt
     7801 atgtaacgcg gaactccata tatgggctat gaactaatga ccccgtaatt gattactatt
     7861 aataactagt caataatcaa tgtcataagc tctaatcgca taatcgagct aagagcgatt
     7921 aatatgctta gctagcttag ctaagctaga tttctctatt ctcccacaat atatagcagc
     7981 tattagctta gcagtccgaa tacgatcgtc cacgatccat aagcgtaaca catttgagta
     8041 gcgactgacg ggcaccggag ccactcgagt ggaatta
//
