LOCUS       sequence-341538-        7867 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express SARS-CoV-2 Nucleocapsid protein tagged with 3xHA and in 
            frame with mcherry (NCP-IRES-mcherry) in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7867)
  AUTHORS   Fareh M, Zhao W, Hu W, Casan JML, Kumar A, Symons J, Zerbato JM, 
            Fong D, Voskoboinik I, Ekert PG, Rudraraju R, Purcell DFJ, Lewin SR,
            Trapani JA
  TITLE     Reprogrammed CRISPR-Cas13b suppresses SARS-CoV-2 replication and 
            circumvents its mutational escape through mismatch tolerance.
  JOURNAL   Nat Commun. 2021 Jul 13;12(1):4270. doi: 10.1038/s41467-021-24577-9.
  PUBMED    34257311
REFERENCE   2  (bases 1 to 7867)
  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 
            Commun."; date: "2021-07-13"; volume: "12(1):4270. doi"; pages: " 
            10.1038/s41467-021-24577-9"
COMMENT     Sequence Label: SARS-CoV-2 Nucleocapsid-3xHA-IRES-mcherry
FEATURES             Location/Qualifiers
     source          1..7867
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    170..511
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             578..994
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     924..946
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     962..978
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     CDS             2282..2362
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     misc_feature    2427..2976
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2571..2588)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2798..2817
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     primer_bind     complement(3124..3142)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3392..3411
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     LTR             3851..4365
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    4527..4543
                     /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(4551..4557,4558..4575,4576..4581))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4596..4617
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4734..4751)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4905..5493)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4985..5004)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5664..6455,6456..6524))
                     /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     6287..6306
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6525..6629)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6697..6715
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6753..6775)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6875..6894
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7088..7110
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7238..7257
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             join(7457..7867,1..106)
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
ORIGIN
        1 tattcccaat aaagcctctt gctgtttgca tccgaatcgt ggactcgctg atccttggga
       61 gggtctcctc agattgattg actgcccacc tcgggggtct ttcatttgga ggttccaccg
      121 agatttggag acccctgccc agggaccacc gacccccccg ccgggaggta agctggccag
      181 cggtcgtttc gtgtctgtct ctgtctttgt gcgtgtttgt gccggcatct aatgtttgcg
      241 cctgcgtctg tactagttag ctaactagct ctgtatctgg cggacccgtg gtggaactga
      301 cgagttctga acacccggcc gcaaccctgg gagacgtccc agggactttg ggggccgttt
      361 ttgtggcccg acctgaggaa gggagtcgat gtggaatccg accccgtcag gatatgtggt
      421 tctggtagga gacgagaacc taaaacagtt cccgcctccg tctgaatttt tgctttcggt
      481 ttggaaccga agccgcgcgt cttgtctgct gcagcgctgc agcatcgttc tgtgttgtct
      541 ctgtctgact gtgtttctgt atttgtctga aaattagggc cagactgtta ccactccctt
      601 aagtttgacc ttaggtcact ggaaagatgt cgagcggatc gctcacaacc agtcggtaga
      661 tgtcaagaag agacgttggg ttaccttctg ctctgcagaa tggccaacct ttaacgtcgg
      721 atggccgcga gacggcacct ttaaccgaga cctcatcacc caggttaaga tcaaggtctt
      781 ttcacctggc ccgcatggac acccagacca ggtcccctac atcgtgacct gggaagcctt
      841 ggcttttgac ccccctccct gggtcaagcc ctttgtacac cctaagcctc cgcctcctct
