LOCUS       sequence-439856-        7396 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac transposon plasmid for doxycycline inducible expression of 
            MSX1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7396)
  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 7396)
  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-MSX1
FEATURES             Location/Qualifiers
     source          1..7396
                     /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"
     regulatory      393..402
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(1311..1335)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1337..1363
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1530..1664
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   1936..1970
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1981..1998)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1981..1997)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1990..2012)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2206..2225)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2327..2349
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2388..2406)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2474..2578
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2579..2647,2648..3439)
                     /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(2797..2816)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3610..4198
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4099..4118
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4352..4369
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4575..4596
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4611..4616,4617..4634,4635..4641)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4649..4665
                     /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     4654..4676
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4673..4689
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4673..4689
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4715..4777
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    4953..5034
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5751..5807)
                     /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(5817..6560)
                     /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(6561..6584)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6584..6593
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6613..6824)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        6825..7204
                     /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 ctgccaccat ggcccccgcg gcagatatga
      421 cctctcttcc cttaggagtt aaggtcgaag attctgcgtt tggtaaaccc gctggtggcg
      481 gggccgggca agctccttcc gctgctgctg cgactgctgc ggcaatggga gcagatgaag
      541 aaggagctaa accaaaggtc agccccagtc tgcttccatt ttccgtcgaa gcactgatgg
      601 cagatcatag aaaacccggc gctaaagaaa gtgcactcgc cccatctgaa ggagtccaag
      661 cagccggcgg aagtgctcaa cctctcggag ttcccccagg ttcccttggc gcgccagatg
      721 ccccaagctc tcctagaccc ttggggcatt ttagtgtcgg tggccttctg aaacttcccg
      781 aggacgctct ggtgaaggct gaatcccctg agaaaccaga aagaacgcca tggatgcaat
      841 ctccacggtt ttcaccacca cccgctagaa ggctctcccc gcctgcatgt actttaagga
      901 agcataaaac taatagaaaa cctagaaccc cttttactac agcacaactt ctcgctctcg
      961 aacgtaaatt taggcagaaa caatatctca gtattgctga acgggctgaa ttttcaagtt
     1021 ccctgtcact tacagaaaca caagtcaaaa tctggtttca aaatcgacga gcgaaagcca
     1081 aacgacttca ggaagccgaa ctcgaaaagt tgaaaatggc agctaaacct atgcttccgc
     1141 ctgcagcttt tggacttagt tttccactgg gcggaccagc cgcagtggca gctgccgccg
     1201 gcgcaagctt gtatggagct tccgggccgt ttcaaagagc agcattaccg gttgcaccag
     1261 tcggcctgta tactgcacac gtaggttatt ccatgtatca tttaacttga acccagcttt
     1321 cttgtacaaa gtggtatacc catacgatgt tccagattac gcttagtaat gagaaatact
     1381 cagaagatgt cactcaccat agtccgcagt gaaagataaa tgatccatat ttagttctcc
     1441 ttcttgtttt agagctagaa atagcaagta ggccactgag caagaactat gaatgagatc
     1501 tttagacata ttgcggccgc tttctccgcg atccagacat gataagatac attgatgagt
     1561 ttggacaaac caaaactaga atgcagtgaa aaaaatgcct tatttgtgaa atttgtgatg
     1621 ctattgcctt atttgtaacc attataagct gcaataaaca agttatctat aacaagaaaa
     1681 tatatatata ataagttatc acgtaagtag aacatgaaat aacaatataa ttatcgtatg
     1741 agttaaatct taaaagtcac gtaaaagata atcatgcgtc attttgactc acgcggtcgt
     1801 tatagttcaa aatcagtgac acttaccgca ttgacaagca cgcctcacgg gagctccaag
     1861 cggcgactga gatgtcctaa atgcacagcg acggattcgc gctatttaga aagagagagc
     1921 aatatttcaa gaatgcatgc gtcaatttta cgcagactat ctttctaggg ttaagaattc
     1981 actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg
     2041 ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg
     2101 cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc ctgatgcggt attttctcct
     2161 tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa tctgctctga
     2221 tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     2281 ttgtctgctc ccggcatccg cttacagaca agctgtgacc atgtctccgg gagctgcatg
     2341 tgtcagaggt tttcaccgtc atcaccgaaa cacgcgagtt cgaaagggcc tcgtgatacg
     2401 cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt
     2461 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     2521 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     2581 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     2641 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     2701 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     2761 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     2821 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     2881 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     2941 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     3001 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     3061 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     3121 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     3181 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     3241 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtggttcacg
     3301 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     3361 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     3421 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     3481 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     3541 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     3601 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     3661 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     3721 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     3781 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     3841 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     3901 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     3961 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     4021 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     4081 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     4141 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     4201 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     4261 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     4321 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggatga
     4381 gcgcccaata cgcaaaccgc ctctcaccgc gcgttggccg attcattaat gcagctggca
     4441 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaaaccgcc tctccccgcg
     4501 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
     4561 gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc tttacacttt
     4621 atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac
     4681 agctatgacc atgattacgc caaggtcgac ttaaccctag aaagataatc atattgtgac
     4741 gtacgttaaa gataatcatg cgtaaaattg acgcatgtgt tttatcggtc tgtatatcga
     4801 ggtttattta ttaatttgaa tagatattaa gttttattat atttacactt acatactaat
     4861 aataaattca acaaacaatt tatttatgtt tatttattta ttaaaaaaaa acaaaaactc
     4921 aaaatttctt ctataaagta acaaaacttt tagcagtgaa aaaaatgctt tatttgtgaa
     4981 atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac
     5041 aacaattgca ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaaagc
     5101 aagtaaaacc tctacaaatg tggtatggct gattatgatc ctctggagat tcatgcgcca
     5161 ggttttctgg tcatgcacca cgttcgtggg ccctcaggaa cctccacgtc agcggtaact
     5221 gtgaagccca gtcgctcgta aaaaggaaga ttccggggag cactggtttc gaggaacgcc
     5281 ggcacgccag cccgttccgc ggcctcaaca ccaggaagaa cgacagcgct tcccaaacct
     5341 ttaccttggt gatctgggct gacaccgaca gtggcaagaa accacgcagg ctcctttggt
     5401 ctatgaggag ccagcaagcc ctccatttgc tgttgtgccg ccaacctact ccctgacagc
     5461 tcggccatcc gcggacctat ttccgcgaac actgcgccgg cttcgacgct ttccggagta
     5521 gtccagaccg caactgcggc accatcgtcc gcaacccaaa ctttaccaat gtccagaccc
     5581 acgcgcgtaa gaaacagctc ttgcaactca gttaccctct ctatgtgtct gtccggatct
     5641 acggtatggc gggtcgctgg ataatctgca aatgctgcgg caagtgttcg gacagcccgt
     5701 ggtacatcgt cacgggtagc gagcctaaca gtgggcttat attccgtcat ggggccgggg
     5761 ttctcctcca cgtcgccggc ctgcttcagc aggctgaagt tggtggcgcc gctgcccccg
     5821 gggagcatgt caaggtcaaa atcgtcaaga gcgtcagcag gcagcatatc aaggtcaaag
     5881 tcgtcaaggg catcggctgg gagcatgtct aagtcaaaat cgtcaagggc gtcggtcggc
     5941 ccgccgcttt cgcactttag ctgtttctcc aggccacata tgattagttc caggccgaaa
     6001 aggaaggcag gttcggctcc ctgccggtcg aacagctcaa ttgcttgttt cagaagtggg
     6061 ggcatagaat cggtggtagg tgtctctctt tcctcttttg ctacttgatg ctcctgttcc
     6121 tccaatacgc agcccagtgt aaagtggccc acggcggaca gagcgtacag tgcgttctcc
     6181 agggagaagc cttgctgaca caggaacgcg agctgatttt ccagggtttc gtactgtttc
     6241 tctgttgggc gggtgccgag atgcacttta gccccgtcac gatgtgagag gagagcacag
     6301 cggtatgact tggcgttgtt ccgcagaaag tcttgccatg actcgccttc cagggggcag
     6361 gagtgggtat gatgcctgtc cagcatctcg attggcaggg catcgagcag ggcccgcttg
     6421 ttcttcacgt gccagtacag ggtaggctgc tcaactccca gcttttgagc gagtttcctt
     6481 gtcgtcaggc cttcgatacc gactccattg agtaattcca gagcagagtt tatgactttg
     6541 ctcttgtcca gtctagacat cttatcgtca tcgtctttgt aatccatggt ggcggatccc
     6601 gcgtcacgac acctgtgttc tggcggcaaa cccgttgcga aaaagaacgt tcacggcgac
     6661 tactgcactt atatacggtt ctcccccacc ctcgggaaaa aggcggagcc agtacacgac
     6721 atcactttcc cagtttaccc cgcgccacct tctctaggca ccggttcaat tgccgacccc
     6781 tccccccaac ttctcgggca ctgtgggcca tgtgcgctct gccccatggt aatagccatg
     6841 actaatacgt agatgtactg ccaagtagga aagtgccata aggtcatgta ctgggcataa
     6901 tgccaggcgg gccatttacc gtcattgacg tcaatagggg gcgtacttgg catatgatac
     6961 acttgatgta ctgccaagtg ggcagtttac cgtaaatact ccacccattg acgtcaatgg
     7021 aaagtgccta ttggcgttac tatgggaaca tacgtcatta ttgacgtcaa tgggcggggg
     7081 tcgttgggcg gtcagccagg cgggccattt accgtaagtt atgtaacgcg gaactccata
     7141 tatgggctat gaactaatga ccccgtaatt gattactatt aataactagt caataatcaa
     7201 tgtcataagc tctaatcgca taatcgagct aagagcgatt aatatgctta gctagcttag
     7261 ctaagctaga tttctctatt ctcccacaat atatagcagc tattagctta gcagtccgaa
     7321 tacgatcgtc cacgatccat aagcgtaaca catttgagta gcgactgacg ggcaccggag
     7381 ccactcgagt ggaatt
//
