LOCUS       sequence-309558-        6693 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 6693)
  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 6693)
  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 Orf8
FEATURES             Location/Qualifiers
     source          1..6693
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(50..69)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        241..620
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        621..824
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     775..795
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    826..844
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    847..865
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     875..899
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    1042..1066
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1436..1460)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1507..1530
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1567..1590
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1621..1647
                     /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             1621..1647
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(1648..1995,1996..1998,1999..2607)
                     /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(2651..2668)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2657..2881
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3014..3033)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3165..3212
                     /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             3220..4242
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3220..4242
                     /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    4372..4493
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4409..4428)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4463..4482
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4542..4558)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4542..4558)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4555..4577)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4566..4582
                     /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(4590..4596,4597..4614,4615..4620))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4635..4656
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4773..4790)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4944..5532)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5024..5043)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5703..6494,6495..6563))
                     /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     6326..6345
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6564..6668)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt acaatctgct
       61 ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt
      121 agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga
      181 atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt
      241 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
      301 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      361 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      421 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      481 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      541 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      601 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      661 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      721 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      781 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctccct atcagtgata
      841 gagatctccc tatcagtgat agagatcgtc gacgagctcg tttagtgaac cgtcagatcg
      901 cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc
      961 tccggactct agcgtttaaa cttaagcttg gtaccgagct cggatccact agtccagtgt
     1021 ggtggaattc tgcagatatc aacaagtttg tacaaaaaag caggctccac catgaagttt
     1081 ctcgtgttct tgggcattat caccacagtc gcggcttttc atcaagaatg ctctttgcag
     1141 tcttgtacac agcatcaacc ttatgtcgtc gatgatcctt gcccgattca cttttacagt
     1201 aaatggtata tcagggtagg ggcccgaaaa agcgcaccat tgattgaatt gtgtgtggac
     1261 gaggcaggga gcaaaagtcc gattcaatac attgacatcg gtaattatac tgtcagttgt
     1321 tcccccttta cgatcaactg ccaagaaccc aaactgggga gcttggtggt ccgatgttcc
     1381 ttctatgaag attttcttga gtatcacgat gtcagggtgg tccttgactt tatagaccca
     1441 gctttcttgt acaaagtggt tgatatcaag gccgccgaca tcaccagcct gtacaagaaa
     1501 gtggggtgga gccatcccca gttcgagaag ggcggaggat ctggcggcgg aagcggcgga
     1561 ggaagctggt cccaccctca gtttgagaaa ggcaccgagc tgggcagcac catggccagc
     1621 tacccctacg acgtgcccga ctacgccaag gacaacaccg tgcccctgaa gctgatcgcc
     1681 ctgctggcca acggcgagtt ccactctggc gagcagctgg gcgagacact gggcatgagc
     1741 agagccgcca tcaacaagca catccagacc ctgcgggact ggggcgtgga cgtgttcaca
     1801 gtgcccggca agggctacag cctgcccgag cccatccagc tgctgaacgc caagcagatc
     1861 ctgggccagc tggatggcgg atctgtggcc gtgctgcccg tgatcgacag caccaaccag
     1921 tatctgctcg accggatcgg cgagctgaag tccggcgacg cctgtatcgc cgagtaccag
     1981 caggccggca gaggcggcag gggcagaaag tggttcagcc ccttcggcgc caacctgtac
     2041 ctgagcatgt tctggagact ggaacagggc ccagccgccg ctattggcct gagcctggtc
     2101 atcggcatcg tgatggccga ggtgctgcgg aagctgggcg ccgacaaagt gcgcgtgaag
     2161 tggcccaacg acctgtatct gcaggaccgg aagctggccg gcatcctggt ggaactgacc
     2221 ggcaaaacag gcgacgccgc ccagatcgtg atcggcgctg gcatcaacat ggccatgcgg
     2281 cgggtggaag agagcgtggt caaccagggc tggatcaccc tgcaggaagc cggcatcaac
     2341 ctggaccgga acaccctggc cgccatgctg atcagagagc tgagagccgc cctggaactg
     2401 ttcgagcagg aaggcctggc cccctacctg agcagatggg agaagctgga caacttcatc
     2461 aaccggcccg tgaaactgat catcggcgac aaagagatct tcggcatcag ccggggcatc
     2521 gacaagcagg gggccctgct cctggaacag gacggcatca tcaagccctg gatgggcggc
     2581 gagatcagcc tgcggagcgc cgagaagtga tgactcgagt ctagagggcc cgtttaaacc
     2641 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     2701 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     2761 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     2821 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg
     2881 gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc
     2941 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     3001 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     3061 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     3121 ctcgacccca aaaaacttga ttagggtgat ggttcacgta cctagaagtt cctattccga
     3181 agttcctatt ctctagaaag tataggaact tccttggcca aaaagcctga actcaccgcg
     3241 acgtctgtcg agaagtttct gatcgaaaag ttcgacagcg tctccgacct gatgcagctc
     3301 tcggagggcg aagaatctcg tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg
     3361 cgggtaaata gctgcgccga tggtttctac aaagatcgtt atgtttatcg gcactttgca
     3421 tcggccgcgc tcccgattcc ggaagtgctt gacattgggg aattcagcga gagcctgacc
     3481 tattgcatct cccgccgtgc acagggtgtc acgttgcaag acctgcctga aaccgaactg
     3541 cccgctgttc tgcagccggt cgcggaggcc atggatgcga tcgctgcggc cgatcttagc
     3601 cagacgagcg ggttcggccc attcggaccg caaggaatcg gtcaatacac tacatggcgt
     3661 gatttcatat gcgcgattgc tgatccccat gtgtatcact ggcaaactgt gatggacgac
     3721 accgtcagtg cgtccgtcgc gcaggctctc gatgagctga tgctttgggc cgaggactgc
     3781 cccgaagtcc ggcacctcgt gcacgcggat ttcggctcca acaatgtcct gacggacaat
     3841 ggccgcataa cagcggtcat tgactggagc gaggcgatgt tcggggattc ccaatacgag
     3901 gtcgccaaca tcttcttctg gaggccgtgg ttggcttgta tggagcagca gacgcgctac
     3961 ttcgagcgga ggcatccgga gcttgcagga tcgccgcggc tccgggcgta tatgctccgc
     4021 attggtcttg accaactcta tcagagcttg gttgacggca atttcgatga tgcagcttgg
     4081 gcgcagggtc gatgcgacgc aatcgtccga tccggagccg ggactgtcgg gcgtacacaa
     4141 atcgcccgca gaagcgcggc cgtctggacc gatggctgtg tagaagtact cgccgatagt
     4201 ggaaaccgac gccccagcac tcgtccgagg gcaaaggaat agcacgtact acgagatttc
     4261 gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc
     4321 tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt
     4381 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     4441 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     4501 tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg
     4561 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     4621 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     4681 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     4741 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     4801 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     4861 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     4921 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     4981 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     5041 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     5101 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     5161 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     5221 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     5281 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     5341 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     5401 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     5461 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     5521 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     5581 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     5641 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     5701 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     5761 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     5821 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     5881 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     5941 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     6001 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     6061 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     6121 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     6181 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     6241 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     6301 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     6361 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     6421 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     6481 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     6541 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     6601 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     6661 gttccgcgca catttccccg aaaagtgcca cct
//
