LOCUS Exported 7155 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION MAC-tagged gene expression. ACCESSION . VERSION . KEYWORDS MAC-SARS-CoV-2 Orf3a SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7155) 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 7155) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7155 /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" protein_bind 586..604 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 607..625 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" primer_bind 635..659 /label=LNCX /note="Human CMV promoter, forward primer" protein_bind 802..826 /gene="mutant version of attB" /label=attB1 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" protein_bind complement(1658..1682) /gene="mutant version of attB" /label=attB2 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" CDS 1729..1752 /codon_start=1 /product="peptide that binds Strep-Tactin(R), an engineered form of streptavidin" /label=Strep-Tag II /translation="WSHPQFEK" CDS 1789..1812 /codon_start=1 /product="peptide that binds Strep-Tactin(R), an engineered form of streptavidin" /label=Strep-Tag II /translation="WSHPQFEK" CDS 1843..1869 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 1870..2829 /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(2873..2890) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2879..3103 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(3236..3255) /label=F1ori-R /note="F1 origin, reverse primer" protein_bind 3387..3434 /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 3442..4464 /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 4594..4715 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4631..4650) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4685..4704 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(4764..4780) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4764..4780) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4777..4799) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4788..4804 /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(4812..4842) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4857..4878 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4995..5012) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5166..5754) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5246..5265) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5925..6785) /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 6548..6567 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6786..6890) /gene="bla" /label=AmpR promoter primer_bind complement(6965..6984) /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 agctctccct atcagtgata 601 gagatctccc tatcagtgat agagatcgtc gacgagctcg tttagtgaac cgtcagatcg 661 cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc 721 tccggactct agcgtttaaa cttaagcttg gtaccgagct cggatccact agtccagtgt 781 ggtggaattc tgcagatatc aacaagtttg tacaaaaaag caggctccac catggacctt 841 tttatgagaa tctttaccat cgggactgtc acactgaaac agggagaaat taaggacgcg 901 accccgagtg actttgtgag ggccactgct acaattccca tccaagcctc tcttccattc 961 ggctggctca tagtgggtgt ggcactgctc gcggtattcc agtcagcgtc taagatcata 1021 acgctgaaga agagatggca gttggctctc agcaaaggtg ttcatttcgt ctgtaacctg 1081 cttcttcttt tcgtcactgt ctattctcac ttgctcttgg ttgcagccgg gctggaagcc 1141 ccgtttctgt atctgtacgc gctcgtttac ttcctccaga gcataaattt cgtacgaatt 1201 attatgagac tctggttgtg ctggaagtgt cggagcaaga atcctctctt gtatgacgcc 1261 aattacttcc tttgttggca cactaattgt tatgattact gcatacctta caatagcgtt 1321 acctcatcca tagtaatcac ctccggtgac ggaacaacgt caccaatctc cgagcatgat 1381 taccagattg gaggctatac ggaaaaatgg gagagtggtg tcaaggactg tgtggtgctg 1441 catagctatt tcacaagcga ttattatcaa ctgtacagta cacagttgtc cacggatacc 1501 ggggtcgagc atgtcacatt ttttatttat aacaagatcg ttgatgagcc agaggagcat 1561 gtgcagatcc acacgataga tggaagctcc ggtgtagtga atccagtcat ggaaccgatc 1621 tatgacgagc ccaccacgac cacatctgtg cccctggacc cagctttctt gtacaaagtg 1681 gttgatatca aggccgccga catcaccagc ctgtacaaga aagtggggtg gagccatccc 1741 cagttcgaga agggcggagg atctggcggc ggaagcggcg gaggaagctg gtcccaccct 1801 cagtttgaga aaggcaccga gctgggcagc accatggcca gctaccccta cgacgtgccc 1861 gactacgcca aggacaacac cgtgcccctg aagctgatcg ccctgctggc caacggcgag 1921 ttccactctg gcgagcagct gggcgagaca ctgggcatga gcagagccgc catcaacaag 1981 cacatccaga ccctgcggga ctggggcgtg gacgtgttca cagtgcccgg caagggctac 2041 agcctgcccg agcccatcca gctgctgaac gccaagcaga tcctgggcca gctggatggc 2101 ggatctgtgg ccgtgctgcc cgtgatcgac agcaccaacc agtatctgct cgaccggatc 2161 ggcgagctga agtccggcga cgcctgtatc gccgagtacc agcaggccgg cagaggcggc 2221 aggggcagaa agtggttcag ccccttcggc gccaacctgt acctgagcat gttctggaga 2281 ctggaacagg gcccagccgc cgctattggc ctgagcctgg tcatcggcat cgtgatggcc 2341 gaggtgctgc ggaagctggg cgccgacaaa gtgcgcgtga agtggcccaa cgacctgtat 2401 ctgcaggacc ggaagctggc cggcatcctg gtggaactga ccggcaaaac aggcgacgcc 2461 gcccagatcg tgatcggcgc tggcatcaac atggccatgc ggcgggtgga agagagcgtg 