LOCUS Exported 7909 bp ds-DNA circular SYN 16-NOV-2022 DEFINITION Barcoded piggybac transposon vector with Dox-inducible expression of PPARG. ACCESSION . VERSION . KEYWORDS pPB-cT3G-cERP2-PPARG SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7909) AUTHORS Smela M, Church GM TITLE Church lab granulosa cell plasmids JOURNAL unpublished REFERENCE 2 (bases 1 to 7909) AUTHORS . TITLE Direct Submission JOURNAL Exported Nov 16, 2022 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7909 /organism="synthetic DNA construct" /mol_type="other DNA" protein_bind 5..23 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 41..59 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 77..95 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 113..131 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 149..167 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 185..203 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 221..239 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" primer_bind 266..290 /label=LNCX /note="Human CMV promoter, forward primer" protein_bind 356..380 /gene="mutant version of attB" /label=attB1 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" protein_bind complement(1812..1836) /gene="mutant version of attB" /label=attB2 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" CDS 1838..1864 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" polyA_signal 2031..2165 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" repeat_region 2437..2471 /label=piggyBac left (5') inverted repeat /note="piggyBac transposon-specific inverted terminal repeat sequence (ITR)" primer_bind complement(2482..2499) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(2482..2498) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(2491..2513) /label=M13/pUC Forward /note="In lacZ gene" primer_bind complement(2707..2726) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 2828..2850 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(2889..2907) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 2975..3079 /gene="bla" /label=AmpR promoter CDS 3080..3940 /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(3298..3317) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 4111..4699 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4600..4619 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 4853..4870 /label=L4440 /note="L4440 vector, forward primer" protein_bind 5076..5097 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 5112..5142 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5150..5166 /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 5155..5177 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 5174..5190 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 5174..5190 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" repeat_region 5216..5278 /label=piggyBac right (3') inverted repeat /note="piggyBac transposon-specific inverted terminal repeat sequence (ITR)" polyA_signal 5454..5535 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" CDS complement(6252..6308) /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(6318..7061) /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(7062..7085) /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" regulatory 7085..7094 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" promoter complement(7114..7325) /label=EF-1-alpha core promoter /note="core promoter for human elongation factor EF-1-alpha" enhancer 7326..7705 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" ORIGIN 1 ttactcccta tcagtgatag agaacgtatg aagagtttac tccctatcag tgatagagaa 61 cgtatgcaga ctttactccc tatcagtgat agagaacgta taaggagttt actccctatc 121 agtgatagag aacgtatgac cagtttactc cctatcagtg atagagaacg tatctacagt 181 ttactcccta tcagtgatag agaacgtata tccagtttac tccctatcag tgatagagaa 241 cgtataagct tgcctatata agcagagctc gtttagtgaa ccgtcagatc gcctggagac 301 gccatccacg ctgttttgac ctccatagaa gacaccggga ccgatccagc ctaccacaag 361 tttgtacaaa aaagttggca tgaccatggt tgacacagag atgccattct ggcccaccaa 421 ctttgggatc agctccgtgg atctctccgt aatggaagac cactcccact cctttgatat 481 caagcccttc actactgttg acttctccag catttctact