LOCUS       sequence-377394-        6997 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of MBP-tagged SARS-CoV-2 Nsp1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6997)
  AUTHORS   Vora SM, Fontana P, Mao T, Leger V, Zhang Y, Fu TM, Lieberman J, 
            Gehrke L, Shi M, Wang L, Iwasaki A, Wu H
  TITLE     Targeting stem-loop 1 of the SARS-CoV-2 5' UTR to suppress viral 
            translation and Nsp1 evasion.
  JOURNAL   Proc Natl Acad Sci U S A. 2022 Mar 1;119(9). pii: 2117198119. doi: 
            10.1073/pnas.2117198119.
  PUBMED    35149555
REFERENCE   2  (bases 1 to 6997)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2022-03-1"; volume: "119(9). pii: 
            2117198119. doi"; pages: " 10.1073/pnas.2117198119"
COMMENT     Sequence Label: pCMV-Dest-MBP-Nsp1
FEATURES             Location/Qualifiers
     source          1..6997
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        141..520
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        521..724
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     675..695
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      771..780
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             789..812
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             822..1922
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     1896..1919
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1977..1997
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     polyA_signal    2700..2834
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2737..2756)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2791..2810
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     mobile_element  2863..3028
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     rep_origin      3212..3666
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3299..3318)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3508..3529
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3692..3796
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3797..3865,3866..4657)
                     /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     complement(4015..4034)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4828..5416
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5317..5336
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5570..5587
                     /label=L4440
                     /note="L4440 vector, forward primer"
     mobile_element  5721..5945
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(6012..6545)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(6734..6739,6740..6756,6757..6762))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
ORIGIN
        1 accccggatg aagtggttcg catcctcggt tttctggaag gcgagcatcg tttgttcgcc
       61 caggactcta gctatagttc tagtggttgg ctacgtatac tccggactgc ttcgcgatgt
      121 acgggccaga tatacgcgtt gacattgatt attgactagt tattaatagt aatcaattac
      181 ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg
      241 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc
      301 catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac
      361 tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa
      421 tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac
      481 ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta
      541 catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga
      601 cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa
      661 ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag
      721 agctctctgg ctaactagag aacccactgc ttactggctt atcgaaagcc gccaccatgg
      781 gttcttctca ccatcaccat caccatcacc atggttcttc tatgaaaatc gaagaaggta
      841 aactggtaat ctggattaac ggcgataaag gctataacgg tctcgctgaa gtcggtaaga
      901 aattcgagaa agataccgga attaaagtca ccgttgagca cccggataaa ctggaagaga
      961 aattcccaca ggttgcggca actggcgatg gccctgacat tatcttctgg gcacacgacc
     1021 gctttggtgg ctacgctcaa tctggcctgt tggctgaaat caccccggac aaagcgttcc
     1081 aggacaagct gtatccgttt acctgggatg ccgtacgtta caacggcaag ctgattgctt
     1141 acccgatcgc tgttgaagcg ttatcgctga tttataacaa agacctgctg ccgaacccgc
     1201 caaaaacctg ggaagagatc ccggcgctgg ataaagaact gaaagcgaaa ggtaagagcg
     1261 cgctgatgtt caacctgcaa gaaccgtact tcacctggcc gctgattgct gctgacgggg
     1321 gttatgcgtt caagtatgaa aacggcaagt acgacattaa agacgtgggc gtggataacg
     1381 ctggcgcgaa agcgggtctg accttcctgg ttgacctgat taaaaacaaa cacatgaatg
     1441 cagacaccga ttactccatc gcagaagctg cctttaataa aggcgaaaca gcgatgacca
     1501 tcaacggccc gtgggcatgg tccaacatcg acaccagcaa agtgaattat ggtgtaacgg
     1561 tactgccgac cttcaagggt caaccatcca aaccgttcgt tggcgtgctg agcgcaggta
     1621 ttaacgccgc cagtccgaac aaagagctgg caaaagagtt cctcgaaaac tatctgctga
     1681 ctgatgaagg tctggaagcg gttaataaag acaaaccgct gggtgccgta gcgctgaagt
     1741 cttacgagga agagttggcg aaagatccac gtattgccgc cactatggaa aacgcccaga
     1801 aaggtgaaat catgccgaac atcccgcaga tgtccgcttt ctggtatgcc gtgcgtactg
     1861 cggtgatcaa cgccgccagc ggtcgtcaga ctgtcgatga agccctgaaa gacgcgcaga
     1921 ctaattcgag ctcgaacaac aacaacaata acaataacaa caacctcggg atcgaggaaa
     1981 acctgtattt tcagggcgcc atgggatcca tggagagtct tgtacccggc tttaacgaga
     2041 aaacacatgt tcaattgtca ttgcctgtgc tgcaagtccg ggacgtactg gttagggggt
     2101 tcggagattc agtagaagag gtgctgtctg aggcgaggca acatttgaaa gacgggacgt
     2161 gcgggctggt tgaggtggaa aagggagtac ttccacagtt ggaacaaccg tatgttttta
     2221 ttaagcggag tgatgccagg actgcgccgc acggtcatgt catggttgag cttgtcgcag
