LOCUS Exported 7635 bp ds-DNA circular SYN 17-OCT-2022 DEFINITION Plasmid ensures constitutive expression of the N-terminal fragment of split ERbeta (aa 261-446), fused to the VPR activation domain, a flexible GS linker and the SV40 NLS signal at the C-terminus. ACCESSION . VERSION . KEYWORDS nERb446-gs10-NLS-VPR SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7635) AUTHORS Rihtar E, Lebar T, Lainscek D, Kores K, Lesnik S, Bren U, Jerala R TITLE Chemically inducible split protein regulators for mammalian cells JOURNAL Nat Chem Biol. 2022 REFERENCE 2 (bases 1 to 7635) AUTHORS . TITLE Direct Submission JOURNAL Exported Oct 17, 2022 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7635 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(45..64) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 236..615 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 616..819 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 770..790 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 864..883 /label=T7 /note="T7 promoter, forward primer" promoter 864..882 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" regulatory 945..954 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1545..1565 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1590..1739 /codon_start=1 /product="tetrameric repeat of the minimal activation domain of herpes simplex virus VP16 (Beerli et al., 1998)" /label=VP64 /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF DLDML" CDS 2208..2564 /codon_start=1 /product="transcriptional activation domain of human RelA, also known as p65 (O'Shea and Perkins, 2008)" /label=RelA (p65) AD /translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD EDFSSIADMDFSALL" CDS 2583..3152 /codon_start=1 /product="transcriptional activation domain from the human herpesvirus 4 (Epstein-Barr virus) replication and transcription activator Rta/BRLF1 (Hardwick et al., 1992; Chavez et al., 2015)" /label=Rta AD /translation="RDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWAN RPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKAL REMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEIL DTFLNDECLLHAMHISTGLSIFDTSLF" promoter complement(3189..3207) /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind complement(3190..3207) /label=SP6 /note="SP6 promoter, forward primer" primer_bind complement(3227..3244) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 3233..3457 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 3503..3931 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3590..3609) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind complement(3940..3960) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 3945..4274 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 4125..4260 /label=SV40 ori /note="SV40 origin of replication" primer_bind 4187..4206 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 4341..5135 /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(4395..4414) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 5005..5024 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 5309..5430 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(5346..5365) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 5400..5419 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(5479..5495) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5479..5495) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5492..5514) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5503..5519 /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)." promoter complement(5527..5557) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5572..5593 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5710..5727) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5881..6469) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5961..5980) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6640..7500) /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 7263..7282 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7501..7605) /gene="bla" /label=AmpR promoter ORIGIN 1 cgacggatcg ggagatctcc cgatccccta tggtgcactc tcagtacaat ctgctctgat 61 gccgcatagt taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc 121 gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg acaattgcat gaagaatctg 181 cttagggtta ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgttgacat 241 tgattattga ctagttatta atagtaatca attacggggt cattagttca tagcccatat 301 atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac 361 ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc 421 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg 481 tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat 541 tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc 601 atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt 661 gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac 721 caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc 781 ggtaggcgtg tacggtggga ggtctatata agcagagctc tctggctaac tagagaaccc 841 actgcttact ggcttatcga aattaatacg actcactata gggagaccca agcttagtac 901 cgagctcgga tccactagta acggccgcca gtgtgctgga attcgccacc atggacgctc 961 tgtcacctga acagctcgtt ttgacgcttt tggaagctga accaccacac gttctgatca 1021 gccgaccctc tgctccattc acagaggcca gtatgatgat gagcctcact aagttggccg 1081 ataaagaact tgtccatatg atttcctggg cgaagaaaat acccggattc gtagagcttt 1141 ccctcttcga tcaggtacgg ctgctcgaga gttgctggat ggaggttctg atgatggggc 1201 tgatgtggag atccatagat caccccggta aacttatctt tgcgccagac cttgtactcg 1261 accgggacga aggaaaatgc gtagaaggga tcctggaaat ttttgatatg ctgctcgcta 1321 ccaccagtcg gttcagggag ctgaaattgc agcacaagga atatctttgc gtgaaagcga 1381 tgatacttct caattcaagt atgtaccccc tcgtgacagc aacccaggac gcagatagct 1441 ccagaaaact tgctcatctt cttaatgcgg tcaccgacgc attggtctgg gttattgcaa 1501 agtctggtat aggcggctct ggcggcggct ccggaggctc tgatccaaaa aagaagagaa 1561 aggtagaggc cagcggttcg ggacgggctg acgcattgga cgattttgat ctggatatgc 1621 tgggaagtga cgccctcgat gattttgacc ttgacatgct tggttcggat gcccttgatg 1681 actttgacct cgacatgctc ggcagtgacg cccttgatga tttcgacctg gacatgctga 1741 ttaacactag tggcggctct ggcggcggct ccggaggctc tagccagtac ctgcccgaca 1801 ccgacgacag gcaccggatc