LOCUS       sequence-437995-        7624 bp    DNA     circular SYN 15-APR-2025
DEFINITION  PiggyBac transposon plasmid for doxycycline inducible expression of 
            STRA8.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7624)
  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 7624)
  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-STRA8
FEATURES             Location/Qualifiers
     source          1..7624
                     /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(1539..1563)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1565..1591
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1758..1892
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   2164..2198
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2209..2226)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2209..2225)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2218..2240)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2434..2453)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2555..2577
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2616..2634)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2702..2806
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2807..2875,2876..3667)
                     /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(3025..3044)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3838..4426
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4327..4346
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4580..4597
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4803..4824
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4839..4844,4845..4862,4863..4869)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4877..4893
                     /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     4882..4904
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4901..4917
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4901..4917
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4943..5005
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    5181..5262
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5979..6035)
                     /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(6045..6788)
                     /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(6789..6812)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6812..6821
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6841..7052)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        7053..7432
                     /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 ggcaacccct gaagaaaaca
      421 gcaatcccca tgacagagca acaccccagc tgccagcaca gctgcaggag cttgagcatc
      481 gggtggcccg gagacggctg tcccaggccc gccaccgagc caccctggca gcgctcttca
      541 acaacctcag gaagacagtg tactctcagt ctgatctcat agcctcaaag tggcaggttc
      601 tgaataaggc aaagagtcat attccagaac tggagcaaac cctggataat ttgctgaagc
      661 tgaaagcatc cttcaacctg gaagatgggc atgcaagcag cttagaggag gtcaagaaag
      721 aatatgccag catgtattct ggaaatgaca gcctgctttc aaacagtttt cctcagaatg
      781 gttcctcccc ttggtgccca actgaggcag tcaggaagga tgctgaggag gaggaagatg
      841 aggaagagga agatcaagaa gaagaggagg aggaagaaga agaggaggag gaggaagagg
      901 aggaagagga agaggaggag gaggaagagg agaaaaaagt gatcttatac tccccaggaa
      961 ctttgtcgcc tgacctcatg gaatttgaac ggtatctcaa cttttacaaa cagacgatgg
     1021 accttctgac tggcagcggg atcattaccc cgcaggaggc ggcgctgccc atcgtctccg
     1081 cggccatctc ccacctgtgg cagaacctct cggaggagag gaaggccagc ctccggcagg
     1141 cctgggcgca gaagcaccgc ggccctgcga ccctggcgga ggcctgccga gagccggcct
     1201 gtgccgaggg cagcgtgaag gacagcggcg tggacagcca gggggccagc tgctcgctgg
     1261 tctccacgcc cgaggagatc ctttttgagg atgcctttga tgtggcaagc ttcctggaca
     1321 aaagtgaggt tccgagtaca tctagctcca gttcagtgct tgccagctgc aacccagaaa
     1381 acccagagga gaagtttcag ctctatatgc agatcatcaa cttttttaaa ggccttagct
     1441 gtgcaaacac tcaagtaaag caggaagcat cctttcccgt tgatgaagag atgatcatgt
     1501 tgcagtgcac agagaccttt gacgatgaag atttgtaaac ccagctttct tgtacaaagt
     1561 ggtataccca tacgatgttc cagattacgc ttagtaatga gaaatactca gaagatgtca
     1621 ctcaccatag tccgcagtga aagataaatg atcgggcagg gtaacgaggg ggggttttag
     1681 agctagaaat agcaagtagg ccactgagca agaactatga atgagatctt tagacatatt
     1741 gcggccgctt tctccgcgat ccagacatga taagatacat tgatgagttt ggacaaacca
     1801 aaactagaat gcagtgaaaa aaatgcctta tttgtgaaat ttgtgatgct attgccttat
     1861 ttgtaaccat tataagctgc aataaacaag ttatctataa caagaaaata tatatataat
     1921 aagttatcac gtaagtagaa catgaaataa caatataatt atcgtatgag ttaaatctta
     1981 aaagtcacgt aaaagataat catgcgtcat tttgactcac gcggtcgtta tagttcaaaa
     2041 tcagtgacac ttaccgcatt gacaagcacg cctcacggga gctccaagcg gcgactgaga
     2101 tgtcctaaat gcacagcgac ggattcgcgc tatttagaaa gagagagcaa tatttcaaga
     2161 atgcatgcgt caattttacg cagactatct ttctagggtt aagaattcac tggccgtcgt
     2221 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca
     2281 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
     2341 gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat tttctcctta cgcatctgtg
     2401 cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     2461 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     2521 ggcatccgct tacagacaag ctgtgaccat gtctccggga gctgcatgtg tcagaggttt
     2581 tcaccgtcat caccgaaaca cgcgagttcg aaagggcctc gtgatacgcc tatttttata
     2641 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
     2701 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     2761 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     2821 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     2881 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     2941 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     3001 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     3061 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     3121 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     3181 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     3241 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     3301 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     3361 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     3421 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     3481 tggctggttt attgctgata aatctggagc cggtgagcgt ggttcacgcg gtatcattgc
     3541 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     3601 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     3661 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     3721 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     3781 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     3841 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     3901 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     3961 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     4021 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     4081 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     4141 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     4201 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     4261 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     4321 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     4381 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     4441 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     4501 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     4561 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggatgagc gcccaatacg
     4621 caaaccgcct ctcaccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
     4681 cgactggaaa gcgggcagtg agcgcaacgc aaaccgcctc tccccgcgcg ttggccgatt
     4741 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
     4801 attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct
     4861 cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat
     4921 gattacgcca aggtcgactt aaccctagaa agataatcat attgtgacgt acgttaaaga
     4981 taatcatgcg taaaattgac gcatgtgttt tatcggtctg tatatcgagg tttatttatt
     5041 aatttgaata gatattaagt tttattatat ttacacttac atactaataa taaattcaac
     5101 aaacaattta tttatgttta tttatttatt aaaaaaaaac aaaaactcaa aatttcttct
     5161 ataaagtaac aaaactttta gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct
     5221 attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt
     5281 cattttatgt ttcaggttca gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc
     5341 tacaaatgtg gtatggctga ttatgatcct ctggagattc atgcgccagg ttttctggtc
     5401 atgcaccacg ttcgtgggcc ctcaggaacc tccacgtcag cggtaactgt gaagcccagt
     5461 cgctcgtaaa aaggaagatt ccggggagca ctggtttcga ggaacgccgg cacgccagcc
     5521 cgttccgcgg cctcaacacc aggaagaacg acagcgcttc ccaaaccttt accttggtga
     5581 tctgggctga caccgacagt ggcaagaaac cacgcaggct cctttggtct atgaggagcc
     5641 agcaagccct ccatttgctg ttgtgccgcc aacctactcc ctgacagctc ggccatccgc
     5701 ggacctattt ccgcgaacac tgcgccggct tcgacgcttt ccggagtagt ccagaccgca
     5761 actgcggcac catcgtccgc aacccaaact ttaccaatgt ccagacccac gcgcgtaaga
     5821 aacagctctt gcaactcagt taccctctct atgtgtctgt ccggatctac ggtatggcgg
     5881 gtcgctggat aatctgcaaa tgctgcggca agtgttcgga cagcccgtgg tacatcgtca
     5941 cgggtagcga gcctaacagt gggcttatat tccgtcatgg ggccggggtt ctcctccacg
     6001 tcgccggcct gcttcagcag gctgaagttg gtggcgccgc tgcccccggg gagcatgtca
     6061 aggtcaaaat cgtcaagagc gtcagcaggc agcatatcaa ggtcaaagtc gtcaagggca
     6121 tcggctggga gcatgtctaa gtcaaaatcg tcaagggcgt cggtcggccc gccgctttcg
     6181 cactttagct gtttctccag gccacatatg attagttcca ggccgaaaag gaaggcaggt
     6241 tcggctccct gccggtcgaa cagctcaatt gcttgtttca gaagtggggg catagaatcg
     6301 gtggtaggtg tctctctttc ctcttttgct acttgatgct cctgttcctc caatacgcag
     6361 cccagtgtaa agtggcccac ggcggacaga gcgtacagtg cgttctccag ggagaagcct
     6421 tgctgacaca ggaacgcgag ctgattttcc agggtttcgt actgtttctc tgttgggcgg
     6481 gtgccgagat gcactttagc cccgtcacga tgtgagagga gagcacagcg gtatgacttg
     6541 gcgttgttcc gcagaaagtc ttgccatgac tcgccttcca gggggcagga gtgggtatga
     6601 tgcctgtcca gcatctcgat tggcagggca tcgagcaggg cccgcttgtt cttcacgtgc
     6661 cagtacaggg taggctgctc aactcccagc ttttgagcga gtttccttgt cgtcaggcct
     6721 tcgataccga ctccattgag taattccaga gcagagttta tgactttgct cttgtccagt
     6781 ctagacatct tatcgtcatc gtctttgtaa tccatggtgg cggatcccgc gtcacgacac
     6841 ctgtgttctg gcggcaaacc cgttgcgaaa aagaacgttc acggcgacta ctgcacttat
     6901 atacggttct cccccaccct cgggaaaaag gcggagccag tacacgacat cactttccca
     6961 gtttaccccg cgccaccttc tctaggcacc ggttcaattg ccgacccctc cccccaactt
     7021 ctcgggcact gtgggccatg tgcgctctgc cccatggtaa tagccatgac taatacgtag
     7081 atgtactgcc aagtaggaaa gtgccataag gtcatgtact gggcataatg ccaggcgggc
     7141 catttaccgt cattgacgtc aatagggggc gtacttggca tatgatacac ttgatgtact
     7201 gccaagtggg cagtttaccg taaatactcc acccattgac gtcaatggaa agtgcctatt
     7261 ggcgttacta tgggaacata cgtcattatt gacgtcaatg ggcgggggtc gttgggcggt
     7321 cagccaggcg ggccatttac cgtaagttat gtaacgcgga actccatata tgggctatga
     7381 actaatgacc ccgtaattga ttactattaa taactagtca ataatcaatg tcataagctc
     7441 taatcgcata atcgagctaa gagcgattaa tatgcttagc tagcttagct aagctagatt
     7501 tctctattct cccacaatat atagcagcta ttagcttagc agtccgaata cgatcgtcca
     7561 cgatccataa gcgtaacaca tttgagtagc gactgacggg caccggagcc actcgagtgg
     7621 aatt
//
