LOCUS       sequence-309556-        7155 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 7155)
  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 7155)
  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 Orf3a
FEATURES             Location/Qualifiers
     source          1..7155
                     /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(1658..1682)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1729..1752
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1789..1812
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1843..1869
                     /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             1843..1869
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(1870..2217,2218..2220,2221..2829)
                     /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(2873..2890)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2879..3103
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3236..3255)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3387..3434
                     /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             3442..4464
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3442..4464
                     /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    4594..4715
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4631..4650)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4685..4704
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4764..4780)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4764..4780)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4777..4799)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4788..4804
                     /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(4812..4818,4819..4836,4837..4842))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4857..4878
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4995..5012)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5166..5754)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5246..5265)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5925..6716,6717..6785))
                     /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     6548..6567
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6786..6890)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6965..6984)
                     /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 caggctccac catggacctt
      841 tttatgagaa tctttaccat cgggactgtc acactgaaac agggagaaat taaggacgcg
      901 accccgagtg actttgtgag ggccactgct acaattccca tccaagcctc tcttccattc
      961 ggctggctca tagtgggtgt ggcactgctc gcggtattcc agtcagcgtc taagatcata
     1021 acgctgaaga agagatggca gttggctctc agcaaaggtg ttcatttcgt ctgtaacctg
     1081 cttcttcttt tcgtcactgt ctattctcac ttgctcttgg ttgcagccgg gctggaagcc
     1141 ccgtttctgt atctgtacgc gctcgtttac ttcctccaga gcataaattt cgtacgaatt
     1201 attatgagac tctggttgtg ctggaagtgt cggagcaaga atcctctctt gtatgacgcc
     1261 aattacttcc tttgttggca cactaattgt tatgattact gcatacctta caatagcgtt
     1321 acctcatcca tagtaatcac ctccggtgac ggaacaacgt caccaatctc cgagcatgat
     1381 taccagattg gaggctatac ggaaaaatgg gagagtggtg tcaaggactg tgtggtgctg
     1441 catagctatt tcacaagcga ttattatcaa ctgtacagta cacagttgtc cacggatacc
     1501 ggggtcgagc atgtcacatt ttttatttat aacaagatcg ttgatgagcc agaggagcat
     1561 gtgcagatcc acacgataga tggaagctcc ggtgtagtga atccagtcat ggaaccgatc
     1621 tatgacgagc ccaccacgac cacatctgtg cccctggacc cagctttctt gtacaaagtg
     1681 gttgatatca aggccgccga catcaccagc ctgtacaaga aagtggggtg gagccatccc
     1741 cagttcgaga agggcggagg atctggcggc ggaagcggcg gaggaagctg gtcccaccct
     1801 cagtttgaga aaggcaccga gctgggcagc accatggcca gctaccccta cgacgtgccc
     1861 gactacgcca aggacaacac cgtgcccctg aagctgatcg ccctgctggc caacggcgag
     1921 ttccactctg gcgagcagct gggcgagaca ctgggcatga gcagagccgc catcaacaag
     1981 cacatccaga ccctgcggga ctggggcgtg gacgtgttca cagtgcccgg caagggctac
     2041 agcctgcccg agcccatcca gctgctgaac gccaagcaga tcctgggcca gctggatggc
     2101 ggatctgtgg ccgtgctgcc cgtgatcgac agcaccaacc agtatctgct cgaccggatc
     2161 ggcgagctga agtccggcga cgcctgtatc gccgagtacc agcaggccgg cagaggcggc
     2221 aggggcagaa agtggttcag ccccttcggc gccaacctgt acctgagcat gttctggaga
     2281 ctggaacagg gcccagccgc cgctattggc ctgagcctgg tcatcggcat cgtgatggcc
     2341 gaggtgctgc ggaagctggg cgccgacaaa gtgcgcgtga agtggcccaa cgacctgtat
     2401 ctgcaggacc ggaagctggc cggcatcctg gtggaactga ccggcaaaac aggcgacgcc
     2461 gcccagatcg tgatcggcgc tggcatcaac atggccatgc ggcgggtgga agagagcgtg
     2521 gtcaaccagg gctggatcac cctgcaggaa gccggcatca acctggaccg gaacaccctg
     2581 gccgccatgc tgatcagaga gctgagagcc gccctggaac tgttcgagca ggaaggcctg
     2641 gccccctacc tgagcagatg ggagaagctg gacaacttca tcaaccggcc cgtgaaactg
     2701 atcatcggcg acaaagagat cttcggcatc agccggggca tcgacaagca gggggccctg
     2761 ctcctggaac aggacggcat catcaagccc tggatgggcg gcgagatcag cctgcggagc
     2821 gccgagaagt gatgactcga gtctagaggg cccgtttaaa cccgctgatc agcctcgact
     2881 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
     2941 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
     3001 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
     3061 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga
     3121 accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg
     3181 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     3241 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     3301 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     3361 gattagggtg atggttcacg tacctagaag ttcctattcc gaagttccta ttctctagaa
     3421 agtataggaa cttccttggc caaaaagcct gaactcaccg cgacgtctgt cgagaagttt
     3481 ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc tctcggaggg cgaagaatct
     3541 cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc
     3601 gatggtttct acaaagatcg ttatgtttat cggcactttg catcggccgc gctcccgatt
     3661 ccggaagtgc ttgacattgg ggaattcagc gagagcctga cctattgcat ctcccgccgt
     3721 gcacagggtg tcacgttgca agacctgcct gaaaccgaac tgcccgctgt tctgcagccg
     3781 gtcgcggagg ccatggatgc gatcgctgcg gccgatctta gccagacgag cgggttcggc
     3841 ccattcggac cgcaaggaat cggtcaatac actacatggc gtgatttcat atgcgcgatt
     3901 gctgatcccc atgtgtatca ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc
     3961 gcgcaggctc tcgatgagct gatgctttgg gccgaggact gccccgaagt ccggcacctc
     4021 gtgcacgcgg atttcggctc caacaatgtc ctgacggaca atggccgcat aacagcggtc
     4081 attgactgga gcgaggcgat gttcggggat tcccaatacg aggtcgccaa catcttcttc
     4141 tggaggccgt ggttggcttg tatggagcag cagacgcgct acttcgagcg gaggcatccg
     4201 gagcttgcag gatcgccgcg gctccgggcg tatatgctcc gcattggtct tgaccaactc
     4261 tatcagagct tggttgacgg caatttcgat gatgcagctt gggcgcaggg tcgatgcgac
     4321 gcaatcgtcc gatccggagc cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg
     4381 gccgtctgga ccgatggctg tgtagaagta ctcgccgata gtggaaaccg acgccccagc
     4441 actcgtccga gggcaaagga atagcacgta ctacgagatt tcgattccac cgccgccttc
     4501 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     4561 ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt
     4621 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     4681 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct
     4741 agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     4801 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     4861 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     4921 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     4981 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     5041 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     5101 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     5161 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     5221 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     5281 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     5341 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     5401 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     5461 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     5521 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     5581 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     5641 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     5701 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     5761 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     5821 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     5881 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     5941 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     6001 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     6061 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     6121 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     6181 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     6241 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     6301 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     6361 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     6421 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     6481 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     6541 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     6601 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     6661 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     6721 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     6781 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     6841 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     6901 cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta tggtgcactc
     6961 tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt
     7021 tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg
     7081 acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg
     7141 ccagatatac gcgtt
//
