LOCUS Exported 5934 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION bacterial expression of his6 and SUMO tagged RRM motif of Xenopus laevis Nup53. ACCESSION . VERSION . KEYWORDS pET28a SUMO Nup53xl 162-267 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5934) AUTHORS Antonin lab TITLE Antonin lab MISTIC plasmid JOURNAL Unpublished REFERENCE 2 (bases 1 to 5934) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5934 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 1261..1452 /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" primer_bind complement(1446..1468) /label=pGEX 3' /note="pGEX vectors, reverse primer" misc_feature 1554..1693 /label=bom /note="basis of mobility region from pBR322" primer_bind 1609..1628 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind complement(1708..1725) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(1879..2467) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(1959..1978) /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind complement(2578..2597) /label=pENTR-R /note="pENTR vectors, reverse primer" CDS 2589..3404 /codon_start=1 /gene="aph(3')-Ia" /product="aminoglycoside phosphotransferase" /label=KanR /note="confers resistance to kanamycin in bacteria or G418 (Geneticin(R)) in eukaryotes" /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" primer_bind complement(2662..2681) /label=Kan-R /note="Kanamycin resistance gene, reverse primer" rep_origin complement(3496..3951) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3633..3654) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 3845..3864 /label=F1ori-R /note="F1 origin, reverse primer" terminator 3988..4035 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" primer_bind 4031..4049 /label=T7 Term /note="T7 terminator, reverse primer" CDS complement(4102..4119) /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS complement(4456..4476) /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" CDS complement(4477..4767) /codon_start=1 /gene="S. cerevisiae SMT3 (truncated)" /product="cleavable ubiquitin-like protein tag" /label=SUMO /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIG" CDS complement(4774..4791) /codon_start=1 /product="thrombin recognition and cleavage site" /label=thrombin site /translation="LVPRGS" CDS complement(4801..4818) /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" RBS 4837..4859 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" protein_bind 4874..4898 /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 complement(4898..4917) /label=T7 /note="T7 promoter, forward primer" promoter complement(4899..4917) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(5001..5020) /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" promoter 5226..5303 /gene="lacI" /label=lacI promoter CDS 5304..452 /codon_start=1 /gene="lacI" /product="lac repressor" /label=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)." /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR ALADSLMQLARQVSRLESGQ" primer_bind complement(5323..5342) /label=LacI-R /note="LacI, reverse primer" ORIGIN 1 agccgatagc ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa caaaccatgc 61 aaatgctgaa tgagggcatc gttcccactg cgatgctggt tgccaacgat cagatggcgc 121 tgggcgcaat gcgcgccatt accgagtccg ggctgcgcgt tggtgcggat atctcggtag 181 tgggatacga cgataccgaa gacagctcat gttatatccc gccgttaacc accatcaaac 241 aggattttcg cctgctgggg caaaccagcg tggaccgctt gctgcaactc tctcagggcc 301 aggcggtgaa gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa accaccctgg 361 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac 421 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt aagttagctc 481 actcattagg caccgggatc tcgaccgatg cccttgagag ccttcaaccc agtcagctcc 541 ttccggtggg cgcggggcat gactatcgtc gccgcactta tgactgtctt ctttatcatg 601 caactcgtag gacaggtgcc ggcagcgctc tgggtcattt tcggcgagga ccgctttcgc 661 tggagcgcga cgatgatcgg cctgtcgctt gcggtattcg gaatcttgca cgccctcgct 721 caagccttcg tcactggtcc cgccaccaaa cgtttcggcg agaagcaggc cattatcgcc 781 ggcatggcgg ccccacgggt gcgcatgatc gtgctcctgt cgttgaggac ccggctaggc 841 tggcggggtt gccttactgg ttagcagaat gaatcaccga tacgcgagcg aacgtgaagc 901 gactgctgct gcaaaacgtc tgcgacctga gcaacaacat gaatggtctt cggtttccgt 961 gtttcgtaaa gtctggaaac gcggaagtca gcgccctgca ccattatgtt ccggatctgc 1021 atcgcaggat gctgctggct accctgtgga acacctacat ctgtattaac gaagcgctgg 1081 cattgaccct gagtgatttt tctctggtcc cgccgcatcc ataccgccag ttgtttaccc 1141 tcacaacgtt ccagtaaccg ggcatgttca tcatcagtaa cccgtatcgt gagcatcctc 1201 tctcgtttca tcggtatcat tacccccatg aacagaaatc ccccttacac ggaggcatca 1261 gtgaccaaac aggaaaaaac cgcccttaac atggcccgct ttatcagaag ccagacatta 1321 acgcttctgg agaaactcaa cgagctggac gcggatgaac aggcagacat ctgtgaatcg 1381 cttcacgacc acgctgatga gctttaccgc agctgcctcg cgcgtttcgg tgatgacggt 1441 gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc 1501 gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc 1561 