LOCUS Exported 6900 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION bacterial expression of USPL1 catalytic domain (aa 212-514) as His-GST-TEVsite fusion. ACCESSION . VERSION . KEYWORDS USPL1cat-pETM30 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6900) AUTHORS Schulz S, Chachami G, Kozaczkiewicz L, Winter U, Stankovic-Valentin N, Haas P, Hofmann K, Urlaub H, Ovaa H, Wittbrodt J, Meulmeester E, Melchior F TITLE Ubiquitin-specific protease-like 1 (USPL1) is a SUMO isopeptidase with essential, non-catalytic functions. JOURNAL EMBO Rep. 2012 Oct;13(10):930-8. doi: 10.1038/embor.2012.125. Epub 2012 Aug 10. PUBMED 22878415 REFERENCE 2 (bases 1 to 6900) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6900 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin 12..467 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(99..118) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 309..330 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(559..1374) /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 1282..1301 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 1366..1385 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 1496..2084 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 1985..2004 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 2238..2255 /label=L4440 /note="L4440 vector, forward primer" misc_feature 2270..2409 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(2335..2354) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 2495..2517 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(2511..2702) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(3511..4593) /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 4555..4574 /label=LacI-R /note="LacI, reverse primer" promoter complement(4594..4671) /gene="lacI" /label=lacI promoter primer_bind 4877..4896 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 4980..4999 /label=T7 /note="T7 promoter, forward primer" promoter 4980..4998 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 4999..5023 /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)." RBS 5038..5060 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 5073..5090 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 5109..5759 /codon_start=1 /product="glutathione S-transferase from Schistosoma japonicum" /label=GST /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPP" primer_bind 5720..5742 /label=pGEX 5' /note="pGEX vectors, Glutathione-S-transferase, forward primer" CDS 5796..5816 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" CDS 6744..6761 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(6814..6832) /label=T7 Term /note="T7 terminator, reverse primer" terminator 6828..6875 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" ORIGIN 1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 61 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 241 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 301 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 361 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 421 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt 481 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat 541 ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt 601 catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa 661 ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg 721 tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa 781 atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca 841 gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc 901 gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca 961 attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt 1021 ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt 1081 ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat 1141 aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc 1201 tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt 1261 cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat 1321 gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc 1381 ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac 1441 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag 1501 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg 1561 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca 1621 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga 1681 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca 1741 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc 1801 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca 1861 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa 1921 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc 1981 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc 2041 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg 2101 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat 2161 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca 2221 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt 2281 attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat 2341 ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc 2401 atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc 2461 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt 2521 tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga 2581 agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc 2641 gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc 2701 actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga 2761 gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt 2821 gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa 2881 tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg 2941 atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa 3001 acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag 3061 tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc 3121 cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg 3181 ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct 3241 tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc 3301 cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt 3361 tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg 3421 aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga 3481 gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg 3541 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg 3601 cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc 3661 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc 3721 ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc 3781 cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc 3841 cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat 3901 ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg 3961 ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac 4021 agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc 4081 cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc 4141 agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc 4201 ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg 4261 caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc 4321 acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc 4381 cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac 4441 gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc 4501 agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata 4561 ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc 4621 cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc 4681 gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt 4741 tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg 4801 ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag 4861 cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc 4921 cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt 4981 aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag aaataatttt 5041 gtttaacttt aagaaggaga tataccatga aacatcacca tcaccatcac aacactagta 5101 gcaattccat gtcccctata ctaggttatt ggaaaattaa gggccttgtg caacccactc 5161 gacttctttt ggaatatctt gaagaaaaat atgaagagca tttgtatgag cgcgatgaag 5221 gtgataaatg gcgaaacaaa aagtttgaat tgggtttgga gtttcccaat cttccttatt 5281 atattgatgg tgatgttaaa ttaacacagt ctatggccat catacgttat atagctgaca 5341 agcacaacat gttgggtggt tgtccaaaag agcgtgcaga gatttcaatg cttgaaggag 5401 cggttttgga tattagatac ggtgtttcga gaattgcata tagtaaagac tttgaaactc 5461 tcaaagttga ttttcttagc aagctacctg aaatgctgaa aatgttcgaa gatcgtttat 5521 gtcataaaac atatttaaat ggtgatcatg taacccatcc tgacttcatg ttgtatgacg 5581 ctcttgatgt tgttttatac atggacccaa tgtgcctgga tgcgttccca aaattagttt 5641 gttttaaaaa acgtattgaa gctatcccac aaattgataa gtacttgaaa tccagcaagt 5701 atatagcatg gcctttgcag ggctggcaag ccacgtttgg tggtggcgac catcctccaa 5761 ctagtggatc tggtggtggt ggcggatgga tgagcgagaa tctttatttt cagggcgcca 5821 tggggatgcc actggagagc aaatgtacat catttcccca ggctttatgt gtccagtgga 5881 aaaatgctta tgctctctgt tggttagact gtatcctgtc agctttggtg cactcggaag 5941 agttaaagaa caccgtgact ggactgtgct cgaaggagga atctatattc tggcggttgc 6001 ttacaaaata taatcaagca aatacacttc tatataccag tcaattgagt ggtgttaaag 6061 atggagattg taaaaaactt acctcagaaa tatttgcaga gatagagacc tgtctgaatg 6121 aagttagaga tgaaattttt attagccttc agccccagct tagatgcaca ttaggtgata 6181 tggaaagccc tgtgtttgca tttcccctgc tcttaaaact agaaacccac attgaaaagc 6241 tcttcctata ttctttttct tgggactttg aatgttcgca gtgtggacac caatatcaaa 6301 acaggcatat gaagagtctg gtcaccttta caaatgtcat ccctgagtgg cacccactta 6361 atgctgccca ttttggtcca tgtaacaatt gcaacagtaa atcacaaata agaaaaatgg 6421 tattagaaaa agtatctccc atattcatgt tgcactttgt agaaggctta ccacagaatg 6481 acttgcagca ctatgcattt cattttgaag gctgtcttta tcagataact tctgtaattc 6541 agtatcgagc aaataatcat tttataacat ggattttaga tgctgatgga agttggctgg 6601 aatgtgatga cttaaaaggc ccatgttctg aaaggcacaa gaaatttgaa gttcctgctt 6661 cagagataca tattgttatt tgggaaagaa aaatatccca agtgacagat aaagaagctg 6721 cctgccttcc actttgactc gagcaccacc accaccacca ctgagatccg gctgctaaca 6781 aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc 6841 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat //