LOCUS       sequence-309561-        6621 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 6621)
  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 6621)
  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 Orf9b
FEATURES             Location/Qualifiers
     source          1..6621
                     /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(1124..1148)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1195..1218
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1255..1278
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1309..1335
                     /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             1309..1335
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(1336..1683,1684..1686,1687..2295)
                     /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(2339..2356)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2345..2569
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2702..2721)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2853..2900
                     /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             2908..3930
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2908..3930
                     /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    4060..4181
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4097..4116)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4151..4170
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4230..4246)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4230..4246)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4243..4265)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4254..4270
                     /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(4278..4284,4285..4302,4303..4308))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4323..4344
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4461..4478)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4632..5220)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4712..4731)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5391..6182,6183..6251))
                     /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     6014..6033
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6252..6356)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6431..6450)
                     /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 catggacccc
      841 aaaatttctg aaatgcaccc ggcgcttcgc ttggtggacc cccaaatcca actcgctgtc
      901 acgcggatgg aaaacgcagt aggacgcgac caaaataatg tcggcccaaa agtgtatccg
      961 attatcctcc gccttggcag tcccctgagc ctgaatatgg ccagaaagac tcttaacagc
     1021 ctggaggaca aagcgtttca gctgaccccc attgcggttc agatgactaa gctggcgaca
     1081 actgaagagt tgcccgacga atttgtggta gtaaccgtaa aggacccagc tttcttgtac
     1141 aaagtggttg atatcaaggc cgccgacatc accagcctgt acaagaaagt ggggtggagc
     1201 catccccagt tcgagaaggg cggaggatct ggcggcggaa gcggcggagg aagctggtcc
     1261 caccctcagt ttgagaaagg caccgagctg ggcagcacca tggccagcta cccctacgac
     1321 gtgcccgact acgccaagga caacaccgtg cccctgaagc tgatcgccct gctggccaac
     1381 ggcgagttcc actctggcga gcagctgggc gagacactgg gcatgagcag agccgccatc
     1441 aacaagcaca tccagaccct gcgggactgg ggcgtggacg tgttcacagt gcccggcaag
     1501 ggctacagcc tgcccgagcc catccagctg ctgaacgcca agcagatcct gggccagctg
     1561 gatggcggat ctgtggccgt gctgcccgtg atcgacagca ccaaccagta tctgctcgac
     1621 cggatcggcg agctgaagtc cggcgacgcc tgtatcgccg agtaccagca ggccggcaga
     1681 ggcggcaggg gcagaaagtg gttcagcccc ttcggcgcca acctgtacct gagcatgttc
     1741 tggagactgg aacagggccc agccgccgct attggcctga gcctggtcat cggcatcgtg
     1801 atggccgagg tgctgcggaa gctgggcgcc gacaaagtgc gcgtgaagtg gcccaacgac
     1861 ctgtatctgc aggaccggaa gctggccggc atcctggtgg aactgaccgg caaaacaggc
     1921 gacgccgccc agatcgtgat cggcgctggc atcaacatgg ccatgcggcg ggtggaagag
     1981 agcgtggtca accagggctg gatcaccctg caggaagccg gcatcaacct ggaccggaac
     2041 accctggccg ccatgctgat cagagagctg agagccgccc tggaactgtt cgagcaggaa
     2101 ggcctggccc cctacctgag cagatgggag aagctggaca acttcatcaa ccggcccgtg
     2161 aaactgatca tcggcgacaa agagatcttc ggcatcagcc ggggcatcga caagcagggg
     2221 gccctgctcc tggaacagga cggcatcatc aagccctgga tgggcggcga gatcagcctg
     2281 cggagcgccg agaagtgatg actcgagtct agagggcccg tttaaacccg ctgatcagcc
     2341 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     2401 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
     2461 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
     2521 gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg
     2581 gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc
     2641 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     2701 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     2761 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     2821 aaacttgatt agggtgatgg ttcacgtacc tagaagttcc tattccgaag ttcctattct
     2881 ctagaaagta taggaacttc cttggccaaa aagcctgaac tcaccgcgac gtctgtcgag
     2941 aagtttctga tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc ggagggcgaa
     3001 gaatctcgtg ctttcagctt cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc
     3061 tgcgccgatg gtttctacaa agatcgttat gtttatcggc actttgcatc ggccgcgctc
     3121 ccgattccgg aagtgcttga cattggggaa ttcagcgaga gcctgaccta ttgcatctcc
     3181 cgccgtgcac agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctg
     3241 cagccggtcg cggaggccat ggatgcgatc gctgcggccg atcttagcca gacgagcggg
     3301 ttcggcccat tcggaccgca aggaatcggt caatacacta catggcgtga tttcatatgc
     3361 gcgattgctg atccccatgt gtatcactgg caaactgtga tggacgacac cgtcagtgcg
     3421 tccgtcgcgc aggctctcga tgagctgatg ctttgggccg aggactgccc cgaagtccgg
     3481 cacctcgtgc acgcggattt cggctccaac aatgtcctga cggacaatgg ccgcataaca
     3541 gcggtcattg actggagcga ggcgatgttc ggggattccc aatacgaggt cgccaacatc
     3601 ttcttctgga ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt cgagcggagg
     3661 catccggagc ttgcaggatc gccgcggctc cgggcgtata tgctccgcat tggtcttgac
     3721 caactctatc agagcttggt tgacggcaat ttcgatgatg cagcttgggc gcagggtcga
     3781 tgcgacgcaa tcgtccgatc cggagccggg actgtcgggc gtacacaaat cgcccgcaga
     3841 agcgcggccg tctggaccga tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc
     3901 cccagcactc gtccgagggc aaaggaatag cacgtactac gagatttcga ttccaccgcc
     3961 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     4021 cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat
     4081 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     4141 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg
     4201 acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     4261 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     4321 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     4381 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     4441 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     4501 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     4561 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     4621 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     4681 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     4741 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     4801 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     4861 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     4921 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     4981 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     5041 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     5101 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     5161 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     5221 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     5281 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     5341 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     5401 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     5461 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     5521 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     5581 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     5641 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     5701 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     5761 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     5821 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     5881 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     5941 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     6001 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     6061 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     6121 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     6181 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     6241 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     6301 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     6361 tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt
     6421 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt
     6481 gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct
     6541 tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg
     6601 tacgggccag atatacgcgt t
//
