LOCUS       sequence-439869-        7954 bp    DNA     circular SYN 15-APR-2025
DEFINITION  PiggyBac transposon plasmid for doxycycline inducible expression of 
            3xFLAG-SMAD1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7954)
  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 7954)
  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-cERP2-3xFLAG-SMAD1-active
FEATURES             Location/Qualifiers
     source          1..7954
                     /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"
     CDS             401..472
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
     protein_bind    complement(1869..1893)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1895..1921
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    2088..2222
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   2494..2528
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2539..2556)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2539..2555)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2548..2570)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2764..2783)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2885..2907
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2946..2964)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3032..3136
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3137..3205,3206..3997)
                     /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(3355..3374)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4168..4756
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4657..4676
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4910..4927
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5133..5154
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5169..5174,5175..5192,5193..5199)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5207..5223
                     /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     5212..5234
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5231..5247
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5231..5247
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   5273..5335
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    5511..5592
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(6309..6365)
                     /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(6375..7118)
                     /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(7119..7142)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      7142..7151
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(7171..7382)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        7383..7762
                     /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 ctgcaccatg gattataagg acgatgatga
      421 caaggactat aaagacgatg atgataaaga ctacaaagat gacgacgata agaacgtgac
      481 aagcttgttt agtttcacat ctcccgccgt gaaacgactg ctgggctgga agcagggaga
      541 cgaggaggag aaatgggctg agaaagccgt tgatgctctg gtgaaaaagc tgaagaagaa
      601 gaagggggcc atggaggagc tggaaaaggc cctctcatgc cccggtcagc cttctaactg
      661 cgtgacgata cctcgatccc tcgatgggag gctgcaggta tcacatcgca aaggcttgcc
      721 tcacgtcatc tactgtagag tgtggcgatg gcctgacctt cagtcccatc acgagctcaa
      781 gccccttgaa tgctgtgaat tccccttcgg cagtaagcag aaggaggtgt gtataaaccc
      841 ctaccactat aaacgggtcg agtcccctgt cttgccacca gtgctggtgc cacggcattc
      901 tgaatacaat ccccagcatt cacttctggc acaatttcga aacctgggcc agaacgaacc
      961 ccatatgcca cttaacgcca cttttccaga ttccttccag cagcccaaca gccatccttt
     1021 tccccacagc ccaaatagtt catatccaaa ttcaccaggt tcttcctcca gtacgtatcc
     1081 acattcaccc acaagctctg accccggatc tccgttccag atgcccgccg acacaccccc
     1141 acccgcctat ctgcctcctg aagatccgat gacccaggac gggtcccagc caatggacac
     1201 gaacatgatg gcccctccgc tcccatcaga gatcaatcgc ggggacgtcc aagcggttgc
     1261 atacgaagag cccaaacact ggtgtagtat cgtgtactac gagcttaaca atagagtggg
     1321 agaagccttc cacgcttcct ctacctctgt actggtcgac gggttcactg atccaagcaa
     1381 caataagaat cggttctgtc ttggcctgct gagcaatgtg aatcgcaatt caacgatcga
     1441 gaatactcga cgacacatcg gtaaaggagt ccacctctac tatgtgggcg gggaggtcta
     1501 cgcagagtgc cttagtgata gttctatctt tgtgcagtca aggaactgca actaccatca
     1561 tggttttcat cccaccaccg tgtgtaaaat cccttctgga tgctctctca agatttttaa
     1621 taaccaggaa tttgctcagt tgttggctca gtccgtgaac cacggcttcg agaccgttta
     1681 cgagctcaca aagatgtgca caatccggat gagcttcgtg aagggatggg gagccgagta
     1741 tcataggcag gacgtgactt ccacaccttg ttggatcgag atccacttgc atggaccact
     1801 tcagtggctg gacaaagttt tgactcagat ggggtcccct cacaatccga tttcagaagt
     1861 ggaatgagac ccagctttct tgtacaaagt ggtataccca tacgatgttc cagattacgc
     1921 ttagtaatga gaaatactca gaagatgtca ctcaccatag tccgcagtga aagataaatg
     1981 atcaatacct atcactatag cttgttttag agctagaaat agcaagtagg ccactgagca
     2041 agaactatga atgagatctt tagacatatt gcggccgctt tctccgcgat ccagacatga
     2101 taagatacat tgatgagttt ggacaaacca aaactagaat gcagtgaaaa aaatgcctta
     2161 tttgtgaaat ttgtgatgct attgccttat ttgtaaccat tataagctgc aataaacaag
     2221 ttatctataa caagaaaata tatatataat aagttatcac gtaagtagaa catgaaataa
     2281 caatataatt atcgtatgag ttaaatctta aaagtcacgt aaaagataat catgcgtcat
     2341 tttgactcac gcggtcgtta tagttcaaaa tcagtgacac ttaccgcatt gacaagcacg
     2401 cctcacggga gctccaagcg gcgactgaga tgtcctaaat gcacagcgac ggattcgcgc
     2461 tatttagaaa gagagagcaa tatttcaaga atgcatgcgt caattttacg cagactatct
     2521 ttctagggtt aagaattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg
     2581 cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga
     2641 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcgcct
     2701 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat ggtgcactct
     2761 cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc
     2821 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccat
     2881 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaaca cgcgagttcg
