LOCUS Exported 6513 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION MAC-tagged gene expression. ACCESSION . VERSION . KEYWORDS MAC-SARS-CoV-2 Orf6 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6513) AUTHORS Liu X, Salokas K, Weldatsadik RG, Gawriyski L, Varjosalo M TITLE Combined proximity labeling and affinity purification-mass spectrometry workflow for mapping and visualizing protein interaction networks. JOURNAL Nat Protoc. 2020 Aug 10. pii: 10.1038/s41596-020-0365-x. doi: 10.1038/s41596-020-0365-x. PUBMED 32778839 REFERENCE 2 (bases 1 to 6513) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6513 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 1..204 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 155..175 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" protein_bind 206..224 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 227..245 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" primer_bind 255..279 /label=LNCX /note="Human CMV promoter, forward primer" protein_bind 422..446 /gene="mutant version of attB" /label=attB1 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" protein_bind complement(636..660) /gene="mutant version of attB" /label=attB2 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" CDS 707..730 /codon_start=1 /product="peptide that binds Strep-Tactin(R), an engineered form of streptavidin" /label=Strep-Tag II /translation="WSHPQFEK" CDS 767..790 /codon_start=1 /product="peptide that binds Strep-Tactin(R), an engineered form of streptavidin" /label=Strep-Tag II /translation="WSHPQFEK" CDS 821..847 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 848..1807 /codon_start=1 /product="promiscuous R118G mutant of the E. coli biotin protein ligase (Cronan, 2005)" /label=BirA(R118G) /note="also known as BirA* or BioID" /translation="KDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDW GVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSGD ACIAEYQQAGRGGRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRKL GADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQGW ITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIGD KEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK" primer_bind complement(1851..1868) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 1857..2081 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(2214..2233) /label=F1ori-R /note="F1 origin, reverse primer" protein_bind 2365..2412 /label=FRT /bound_moiety="FLP recombinase from the Saccharomyces cerevisiae 2u plasmid" /note="FLP-mediated recombination occurs in the 8-bp core sequence TCTAGAAA (Turan and Bode, 2011)." CDS 2420..3442 /codon_start=1 /gene="aph(4)-Ia" /product="aminoglycoside phosphotransferase from E. coli" /label=HygR /note="confers resistance to hygromycin" /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE " polyA_signal 3572..3693 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3609..3628) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 3663..3682 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(3742..3758) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3742..3758) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(3755..3777) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 3766..3782 /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(3790..3820) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 3835..3856 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(3973..3990) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4144..4732) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4224..4243) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(4903..5763) /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 5526..5545 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5764..5868) /gene="bla" /label=AmpR promoter primer_bind complement(5943..5962) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 6134..6513 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" ORIGIN 1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt 61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac 121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg 181 tgggaggtct atataagcag agctctccct atcagtgata gagatctccc tatcagtgat 241 agagatcgtc gacgagctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc 301 tgttttgacc tccatagaag acaccgggac cgatccagcc tccggactct agcgtttaaa 361 cttaagcttg gtaccgagct cggatccact agtccagtgt ggtggaattc tgcagatatc 421 aacaagtttg tacaaaaaag caggctccac catgtttcat cttgtcgatt ttcaagttac 481 gattgcggag atactcctca taatcatgcg gacatttaag gtttccattt ggaaccttga 541 ttacattatt aacctgatta tcaagaatct ctccaagagc ctgaccgaga ataagtattc 601 tcagttggac gaggagcagc ctatggaaat agacgaccca gctttcttgt acaaagtggt 661 tgatatcaag gccgccgaca tcaccagcct gtacaagaaa gtggggtgga gccatcccca 721 gttcgagaag ggcggaggat ctggcggcgg aagcggcgga ggaagctggt cccaccctca 781 gtttgagaaa ggcaccgagc tgggcagcac catggccagc tacccctacg acgtgcccga 841 ctacgccaag gacaacaccg tgcccctgaa gctgatcgcc ctgctggcca acggcgagtt 901 ccactctggc gagcagctgg gcgagacact gggcatgagc agagccgcca tcaacaagca 961 catccagacc ctgcgggact ggggcgtgga cgtgttcaca gtgcccggca agggctacag 1021 cctgcccgag cccatccagc tgctgaacgc caagcagatc ctgggccagc tggatggcgg 1081 atctgtggcc gtgctgcccg tgatcgacag caccaaccag tatctgctcg accggatcgg 1141 cgagctgaag tccggcgacg cctgtatcgc cgagtaccag caggccggca gaggcggcag 1201 gggcagaaag tggttcagcc ccttcggcgc caacctgtac ctgagcatgt tctggagact 1261 ggaacagggc ccagccgccg ctattggcct gagcctggtc atcggcatcg tgatggccga 1321 ggtgctgcgg aagctgggcg ccgacaaagt gcgcgtgaag tggcccaacg acctgtatct 