      901 tcctccatcc gccccgtctc tcccccttga acctcctcgt tcgaccccgc ctcgatcctc
      961 cctttatcca gccctcactc cttctctagg cgccggaatt cgtttaaacg gatccatgtc
     1021 tgataatgga ccccaaaatc agcgaaatgc accccgcatt acgtttggtg gaccctcaga
     1081 ttcaactggc agtaaccaga atggagaacg cagtggggcg cgatcaaaac aacgtcggcc
     1141 ccaaggttta cccaataata ctgcgtcttg gttcaccgct ctcactcaac atggcaagga
     1201 agaccttaaa ttccctcgag gacaaggcgt tccaattaac accaatagca gtccagatga
     1261 ccaaattggc tactaccgaa gagctaccag acgaattcgt ggtggtgacg gtaaaatgaa
     1321 agatctcagt ccaagatggt atttctacta cctaggaact gggccagaag ctggacttcc
     1381 ctatggtgct aacaaagacg gcatcatatg ggttgcaact gagggagcct tgaatacacc
     1441 aaaagatcac attggcaccc gcaatcctgc taacaatgct gcaatcgtgc tacaacttcc
     1501 tcaaggaaca acattgccaa aaggcttcta cgcagaaggg agcagaggcg gcagtcaagc
     1561 ctcttctcgt tcctcatcac gtagtcgcaa cagttcaaga aattcaactc caggcagcag
     1621 taggggaact tctcctgcta gaatggctgg caatggcggt gatgctgctc ttgctttgct
     1681 gctgcttgac agattgaacc agcttgagag caaaatgtct ggtaaaggcc aacaacaaca
     1741 aggccaaact gtcactaaga aatctgctgc tgaggcttct aagaagcctc ggcaaaaacg
     1801 tactgccact aaagcataca atgtaacaca agctttcggc agacgtggtc cagaacaaac
     1861 ccaaggaaat tttggggacc aggaactaat cagacaagga actgattaca aacattggcc
     1921 gcaaattgca caatttgccc ccagcgcttc agcgttcttc ggaatgtcgc gcattggcat
     1981 ggaagtcaca ccttcgggaa cgtggttgac ctacacaggt gccatcaaat tggatgacaa
     2041 agatccaaat ttcaaagatc aagtcatttt gctgaataag catattgacg catacaaaac
     2101 attcccacca acagagccta aaaaggacaa aaagaagaag gctgatgaaa ctcaagcctt
     2161 accgcagaga cagaagaaac agcaaactgt gactcttctt cctgctgcag atttggatga
     2221 tttctccaaa caattgcaac aatccatgag cagtgctgac tcaactcagg ccggtagttc
     2281 ctacccatac gatgttccag attacgctta tccctacgac gtgcctgatt atgcataccc
     2341 atatgatgtc cccgactatg cctaaggatc cagatctcca gcacagtgga cgcgtctcga
     2401 gcgggatcaa ttccgccccc cccctaacgt tactggccga agccgcttgg aataaggccg
     2461 gtgtgcgttt gtctatatgt tattttccac catattgccg tcttttggca atgtgagggc
     2521 ccggaaacct ggccctgtct tcttgacgag cattcctagg ggtctttccc ctctcgccaa
     2581 aggaatgcaa ggtctgttga atgtcgtgaa ggaagcagtt cctctggaag cttcttgaag
     2641 acaaacaacg tctgtagcga ccctttgcag gcagcggaac cccccacctg gcgacaggtg
     2701 cctctgcggc caaaagccac gtgtataaga tacacctgca aaggcggcac aaccccagtg
     2761 ccacgttgtg agttggatag ttgtggaaag agtcaaatgg ctctcctcaa gcgtattcaa
     2821 caaggggctg aaggatgccc agaaggtacc ccattgtatg ggatctgatc tggggcctcg
     2881 gtgcacatgc tttacatgtg tttagtcgag gttaaaaaaa cgtctaggcc ccccgaacca
     2941 cggggacgtg gttttccttt gaaaaacacg ataataccat ggtgagcatg gacgaggagg
     3001 ataacatggc catcatcaag gagttcatgc gcttcaaggt gcacatggag ggctccgtga
     3061 acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga
     3121 ccgccaagct gaaggtgacc aagggtggcc ccctgccctt cgcctgggac atcctgtccc
     3181 ctcagttcat gtacggctcc aaggcctacg tgaagcaccc cgccgacatc cccgactact
     3241 tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg
     3301 gcgtggtgac cgtgacccag gactcctccc tgcaggacgg cgagttcatc tacaaggtga
     3361 agctgcgcgg caccaacttc ccctccgacg gccccgtaat gcagaagaag accatgggct
     3421 gggaggcctc ctccgagcgg atgtaccccg aggacggcgc cctgaagggc gagatcaagc
     3481 agaggctgaa gctgaaggac ggcggccact acgacgctga ggtcaagacc acctacaagg
     3541 ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa catcaagttg gacatcacct
     3601 cccacaacga ggactacacc atcgtggaac agtacgaacg cgccgagggc cgccactcca
     3661 ccggcggcat ggacgagctg tacaagtaaa gcggccgcga ctctagagtc gacctgcagg
     3721 catgcaagct tcaggtagcc ggctaacgtt aacaaccggt acctctagaa ctatagctag
     3781 catgcgcaaa tttaaagcgc tgatatcgat aaaataaaag attttattta gtctccagaa
     3841 aaagggggga atgaaagacc ccacctgtag gtttggcaag ctagcttaag taacgccatt
     3901 ttgcaaggca tggaaaatac ataactgaga atagagaagt tcagatcaag gttaggaaca
     3961 gagagacagc agaatatggg ccaaacagga tatctgtggt aagcagttcc tgccccggct
     4021 cagggccaag aacagatggt ccccagatgc ggtcccgccc tcagcagttt ctagagaacc
     4081 atcagatgtt tccagggtgc cccaaggacc tgaaatgacc ctgtgcctta tttgaactaa
     4141 ccaatcagtt cgcttctcgc ttctgttcgc gcgcttctgc tccccgagct caataaaaga
