LOCUS       sequence-316756-        7245 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 7245)
  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 7245)
  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 NSP5
FEATURES             Location/Qualifiers
     source          1..7245
                     /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(1748..1772)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1819..1842
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1879..1902
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1933..1959
                     /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             1933..1959
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(1960..2307,2308..2310,2311..2919)
                     /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(2963..2980)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2969..3193
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3326..3345)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3477..3524
                     /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             3532..4554
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3532..4554
                     /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    4684..4805
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4721..4740)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4775..4794
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4854..4870)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4854..4870)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4867..4889)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4878..4894
                     /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(4902..4908,4909..4926,4927..4932))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4947..4968
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5085..5102)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5256..5844)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5336..5355)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6015..6806,6807..6875))
                     /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     6638..6657
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6876..6980)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7055..7074)
                     /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 ttggcatgtc cggattccgc
      841 aagatggcct tcccttctgg caaggtggag ggctgcatgg tgcaggtgac ctgcggaacc
      901 accaccctga acggtctgtg gctggacgac gtggtgtact gccccaggca cgtgatctgc
      961 acctctgagg acatgctgaa ccctaactac gaagacctgc tgatcagaaa gagcaaccac
     1021 aacttcctgg tgcaggccgg taacgtgcag ctgcgtgtga tcggccactc tatgcagaac
     1081 tgcgtgctga agctgaaggt ggacaccgct aaccccaaga cccctaagta caagttcgtg
     1141 agaatccagc ctggtcagac cttctccgtg ctggcctgct acaacggaag cccatccggc
     1201 gtgtaccagt gcgctatgcg tcccaacttc accatcaagg gcagcttcct gaacggctct
     1261 tgcggaagcg tgggtttcaa catcgactac gactgcgtgt ccttctgcta catgcaccac
     1321 atggagctgc caactggagt gcacgctgga actgacctgg agggtaactt ctacggcccc
     1381 ttcgtggaca gacagactgc tcaggctgct ggcaccgaca ccaccatcac cgtgaacgtg
     1441 ctggcttggc tgtacgccgc tgtgatcaac ggagaccgct ggttcctgaa caggttcacc
     1501 accaccctga acgacttcaa cctggtggct atgaagtaca actacgagcc tctgacccag
     1561 gaccacgtgg atatcctggg tccactgagc gcccagactg gaatcgctgt gctggacatg
     1621 tgcgcctccc tgaaggaact gctgcagaac ggcatgaacg gacgcaccat cctgggatcc
     1681 gctctgctgg aggacgaatt cacccccttc gacgtggtga ggcagtgctc tggtgtgacc
     1741 ttccagtacc caactttctt gtacaaagtg gttgatatca aggccgccga catcaccagc
     1801 ctgtacaaga aagtggggtg gagccatccc cagttcgaga agggcggagg atctggcggc
     1861 ggaagcggcg gaggaagctg gtcccaccct cagtttgaga aaggcaccga gctgggcagc
     1921 accatggcca gctaccccta cgacgtgccc gactacgcca aggacaacac cgtgcccctg
     1981 aagctgatcg ccctgctggc caacggcgag ttccactctg gcgagcagct gggcgagaca
     2041 ctgggcatga gcagagccgc catcaacaag cacatccaga ccctgcggga ctggggcgtg
     2101 gacgtgttca cagtgcccgg caagggctac agcctgcccg agcccatcca gctgctgaac
     2161 gccaagcaga tcctgggcca gctggatggc ggatctgtgg ccgtgctgcc cgtgatcgac
     2221 agcaccaacc agtatctgct cgaccggatc ggcgagctga agtccggcga cgcctgtatc
     2281 gccgagtacc agcaggccgg cagaggcggc aggggcagaa agtggttcag ccccttcggc
     2341 gccaacctgt acctgagcat gttctggaga ctggaacagg gcccagccgc cgctattggc
     2401 ctgagcctgg tcatcggcat cgtgatggcc gaggtgctgc ggaagctggg cgccgacaaa
     2461 gtgcgcgtga agtggcccaa cgacctgtat ctgcaggacc ggaagctggc cggcatcctg
     2521 gtggaactga ccggcaaaac aggcgacgcc gcccagatcg tgatcggcgc tggcatcaac
     2581 atggccatgc ggcgggtgga agagagcgtg gtcaaccagg gctggatcac cctgcaggaa
     2641 gccggcatca acctggaccg gaacaccctg gccgccatgc tgatcagaga gctgagagcc
     2701 gccctggaac tgttcgagca ggaaggcctg gccccctacc tgagcagatg ggagaagctg
     2761 gacaacttca tcaaccggcc cgtgaaactg atcatcggcg acaaagagat cttcggcatc
     2821 agccggggca tcgacaagca gggggccctg ctcctggaac aggacggcat catcaagccc
     2881 tggatgggcg gcgagatcag cctgcggagc gccgagaagt gatgactcga gtctagaggg
     2941 cccgtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt
     3001 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     3061 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
     3121 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg
     3181 gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac
     3241 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     3301 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     3361 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     3421 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tacctagaag
     3481 ttcctattcc gaagttccta ttctctagaa agtataggaa cttccttggc caaaaagcct
     3541 gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag cgtctccgac
     3601 ctgatgcagc tctcggaggg cgaagaatct cgtgctttca gcttcgatgt aggagggcgt
     3661 ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg ttatgtttat
     3721 cggcactttg catcggccgc gctcccgatt ccggaagtgc ttgacattgg ggaattcagc
     3781 gagagcctga cctattgcat ctcccgccgt gcacagggtg tcacgttgca agacctgcct
     3841 gaaaccgaac tgcccgctgt tctgcagccg gtcgcggagg ccatggatgc gatcgctgcg
     3901 gccgatctta gccagacgag cgggttcggc ccattcggac cgcaaggaat cggtcaatac
     3961 actacatggc gtgatttcat atgcgcgatt gctgatcccc atgtgtatca ctggcaaact
     4021 gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg
     4081 gccgaggact gccccgaagt ccggcacctc gtgcacgcgg atttcggctc caacaatgtc
     4141 ctgacggaca atggccgcat aacagcggtc attgactgga gcgaggcgat gttcggggat
     4201 tcccaatacg aggtcgccaa catcttcttc tggaggccgt ggttggcttg tatggagcag
     4261 cagacgcgct acttcgagcg gaggcatccg gagcttgcag gatcgccgcg gctccgggcg
     4321 tatatgctcc gcattggtct tgaccaactc tatcagagct tggttgacgg caatttcgat
     4381 gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc
     4441 gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg tgtagaagta
     4501 ctcgccgata gtggaaaccg acgccccagc actcgtccga gggcaaagga atagcacgta
     4561 ctacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc
     4621 cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac
     4681 cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     4741 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     4801 tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag
     4861 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     4921 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     4981 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     5041 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     5101 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     5161 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     5221 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     5281 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     5341 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     5401 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     5461 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     5521 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     5581 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     5641 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     5701 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     5761 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     5821 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     5881 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     5941 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     6001 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     6061 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     6121 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     6181 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     6241 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     6301 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     6361 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     6421 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     6481 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     6541 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     6601 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     6661 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     6721 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     6781 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     6841 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     6901 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     6961 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgacggatcg
     7021 ggagatctcc cgatccccta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     7081 taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa
     7141 tttaagctac aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta
     7201 ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgtt
//