     2941 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta
     3001 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     3061 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     3121 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     3181 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     3241 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     3301 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     3361 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     3421 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     3481 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     3541 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     3601 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     3661 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     3721 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     3781 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     3841 cggtgagcgt ggttcacgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     3901 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     3961 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     4021 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     4081 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     4141 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     4201 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     4261 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     4321 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     4381 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     4441 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     4501 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     4561 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     4621 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     4681 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     4741 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     4801 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     4861 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     4921 gagcgaggaa gcggatgagc gcccaatacg caaaccgcct ctcaccgcgc gttggccgat
     4981 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
     5041 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
     5101 gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca
     5161 ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa
     5221 caatttcaca caggaaacag ctatgaccat gattacgcca aggtcgactt aaccctagaa
     5281 agataatcat attgtgacgt acgttaaaga taatcatgcg taaaattgac gcatgtgttt
     5341 tatcggtctg tatatcgagg tttatttatt aatttgaata gatattaagt tttattatat
     5401 ttacacttac atactaataa taaattcaac aaacaattta tttatgttta tttatttatt
     5461 aaaaaaaaac aaaaactcaa aatttcttct ataaagtaac aaaactttta gcagtgaaaa
     5521 aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc
     5581 aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggaggtg
     5641 tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg gtatggctga ttatgatcct
     5701 ctggagattc atgcgccagg ttttctggtc atgcaccacg ttcgtgggcc ctcaggaacc
     5761 tccacgtcag cggtaactgt gaagcccagt cgctcgtaaa aaggaagatt ccggggagca
     5821 ctggtttcga ggaacgccgg cacgccagcc cgttccgcgg cctcaacacc aggaagaacg
     5881 acagcgcttc ccaaaccttt accttggtga tctgggctga caccgacagt ggcaagaaac
     5941 cacgcaggct cctttggtct atgaggagcc agcaagccct ccatttgctg ttgtgccgcc
     6001 aacctactcc ctgacagctc ggccatccgc ggacctattt ccgcgaacac tgcgccggct
     6061 tcgacgcttt ccggagtagt ccagaccgca actgcggcac catcgtccgc aacccaaact
     6121 ttaccaatgt ccagacccac gcgcgtaaga aacagctctt gcaactcagt taccctctct
     6181 atgtgtctgt ccggatctac ggtatggcgg gtcgctggat aatctgcaaa tgctgcggca
     6241 agtgttcgga cagcccgtgg tacatcgtca cgggtagcga gcctaacagt gggcttatat
     6301 tccgtcatgg ggccggggtt ctcctccacg tcgccggcct gcttcagcag gctgaagttg
     6361 gtggcgccgc tgcccccggg gagcatgtca aggtcaaaat cgtcaagagc gtcagcaggc
     6421 agcatatcaa ggtcaaagtc gtcaagggca tcggctggga gcatgtctaa gtcaaaatcg
     6481 tcaagggcgt cggtcggccc gccgctttcg cactttagct gtttctccag gccacatatg
     6541 attagttcca ggccgaaaag gaaggcaggt tcggctccct gccggtcgaa cagctcaatt
     6601 gcttgtttca gaagtggggg catagaatcg gtggtaggtg tctctctttc ctcttttgct
     6661 acttgatgct cctgttcctc caatacgcag cccagtgtaa agtggcccac ggcggacaga
     6721 gcgtacagtg cgttctccag ggagaagcct tgctgacaca ggaacgcgag ctgattttcc
     6781 agggtttcgt actgtttctc tgttgggcgg gtgccgagat gcactttagc cccgtcacga
     6841 tgtgagagga gagcacagcg gtatgacttg gcgttgttcc gcagaaagtc ttgccatgac
     6901 tcgccttcca gggggcagga gtgggtatga tgcctgtcca gcatctcgat tggcagggca
     6961 tcgagcaggg cccgcttgtt cttcacgtgc cagtacaggg taggctgctc aactcccagc
     7021 ttttgagcga gtttccttgt cgtcaggcct tcgataccga ctccattgag taattccaga
     7081 gcagagttta tgactttgct cttgtccagt ctagacatct tatcgtcatc gtctttgtaa
     7141 tccatggtgg cggatcccgc gtcacgacac ctgtgttctg gcggcaaacc cgttgcgaaa
     7201 aagaacgttc acggcgacta ctgcacttat atacggttct cccccaccct cgggaaaaag
     7261 gcggagccag tacacgacat cactttccca gtttaccccg cgccaccttc tctaggcacc
     7321 ggttcaattg ccgacccctc cccccaactt ctcgggcact gtgggccatg tgcgctctgc
     7381 cccatggtaa tagccatgac taatacgtag atgtactgcc aagtaggaaa gtgccataag
     7441 gtcatgtact gggcataatg ccaggcgggc catttaccgt cattgacgtc aatagggggc
     7501 gtacttggca tatgatacac ttgatgtact gccaagtggg cagtttaccg taaatactcc
     7561 acccattgac gtcaatggaa agtgcctatt ggcgttacta tgggaacata cgtcattatt
     7621 gacgtcaatg ggcgggggtc gttgggcggt cagccaggcg ggccatttac cgtaagttat
     7681 gtaacgcgga actccatata tgggctatga actaatgacc ccgtaattga ttactattaa
     7741 taactagtca ataatcaatg tcataagctc taatcgcata atcgagctaa gagcgattaa
     7801 tatgcttagc tagcttagct aagctagatt tctctattct cccacaatat atagcagcta
     7861 ttagcttagc agtccgaata cgatcgtcca cgatccataa gcgtaacaca tttgagtagc
     7921 gactgacggg caccggagcc actcgagtgg aatt
//