gaggaaaagc ggaagcggac ctacgagaca ttcaagagca 1861 tcatgaagaa gtcccccttc agcggcccca ccgaccctag acctccacct agaagaatcg 1921 ccgtgcccag cagatccagc gccagcgtgc caaaacctgc cccccagcct taccccttca 1981 ccagcagcct gagcaccatc aactacgacg agttccctac catggtgttc cccagcggcc 2041 agatctctca ggcctctgct ctggctccag cccctcctca ggtgctgcct caggctcctg 2101 ctcctgcacc agctccagcc atggtgtctg cactggctca ggcaccagca cccgtgcctg 2161 tgctggctcc tggacctcca caggctgtgg ctccaccagc ccctaaacct acacaggccg 2221 gcgagggcac actgtctgaa gctctgctgc agctgcagtt cgacgacgag gatctgggag 2281 ccctgctggg aaacagcacc gatcctgccg tgttcaccga cctggccagc gtggacaaca 2341 gcgagttcca gcagctgctg aaccagggca tccctgtggc ccctcacacc accgagccca 2401 tgctgatgga ataccccgag gccatcaccc ggctcgtgac aggcgctcag aggcctcctg 2461 atccagctcc tgcccctctg ggagcaccag gcctgcctaa tggactgctg tctggcgacg 2521 aggacttcag ctctatcgcc gatatggatt tctcagcctt gctgggctct ggcagcggca 2581 gccgggattc cagggaaggg atgtttttgc cgaagcctga ggccggctcc gctattagtg 2641 acgtgtttga gggccgcgag gtgtgccagc caaaacgaat ccggccattt catcctccag 2701 gaagtccatg ggccaaccgc ccactccccg ccagcctcgc accaacacca accggtccag 2761 tacatgagcc agtcgggtca ctgaccccgg caccagtccc tcagccactg gacccagcgc 2821 ccgcagtgac tcccgaggcc agtcacctgt tggaggaccc cgatgaagag acgagccagg 2881 ctgtcaaagc ccttcgggag atggccgata ctgtgattcc ccagaaggaa gaggctgcaa 2941 tctgtggcca aatggacctt tcccatccgc ccccaagggg ccatctggat gagctgacaa 3001 ccacacttga gtccatgacc gaggatctga acctggactc acccctgacc ccggaattga 3061 acgagattct ggataccttc ctgaacgacg agtgcctctt gcatgccatg catatcagca 3121 caggactgtc catcttcgac acatctctgt ttgacacata caggtatatc taatctagag 3181 ggccctattc tatagtgtca cctaaatgct agagctcgct gatcagcctc gactgtgcct 3241 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt 3301 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg 3361 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac 3421 aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc 3481 tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg 3541 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct 3601 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg 3661 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag 3721 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgcct tttgacgttg 3781 gagtccacgt tctttaatag tggactcttg ttccaaacta aaacaacact caaccctatc 3841 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat 3901 gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttagggt 3961 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt 4021 cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc 4081 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc 4141 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg 4201 ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc 4261 taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct gatcaagaga 4321 caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg 4381 cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg 4441 ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt 4501 ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg 4561 gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat 4621 tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat 4681 ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg 4741 accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg 4801 atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc 4861 tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc 4921 cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg 4981 tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg 5041 gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca 5101 tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac 5161 cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga 5221 aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga 5281 tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa 5341 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg 5401 tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta 5461 gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat 5521 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag 5581 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg 5641 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc 5701 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc 5761 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa 5821 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt 5881 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 5941 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 6001 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 6061 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 6121 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 6181 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 6241 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 6301 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac 6361 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg 6421 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt 6481 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt 6541 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa 6601 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga 6661 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt 6721 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg 6781 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga 6841 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga 6901 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg 6961 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc 7021 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc 7081 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca 7141 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac 7201 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg 7261 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc 7321 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg 7381 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac 7441 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat 7501 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata 7561 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa 7621 agtgccacct gacgt //