LOCUS Exported 6250 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Bacterial expression for structure determination; may not be full ORF. ACCESSION . VERSION . KEYWORDS 5UBB (LOC149281) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6250) TITLE SGC Toronto Q4 2016, Q1 Q2 2017 REFERENCE 2 (bases 1 to 6250) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6250 /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" primer_bind 1942..1961 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 2195..2212 /label=L4440 /note="L4440 vector, forward primer" misc_feature 2227..2366 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(2292..2311) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 2452..2474 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(2468..2659) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(3468..4550) /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 4512..4531 /label=LacI-R /note="LacI, reverse primer" promoter complement(4551..4628) /gene="lacI" /label=lacI promoter primer_bind 4834..4853 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 4937..4956 /label=T7 /note="T7 promoter, forward primer" promoter 4937..4955 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 4956..4980 /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 4995..5017 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 5033..5056 /codon_start=1 /product="8xHis affinity tag" /label=8xHis /translation="HHHHHHHH" CDS 5066..5083 /codon_start=1 /product="thrombin recognition and cleavage site" /label=thrombin site /translation="LVPRGS" CDS 5084..5374 /codon_start=1 /gene="S. cerevisiae SMT3 (truncated)" /product="cleavable ubiquitin-like protein tag" /label=SUMO /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIG" CDS 5381..5401 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" CDS 6094..6111 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(6164..6182) /label=T7 Term /note="T7 terminator, reverse primer" terminator 6178..6225 /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 aggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga 1921 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg 1981 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa 2041 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat 2101 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc 2161 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga 2221 agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg 2281 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat 2341 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct 2401 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 2461 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct 2521 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt 2581 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg 2641 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa 2701 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc 2761 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa 2821 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta 2881 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg 2941 tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag 3001 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac 3061 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca 3121 cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg 3181 gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc 3241 cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg 3301 gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca 3361 tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag 3421 atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt 3481 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag 3541 gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc 3601 tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc 3661 cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct 3721 tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta 3781 atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg 3841 atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct 3901 tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga 3961 cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc 4021 aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg 4081 ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct 4141 tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt 4201 tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc 4261 gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc 4321 gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc 4381 gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact 4441 ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga 4501 taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc 4561 ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg 4621 atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag 4681 tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc 4741 gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat 4801 gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc 4861 aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat 4921 ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc 4981 cctctagaaa taattttgtt taactttaag aaggagatat accatgaaaa aacatcacca 5041 tcatcatcat catcacagca gcggcctggt tccgcgtggt agtatgtcgg actcagaagt 5101 caatcaagaa gctaagccag aggtcaagcc agaagtcaag cctgagactc acatcaattt 5161 aaaggtgtcc gatggatctt cagagatctt cttcaagatc aaaaagacca ctcctttaag 5221 aaggctgatg gaagcgttcg ctaaaagaca gggtaaggaa atggactcct taagattctt 5281 gtacgacggt attagaatcc aagctgatca gacccctgaa gatttggaca tggaggataa 5341 cgatattatt gaggctcaca gagaacagat tggtggaaca gaaaacttgt atttccaggg 5401 cacgtcccag gttattaacg gtgaaatgca gttctacgcg cgggccaagc ttttttatca 5461 agaggtccca gcaactgaag aagggatgat gggaaatttc atagagctgt cgagccctga 5521 cattcaggct tctcaaaaat ttctgcgcaa attcgtgggc ggtcccggga gagcgggaac 5581 agattgtgcc ttagactgcg gcagtggtat cggccgagta tcaaagcatg ttttgcttcc 5641 ggtctttaac tccgtagaac tcgtggatat gatggagtcg tttttattgg aagcacagaa 5701 ttaccttcaa gttaaaggtg ataaagtcga aagctatcac tgttactctc tccaggagtt 5761 caccccacct tttcggcgtt atgacgtgat atggattcag tgggtaagtg ggcatttaac 5821 ggataaggac ctgttggctt tcctttcacg ctgcagagat ggactgaaag aaaacggcat 5881 cattattctg aaagacaatg tggcgcgaga gggttgtatc ctcgatttat ccgattcgag 5941 cgttactcgt gacatggata tactgcgctc tttgatccgg aagagtggcc ttgtcgttct 6001 gggtcaagaa aaacaggacg ggtttccgga acaatgcatt cccgtatgga tgttcgccct 6061 gcactgatga cgaagcttgc ggccgcactc gagcaccacc accaccacca ctgagatccg 6121 gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta 6181 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact 6241 atatccggat //