ccacattacg aagacattcc 541 attcacaaga acagatccag tggttgcaga ttacaagtat gacctgaaac ttcaagagta 601 ccaaagtgca atcaaagtgg agcctgcatc tccaccttat tattctgaga agactcagct 661 ctacaataag cctcatgaag agccttccaa ctccctcatg gcaattgaat gtcgtgtctg 721 tggagataaa gcttctggat ttcactatgg agttcatgct tgtgaaggat gcaagggttt 781 cttccggaga acaatcagat tgaagcttat ctatgacaga tgtgatctta actgtcggat 841 ccacaaaaaa agtagaaata aatgtcagta ctgtcggttt cagaaatgcc ttgcagtggg 901 gatgtctcat aatgccatca ggtttgggcg gatgccacag gccgagaagg agaagctgtt 961 ggcggagatc tccagtgata tcgaccagct gaatccagag tccgctgacc tccgggccct 1021 ggcaaaacat ttgtatgact catacataaa gtccttcccg ctgaccaaag caaaggcgag 1081 ggcgatcttg acaggaaaga caacagacaa atcaccattc gttatctatg acatgaattc 1141 cttaatgatg ggagaagata aaatcaagtt caaacacatc acccccctgc aggagcagag 1201 caaagaggtg gccatccgca tctttcaggg ctgccagttt cgctccgtgg aggctgtgca 1261 ggagatcaca gagtatgcca aaagcattcc tggttttgta aatcttgact tgaacgacca 1321 agtaactctc ctcaaatatg gagtccacga gatcatttac acaatgctgg cctccttgat 1381 gaataaagat ggggttctca tatccgaggg ccaaggcttc atgacaaggg agtttctaaa 1441 gagcctgcga aagccttttg gtgactttat ggagcccaag tttgagtttg ctgtgaagtt 1501 caatgcactg gaattagatg acagcgactt ggcaatattt attgctgtca ttattctcag 1561 tggagaccgc ccaggtttgc tgaatgtgaa gcccattgaa gacattcaag acaacctgct 1621 acaagccctg gagctccagc tgaagctgaa ccaccctgag tcctcacagc tgtttgccaa 1681 gctgctccag aaaatgacag acctcagaca gattgtcacg gaacacgtgc agctactgca 1741 ggtgatcaag aagacggaga cagacatgag tcttcacccg ctcctgcagg agatctacaa 1801 ggacttgtac tacccaactt tcttgtacaa agtggtatac ccatacgatg ttccagatta 1861 cgcttagtaa tgagaaatac tcagaagatg tcactcacca tagtccgcag tgaaagataa 1921 atgatcggaa aaatctttat agaggggttt tagagctaga aatagcaagt aggccactga 1981 gcaagaacta tgaatgagat ctttagacat attgcggccg ctttctccgc gatccagaca 2041 tgataagata cattgatgag tttggacaaa ccaaaactag aatgcagtga aaaaaatgcc 2101 ttatttgtga aatttgtgat gctattgcct tatttgtaac cattataagc tgcaataaac 2161 aagttatcta taacaagaaa atatatatat aataagttat cacgtaagta gaacatgaaa 2221 taacaatata attatcgtat gagttaaatc ttaaaagtca cgtaaaagat aatcatgcgt 2281 cattttgact cacgcggtcg ttatagttca aaatcagtga cacttaccgc attgacaagc 2341 acgcctcacg ggagctccaa gcggcgactg agatgtccta aatgcacagc gacggattcg 2401 cgctatttag aaagagagag caatatttca agaatgcatg cgtcaatttt acgcagacta 2461 tctttctagg gttaagaatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc 2521 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag 2581 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggcg 2641 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac 2701 tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc 2761 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac 2821 catgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acacgcgagt 2881 tcgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc 2941 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt 3001 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata 3061 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt 3121 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc 3181 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat 3241 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct 3301 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca 3361 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg 3421 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa 3481 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg 3541 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga 3601 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg 3661 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt 3721 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg 3781 agccggtgag cgtggttcac gcggtatcat tgcagcactg gggccagatg gtaagccctc 3841 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca 3901 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc 3961 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat 4021 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc 4081 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg 4141 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct 4201 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct 4261 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct 4321 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg 4381 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc 4441 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga 4501 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg 4561 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta 4621 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg 4681 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg 4741 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat 4801 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc 4861 agtgagcgag gaagcggatg agcgcccaat acgcaaaccg cctctcaccg cgcgttggcc 4921 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa 4981 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt 5041 cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag 5101 gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga 5161 taacaatttc acacaggaaa cagctatgac catgattacg ccaaggtcga cttaacccta 5221 gaaagataat catattgtga cgtacgttaa agataatcat gcgtaaaatt gacgcatgtg 5281 ttttatcggt ctgtatatcg aggtttattt attaatttga atagatatta agttttatta 5341 tatttacact tacatactaa taataaattc aacaaacaat ttatttatgt ttatttattt 5401 attaaaaaaa aacaaaaact caaaatttct tctataaagt aacaaaactt ttagcagtga 5461 aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc 5521 tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag 5581 gtgtgggagg ttttttaaag caagtaaaac ctctacaaat gtggtatggc tgattatgat 5641 cctctggaga ttcatgcgcc aggttttctg gtcatgcacc acgttcgtgg gccctcagga 5701 acctccacgt cagcggtaac tgtgaagccc agtcgctcgt aaaaaggaag attccgggga 5761 gcactggttt cgaggaacgc cggcacgcca gcccgttccg cggcctcaac accaggaaga 5821 acgacagcgc ttcccaaacc tttaccttgg tgatctgggc tgacaccgac agtggcaaga 5881 aaccacgcag gctcctttgg tctatgagga gccagcaagc cctccatttg ctgttgtgcc 5941 gccaacctac tccctgacag ctcggccatc cgcggaccta tttccgcgaa cactgcgccg 6001 gcttcgacgc tttccggagt agtccagacc gcaactgcgg caccatcgtc cgcaacccaa 6061 actttaccaa tgtccagacc cacgcgcgta agaaacagct cttgcaactc agttaccctc 6121 tctatgtgtc tgtccggatc tacggtatgg cgggtcgctg gataatctgc aaatgctgcg 6181 gcaagtgttc ggacagcccg tggtacatcg tcacgggtag cgagcctaac agtgggctta 6241 tattccgtca tggggccggg gttctcctcc acgtcgccgg cctgcttcag caggctgaag 6301 ttggtggcgc cgctgccccc ggggagcatg tcaaggtcaa aatcgtcaag agcgtcagca 6361 ggcagcatat caaggtcaaa gtcgtcaagg gcatcggctg ggagcatgtc taagtcaaaa 6421 tcgtcaaggg cgtcggtcgg cccgccgctt tcgcacttta gctgtttctc caggccacat 6481 atgattagtt ccaggccgaa aaggaaggca ggttcggctc cctgccggtc gaacagctca 6541 attgcttgtt tcagaagtgg gggcatagaa tcggtggtag gtgtctctct ttcctctttt 6601 gctacttgat gctcctgttc ctccaatacg cagcccagtg taaagtggcc cacggcggac 6661 agagcgtaca gtgcgttctc cagggagaag ccttgctgac acaggaacgc gagctgattt 6721 tccagggttt cgtactgttt ctctgttggg cgggtgccga gatgcacttt agccccgtca 6781 cgatgtgaga ggagagcaca gcggtatgac ttggcgttgt tccgcagaaa gtcttgccat 6841 gactcgcctt ccagggggca ggagtgggta tgatgcctgt ccagcatctc gattggcagg 6901 gcatcgagca gggcccgctt gttcttcacg tgccagtaca gggtaggctg ctcaactccc 6961 agcttttgag cgagtttcct tgtcgtcagg ccttcgatac cgactccatt gagtaattcc 7021 agagcagagt ttatgacttt gctcttgtcc agtctagaca tcttatcgtc atcgtctttg 7081 taatccatgg tggcggatcc cgcgtcacga cacctgtgtt ctggcggcaa acccgttgcg 7141 aaaaagaacg ttcacggcga ctactgcact tatatacggt tctcccccac cctcgggaaa 7201 aaggcggagc cagtacacga catcactttc ccagtttacc ccgcgccacc ttctctaggc 7261 accggttcaa ttgccgaccc ctccccccaa cttctcgggc actgtgggcc atgtgcgctc 7321 tgccccatgg taatagccat gactaatacg tagatgtact gccaagtagg aaagtgccat 7381 aaggtcatgt actgggcata atgccaggcg ggccatttac cgtcattgac gtcaataggg 7441 ggcgtacttg gcatatgata cacttgatgt actgccaagt gggcagttta ccgtaaatac 7501 tccacccatt gacgtcaatg gaaagtgcct attggcgtta ctatgggaac atacgtcatt 7561 attgacgtca atgggcgggg gtcgttgggc ggtcagccag gcgggccatt taccgtaagt 7621 tatgtaacgc ggaactccat atatgggcta tgaactaatg accccgtaat tgattactat 7681 taataactag tcaataatca atgtcataag ctctaatcgc ataatcgagc taagagcgat 7741 taatatgctt agctagctta gctaagctag atttctctat tctcccacaa tatatagcag 7801 ctattagctt agcagtccga atacgatcgt ccacgatcca taagcgtaac acatttgagt 7861 agcgactgac gggcaccgga gccactcgag tggaattatc acctcgagt //