LOCUS Exported 6253 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Expresses FLAG-OTUB1 T134R mutant in mammalian cells. ACCESSION . VERSION . KEYWORDS FLAG-OTUB1 T134R-pcDNA3.1 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6253) AUTHORS Pasupala N, Morrow ME, Que LT, Malynn BA, Ma A, Wolberger C TITLE OTUB1 non-catalytically stabilizes the E2 ubiquitin-conjugating enzyme UBE2E1 by preventing its autoubiquitination. JOURNAL J Biol Chem. 2018 Nov 23;293(47):18285-18295. doi: 10.1074/jbc.RA118.004677. Epub 2018 Oct 3. PUBMED 30282802 REFERENCE 2 (bases 1 to 6253) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6253 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 1..380 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 381..584 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 535..555 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 629..648 /label=T7 /note="T7 promoter, forward primer" promoter 629..647 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 686..751 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" primer_bind complement(1610..1627) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 1616..1840 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 1886..2314 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(1973..1992) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2183..2204 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(2323..2343) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 2328..2657 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 2508..2643 /label=SV40 ori /note="SV40 origin of replication" primer_bind 2570..2589 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 2724..3518 /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(2778..2797) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 3388..3407 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 3692..3813 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3729..3748) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 3783..3802 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(3862..3878) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3862..3878) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(3875..3897) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 3886..3902 /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(3910..3940) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 3955..3976 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4093..4110) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4264..4852) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4344..4363) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5023..5883) /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 5646..5665 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5884..5988) /gene="bla" /label=AmpR promoter primer_bind complement(6063..6082) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc 421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa 541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag 601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg 661 gctagcgttt aaacttaagc ttatggacta caaagaccat gacggtgatt ataaagatca 721 tgacatcgat tacaaggatg acgatgacaa gaattcaatg gcggcggagg aacctcagca 781 gcagaagcag gagccgctgg gcagcgactc cgaaggtgtt aactgtctgg cctatgatga 841 agccatcatg gctcagcagg accgaattca gcaagagatt gctgtgcaga accctctggt 901 gtcagagcgg ctggagctct cggtcctata caaggagtat gctgaagatg acaacatcta 961 tcaacagaag atcaaggacc tccacaaaaa gtactcgtac atccgcaaga ccaggcctga 1021 cggcaactgt ttctatcggg ctttcggatt ctcccacttg gaggcactgc tggatgacag 1081 caaggagttg cagcggttca aggctgtgtc tgccaagagc aaggaagacc tggtgtccca 1141 gggcttcact gaattcagaa ttgaggattt ccacaacacg ttcatggacc tgattgagca 1201 ggtggagaag cagacctctg tcgccgacct gctggcctcc ttcaatgacc agagcacctc 1261 cgactacctt gtggtctacc tgcggctgct cacctcgggc tacctgcagc gcgagagcaa 1321 gttcttcgag cacttcatcg agggtggacg gactgtcaag gagttctgcc agcaggaggt 1381 ggagcccatg tgcaaggaga gcgaccacat ccacatcatt gcgctggccc aggccctcag 1441 cgtgtccatc caggtggagt acatggaccg cggcgagggc ggcaccacca atccgcacat 1501 cttccctgag ggctccgagc ccaaggtcta ccttctctac cggcctggac actacgatat 1561 cctctacaaa tagtcgactc tagagggccc gtttaaaccc gctgatcagc ctcgactgtg 1621 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa 1681 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt 1741 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa 1801 gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc 1861 agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt 1921 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc 1981 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg 2041 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat 2101 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg 2161 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct 2221 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa 2281 aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag 2341 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt 2401 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca 2461 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa 2521 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag 2581 aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag 2641 gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcaag 2701 agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg 2761 ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg 2821 atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc 2881 tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga 2941 cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc 3001 tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag 3061 tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat 3121 tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg 3181 tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca 3241 ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct 3301 tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg 3361 gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg 3421 gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc 3481 gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat 3541 gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta 3601 tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg 3661 ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta 3721 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag 3781 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag 3841 ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac 3901 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt 3961 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc 4021 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg 4081 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt 4141 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa 4201 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc 4261 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag 4321 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt 4381 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg 4441 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg 4501 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg 4561 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac 4621 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg 4681 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt 4741 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg 4801 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc 4861 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt 4921 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt 4981 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag 5041 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt 5101 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc 5161 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc 5221 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg 5281 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac 5341 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg 5401 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc 5461 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact 5521 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc 5581 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat 5641 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc 5701 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac 5761 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa 5821 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact 5881 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg 5941 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg 6001 aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg gtgcactctc 6061 agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg 6121 gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac 6181 aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc 6241 agatatacgc gtt //