     4201 gcccacaacc cctcactcgg cgcgccagtc ctccgataga ctgcgtcgcc cgggtacccg
     4261 tgtatccaat aaaccctctt gcagttgcat ccgacttgtg gtctcgctgt tccttgggag
     4321 ggtctcctct gagtgattga ctacccgtca gcgggggtct ttcatgggta acagtttctt
     4381 gaagttggag aacaacattc tgagggtagg agtcgaatat taagtaatcc tgactcaatt
     4441 agccactgtt ttgaatccac atactccaat actcctgaaa tagttcatta tggacagcgc
     4501 agaaagagct ggggagaatt gtgaaattgt tatccgctca caattccaca caacatacga
     4561 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     4621 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     4681 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     4741 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     4801 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     4861 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     4921 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     4981 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     5041 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     5101 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     5161 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     5221 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     5281 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     5341 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     5401 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     5461 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     5521 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     5581 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     5641 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     5701 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     5761 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     5821 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     5881 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     5941 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     6001 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     6061 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     6121 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     6181 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     6241 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     6301 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     6361 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     6421 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     6481 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     6541 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     6601 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     6661 taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt
     6721 cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg
     6781 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg
     6841 ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga
     6901 gtgcaccata tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg
     6961 cgccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg
     7021 ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca
     7081 gggttttccc agtcacgacg ttgtaaaacg acggcgcaag gaatggtgca tgcaaggaga
     7141 tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc
     7201 tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc
     7261 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggcgat
     7321 tagtccaatt tgttaaagac aggatatcag tggtccaggc tctagttttg actcaacaat
     7381 atcaccagct gaagcctata gagtacgagc catagataaa ataaaagatt ttatttagtc
     7441 tccagaaaaa ggggggaatg aaagacccca cctgtaggtt tggcaagcta gcttaagtaa
     7501 cgccattttg caaggcatgg aaaatacata actgagaata gagaagttca gatcaaggtt
     7561 aggaacagag agacagcaga atatgggcca aacaggatat ctgtggtaag cagttcctgc
     7621 cccggctcag ggccaagaac agatggtccc cagatgcggt cccgccctca gcagtttcta
     7681 gagaaccatc agatgtttcc agggtgcccc aaggacctga aatgaccctg tgccttattt
     7741 gaactaacca atcagttcgc ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa
     7801 taaaagagcc cacaacccct cactcggcgc gccagtcctc cgatagactg cgtcgcccgg
     7861 gtacccg
//
