LOCUS       sequence-309538-        6927 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 6927)
  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 6927)
  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 NSP8
FEATURES             Location/Qualifiers
     source          1..6927
                     /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(1430..1454)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1501..1524
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1561..1584
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1615..1641
                     /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             1615..1641
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(1642..1989,1990..1992,1993..2601)
                     /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(2645..2662)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2651..2875
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3008..3027)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3159..3206
                     /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             3214..4236
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3214..4236
                     /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    4366..4487
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4403..4422)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4457..4476
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4536..4552)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4536..4552)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4549..4571)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4560..4576
                     /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(4584..4590,4591..4608,4609..4614))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4629..4650
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4767..4784)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4938..5526)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5018..5037)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5697..6488,6489..6557))
                     /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     6320..6339
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6558..6662)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6737..6756)
                     /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 catggcaatc
      841 gcatctgaat tttctagtct cccctcctat gcagcgtttg caactgccca ggaggcatac
      901 gagcaggctg ttgcgaacgg cgactccgaa gtggtgctga agaagctgaa aaagtctctg
      961 aacgtcgcaa aatccgagtt tgatagagat gcagcaatgc aaagaaagct ggagaaaatg
     1021 gcagatcaag ctatgacaca aatgtacaag caggcccgct cagaggataa acgagctaaa
     1081 gttacctccg caatgcaaac tatgctgttc accatgctga gaaagctgga caacgacgcg
     1141 ctgaacaata taattaataa tgcgcgggac gggtgcgtac ccttgaatat tattcccctt
     1201 acgactgccg cgaagctcat ggttgtcata ccagattata acacttacaa gaatacgtgt
     1261 gatggtacga cgttcactta tgcgtctgca ttgtgggaaa ttcagcaagt ggtagacgct
     1321 gattccaaga tagtacagtt gtccgagatt agcatggaca acagtcccaa tctcgcttgg
     1381 cctctgatcg ttaccgcatt gagggctaac agcgcagtca aactgcagga cccagctttc
     1441 ttgtacaaag tggttgatat caaggccgcc gacatcacca gcctgtacaa gaaagtgggg
     1501 tggagccatc cccagttcga gaagggcgga ggatctggcg gcggaagcgg cggaggaagc
     1561 tggtcccacc ctcagtttga gaaaggcacc gagctgggca gcaccatggc cagctacccc
     1621 tacgacgtgc ccgactacgc caaggacaac accgtgcccc tgaagctgat cgccctgctg
     1681 gccaacggcg agttccactc tggcgagcag ctgggcgaga cactgggcat gagcagagcc
     1741 gccatcaaca agcacatcca gaccctgcgg gactggggcg tggacgtgtt cacagtgccc
     1801 ggcaagggct acagcctgcc cgagcccatc cagctgctga acgccaagca gatcctgggc
     1861 cagctggatg gcggatctgt ggccgtgctg cccgtgatcg acagcaccaa ccagtatctg
     1921 ctcgaccgga tcggcgagct gaagtccggc gacgcctgta tcgccgagta ccagcaggcc
     1981 ggcagaggcg gcaggggcag aaagtggttc agccccttcg gcgccaacct gtacctgagc
     2041 atgttctgga gactggaaca gggcccagcc gccgctattg gcctgagcct ggtcatcggc
     2101 atcgtgatgg ccgaggtgct gcggaagctg ggcgccgaca aagtgcgcgt gaagtggccc
     2161 aacgacctgt atctgcagga ccggaagctg gccggcatcc tggtggaact gaccggcaaa
     2221 acaggcgacg ccgcccagat cgtgatcggc gctggcatca acatggccat gcggcgggtg
     2281 gaagagagcg tggtcaacca gggctggatc accctgcagg aagccggcat caacctggac
     2341 cggaacaccc tggccgccat gctgatcaga gagctgagag ccgccctgga actgttcgag
     2401 caggaaggcc tggcccccta cctgagcaga tgggagaagc tggacaactt catcaaccgg
     2461 cccgtgaaac tgatcatcgg cgacaaagag atcttcggca tcagccgggg catcgacaag
     2521 cagggggccc tgctcctgga acaggacggc atcatcaagc cctggatggg cggcgagatc
     2581 agcctgcgga gcgccgagaa gtgatgactc gagtctagag ggcccgttta aacccgctga
     2641 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     2701 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     2761 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     2821 ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct
     2881 gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca
     2941 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     3001 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     3061 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     3121 cccaaaaaac ttgattaggg tgatggttca cgtacctaga agttcctatt ccgaagttcc
     3181 tattctctag aaagtatagg aacttccttg gccaaaaagc ctgaactcac cgcgacgtct
     3241 gtcgagaagt ttctgatcga aaagttcgac agcgtctccg acctgatgca gctctcggag
     3301 ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt cctgcgggta
     3361 aatagctgcg ccgatggttt ctacaaagat cgttatgttt atcggcactt tgcatcggcc
     3421 gcgctcccga ttccggaagt gcttgacatt ggggaattca gcgagagcct gacctattgc
     3481 atctcccgcc gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga actgcccgct
     3541 gttctgcagc cggtcgcgga ggccatggat gcgatcgctg cggccgatct tagccagacg
     3601 agcgggttcg gcccattcgg accgcaagga atcggtcaat acactacatg gcgtgatttc
     3661 atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc
     3721 agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga ctgccccgaa
     3781 gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga caatggccgc
     3841 ataacagcgg tcattgactg gagcgaggcg atgttcgggg attcccaata cgaggtcgcc
     3901 aacatcttct tctggaggcc gtggttggct tgtatggagc agcagacgcg ctacttcgag
     3961 cggaggcatc cggagcttgc aggatcgccg cggctccggg cgtatatgct ccgcattggt
     4021 cttgaccaac tctatcagag cttggttgac ggcaatttcg atgatgcagc ttgggcgcag
     4081 ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg tcgggcgtac acaaatcgcc
     4141 cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag tactcgccga tagtggaaac
     4201 cgacgcccca gcactcgtcc gagggcaaag gaatagcacg tactacgaga tttcgattcc
     4261 accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg
     4321 atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca
     4381 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     4441 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata
     4501 ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     4561 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     4621 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     4681 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     4741 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     4801 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     4861 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     4921 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     4981 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     5041 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     5101 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     5161 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     5221 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     5281 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     5341 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     5401 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     5461 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     5521 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     5581 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     5641 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     5701 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     5761 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     5821 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     5881 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     5941 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     6001 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     6061 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     6121 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     6181 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     6241 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     6301 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     6361 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     6421 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     6481 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     6541 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     6601 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     6661 cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatct cccgatcccc
     6721 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc
     6781 ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc
     6841 aaggcttgac cgacaattgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc
     6901 gcgatgtacg ggccagatat acgcgtt
//
