LOCUS Exported 6750 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION MAC-tagged gene expression. ACCESSION . VERSION . KEYWORDS MAC-SARS-CoV-2 NSP10 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6750) 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 6750) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6750 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(31..48) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(202..790) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(282..301) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(961..1821) /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 1584..1603 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(1822..1926) /gene="bla" /label=AmpR promoter primer_bind complement(2001..2020) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 2192..2571 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 2572..2775 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 2726..2746 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" protein_bind 2777..2795 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 2798..2816 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" primer_bind 2826..2850 /label=LNCX /note="Human CMV promoter, forward primer" protein_bind 2993..3017 /gene="mutant version of attB" /label=attB1 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" protein_bind complement(3444..3468) /gene="mutant version of attB" /label=attB2 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" CDS 3515..3538 /codon_start=1 /product="peptide that binds Strep-Tactin(R), an engineered form of streptavidin" /label=Strep-Tag II /translation="WSHPQFEK" CDS 3575..3598 /codon_start=1 /product="peptide that binds Strep-Tactin(R), an engineered form of streptavidin" /label=Strep-Tag II /translation="WSHPQFEK" CDS 3629..3655 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 3656..4615 /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(4659..4676) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 4665..4889 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(5022..5041) /label=F1ori-R /note="F1 origin, reverse primer" protein_bind 5173..5220 /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 5228..6250 /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 6380..6501 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(6417..6436) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 6471..6490 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(6550..6566) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(6550..6566) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(6563..6585) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 6574..6590 /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(6598..6628) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6643..6664 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." ORIGIN 1 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt 61 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc 121 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa 181 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa 241 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc 301 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc 361 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag 421 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga 481 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc 541 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac 601 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg 661 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca 721 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa 781 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa 841 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt 901 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag 961 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat 1021 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc 1081 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa 1141 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca 1201 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa 1261 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt 1321 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc 1381 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact 1441 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc 1501 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg 1561 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct 1621 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc 1681 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag 1741 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac 1801 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg 1861 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt 1921 tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat 1981 cccctatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg 2041 ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca 2101 aggcaaggct tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg 2161 cttcgcgatg tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag 2221 taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt 2281 acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg 2341 acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat 2401 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct 2461 attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg 2521 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg 2581 ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc 2641 caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa 2701 tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc 2761 tatataagca gagctctccc tatcagtgat agagatctcc ctatcagtga tagagatcgt 2821 cgacgagctc gtttagtgaa ccgtcagatc gcctggagac gccatccacg ctgttttgac 2881 ctccatagaa gacaccggga ccgatccagc ctccggactc tagcgtttaa acttaagctt 2941 ggtaccgagc tcggatccac tagtccagtg tggtggaatt ctgcagatat caacaagttt 3001 gtacaaaaaa gcaggctcca ccatggctgg taatgcaact gaggttcccg cgaatagtac 3061 ggttctgagc ttttgcgcct ttgctgttga cgcagctaag gcatataagg actacttggc 3121 ctccgggggc cagccaatca caaactgcgt caagatgttg tgtacgcaca cgggcacagg 3181 tcaggcaatc actgtcacac ctgaagctaa catggatcag gaatcttttg gtggtgccag 3241 ttgttgtctg tactgtcgat gtcatataga tcatcccaac ccgaaagggt tttgtgatct 3301 gaaaggaaaa tacgtccaaa tcccgaccac gtgcgcgaac gacccggttg ggtttacgtt 3361 gaaaaatacc gtatgtactg tctgtggtat gtggaagggc tatggctgtt catgcgatca 3421 attgcgagag ccgatgttgc aggacccagc tttcttgtac aaagtggttg atatcaaggc 3481 cgccgacatc accagcctgt acaagaaagt ggggtggagc catccccagt tcgagaaggg 3541 cggaggatct ggcggcggaa gcggcggagg aagctggtcc caccctcagt ttgagaaagg 3601 caccgagctg ggcagcacca tggccagcta cccctacgac gtgcccgact acgccaagga 3661 caacaccgtg cccctgaagc tgatcgccct gctggccaac ggcgagttcc actctggcga 3721 gcagctgggc gagacactgg gcatgagcag agccgccatc aacaagcaca tccagaccct 3781 gcgggactgg ggcgtggacg tgttcacagt gcccggcaag ggctacagcc tgcccgagcc 3841 catccagctg ctgaacgcca agcagatcct gggccagctg gatggcggat ctgtggccgt 3901 gctgcccgtg atcgacagca ccaaccagta tctgctcgac cggatcggcg agctgaagtc 3961 cggcgacgcc tgtatcgccg agtaccagca ggccggcaga ggcggcaggg gcagaaagtg 4021 gttcagcccc ttcggcgcca acctgtacct gagcatgttc tggagactgg aacagggccc 4081 agccgccgct attggcctga gcctggtcat cggcatcgtg atggccgagg tgctgcggaa 4141 gctgggcgcc gacaaagtgc gcgtgaagtg gcccaacgac ctgtatctgc aggaccggaa 4201 gctggccggc atcctggtgg aactgaccgg caaaacaggc gacgccgccc agatcgtgat 4261 cggcgctggc atcaacatgg ccatgcggcg ggtggaagag agcgtggtca accagggctg 4321 gatcaccctg caggaagccg gcatcaacct ggaccggaac accctggccg ccatgctgat 4381 cagagagctg agagccgccc tggaactgtt cgagcaggaa ggcctggccc cctacctgag 4441 cagatgggag aagctggaca acttcatcaa ccggcccgtg aaactgatca tcggcgacaa 4501 agagatcttc ggcatcagcc ggggcatcga caagcagggg gccctgctcc tggaacagga 4561 cggcatcatc aagccctgga tgggcggcga gatcagcctg cggagcgccg agaagtgatg 4621 actcgagtct agagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg 4681 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc 4741 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc 4801 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag 4861 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc 4921 tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac 4981 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc 5041 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt 5101 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg 5161 ttcacgtacc tagaagttcc tattccgaag ttcctattct ctagaaagta taggaacttc 5221 cttggccaaa aagcctgaac tcaccgcgac gtctgtcgag aagtttctga tcgaaaagtt 5281 cgacagcgtc tccgacctga tgcagctctc ggagggcgaa gaatctcgtg ctttcagctt 5341 cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg gtttctacaa 5401 agatcgttat gtttatcggc actttgcatc ggccgcgctc ccgattccgg aagtgcttga 5461 cattggggaa ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac 5521 gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg cggaggccat 5581 ggatgcgatc gctgcggccg atcttagcca gacgagcggg ttcggcccat tcggaccgca 5641 aggaatcggt caatacacta catggcgtga tttcatatgc gcgattgctg atccccatgt 5701 gtatcactgg caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc aggctctcga 5761 tgagctgatg ctttgggccg aggactgccc cgaagtccgg cacctcgtgc acgcggattt 5821 cggctccaac aatgtcctga cggacaatgg ccgcataaca gcggtcattg actggagcga 5881 ggcgatgttc ggggattccc aatacgaggt cgccaacatc ttcttctgga ggccgtggtt 5941 ggcttgtatg gagcagcaga cgcgctactt cgagcggagg catccggagc ttgcaggatc 6001 gccgcggctc cgggcgtata tgctccgcat tggtcttgac caactctatc agagcttggt 6061 tgacggcaat ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa tcgtccgatc 6121 cggagccggg actgtcgggc gtacacaaat cgcccgcaga agcgcggccg tctggaccga 6181 tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc cccagcactc gtccgagggc 6241 aaaggaatag cacgtactac gagatttcga ttccaccgcc gccttctatg aaaggttggg 6301 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct 6361 ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa 6421 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc 6481 caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc 6541 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa 6601 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac 6661 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca 6721 ttaatgaatc ggccaacgcg cggggagagg //