LOCUS       sequence-309502-        7197 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  MAC-tagged gene expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7197)
  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 7197)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat Protoc.
            2020 Aug 10. pii: 10.1038/s41596-020-0365-x. doi: 
            10.1038/s41596-020-0365-x."
COMMENT     Sequence Label: MAC-SARS-CoV-2 NSP6
FEATURES             Location/Qualifiers
     source          1..7197
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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(1700..1724)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1771..1794
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1831..1854
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1885..1911
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     CDS             1885..1911
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(1912..2259,2260..2262,2263..2871)
                     /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(2915..2932)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2921..3145
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3278..3297)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3429..3476
                     /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             3484..4506
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3484..4506
                     /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    4636..4757
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4673..4692)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4727..4746
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4806..4822)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4806..4822)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4819..4841)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4830..4846
                     /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(join(4854..4860,4861..4878,4879..4884))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4899..4920
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5037..5054)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5208..5796)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5288..5307)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5967..6758,6759..6827))
                     /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     6590..6609
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6828..6932)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7007..7026)
                     /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 ttggcatgag cgccgtgaag
      841 agaaccatca agggaaccca ccactggctg ctgctgacca tcctgacctc cctgctggtg
      901 ctggtgcagt ctacccagtg gagcctgttc ttcttcctgt acgagaacgc cttcctgccc
      961 ttcgctatgg gtatcatcgc catgtccgcc ttcgctatga tgttcgtgaa gcacaagcac
     1021 gctttcctgt gcctgttcct gctgccatcc ctggccaccg tggcttactt caacatggtg
     1081 tacatgcccg cctcttgggt catgcgcatc atgacctggc tggacatggt ggacaccagc
     1141 ctgtccggct tcaagctgaa ggactgcgtg atgtacgcct ctgctgtggt gctgctgatc
     1201 ctgatgaccg ctagaaccgt gtacgatgat ggtgctagaa gggtgtggac cctgatgaac
     1261 gtgctgaccc tggtgtacaa ggtgtactac ggcaacgccc tggaccaggc tatcagcatg
     1321 tgggctctga tcatctctgt gaccagcaac tactccggag tggtgaccac cgtgatgttc
     1381 ctggccagag gtatcgtgtt catgtgcgtg gagtactgcc ctatcttctt catcaccggc
     1441 aacaccctgc agtgcatcat gctggtgtac tgcttcctgg gttacttctg cacctgctat
     1501 ttcggcctgt tctgcctgct gaacagatac ttccgtctga ccctgggcgt gtacgactac
     1561 ctggtgtcca cccaggaatt ccgttacatg aactctcagg gactgctgcc ccctaagaac
     1621 agcatcgacg ctttcaagct gaacatcaag ctgctgggag tgggcggaaa gccatgcatc
     1681 aaggtggcca ccgtgcagta cccaactttc ttgtacaaag tggttgatat caaggccgcc
     1741 gacatcacca gcctgtacaa gaaagtgggg tggagccatc cccagttcga gaagggcgga
     1801 ggatctggcg gcggaagcgg cggaggaagc tggtcccacc ctcagtttga gaaaggcacc
     1861 gagctgggca gcaccatggc cagctacccc tacgacgtgc ccgactacgc caaggacaac
     1921 accgtgcccc tgaagctgat cgccctgctg gccaacggcg agttccactc tggcgagcag
     1981 ctgggcgaga cactgggcat gagcagagcc gccatcaaca agcacatcca gaccctgcgg
     2041 gactggggcg tggacgtgtt cacagtgccc ggcaagggct acagcctgcc cgagcccatc
     2101 cagctgctga acgccaagca gatcctgggc cagctggatg gcggatctgt ggccgtgctg
     2161 cccgtgatcg acagcaccaa ccagtatctg ctcgaccgga tcggcgagct gaagtccggc
     2221 gacgcctgta tcgccgagta ccagcaggcc ggcagaggcg gcaggggcag aaagtggttc
     2281 agccccttcg gcgccaacct gtacctgagc atgttctgga gactggaaca gggcccagcc
     2341 gccgctattg gcctgagcct ggtcatcggc atcgtgatgg ccgaggtgct gcggaagctg
     2401 ggcgccgaca aagtgcgcgt gaagtggccc aacgacctgt atctgcagga ccggaagctg
     2461 gccggcatcc tggtggaact gaccggcaaa acaggcgacg ccgcccagat cgtgatcggc
     2521 gctggcatca acatggccat gcggcgggtg gaagagagcg tggtcaacca gggctggatc
     2581 accctgcagg aagccggcat caacctggac cggaacaccc tggccgccat gctgatcaga
     2641 gagctgagag ccgccctgga actgttcgag caggaaggcc tggcccccta cctgagcaga
     2701 tgggagaagc tggacaactt catcaaccgg cccgtgaaac tgatcatcgg cgacaaagag
     2761 atcttcggca tcagccgggg catcgacaag cagggggccc tgctcctgga acaggacggc
     2821 atcatcaagc cctggatggg cggcgagatc agcctgcgga gcgccgagaa gtgatgactc
     2881 gagtctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag
     2941 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     3001 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     3061 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     3121 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg
     3181 gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     3241 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     3301 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     3361 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     3421 cgtacctaga agttcctatt ccgaagttcc tattctctag aaagtatagg aacttccttg
     3481 gccaaaaagc ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac
     3541 agcgtctccg acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat
     3601 gtaggagggc gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat
     3661 cgttatgttt atcggcactt tgcatcggcc gcgctcccga ttccggaagt gcttgacatt
     3721 ggggaattca gcgagagcct gacctattgc atctcccgcc gtgcacaggg tgtcacgttg
     3781 caagacctgc ctgaaaccga actgcccgct gttctgcagc cggtcgcgga ggccatggat
     3841 gcgatcgctg cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga
     3901 atcggtcaat acactacatg gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat
     3961 cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag
     4021 ctgatgcttt gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc
     4081 tccaacaatg tcctgacgga caatggccgc ataacagcgg tcattgactg gagcgaggcg
     4141 atgttcgggg attcccaata cgaggtcgcc aacatcttct tctggaggcc gtggttggct
     4201 tgtatggagc agcagacgcg ctacttcgag cggaggcatc cggagcttgc aggatcgccg
     4261 cggctccggg cgtatatgct ccgcattggt cttgaccaac tctatcagag cttggttgac
     4321 ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga
     4381 gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc
     4441 tgtgtagaag tactcgccga tagtggaaac cgacgcccca gcactcgtcc gagggcaaag
     4501 gaatagcacg tactacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc
     4561 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag
     4621 ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     4681 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     4741 ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa
     4801 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     4861 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     4921 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     4981 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     5041 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     5101 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     5161 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     5221 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     5281 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     5341 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     5401 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     5461 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     5521 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     5581 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     5641 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     5701 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     5761 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     5821 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     5881 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     5941 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     6001 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     6061 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     6121 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     6181 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     6241 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     6301 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     6361 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     6421 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     6481 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     6541 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     6601 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     6661 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     6721 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     6781 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     6841 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     6901 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     6961 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
     7021 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
     7081 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
     7141 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgtt
//