1381 gcaggaccgg aagctggccg gcatcctggt ggaactgacc ggcaaaacag gcgacgccgc 1441 ccagatcgtg atcggcgctg gcatcaacat ggccatgcgg cgggtggaag agagcgtggt 1501 caaccagggc tggatcaccc tgcaggaagc cggcatcaac ctggaccgga acaccctggc 1561 cgccatgctg atcagagagc tgagagccgc cctggaactg ttcgagcagg aaggcctggc 1621 cccctacctg agcagatggg agaagctgga caacttcatc aaccggcccg tgaaactgat 1681 catcggcgac aaagagatct tcggcatcag ccggggcatc gacaagcagg gggccctgct 1741 cctggaacag gacggcatca tcaagccctg gatgggcggc gagatcagcc tgcggagcgc 1801 cgagaagtga tgactcgagt ctagagggcc cgtttaaacc cgctgatcag cctcgactgt 1861 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga 1921 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag 1981 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga 2041 agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac 2101 cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg 2161 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt 2221 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg 2281 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga 2341 ttagggtgat ggttcacgta cctagaagtt cctattccga agttcctatt ctctagaaag 2401 tataggaact tccttggcca aaaagcctga actcaccgcg acgtctgtcg agaagtttct 2461 gatcgaaaag ttcgacagcg tctccgacct gatgcagctc tcggagggcg aagaatctcg 2521 tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata gctgcgccga 2581 tggtttctac aaagatcgtt atgtttatcg gcactttgca tcggccgcgc tcccgattcc 2641 ggaagtgctt gacattgggg aattcagcga gagcctgacc tattgcatct cccgccgtgc 2701 acagggtgtc acgttgcaag acctgcctga aaccgaactg cccgctgttc tgcagccggt 2761 cgcggaggcc atggatgcga tcgctgcggc cgatcttagc cagacgagcg ggttcggccc 2821 attcggaccg caaggaatcg gtcaatacac tacatggcgt gatttcatat gcgcgattgc 2881 tgatccccat gtgtatcact ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc 2941 gcaggctctc gatgagctga tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt 3001 gcacgcggat ttcggctcca acaatgtcct gacggacaat ggccgcataa cagcggtcat 3061 tgactggagc gaggcgatgt tcggggattc ccaatacgag gtcgccaaca tcttcttctg 3121 gaggccgtgg ttggcttgta tggagcagca gacgcgctac ttcgagcgga ggcatccgga 3181 gcttgcagga tcgccgcggc tccgggcgta tatgctccgc attggtcttg accaactcta 3241 tcagagcttg gttgacggca atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc 3301 aatcgtccga tccggagccg ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc 3361 cgtctggacc gatggctgtg tagaagtact cgccgatagt ggaaaccgac gccccagcac 3421 tcgtccgagg gcaaaggaat agcacgtact acgagatttc gattccaccg ccgccttcta 3481 tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg 3541 ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta 3601 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag 3661 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag 3721 ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac 3781 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt 3841 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc 3901 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg 3961 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt 4021 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa 4081 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc 4141 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag 4201 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt 4261 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg 4321 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg 4381 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg 4441 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac 4501 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg 4561 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt 4621 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg 4681 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc 4741 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt 4801 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt 4861 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag 4921 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt 4981 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc 5041 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc 5101 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg 5161 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac 5221 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg 5281 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc 5341 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact 5401 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc 5461 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat 5521 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc 5581 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac 5641 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa 5701 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact 5761 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg 5821 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg 5881 aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg gtgcactctc 5941 agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg 6001 gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac 6061 aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc 6121 agatatacgc gttgacattg attattgact agttattaat agtaatcaat tacggggtca 6181 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct 6241 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta 6301 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac 6361 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt 6421 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag 6481 tacatctacg tattagtcat cgctattacc atg //