2521 gtcaaccagg gctggatcac cctgcaggaa gccggcatca acctggaccg gaacaccctg 2581 gccgccatgc tgatcagaga gctgagagcc gccctggaac tgttcgagca ggaaggcctg 2641 gccccctacc tgagcagatg ggagaagctg gacaacttca tcaaccggcc cgtgaaactg 2701 atcatcggcg acaaagagat cttcggcatc agccggggca tcgacaagca gggggccctg 2761 ctcctggaac aggacggcat catcaagccc tggatgggcg gcgagatcag cctgcggagc 2821 gccgagaagt gatgactcga gtctagaggg cccgtttaaa cccgctgatc agcctcgact 2881 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg 2941 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg 3001 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg 3061 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga 3121 accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg 3181 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct 3241 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat 3301 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt 3361 gattagggtg atggttcacg tacctagaag ttcctattcc gaagttccta ttctctagaa 3421 agtataggaa cttccttggc caaaaagcct gaactcaccg cgacgtctgt cgagaagttt 3481 ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc tctcggaggg cgaagaatct 3541 cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc 3601 gatggtttct acaaagatcg ttatgtttat cggcactttg catcggccgc gctcccgatt 3661 ccggaagtgc ttgacattgg ggaattcagc gagagcctga cctattgcat ctcccgccgt 3721 gcacagggtg tcacgttgca agacctgcct gaaaccgaac tgcccgctgt tctgcagccg 3781 gtcgcggagg ccatggatgc gatcgctgcg gccgatctta gccagacgag cgggttcggc 3841 ccattcggac cgcaaggaat cggtcaatac actacatggc gtgatttcat atgcgcgatt 3901 gctgatcccc atgtgtatca ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc 3961 gcgcaggctc tcgatgagct gatgctttgg gccgaggact gccccgaagt ccggcacctc 4021 gtgcacgcgg atttcggctc caacaatgtc ctgacggaca atggccgcat aacagcggtc 4081 attgactgga gcgaggcgat gttcggggat tcccaatacg aggtcgccaa catcttcttc 4141 tggaggccgt ggttggcttg tatggagcag cagacgcgct acttcgagcg gaggcatccg 4201 gagcttgcag gatcgccgcg gctccgggcg tatatgctcc gcattggtct tgaccaactc 4261 tatcagagct tggttgacgg caatttcgat gatgcagctt gggcgcaggg tcgatgcgac 4321 gcaatcgtcc gatccggagc cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg 4381 gccgtctgga ccgatggctg tgtagaagta ctcgccgata gtggaaaccg acgccccagc 4441 actcgtccga gggcaaagga atagcacgta ctacgagatt tcgattccac cgccgccttc 4501 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc 4561 ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt 4621 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct 4681 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct 4741 agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc 4801 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga 4861 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg 4921 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg 4981 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 5041 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 5101 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 5161 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 5221 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 5281 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 5341 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 5401 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 5461 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 5521 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 5581 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca 5641 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 5701 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 5761 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 5821 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 5881 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 5941 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc 6001 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata 6061 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg 6121 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc 6181 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct 6241 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa 6301 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt 6361 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca 6421 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac 6481 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca 6541 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt 6601 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc 6661 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca 6721 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 6781 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc 6841 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 6901 cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta tggtgcactc 6961 tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt 7021 tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg 7081 acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg 7141 ccagatatac gcgtt //