LOCUS       sequence-309623-        6492 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 6492)
  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 6492)
  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 Orf3b
FEATURES             Location/Qualifiers
     source          1..6492
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(92..116)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(154..171)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    160..384
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(517..536)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    668..715
                     /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             723..1745
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             723..1745
                     /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    1875..1996
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1912..1931)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1966..1985
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2045..2061)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2045..2061)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2058..2080)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2069..2085
                     /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(2093..2099,2100..2117,2118..2123))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2138..2159
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2276..2293)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2447..3035)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2527..2546)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3206..3997,3998..4066))
                     /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     3829..3848
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4067..4171)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4246..4265)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4437..4816
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4817..5020
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4971..4991
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    5022..5040
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5043..5061
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     5071..5095
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             join(5263..5613,5614..5616,5617..6225)
                     /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="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGGRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             6226..6252
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             6283..6306
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             6343..6366
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     protein_bind    6379..6403
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
ORIGIN
        1 accttgcagg gatgctcatt gtgcttgcaa ctcgcggtcg ttgtatgcaa tagcctgctt
       61 acccctttcg ctcgatgctg ctggccgtga gacccagctt tcttgtacaa agtggtgacg
      121 taagctaggg gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc
      181 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
      241 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
      301 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg
      361 ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg
      421 ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
      481 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
      541 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
      601 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
      661 gtacctagaa gttcctattc cgaagttcct attctctaga aagtatagga acttccttgg
      721 ccaaaaagcc tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca
      781 gcgtctccga cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg
      841 taggagggcg tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc
      901 gttatgttta tcggcacttt gcatcggccg cgctcccgat tccggaagtg cttgacattg
      961 gggaattcag cgagagcctg acctattgca tctcccgccg tgcacagggt gtcacgttgc
     1021 aagacctgcc tgaaaccgaa ctgcccgctg ttctgcagcc ggtcgcggag gccatggatg
     1081 cgatcgctgc ggccgatctt agccagacga gcgggttcgg cccattcgga ccgcaaggaa
     1141 tcggtcaata cactacatgg cgtgatttca tatgcgcgat tgctgatccc catgtgtatc
     1201 actggcaaac tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc
     1261 tgatgctttg ggccgaggac tgccccgaag tccggcacct cgtgcacgcg gatttcggct
     1321 ccaacaatgt cctgacggac aatggccgca taacagcggt cattgactgg agcgaggcga
     1381 tgttcgggga ttcccaatac gaggtcgcca acatcttctt ctggaggccg tggttggctt
     1441 gtatggagca gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc
     1501 ggctccgggc gtatatgctc cgcattggtc ttgaccaact ctatcagagc ttggttgacg
     1561 gcaatttcga tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag
     1621 ccgggactgt cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg accgatggct
     1681 gtgtagaagt actcgccgat agtggaaacc gacgccccag cactcgtccg agggcaaagg
     1741 aatagcacgt actacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg
     1801 gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt
     1861 tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     1921 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     1981 tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat
     2041 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     2101 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     2161 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     2221 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     2281 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     2341 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     2401 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     2461 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     2521 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     2581 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     2641 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     2701 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     2761 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     2821 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     2881 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     2941 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     3001 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     3061 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     3121 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     3181 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     3241 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     3301 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     3361 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     3421 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     3481 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     3541 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     3601 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     3661 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     3721 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     3781 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     3841 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     3901 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     3961 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     4021 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     4081 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     4141 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     4201 tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga
     4261 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg
     4321 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct
     4381 gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca
     4441 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
     4501 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
     4561 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
     4621 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
     4681 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
     4741 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
     4801 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
     4861 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
     4921 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
     4981 cggtaggcgt gtacggtggg aggtctatat aagcagagct ctccctatca gtgatagaga
     5041 tctccctatc agtgatagag atcgtcgacg agctcgttta gtgaaccgtc agatcgcctg
     5101 gagacgccat ccacgctgtt ttgacctcca tagaagacac cgggaccgat ccagcctccg
     5161 gactctagcg tttaaactta agcttggtac cgagctcgga tccactagtc cagtgtggtg
     5221 gaattctgca gatatccagc acagtggcgg ccgctcgaga ccatgaagga caacaccgtg
     5281 cccctgaagc tgatcgccct gctggccaac ggcgagttcc actctggcga gcagctgggc
     5341 gagacactgg gcatgagcag agccgccatc aacaagcaca tccagaccct gcgggactgg
     5401 ggcgtggacg tgttcacagt gcccggcaag ggctacagcc tgcccgagcc catccagctg
     5461 ctgaacgcca agcagatcct gggccagctg gatggcggat ctgtggccgt gctgcccgtg
     5521 atcgacagca ccaaccagta tctgctcgac cggatcggcg agctgaagtc cggcgacgcc
     5581 tgtatcgccg agtaccagca ggccggcaga ggcggcaggg gcagaaagtg gttcagcccc
     5641 ttcggcgcca acctgtacct gagcatgttc tggagactgg aacagggccc agccgccgct
     5701 attggcctga gcctggtcat cggcatcgtg atggccgagg tgctgcggaa gctgggcgcc
     5761 gacaaagtgc gcgtgaagtg gcccaacgac ctgtatctgc aggaccggaa gctggccggc
     5821 atcctggtgg aactgaccgg caaaacaggc gacgccgccc agatcgtgat cggcgctggc
     5881 atcaacatgg ccatgcggcg ggtggaagag agcgtggtca accagggctg gatcaccctg
     5941 caggaagccg gcatcaacct ggaccggaac accctggccg ccatgctgat cagagagctg
     6001 agagccgccc tggaactgtt cgagcaggaa ggcctggccc cctacctgag cagatgggag
     6061 aagctggaca acttcatcaa ccggcccgtg aaactgatca tcggcgacaa agagatcttc
     6121 ggcatcagcc ggggcatcga caagcagggg gccctgctcc tggaacagga cggcatcatc
     6181 aagccctgga tgggcggcga gatcagcctg cggagcgccg agaagtaccc atacgatgtt
     6241 cctgactatg ccggtaccga gctcggatcc accatggcta gctggagcca cccgcagttc
     6301 gagaaaggtg gaggttccgg aggtggatcg ggaggtggat cgtggagcca cccgcagttc
     6361 gaaaaagcgg ccgatatcac aagtttgtac aaaaaagcag gctccaccat ggcatactgc
     6421 tggagatgca caagctgttg ttttagtgag cgattccaga atcacaaccc acaaaaagaa
     6481 atggcgacct ca
//
