LOCUS       sequence-310077-        6672 bp    DNA     circular SYN 14-APR-2025
DEFINITION  MAC-tagged gene expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6672)
  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 6672)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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 NSP9
FEATURES             Location/Qualifiers
     source          1..6672
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        77..456
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        457..660
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     611..631
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    662..680
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    683..701
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     711..735
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    878..902
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1251..1275)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1322..1345
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1382..1405
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1436..1462
                     /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             1436..1462
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(1463..1810,1811..1813,1814..2422)
                     /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(2466..2483)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2472..2696
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2829..2848)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2980..3027
                     /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             3035..4057
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3035..4057
                     /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    4187..4308
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4224..4243)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4278..4297
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4357..4373)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4357..4373)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4370..4392)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4381..4397
                     /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(4405..4411,4412..4429,4430..4435))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4450..4471
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4588..4605)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4759..5347)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4839..4858)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5518..6309,6310..6378))
                     /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     6141..6160
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6379..6483)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6558..6577)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacaattgca tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg
       61 gccagatata cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg
      121 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg
      181 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
      241 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
      301 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
      361 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg
      421 cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc
      481 aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc
      541 aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc
      601 gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct
      661 ctccctatca gtgatagaga tctccctatc agtgatagag atcgtcgacg agctcgttta
      721 gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagacac
      781 cgggaccgat ccagcctccg gactctagcg tttaaactta agcttggtac cgagctcgga
      841 tccactagtc cagtgtggtg gaattctgca gatatcaaca agtttgtaca aaaaagcagg
      901 ctccaccatg aataatgagc tttctccggt tgctctgcga cagatgtcct gcgcggctgg
      961 aacaacacaa actgcctgta ctgacgacaa cgcactggcg tactataata ccaccaaagg
     1021 aggtcgattt gtattggcac ttctgtccga cttgcaggac cttaaatggg cgaggtttcc
     1081 caagagtgac ggaacgggca caatttacac ggagctggag cccccatgcc ggtttgtgac
     1141 tgatactcca aaaggtccta aagtaaaata cttgtatttt attaaaggtt tgaataattt
     1201 gaacaggggt atggtactcg gctccctggc tgcgaccgtg cgactccaag acccagcttt
     1261 cttgtacaaa gtggttgata tcaaggccgc cgacatcacc agcctgtaca agaaagtggg
     1321 gtggagccat ccccagttcg agaagggcgg aggatctggc ggcggaagcg gcggaggaag
     1381 ctggtcccac cctcagtttg agaaaggcac cgagctgggc agcaccatgg ccagctaccc
     1441 ctacgacgtg cccgactacg ccaaggacaa caccgtgccc ctgaagctga tcgccctgct
     1501 ggccaacggc gagttccact ctggcgagca gctgggcgag acactgggca tgagcagagc
     1561 cgccatcaac aagcacatcc agaccctgcg ggactggggc gtggacgtgt tcacagtgcc
     1621 cggcaagggc tacagcctgc ccgagcccat ccagctgctg aacgccaagc agatcctggg
     1681 ccagctggat ggcggatctg tggccgtgct gcccgtgatc gacagcacca accagtatct
     1741 gctcgaccgg atcggcgagc tgaagtccgg cgacgcctgt atcgccgagt accagcaggc
     1801 cggcagaggc ggcaggggca gaaagtggtt cagccccttc ggcgccaacc tgtacctgag
     1861 catgttctgg agactggaac agggcccagc cgccgctatt ggcctgagcc tggtcatcgg
     1921 catcgtgatg gccgaggtgc tgcggaagct gggcgccgac aaagtgcgcg tgaagtggcc
     1981 caacgacctg tatctgcagg accggaagct ggccggcatc ctggtggaac tgaccggcaa
     2041 aacaggcgac gccgcccaga tcgtgatcgg cgctggcatc aacatggcca tgcggcgggt
     2101 ggaagagagc gtggtcaacc agggctggat caccctgcag gaagccggca tcaacctgga
     2161 ccggaacacc ctggccgcca tgctgatcag agagctgaga gccgccctgg aactgttcga
     2221 gcaggaaggc ctggccccct acctgagcag atgggagaag ctggacaact tcatcaaccg
     2281 gcccgtgaaa ctgatcatcg gcgacaaaga gatcttcggc atcagccggg gcatcgacaa
     2341 gcagggggcc ctgctcctgg aacaggacgg catcatcaag ccctggatgg gcggcgagat
     2401 cagcctgcgg agcgccgaga agtgatgact cgagtctaga gggcccgttt aaacccgctg
     2461 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     2521 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     2581 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     2641 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc
     2701 tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc
     2761 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     2821 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     2881 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     2941 ccccaaaaaa cttgattagg gtgatggttc acgtacctag aagttcctat tccgaagttc
     3001 ctattctcta gaaagtatag gaacttcctt ggccaaaaag cctgaactca ccgcgacgtc
     3061 tgtcgagaag tttctgatcg aaaagttcga cagcgtctcc gacctgatgc agctctcgga
     3121 gggcgaagaa tctcgtgctt tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt
     3181 aaatagctgc gccgatggtt tctacaaaga tcgttatgtt tatcggcact ttgcatcggc
     3241 cgcgctcccg attccggaag tgcttgacat tggggaattc agcgagagcc tgacctattg
     3301 catctcccgc cgtgcacagg gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc
     3361 tgttctgcag ccggtcgcgg aggccatgga tgcgatcgct gcggccgatc ttagccagac
     3421 gagcgggttc ggcccattcg gaccgcaagg aatcggtcaa tacactacat ggcgtgattt
     3481 catatgcgcg attgctgatc cccatgtgta tcactggcaa actgtgatgg acgacaccgt
     3541 cagtgcgtcc gtcgcgcagg ctctcgatga gctgatgctt tgggccgagg actgccccga
     3601 agtccggcac ctcgtgcacg cggatttcgg ctccaacaat gtcctgacgg acaatggccg
     3661 cataacagcg gtcattgact ggagcgaggc gatgttcggg gattcccaat acgaggtcgc
     3721 caacatcttc ttctggaggc cgtggttggc ttgtatggag cagcagacgc gctacttcga
     3781 gcggaggcat ccggagcttg caggatcgcc gcggctccgg gcgtatatgc tccgcattgg
     3841 tcttgaccaa ctctatcaga gcttggttga cggcaatttc gatgatgcag cttgggcgca
     3901 gggtcgatgc gacgcaatcg tccgatccgg agccgggact gtcgggcgta cacaaatcgc
     3961 ccgcagaagc gcggccgtct ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa
     4021 ccgacgcccc agcactcgtc cgagggcaaa ggaatagcac gtactacgag atttcgattc
     4081 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     4141 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc
     4201 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     4261 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat
     4321 accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     4381 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     4441 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     4501 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     4561 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     4621 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     4681 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     4741 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     4801 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     4861 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     4921 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     4981 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     5041 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     5101 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     5161 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     5221 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     5281 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     5341 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     5401 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     5461 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     5521 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     5581 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     5641 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     5701 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     5761 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     5821 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     5881 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     5941 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     6001 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     6061 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     6121 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     6181 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     6241 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     6301 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     6361 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     6421 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     6481 gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc tcccgatccc
     6541 ctatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc
     6601 cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg
     6661 caaggcttga cc
//
