LOCUS       sequence-442480-        7597 bp    DNA     circular SYN 15-APR-2025
DEFINITION  PiggyBac transposon plasmid for doxycycline inducible expression of 
            DMRT1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7597)
  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 7597)
  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-DMRT1
FEATURES             Location/Qualifiers
     source          1..7597
                     /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(1511..1535)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1537..1563
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1730..1864
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   2136..2170
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2181..2198)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2181..2197)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2190..2212)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2406..2425)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2527..2549
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2588..2606)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2674..2778
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2779..2847,2848..3639)
                     /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(2997..3016)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3810..4398
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4299..4318
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4552..4569
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4775..4796
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4811..4816,4817..4834,4835..4841)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4849..4865
                     /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     4854..4876
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4873..4889
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4873..4889
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4915..4977
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    5153..5234
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5951..6007)
                     /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(6017..6760)
                     /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(6761..6784)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6784..6793
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6813..7024)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        7025..7404
                     /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 atgcccaacg acgaggcatt cagcaagccc
      421 tctacaccgt cggaagcccc tcacgccccc ggggtaccgc cgcagggcag agccgggggc
      481 tttggcaaag cgtctggggc gctagtgggg gcggccagcg gctcgagcgc cgggggcagc
      541 agcagaggag gcggctccgg ctccggggcg tcggacctgg gtgccgggag caagaagtcc
      601 ccgcggctgc ccaagtgcgc acgctgcagg aaccacggct acgcctcgcc gctcaagggc
      661 cacaagcgct tctgcatgtg gcgcgactgc cagtgcaaga agtgcaacct gatcgccgag
      721 aggcagcgcg tgatggccgc gcaggtggcc ctgagaaggc agcaggccca ggaggaggaa
      781 ttgggtatca gccaccccat cccactgccc agtgcggccg agctgcttgt caaaagagag
      841 aacaatggca gtaacccgtg cctcatgact gagtgcagtg gcacctctca gccaccgccg
      901 gccagtgtcc ccaccactgc agcttcagag ggacgtatgg tcatccagga tattcctgct
      961 gtcaccagca gagggcatgt ggagaacaca cctgacctgg tttcagactc cacctactac
     1021 agcagcttct accagccgtc tctgtttcct tattacaaca atctatacaa ctgcccgcag
     1081 tactccatgg ccttggctgc tgattctgct tctggggagg tgggaaatcc cctcggggga
     1141 tcccctgtga agaacagcct tcggggcctc cccggacctt atgtgcctgg tcagacagga
     1201 aaccagtggc agatgaagaa catggagaac cgccatgcaa tgagctccca gtacaggatg
     1261 cattcttact acccgcctcc ctcttacctg ggccagagcg tgccccagtt cttcactttt
     1321 gaggatgctc cctcttaccc ggaagccagg gcgagcgtat tctcgccgcc cagcagtcaa
     1381 gattctggct tggtttccct ctcgagcagc tctcctatta gtaacaagag cacaaaggca
     1441 gtgcttgaat gtgagcctgc gtcggagccc agcagcttca cagtcactcc cgtcatcgag
     1501 gaggacgagt gcccaacttt cttgtacaaa gtggtatacc catacgatgt tccagattac
     1561 gcttagtaat gagaaatact cagaagatgt cactcaccat agtccgcagt gaaagataaa
     1621 tgatcttatt ggagacaaca acgcagtttt agagctagaa atagcaagta ggccactgag
     1681 caagaactat gaatgagatc tttagacata ttgcggccgc tttctccgcg atccagacat
     1741 gataagatac attgatgagt ttggacaaac caaaactaga atgcagtgaa aaaaatgcct
     1801 tatttgtgaa atttgtgatg ctattgcctt atttgtaacc attataagct gcaataaaca
     1861 agttatctat aacaagaaaa tatatatata ataagttatc acgtaagtag aacatgaaat
     1921 aacaatataa ttatcgtatg agttaaatct taaaagtcac gtaaaagata atcatgcgtc
     1981 attttgactc acgcggtcgt tatagttcaa aatcagtgac acttaccgca ttgacaagca
     2041 cgcctcacgg gagctccaag cggcgactga gatgtcctaa atgcacagcg acggattcgc
     2101 gctatttaga aagagagagc aatatttcaa gaatgcatgc gtcaatttta cgcagactat
     2161 ctttctaggg ttaagaattc actggccgtc gttttacaac gtcgtgactg ggaaaaccct
     2221 ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc
     2281 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc
     2341 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact
     2401 ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc
     2461 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     2521 atgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cacgcgagtt
     2581 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct
     2641 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     2701 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     2761 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     2821 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     2881 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     2941 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     3001 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     3061 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     3121 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     3181 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     3241 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     3301 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     3361 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     3421 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     3481 gccggtgagc gtggttcacg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     3541 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     3601 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     3661 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     3721 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     3781 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     3841 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     3901 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     3961 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     4021 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     4081 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     4141 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     4201 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     4261 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     4321 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     4381 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     4441 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     4501 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     4561 gtgagcgagg aagcggatga gcgcccaata cgcaaaccgc ctctcaccgc gcgttggccg
     4621 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac
     4681 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
     4741 ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg
     4801 caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat
     4861 aacaatttca cacaggaaac agctatgacc atgattacgc caaggtcgac ttaaccctag
     4921 aaagataatc atattgtgac gtacgttaaa gataatcatg cgtaaaattg acgcatgtgt
     4981 tttatcggtc tgtatatcga ggtttattta ttaatttgaa tagatattaa gttttattat
     5041 atttacactt acatactaat aataaattca acaaacaatt tatttatgtt tatttattta
     5101 ttaaaaaaaa acaaaaactc aaaatttctt ctataaagta acaaaacttt tagcagtgaa
     5161 aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct
     5221 gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg
     5281 tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtatggct gattatgatc
     5341 ctctggagat tcatgcgcca ggttttctgg tcatgcacca cgttcgtggg ccctcaggaa
     5401 cctccacgtc agcggtaact gtgaagccca gtcgctcgta aaaaggaaga ttccggggag
     5461 cactggtttc gaggaacgcc ggcacgccag cccgttccgc ggcctcaaca ccaggaagaa
     5521 cgacagcgct tcccaaacct ttaccttggt gatctgggct gacaccgaca gtggcaagaa
     5581 accacgcagg ctcctttggt ctatgaggag ccagcaagcc ctccatttgc tgttgtgccg
     5641 ccaacctact ccctgacagc tcggccatcc gcggacctat ttccgcgaac actgcgccgg
     5701 cttcgacgct ttccggagta gtccagaccg caactgcggc accatcgtcc gcaacccaaa
     5761 ctttaccaat gtccagaccc acgcgcgtaa gaaacagctc ttgcaactca gttaccctct
     5821 ctatgtgtct gtccggatct acggtatggc gggtcgctgg ataatctgca aatgctgcgg
     5881 caagtgttcg gacagcccgt ggtacatcgt cacgggtagc gagcctaaca gtgggcttat
     5941 attccgtcat ggggccgggg ttctcctcca cgtcgccggc ctgcttcagc aggctgaagt
     6001 tggtggcgcc gctgcccccg gggagcatgt caaggtcaaa atcgtcaaga gcgtcagcag
     6061 gcagcatatc aaggtcaaag tcgtcaaggg catcggctgg gagcatgtct aagtcaaaat
     6121 cgtcaagggc gtcggtcggc ccgccgcttt cgcactttag ctgtttctcc aggccacata
     6181 tgattagttc caggccgaaa aggaaggcag gttcggctcc ctgccggtcg aacagctcaa
     6241 ttgcttgttt cagaagtggg ggcatagaat cggtggtagg tgtctctctt tcctcttttg
     6301 ctacttgatg ctcctgttcc tccaatacgc agcccagtgt aaagtggccc acggcggaca
     6361 gagcgtacag tgcgttctcc agggagaagc cttgctgaca caggaacgcg agctgatttt
     6421 ccagggtttc gtactgtttc tctgttgggc gggtgccgag atgcacttta gccccgtcac
     6481 gatgtgagag gagagcacag cggtatgact tggcgttgtt ccgcagaaag tcttgccatg
     6541 actcgccttc cagggggcag gagtgggtat gatgcctgtc cagcatctcg attggcaggg
     6601 catcgagcag ggcccgcttg ttcttcacgt gccagtacag ggtaggctgc tcaactccca
     6661 gcttttgagc gagtttcctt gtcgtcaggc cttcgatacc gactccattg agtaattcca
     6721 gagcagagtt tatgactttg ctcttgtcca gtctagacat cttatcgtca tcgtctttgt
     6781 aatccatggt ggcggatccc gcgtcacgac acctgtgttc tggcggcaaa cccgttgcga
     6841 aaaagaacgt tcacggcgac tactgcactt atatacggtt ctcccccacc ctcgggaaaa
     6901 aggcggagcc agtacacgac atcactttcc cagtttaccc cgcgccacct tctctaggca
     6961 ccggttcaat tgccgacccc tccccccaac ttctcgggca ctgtgggcca tgtgcgctct
     7021 gccccatggt aatagccatg actaatacgt agatgtactg ccaagtagga aagtgccata
     7081 aggtcatgta ctgggcataa tgccaggcgg gccatttacc gtcattgacg tcaatagggg
     7141 gcgtacttgg catatgatac acttgatgta ctgccaagtg ggcagtttac cgtaaatact
     7201 ccacccattg acgtcaatgg aaagtgccta ttggcgttac tatgggaaca tacgtcatta
     7261 ttgacgtcaa tgggcggggg tcgttgggcg gtcagccagg cgggccattt accgtaagtt
     7321 atgtaacgcg gaactccata tatgggctat gaactaatga ccccgtaatt gattactatt
     7381 aataactagt caataatcaa tgtcataagc tctaatcgca taatcgagct aagagcgatt
     7441 aatatgctta gctagcttag ctaagctaga tttctctatt ctcccacaat atatagcagc
     7501 tattagctta gcagtccgaa tacgatcgtc cacgatccat aagcgtaaca catttgagta
     7561 gcgactgacg ggcaccggag ccactcgagt ggaatta
//
