LOCUS       sequence-309515-        6750 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 6750)
  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 6750)
  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 NSP10
FEATURES             Location/Qualifiers
     source          1..6750
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(31..48)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(202..790)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(282..301)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(961..1752,1753..1821))
                     /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     1584..1603
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1822..1926)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2001..2020)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2192..2571
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2572..2775
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2726..2746
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    2777..2795
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2798..2816
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2826..2850
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    2993..3017
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(3444..3468)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3515..3538
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             3575..3598
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             3629..3655
                     /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             3629..3655
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(3656..4003,4004..4006,4007..4615)
                     /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(4659..4676)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4665..4889
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5022..5041)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    5173..5220
                     /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             5228..6250
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             5228..6250
                     /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    6380..6501
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6417..6436)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6471..6490
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6550..6566)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6550..6566)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6563..6585)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6574..6590
                     /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(6598..6604,6605..6622,6623..6628))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6643..6664
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
       61 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
      121 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
      181 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
      241 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
      301 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
      361 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
      421 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
      481 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
      541 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
      601 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
      661 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
      721 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
      781 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
      841 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
      901 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
      961 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     1021 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     1081 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     1141 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     1201 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     1261 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     1321 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     1381 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     1441 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     1501 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     1561 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     1621 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     1681 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     1741 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     1801 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     1861 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     1921 tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat
     1981 cccctatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg
     2041 ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca
     2101 aggcaaggct tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg
     2161 cttcgcgatg tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag
     2221 taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt
     2281 acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg
     2341 acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat
     2401 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct
     2461 attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg
     2521 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg
     2581 ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc
     2641 caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa
     2701 tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc
     2761 tatataagca gagctctccc tatcagtgat agagatctcc ctatcagtga tagagatcgt
     2821 cgacgagctc gtttagtgaa ccgtcagatc gcctggagac gccatccacg ctgttttgac
     2881 ctccatagaa gacaccggga ccgatccagc ctccggactc tagcgtttaa acttaagctt
     2941 ggtaccgagc tcggatccac tagtccagtg tggtggaatt ctgcagatat caacaagttt
     3001 gtacaaaaaa gcaggctcca ccatggctgg taatgcaact gaggttcccg cgaatagtac
     3061 ggttctgagc ttttgcgcct ttgctgttga cgcagctaag gcatataagg actacttggc
     3121 ctccgggggc cagccaatca caaactgcgt caagatgttg tgtacgcaca cgggcacagg
     3181 tcaggcaatc actgtcacac ctgaagctaa catggatcag gaatcttttg gtggtgccag
     3241 ttgttgtctg tactgtcgat gtcatataga tcatcccaac ccgaaagggt tttgtgatct
     3301 gaaaggaaaa tacgtccaaa tcccgaccac gtgcgcgaac gacccggttg ggtttacgtt
     3361 gaaaaatacc gtatgtactg tctgtggtat gtggaagggc tatggctgtt catgcgatca
     3421 attgcgagag ccgatgttgc aggacccagc tttcttgtac aaagtggttg atatcaaggc
     3481 cgccgacatc accagcctgt acaagaaagt ggggtggagc catccccagt tcgagaaggg
     3541 cggaggatct ggcggcggaa gcggcggagg aagctggtcc caccctcagt ttgagaaagg
     3601 caccgagctg ggcagcacca tggccagcta cccctacgac gtgcccgact acgccaagga
     3661 caacaccgtg cccctgaagc tgatcgccct gctggccaac ggcgagttcc actctggcga
     3721 gcagctgggc gagacactgg gcatgagcag agccgccatc aacaagcaca tccagaccct
     3781 gcgggactgg ggcgtggacg tgttcacagt gcccggcaag ggctacagcc tgcccgagcc
     3841 catccagctg ctgaacgcca agcagatcct gggccagctg gatggcggat ctgtggccgt
     3901 gctgcccgtg atcgacagca ccaaccagta tctgctcgac cggatcggcg agctgaagtc
     3961 cggcgacgcc tgtatcgccg agtaccagca ggccggcaga ggcggcaggg gcagaaagtg
     4021 gttcagcccc ttcggcgcca acctgtacct gagcatgttc tggagactgg aacagggccc
     4081 agccgccgct attggcctga gcctggtcat cggcatcgtg atggccgagg tgctgcggaa
     4141 gctgggcgcc gacaaagtgc gcgtgaagtg gcccaacgac ctgtatctgc aggaccggaa
     4201 gctggccggc atcctggtgg aactgaccgg caaaacaggc gacgccgccc agatcgtgat
     4261 cggcgctggc atcaacatgg ccatgcggcg ggtggaagag agcgtggtca accagggctg
     4321 gatcaccctg caggaagccg gcatcaacct ggaccggaac accctggccg ccatgctgat
     4381 cagagagctg agagccgccc tggaactgtt cgagcaggaa ggcctggccc cctacctgag
     4441 cagatgggag aagctggaca acttcatcaa ccggcccgtg aaactgatca tcggcgacaa
     4501 agagatcttc ggcatcagcc ggggcatcga caagcagggg gccctgctcc tggaacagga
     4561 cggcatcatc aagccctgga tgggcggcga gatcagcctg cggagcgccg agaagtgatg
     4621 actcgagtct agagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg
     4681 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     4741 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     4801 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
     4861 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc
     4921 tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     4981 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     5041 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     5101 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     5161 ttcacgtacc tagaagttcc tattccgaag ttcctattct ctagaaagta taggaacttc
     5221 cttggccaaa aagcctgaac tcaccgcgac gtctgtcgag aagtttctga tcgaaaagtt
     5281 cgacagcgtc tccgacctga tgcagctctc ggagggcgaa gaatctcgtg ctttcagctt
     5341 cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg gtttctacaa
     5401 agatcgttat gtttatcggc actttgcatc ggccgcgctc ccgattccgg aagtgcttga
     5461 cattggggaa ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac
     5521 gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg cggaggccat
     5581 ggatgcgatc gctgcggccg atcttagcca gacgagcggg ttcggcccat tcggaccgca
     5641 aggaatcggt caatacacta catggcgtga tttcatatgc gcgattgctg atccccatgt
     5701 gtatcactgg caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc aggctctcga
     5761 tgagctgatg ctttgggccg aggactgccc cgaagtccgg cacctcgtgc acgcggattt
     5821 cggctccaac aatgtcctga cggacaatgg ccgcataaca gcggtcattg actggagcga
     5881 ggcgatgttc ggggattccc aatacgaggt cgccaacatc ttcttctgga ggccgtggtt
     5941 ggcttgtatg gagcagcaga cgcgctactt cgagcggagg catccggagc ttgcaggatc
     6001 gccgcggctc cgggcgtata tgctccgcat tggtcttgac caactctatc agagcttggt
     6061 tgacggcaat ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa tcgtccgatc
     6121 cggagccggg actgtcgggc gtacacaaat cgcccgcaga agcgcggccg tctggaccga
     6181 tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc cccagcactc gtccgagggc
     6241 aaaggaatag cacgtactac gagatttcga ttccaccgcc gccttctatg aaaggttggg
     6301 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     6361 ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa
     6421 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     6481 caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc
     6541 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     6601 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     6661 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     6721 ttaatgaatc ggccaacgcg cggggagagg
//