     2281 agcttgaagg cattcaatat gggcgctccg gtgaaactct tggagtactt gtgccgcacg
     2341 taggcgaaat tccagtggca tatcggaagg tgctgttgag aaaaaatggg aacaagggcg
     2401 cagggggcca cagttatgga gccgacctca aatcctttga ccttggcgac gagttgggga
     2461 cagatcccta cgaggatttc caggaaaact ggaatacgaa acatagcagt ggcgttactc
     2521 gcgagttgat gcgcgaactg aacggcggct aactcgaggc atgcggtacc aagcttgtcg
     2581 agaagtacta gaggatcata atcagccata ccacatttgt agaggtttta cttgctttaa
     2641 aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta
     2701 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     2761 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     2821 atcatgtctg gatctgatca ctgcttgagc ctaggagatc cgaaccagat aagtgaaatc
     2881 tagttccaaa ctattttgtc atttttaatt ttcgtattag cttacgacgc tacacccagt
     2941 tcccatctat tttgtcactc ttccctaaat aatccttaaa aactccattt ccacccctcc
     3001 cagttcccaa ctattttgtc cgcccacagc ggggcatttt tcttcctgtt atgtttttaa
     3061 tcaaacatcc tgccaactcc atgtgacaaa ccgtcatctt cggctacttt ttctctgtca
     3121 cagaatgaaa atttttctgt catctcttcg ttattaatgt ttgtaattga ctgaatatca
     3181 acgcttattt gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg
     3241 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     3301 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     3361 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     3421 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     3481 ttgacgttgg agtccacgtt cttaatagtg gactcttgtt ccaaactgga acaacactca
     3541 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     3601 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta
     3661 caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     3721 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     3781 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     3841 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     3901 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     3961 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     4021 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     4081 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     4141 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     4201 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     4261 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     4321 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     4381 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     4441 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     4501 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     4561 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     4621 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     4681 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     4741 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     4801 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     4861 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     4921 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     4981 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     5041 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     5101 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     5161 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     5221 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     5281 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     5341 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     5401 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     5461 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     5521 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     5581 gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     5641 ttcacaccgc atagaccagc cgcgtaacct ggcaaaatcg gttacggttg agtaataaat
     5701 ggatgccctg cgtaagcggg tgtgggcgga caataaagtc ttaaactgaa caaaatagat
     5761 ctaaactatg acaataaagt cttaaactag acagaatagt tgtaaactga aatcagtcca
     5821 gttatgctgt gaaaaagcat actggacttt tgttatggct aaagcaaact cttcattttc
     5881 tgaagtgcaa attgcccgtc gtattaaaga ggggcgtggc caagggcatg gtaaagacta
     5941 tattcgcggc gttgtgacaa tttaccgaac aactccgcgg ccgggaagcc gatctcggct
     6001 tgaacgaatt gttaggtggc ggtacttggg tcgatatcaa agtgcatcac ttcttcccgt
     6061 atgcccaact ttgtatagag agccactgcg ggatcgtcac cgtaatctgc ttgcacgtag
     6121 atcacataag caccaagcgc gttggcctca tgcttgagga gattgatgag cgcggtggca
     6181 atgccctgcc tccggtgctc gccggagact gcgagatcat agatatagat ctcactacgc
     6241 ggctgctcaa acttgggcag aacgtaagcc gcgagagcgc caacaaccgc ttcttggtcg
     6301 aaggcagcaa gcgcgatgaa tgtcttacta cggagcaagt tcccgaggta atcggagtcc
     6361 ggctgatgtt gggagtaggt ggctacgtct ccgaactcac gaccgaaaag atcaagagca
     6421 gcccgcatgg atttgacttg gtcagggccg agcctacatg tgcgaatgat gcccatactt
     6481 gagccaccta actttgtttt agggcgactg ccctgctgcg taacatcgtt gctgctgcgt
     6541 aacatcgttg ctgctccata acatcaaaca tcgacccacg gcgtaacgcg cttgctgctt
     6601 ggatgcccga ggcatagact gtacaaaaaa acagtcataa caagccatga aaaccgccac
     6661 tgcgccgtta ccaccgctgc gttcggtcaa ggttctggac cagttgcgtg agcgcatacg
     6721 ctacttgcat tacagtttac gaaccgaaca ggcttatgtc aactgggttc gtgccttcat
     6781 ccgtttccac ggtgtgcgtc acccggcaac cttgggcagc agcgaagtcg aggcatttct
     6841 gtcctggctg gcgaacgagc gcaaggtttc ggtctccacg catcgtcagg cattggcggc
     6901 cttgctgttc ttctacggca aggtgctgtg cacggatctg ccctggcttc aggagatcgg
     6961 aagacctcgg ccgtcgcggc gcttgccggt ggtgctg
//
