LOCUS       sequence-309521-        6582 bp    DNA     circular SYN 15-APR-2025
DEFINITION  MAC-tagged gene expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6582)
  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 6582)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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 NSP7
FEATURES             Location/Qualifiers
     source          1..6582
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(48..636)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(128..147)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(807..1598,1599..1667))
                     /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     1430..1449
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1668..1772)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1847..1866)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2038..2417
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2418..2621
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2572..2592
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    2623..2641
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2644..2662
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2672..2696
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    2839..2863
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(3122..3146)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3193..3216
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             3253..3276
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             3307..3333
                     /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             3307..3333
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(3334..3681,3682..3684,3685..4293)
                     /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(4337..4354)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4343..4567
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4700..4719)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4851..4898
                     /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             4906..5928
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4906..5928
                     /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    6058..6179
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6095..6114)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6149..6168
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6228..6244)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6228..6244)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6241..6263)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6252..6268
                     /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(6276..6282,6283..6300,6301..6306))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6321..6342
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6459..6476)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
       61 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
      121 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
      181 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
      241 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
      301 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
      361 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
      421 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
      481 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
      541 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
      601 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
      661 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
      721 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
      781 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
      841 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
      901 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
      961 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     1021 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     1081 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     1141 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     1201 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     1261 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     1321 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     1381 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     1441 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     1501 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     1561 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     1621 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     1681 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     1741 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     1801 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
     1861 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
     1921 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
     1981 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
     2041 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
     2101 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
     2161 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
     2221 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
     2281 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
     2341 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
     2401 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
     2461 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
     2521 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
     2581 gcggtaggcg tgtacggtgg gaggtctata taagcagagc tctccctatc agtgatagag
     2641 atctccctat cagtgataga gatcgtcgac gagctcgttt agtgaaccgt cagatcgcct
     2701 ggagacgcca tccacgctgt tttgacctcc atagaagaca ccgggaccga tccagcctcc
     2761 ggactctagc gtttaaactt aagcttggta ccgagctcgg atccactagt ccagtgtggt
     2821 ggaattctgc agatatcaac aagtttgtac aaaaaagcag gctccaccat gagcaagatg
     2881 agcgacgtaa aatgcacgtc agttgttctc ttgagcgtcc tgcaacaact cagagtggag
     2941 tctagttcca agctctgggc tcaatgcgtt cagcttcaca atgacattct gttggcgaaa
     3001 gataccacgg aggcattcga gaagatggtc tcacttctga gcgttctcct gtcaatgcaa
     3061 ggggcggtcg atatcaataa attgtgtgag gaaatgctcg acaaccgggc gacattgcag
     3121 gacccagctt tcttgtacaa agtggttgat atcaaggccg ccgacatcac cagcctgtac
     3181 aagaaagtgg ggtggagcca tccccagttc gagaagggcg gaggatctgg cggcggaagc
     3241 ggcggaggaa gctggtccca ccctcagttt gagaaaggca ccgagctggg cagcaccatg
     3301 gccagctacc cctacgacgt gcccgactac gccaaggaca acaccgtgcc cctgaagctg
     3361 atcgccctgc tggccaacgg cgagttccac tctggcgagc agctgggcga gacactgggc
     3421 atgagcagag ccgccatcaa caagcacatc cagaccctgc gggactgggg cgtggacgtg
     3481 ttcacagtgc ccggcaaggg ctacagcctg cccgagccca tccagctgct gaacgccaag
     3541 cagatcctgg gccagctgga tggcggatct gtggccgtgc tgcccgtgat cgacagcacc
     3601 aaccagtatc tgctcgaccg gatcggcgag ctgaagtccg gcgacgcctg tatcgccgag
     3661 taccagcagg ccggcagagg cggcaggggc agaaagtggt tcagcccctt cggcgccaac
     3721 ctgtacctga gcatgttctg gagactggaa cagggcccag ccgccgctat tggcctgagc
     3781 ctggtcatcg gcatcgtgat ggccgaggtg ctgcggaagc tgggcgccga caaagtgcgc
     3841 gtgaagtggc ccaacgacct gtatctgcag gaccggaagc tggccggcat cctggtggaa
     3901 ctgaccggca aaacaggcga cgccgcccag atcgtgatcg gcgctggcat caacatggcc
     3961 atgcggcggg tggaagagag cgtggtcaac cagggctgga tcaccctgca ggaagccggc
     4021 atcaacctgg accggaacac cctggccgcc atgctgatca gagagctgag agccgccctg
     4081 gaactgttcg agcaggaagg cctggccccc tacctgagca gatgggagaa gctggacaac
     4141 ttcatcaacc ggcccgtgaa actgatcatc ggcgacaaag agatcttcgg catcagccgg
     4201 ggcatcgaca agcagggggc cctgctcctg gaacaggacg gcatcatcaa gccctggatg
     4261 ggcggcgaga tcagcctgcg gagcgccgag aagtgatgac tcgagtctag agggcccgtt
     4321 taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     4381 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     4441 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     4501 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc
     4561 tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc
     4621 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     4681 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     4741 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     4801 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtaccta gaagttccta
     4861 ttccgaagtt cctattctct agaaagtata ggaacttcct tggccaaaaa gcctgaactc
     4921 accgcgacgt ctgtcgagaa gtttctgatc gaaaagttcg acagcgtctc cgacctgatg
     4981 cagctctcgg agggcgaaga atctcgtgct ttcagcttcg atgtaggagg gcgtggatat
     5041 gtcctgcggg taaatagctg cgccgatggt ttctacaaag atcgttatgt ttatcggcac
     5101 tttgcatcgg ccgcgctccc gattccggaa gtgcttgaca ttggggaatt cagcgagagc
     5161 ctgacctatt gcatctcccg ccgtgcacag ggtgtcacgt tgcaagacct gcctgaaacc
     5221 gaactgcccg ctgttctgca gccggtcgcg gaggccatgg atgcgatcgc tgcggccgat
     5281 cttagccaga cgagcgggtt cggcccattc ggaccgcaag gaatcggtca atacactaca
     5341 tggcgtgatt tcatatgcgc gattgctgat ccccatgtgt atcactggca aactgtgatg
     5401 gacgacaccg tcagtgcgtc cgtcgcgcag gctctcgatg agctgatgct ttgggccgag
     5461 gactgccccg aagtccggca cctcgtgcac gcggatttcg gctccaacaa tgtcctgacg
     5521 gacaatggcc gcataacagc ggtcattgac tggagcgagg cgatgttcgg ggattcccaa
     5581 tacgaggtcg ccaacatctt cttctggagg ccgtggttgg cttgtatgga gcagcagacg
     5641 cgctacttcg agcggaggca tccggagctt gcaggatcgc cgcggctccg ggcgtatatg
     5701 ctccgcattg gtcttgacca actctatcag agcttggttg acggcaattt cgatgatgca
     5761 gcttgggcgc agggtcgatg cgacgcaatc gtccgatccg gagccgggac tgtcgggcgt
     5821 acacaaatcg cccgcagaag cgcggccgtc tggaccgatg gctgtgtaga agtactcgcc
     5881 gatagtggaa accgacgccc cagcactcgt ccgagggcaa aggaatagca cgtactacga
     5941 gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac
     6001 gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac
     6061 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     6121 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     6181 catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt
     6241 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     6301 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     6361 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     6421 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     6481 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     6541 acagaatcag gggataacgc aggaaagaac atgtgagcaa aa
//