atgacccagt cacgtagcga tagcggagtg tatactggct taactatgcg gcatcagagc 1621 agattgtact gagagtgcac cattgcggtg tgaaataccg cacagatgcg taaggagaaa 1681 ataccgcatc aggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 1741 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg 1801 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 1861 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg 1921 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc 1981 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 2041 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc 2101 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 2161 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc 2221 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga 2281 gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc 2341 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 2401 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg 2461 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc 2521 acgttaaggg attttggtca tgaacaataa aactgtctgc ttacataaac agtaatacaa 2581 ggggtgttat gagccatatt caacgggaaa cgtcttgctc taggccgcga ttaaattcca 2641 acatggatgc tgatttatat gggtataaat gggctcgcga taatgtcggg caatcaggtg 2701 cgacaatcta tcgattgtat gggaagcccg atgcgccaga gttgtttctg aaacatggca 2761 aaggtagcgt tgccaatgat gttacagatg agatggtcag actaaactgg ctgacggaat 2821 ttatgcctct tccgaccatc aagcatttta tccgtactcc tgatgatgca tggttactca 2881 ccactgcgat ccccgggaaa acagcattcc aggtattaga agaatatcct gattcaggtg 2941 aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta 3001 attgtccttt taacagcgat cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata 3061 acggtttggt tgatgcgagt gattttgatg acgagcgtaa tggctggcct gttgaacaag 3121 tctggaaaga aatgcataaa cttttgccat tctcaccgga ttcagtcgtc actcatggtg 3181 atttctcact tgataacctt atttttgacg aggggaaatt aataggttgt attgatgttg 3241 gacgagtcgg aatcgcagac cgataccagg atcttgccat cctatggaac tgcctcggtg 3301 agttttctcc ttcattacag aaacggcttt ttcaaaaata tggtattgat aatcctgata 3361 tgaataaatt gcagtttcat ttgatgctcg atgagttttt ctaagaatta attcatgagc 3421 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 3481 cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt 3541 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa 3601 aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa 3661 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac 3721 gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga 3781 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa 3841 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc 3901 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc 3961 caatccggat atagttcctc ctttcagcaa aaaacccctc aagacccgtt tagaggcccc 4021 aaggggttat gctagttatt gctcagcggt ggcagcagcc aactcagctt cctttcgggc 4081 tttgttagca gccggatctc agtggtggtg gtggtggtgc tcgagctatc acggggtacg 4141 aatgctttta accggaggtg taaaaacgct gctaacgctc ggggtgctaa ctttttcggt 4201 tgcttccata acgcttttat caatgcacgg tttcacacca atcataatgc tttcaccaaa 4261 aatacgacca tctttgctca gtgctttacg tgcctgcagt ttgctctgat actgaatatg 4321 catccaattg ccattattgc tcatcacatg tttaataata ttgccatact gtgcaaactg 4381 cagcagaata tagcttgcgc ttgcctgcgg aaaaccaaaa acggtaaccc aggtatcatc 4441 cagctgatcc atatggccct gaaaataaag attctcacca atctgttctc tgtgagcctc 4501 aataatatcg ttatcctcca tgtccaaatc ttcaggggtc tgatcagctt gaattctaat 4561 accgtcgtac aagaatctta aggagtccat ttccttaccc tgtcttttag cgaacgcttc 4621 catcagcctt cttaaaggag tggtcttttt gatcttgaag aagatctctg aagatccatc 4681 ggacaccttt aaattgatgt gagtctcagg cttgacttct ggcttgacct ctggcttagc 4741 ttcttgattg acttctgagt ccgacatatt atggctgccg cgcggcacca ggccgctgct 4801 gtgatgatga tgatgatggc tgctgcccat ggtatatctc cttcttaaag ttaaacaaaa 4861 ttatttctag aggggaattg ttatccgctc acaattcccc tatagtgagt cgtattaatt 4921 tcgcgggatc gagatctcga tcctctacgc cggacgcatc gtggccggca tcaccggcgc 4981 cacaggtgcg gttgctggcg cctatatcgc cgacatcacc gatggggaag atcgggctcg 5041 ccacttcggg ctcatgagcg cttgtttcgg cgtgggtatg gtggcaggcc ccgtggccgg 5101 gggactgttg ggcgccatct ccttgcatgc accattcctt gcggcggcgg tgctcaacgg 5161 cctcaaccta ctactgggct gcttcctaat gcaggagtcg cataagggag agcgtcgaga 5221 tcccggacac catcgaatgg cgcaaaacct ttcgcggtat ggcatgatag cgcccggaag 5281 agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg 5341 ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca ggccagccac gtttctgcga 5401 aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg 5461 cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc agtctggccc 5521 tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca 5581 gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca 5641 atcttctcgc gcaacgcgtc agtgggctga tcattaacta tccgctggat gaccaggatg 5701 ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt atttcttgat gtctctgacc 5761 agacacccat caacagtatt attttctccc atgaagacgg tacgcgactg ggcgtggagc 5821 atctggtcgc attgggtcac cagcaaatcg cgctgttagc gggcccatta agttctgtct 5881 cggcgcgtct gcgtctggct ggctggcata aatatctcac tcgcaatcaa attc //