LOCUS       sequence-316754-        7365 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 7365)
  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 7365)
  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 NSP15
FEATURES             Location/Qualifiers
     source          1..7365
                     /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(1868..1892)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1939..1962
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1999..2022
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             2053..2079
                     /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             2053..2079
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(2080..2427,2428..2430,2431..3039)
                     /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(3083..3100)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3089..3313
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3446..3465)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3597..3644
                     /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             3652..4674
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3652..4674
                     /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    4804..4925
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4841..4860)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4895..4914
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4974..4990)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4974..4990)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4987..5009)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4998..5014
                     /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(5022..5028,5029..5046,5047..5052))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5067..5088
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5205..5222)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5376..5964)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5456..5475)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6135..6926,6927..6995))
                     /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     6758..6777
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6996..7100)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7175..7194)
                     /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 tctggagaac
      841 gtggccttca acgtggtgaa caagggtcac ttcgacggtc agcagggcga agtgcctgtg
      901 agcatcatca acaacaccgt gtacaccaag gtggacggcg tggacgtgga gctgttcgaa
      961 aacaagacca ccctgccagt gaacgtggcc ttcgagctgt gggctaagcg caacatcaag
     1021 cccgtgcctg aagtgaagat cctgaacaac ctgggagtgg acatcgccgc taacaccgtg
     1081 atctgggact acaagaggga cgcccccgct cacatcagca ccatcggcgt gtgctccatg
     1141 accgacatcg ccaagaagcc taccgagacc atctgcgctc cactgaccgt gttcttcgac
     1201 ggaagagtgg acggtcaggt ggacctgttc cgcaacgcta ggaacggcgt gctgatcacc
     1261 gagggctctg tgaagggact gcagccaagc gtgggaccca agcaggcctc cctgaacggt
     1321 gtgaccctga tcggcgaagc tgtgaagacc cagttcaact actacaagaa ggtggacggc
     1381 gtggtgcagc agctgccaga gacctacttc acccagtcta gaaacctgca ggaattcaag
     1441 ccccgtagcc agatggagat cgacttcctg gaactggcca tggacgagtt catcgaaaga
     1501 tacaagctgg agggatacgc tttcgaacac atcgtgtacg gtgacttctc tcacagccag
     1561 ctgggcggac tgcacctgct gatcggtctg gccaagcgtt tcaaggagtc ccccttcgag
     1621 ctggaagact tcatccctat ggactctacc gtgaagaact acttcatcac cgacgctcag
     1681 accggaagct ccaagtgcgt gtgcagcgtg atcgacctgc tgctggacga cttcgtggag
     1741 atcatcaagt cccaggacct gtccgtggtg tctaaggtgg tgaaggtgac catcgactac
     1801 accgagatct ccttcatgct gtggtgcaag gacggccacg tggaaacctt ctaccctaag
     1861 ctgcagtacc caactttctt gtacaaagtg gttgatatca aggccgccga catcaccagc
     1921 ctgtacaaga aagtggggtg gagccatccc cagttcgaga agggcggagg atctggcggc
     1981 ggaagcggcg gaggaagctg gtcccaccct cagtttgaga aaggcaccga gctgggcagc
     2041 accatggcca gctaccccta cgacgtgccc gactacgcca aggacaacac cgtgcccctg
     2101 aagctgatcg ccctgctggc caacggcgag ttccactctg gcgagcagct gggcgagaca
     2161 ctgggcatga gcagagccgc catcaacaag cacatccaga ccctgcggga ctggggcgtg
     2221 gacgtgttca cagtgcccgg caagggctac agcctgcccg agcccatcca gctgctgaac
     2281 gccaagcaga tcctgggcca gctggatggc ggatctgtgg ccgtgctgcc cgtgatcgac
     2341 agcaccaacc agtatctgct cgaccggatc ggcgagctga agtccggcga cgcctgtatc
     2401 gccgagtacc agcaggccgg cagaggcggc aggggcagaa agtggttcag ccccttcggc
     2461 gccaacctgt acctgagcat gttctggaga ctggaacagg gcccagccgc cgctattggc
     2521 ctgagcctgg tcatcggcat cgtgatggcc gaggtgctgc ggaagctggg cgccgacaaa
     2581 gtgcgcgtga agtggcccaa cgacctgtat ctgcaggacc ggaagctggc cggcatcctg
     2641 gtggaactga ccggcaaaac aggcgacgcc gcccagatcg tgatcggcgc tggcatcaac
     2701 atggccatgc ggcgggtgga agagagcgtg gtcaaccagg gctggatcac cctgcaggaa
     2761 gccggcatca acctggaccg gaacaccctg gccgccatgc tgatcagaga gctgagagcc
     2821 gccctggaac tgttcgagca ggaaggcctg gccccctacc tgagcagatg ggagaagctg
     2881 gacaacttca tcaaccggcc cgtgaaactg atcatcggcg acaaagagat cttcggcatc
     2941 agccggggca tcgacaagca gggggccctg ctcctggaac aggacggcat catcaagccc
     3001 tggatgggcg gcgagatcag cctgcggagc gccgagaagt gatgactcga gtctagaggg
     3061 cccgtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt
     3121 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     3181 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
     3241 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg
     3301 gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac
     3361 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     3421 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     3481 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     3541 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tacctagaag
     3601 ttcctattcc gaagttccta ttctctagaa agtataggaa cttccttggc caaaaagcct
     3661 gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag cgtctccgac
     3721 ctgatgcagc tctcggaggg cgaagaatct cgtgctttca gcttcgatgt aggagggcgt
     3781 ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg ttatgtttat
     3841 cggcactttg catcggccgc gctcccgatt ccggaagtgc ttgacattgg ggaattcagc
     3901 gagagcctga cctattgcat ctcccgccgt gcacagggtg tcacgttgca agacctgcct
     3961 gaaaccgaac tgcccgctgt tctgcagccg gtcgcggagg ccatggatgc gatcgctgcg
     4021 gccgatctta gccagacgag cgggttcggc ccattcggac cgcaaggaat cggtcaatac
     4081 actacatggc gtgatttcat atgcgcgatt gctgatcccc atgtgtatca ctggcaaact
     4141 gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg
     4201 gccgaggact gccccgaagt ccggcacctc gtgcacgcgg atttcggctc caacaatgtc
     4261 ctgacggaca atggccgcat aacagcggtc attgactgga gcgaggcgat gttcggggat
     4321 tcccaatacg aggtcgccaa catcttcttc tggaggccgt ggttggcttg tatggagcag
     4381 cagacgcgct acttcgagcg gaggcatccg gagcttgcag gatcgccgcg gctccgggcg
     4441 tatatgctcc gcattggtct tgaccaactc tatcagagct tggttgacgg caatttcgat
     4501 gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc
     4561 gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg tgtagaagta
     4621 ctcgccgata gtggaaaccg acgccccagc actcgtccga gggcaaagga atagcacgta
     4681 ctacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc
     4741 cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac
     4801 cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     4861 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     4921 tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag
     4981 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     5041 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     5101 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     5161 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     5221 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     5281 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     5341 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     5401 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     5461 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     5521 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     5581 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     5641 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     5701 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     5761 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     5821 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     5881 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     5941 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     6001 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     6061 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     6121 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     6181 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     6241 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     6301 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     6361 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     6421 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     6481 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     6541 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     6601 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     6661 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     6721 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     6781 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     6841 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     6901 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     6961 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     7021 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     7081 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgacggatcg
     7141 ggagatctcc cgatccccta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     7201 taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa
     7261 tttaagctac aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta
     7321 ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgtt
//
