LOCUS       sequence-309511-        6870 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 6870)
  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 6870)
  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 NSP1
FEATURES             Location/Qualifiers
     source          1..6870
                     /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(1373..1397)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1444..1467
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1504..1527
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1558..1584
                     /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             1558..1584
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(1585..1932,1933..1935,1936..2544)
                     /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(2588..2605)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2594..2818
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2951..2970)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3102..3149
                     /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             3157..4179
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3157..4179
                     /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    4309..4430
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4346..4365)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4400..4419
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4479..4495)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4479..4495)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4492..4514)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4503..4519
                     /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(4527..4533,4534..4551,4552..4557))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4572..4593
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4710..4727)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4881..5469)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4961..4980)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5640..6431,6432..6500))
                     /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     6263..6282
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6501..6605)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6680..6699)
                     /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 catggagagt
      841 cttgtacccg gctttaacga gaaaacacat gttcaattgt cattgcctgt gctgcaagtc
      901 cgggacgtac tggttagggg gttcggagat tcagtagaag aggtgctgtc tgaggcgagg
      961 caacatttga aagacgggac gtgcgggctg gttgaggtgg aaaagggagt acttccacag
     1021 ttggaacaac cgtatgtttt tattaagcgg agtgatgcca ggactgcgcc gcacggtcat
     1081 gtcatggttg agcttgtcgc agagcttgaa ggcattcaat atgggcgctc cggtgaaact
     1141 cttggagtac ttgtgccgca cgtaggcgaa attccagtgg catatcggaa ggtgctgttg
     1201 agaaaaaatg ggaacaaggg cgcagggggc cacagttatg gagccgacct caaatccttt
     1261 gaccttggcg acgagttggg gacagatccc tacgaggatt tccaggaaaa ctggaatacg
     1321 aaacatagca gtggcgttac tcgcgagttg atgcgcgaac tgaacggcgg cgacccagct
     1381 ttcttgtaca aagtggttga tatcaaggcc gccgacatca ccagcctgta caagaaagtg
     1441 gggtggagcc atccccagtt cgagaagggc ggaggatctg gcggcggaag cggcggagga
     1501 agctggtccc accctcagtt tgagaaaggc accgagctgg gcagcaccat ggccagctac
     1561 ccctacgacg tgcccgacta cgccaaggac aacaccgtgc ccctgaagct gatcgccctg
     1621 ctggccaacg gcgagttcca ctctggcgag cagctgggcg agacactggg catgagcaga
     1681 gccgccatca acaagcacat ccagaccctg cgggactggg gcgtggacgt gttcacagtg
     1741 cccggcaagg gctacagcct gcccgagccc atccagctgc tgaacgccaa gcagatcctg
     1801 ggccagctgg atggcggatc tgtggccgtg ctgcccgtga tcgacagcac caaccagtat
     1861 ctgctcgacc ggatcggcga gctgaagtcc ggcgacgcct gtatcgccga gtaccagcag
     1921 gccggcagag gcggcagggg cagaaagtgg ttcagcccct tcggcgccaa cctgtacctg
     1981 agcatgttct ggagactgga acagggccca gccgccgcta ttggcctgag cctggtcatc
     2041 ggcatcgtga tggccgaggt gctgcggaag ctgggcgccg acaaagtgcg cgtgaagtgg
     2101 cccaacgacc tgtatctgca ggaccggaag ctggccggca tcctggtgga actgaccggc
     2161 aaaacaggcg acgccgccca gatcgtgatc ggcgctggca tcaacatggc catgcggcgg
     2221 gtggaagaga gcgtggtcaa ccagggctgg atcaccctgc aggaagccgg catcaacctg
     2281 gaccggaaca ccctggccgc catgctgatc agagagctga gagccgccct ggaactgttc
     2341 gagcaggaag gcctggcccc ctacctgagc agatgggaga agctggacaa cttcatcaac
     2401 cggcccgtga aactgatcat cggcgacaaa gagatcttcg gcatcagccg gggcatcgac
     2461 aagcaggggg ccctgctcct ggaacaggac ggcatcatca agccctggat gggcggcgag
     2521 atcagcctgc ggagcgccga gaagtgatga ctcgagtcta gagggcccgt ttaaacccgc
     2581 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     2641 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     2701 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     2761 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct
     2821 tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc
     2881 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     2941 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     3001 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     3061 gaccccaaaa aacttgatta gggtgatggt tcacgtacct agaagttcct attccgaagt
     3121 tcctattctc tagaaagtat aggaacttcc ttggccaaaa agcctgaact caccgcgacg
     3181 tctgtcgaga agtttctgat cgaaaagttc gacagcgtct ccgacctgat gcagctctcg
     3241 gagggcgaag aatctcgtgc tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg
     3301 gtaaatagct gcgccgatgg tttctacaaa gatcgttatg tttatcggca ctttgcatcg
     3361 gccgcgctcc cgattccgga agtgcttgac attggggaat tcagcgagag cctgacctat
     3421 tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc
     3481 gctgttctgc agccggtcgc ggaggccatg gatgcgatcg ctgcggccga tcttagccag
     3541 acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc aatacactac atggcgtgat
     3601 ttcatatgcg cgattgctga tccccatgtg tatcactggc aaactgtgat ggacgacacc
     3661 gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc tttgggccga ggactgcccc
     3721 gaagtccggc acctcgtgca cgcggatttc ggctccaaca atgtcctgac ggacaatggc
     3781 cgcataacag cggtcattga ctggagcgag gcgatgttcg gggattccca atacgaggtc
     3841 gccaacatct tcttctggag gccgtggttg gcttgtatgg agcagcagac gcgctacttc
     3901 gagcggaggc atccggagct tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt
     3961 ggtcttgacc aactctatca gagcttggtt gacggcaatt tcgatgatgc agcttgggcg
     4021 cagggtcgat gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc
     4081 gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc cgatagtgga
     4141 aaccgacgcc ccagcactcg tccgagggca aaggaatagc acgtactacg agatttcgat
     4201 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
     4261 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt
     4321 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     4381 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt
     4441 ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga
     4501 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     4561 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     4621 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     4681 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     4741 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     4801 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     4861 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     4921 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     4981 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     5041 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     5101 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     5161 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     5221 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     5281 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     5341 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     5401 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     5461 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     5521 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     5581 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     5641 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     5701 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     5761 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     5821 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     5881 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     5941 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     6001 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     6061 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     6121 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     6181 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     6241 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     6301 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     6361 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     6421 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     6481 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     6541 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     6601 ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tctcccgatc
     6661 ccctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc
     6721 tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa
     6781 ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc
     6841 ttcgcgatgt acgggccaga tatacgcgtt
//
