LOCUS       sequence-309566-        7221 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 7221)
  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 7221)
  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 NSP16
FEATURES             Location/Qualifiers
     source          1..7221
                     /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(1724..1748)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1795..1818
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1855..1878
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1909..1935
                     /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             1909..1935
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(1936..2283,2284..2286,2287..2895)
                     /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(2939..2956)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2945..3169
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3302..3321)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3453..3500
                     /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             3508..4530
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3508..4530
                     /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    4660..4781
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4697..4716)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4751..4770
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4830..4846)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4830..4846)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4843..4865)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4854..4870
                     /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(4878..4884,4885..4902,4903..4908))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4923..4944
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5061..5078)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5232..5820)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5312..5331)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5991..6782,6783..6851))
                     /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     6614..6633
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6852..6956)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7031..7050)
                     /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 ttcccaggct
      841 tggcagcctg gagtggctat gcctaacctg tacaagatgc agcgtatgct gctggagaag
      901 tgcgacctgc agaactacgg agactccgcc accctgccca agggtatcat gatgaacgtg
      961 gctaagtaca cccagctgtg ccagtacctg aacaccctga ccctggccgt gccttacaac
     1021 atgcgcgtga tccacttcgg tgctggttct gacaagggag tggctccagg taccgctgtg
     1081 ctgagacagt ggctgcccac cggtaccctg ctggtggact ctgacctgaa cgacttcgtg
     1141 tctgatgctg acagcaccct gatcggcgac tgtgctactg tgcacaccgc taacaagtgg
     1201 gacctgatca tctctgacat gtacgaccca aagaccaaga acgtgaccaa ggagaacgac
     1261 agcaaggaag gtttcttcac ctacatctgc ggcttcatcc agcagaagct ggccctgggc
     1321 ggatctgtgg ctatcaagat caccgaacac tcctggaacg ctgacctgta caagctgatg
     1381 ggacacttcg cctggtggac cgctttcgtg accaacgtga acgcctctag ctccgaggct
     1441 ttcctgatcg gctgcaacta cctgggaaag ccccgtgaac agatcgacgg ttacgtgatg
     1501 cacgccaact acatcttctg gcgcaacacc aaccctatcc agctgtctag ctactccctg
     1561 ttcgacatgt ctaagttccc actgaagctg agaggcaccg ctgtgatgag cctgaaggag
     1621 ggacagatca acgacatgat cctgagcctg ctgtccaagg gacgcctgat catcagggaa
     1681 aacaacagag tggtcatctc ctctgacgtg ctggtgaaca actacccaac tttcttgtac
     1741 aaagtggttg atatcaaggc cgccgacatc accagcctgt acaagaaagt ggggtggagc
     1801 catccccagt tcgagaaggg cggaggatct ggcggcggaa gcggcggagg aagctggtcc
     1861 caccctcagt ttgagaaagg caccgagctg ggcagcacca tggccagcta cccctacgac
     1921 gtgcccgact acgccaagga caacaccgtg cccctgaagc tgatcgccct gctggccaac
     1981 ggcgagttcc actctggcga gcagctgggc gagacactgg gcatgagcag agccgccatc
     2041 aacaagcaca tccagaccct gcgggactgg ggcgtggacg tgttcacagt gcccggcaag
     2101 ggctacagcc tgcccgagcc catccagctg ctgaacgcca agcagatcct gggccagctg
     2161 gatggcggat ctgtggccgt gctgcccgtg atcgacagca ccaaccagta tctgctcgac
     2221 cggatcggcg agctgaagtc cggcgacgcc tgtatcgccg agtaccagca ggccggcaga
     2281 ggcggcaggg gcagaaagtg gttcagcccc ttcggcgcca acctgtacct gagcatgttc
     2341 tggagactgg aacagggccc agccgccgct attggcctga gcctggtcat cggcatcgtg
     2401 atggccgagg tgctgcggaa gctgggcgcc gacaaagtgc gcgtgaagtg gcccaacgac
     2461 ctgtatctgc aggaccggaa gctggccggc atcctggtgg aactgaccgg caaaacaggc
     2521 gacgccgccc agatcgtgat cggcgctggc atcaacatgg ccatgcggcg ggtggaagag
     2581 agcgtggtca accagggctg gatcaccctg caggaagccg gcatcaacct ggaccggaac
     2641 accctggccg ccatgctgat cagagagctg agagccgccc tggaactgtt cgagcaggaa
     2701 ggcctggccc cctacctgag cagatgggag aagctggaca acttcatcaa ccggcccgtg
     2761 aaactgatca tcggcgacaa agagatcttc ggcatcagcc ggggcatcga caagcagggg
     2821 gccctgctcc tggaacagga cggcatcatc aagccctgga tgggcggcga gatcagcctg
     2881 cggagcgccg agaagtgatg actcgagtct agagggcccg tttaaacccg ctgatcagcc
     2941 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     3001 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
     3061 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
     3121 gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg
     3181 gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc
     3241 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     3301 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     3361 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     3421 aaacttgatt agggtgatgg ttcacgtacc tagaagttcc tattccgaag ttcctattct
     3481 ctagaaagta taggaacttc cttggccaaa aagcctgaac tcaccgcgac gtctgtcgag
     3541 aagtttctga tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc ggagggcgaa
     3601 gaatctcgtg ctttcagctt cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc
     3661 tgcgccgatg gtttctacaa agatcgttat gtttatcggc actttgcatc ggccgcgctc
     3721 ccgattccgg aagtgcttga cattggggaa ttcagcgaga gcctgaccta ttgcatctcc
     3781 cgccgtgcac agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctg
     3841 cagccggtcg cggaggccat ggatgcgatc gctgcggccg atcttagcca gacgagcggg
     3901 ttcggcccat tcggaccgca aggaatcggt caatacacta catggcgtga tttcatatgc
     3961 gcgattgctg atccccatgt gtatcactgg caaactgtga tggacgacac cgtcagtgcg
     4021 tccgtcgcgc aggctctcga tgagctgatg ctttgggccg aggactgccc cgaagtccgg
     4081 cacctcgtgc acgcggattt cggctccaac aatgtcctga cggacaatgg ccgcataaca
     4141 gcggtcattg actggagcga ggcgatgttc ggggattccc aatacgaggt cgccaacatc
     4201 ttcttctgga ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt cgagcggagg
     4261 catccggagc ttgcaggatc gccgcggctc cgggcgtata tgctccgcat tggtcttgac
     4321 caactctatc agagcttggt tgacggcaat ttcgatgatg cagcttgggc gcagggtcga
     4381 tgcgacgcaa tcgtccgatc cggagccggg actgtcgggc gtacacaaat cgcccgcaga
     4441 agcgcggccg tctggaccga tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc
     4501 cccagcactc gtccgagggc aaaggaatag cacgtactac gagatttcga ttccaccgcc
     4561 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     4621 cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat
     4681 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     4741 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg
     4801 acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     4861 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     4921 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     4981 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     5041 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     5101 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     5161 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     5221 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     5281 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     5341 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     5401 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     5461 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     5521 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     5581 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     5641 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     5701 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     5761 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     5821 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     5881 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     5941 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     6001 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     6061 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     6121 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     6181 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     6241 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     6301 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     6361 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     6421 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     6481 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     6541 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     6601 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     6661 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     6721 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     6781 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     6841 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     6901 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     6961 tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt
     7021 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt
     7081 gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct
     7141 tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg
     7201 tacgggccag atatacgcgt t
//
