LOCUS       sequence-345357-        5688 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses R99A CoV2 nsp1 in mammalian cells. This contains a 
            C-terminal 3xFLAG fusion to nsp1..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5688)
  AUTHORS   Mendez AS, Ly M, Gonzalez-Sanchez AM, Hartenian E, Ingolia NT, Cate 
            JH, Glaunsinger BA
  TITLE     The N-terminal domain of SARS-CoV-2 nsp1 plays key roles in 
            suppression of cellular gene expression and preservation of viral 
            gene expression.
  JOURNAL   Cell Rep. 2021 Oct 19;37(3):109841. doi: 
            10.1016/j.celrep.2021.109841. Epub 2021 Sep 30.
  PUBMED    34624207
REFERENCE   2  (bases 1 to 5688)
  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: "Cell Rep.";
            date: "2021-10-19"; volume: "37(3):109841. doi"; pages: " 
            10.1016/j.celrep.2021.109841. Epub 2021 Sep 30"
COMMENT     Sequence Label: pCDNA4TO R99A CoV-2 nsp1 3xFLAG
FEATURES             Location/Qualifiers
     source          1..5688
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(69..88)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        260..639
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        640..843
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     794..814
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    845..863
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    866..884
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     894..918
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1643..1708
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(1734..1751)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1740..1964
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2010..2438
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2097..2116)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2307..2328
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2447..2467)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2452..2781
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2632..2767
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2694..2713
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2882..3256
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    3386..3507
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3423..3442)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3477..3496
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3556..3572)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3556..3572)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3569..3591)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3580..3596
                     /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(3604..3610,3611..3628,3629..3634))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3649..3670
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3787..3804)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3958..4546)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4038..4057)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4717..5508,5509..5577))
                     /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     5340..5359
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5578..5682)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc cctatggtgc
       61 actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct ccctgcttgt
      121 gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg
      181 accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta
      241 cgggccagat atacgcgttg acattgatta ttgactagtt attaatagta atcaattacg
      301 gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc
      361 ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc
      421 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact
      481 gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
      541 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact
      601 tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac
      661 atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac
      721 gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac
      781 tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga
      841 gctctcccta tcagtgatag agatctccct atcagtgata gagatcgtcg acgagctcgt
      901 ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga
      961 caccgggacc gatccagcct ccggactcta gcgtttaaac ttaagcttgg taccgagctc
     1021 ggatccacta gtccagtgtg gtggaattct gcagatatcc agcacagtgg cggccatgga
     1081 gagccttgtc cctggtttca acgagaaaac acacgtccaa ctcagtttgc ctgttttaca
     1141 ggttcgcgac gtgctcgtac gtggctttgg agactccgtg gaggaggtct tatcagaggc
     1201 acgtcaacat cttaaagatg gcacttgtgg cttagtagaa gttgaaaaag gcgttttgcc
     1261 tcaacttgaa cagccctatg tgttcatcaa acgttcggat gctcgaactg cacctcatgg
     1321 tcatgttatg gttgagctgg tagcagaact cgaaggcatt cagtacggtg caagtggtga
     1381 gacacttggt gtccttgtcc ctcatgtggg cgaaatacca gtggcttacc gcaaggttct
     1441 tcttcgtaag aacggtaata aaggagctgg tggccatagt tacggcgccg atctaaagtc
     1501 atttgactta ggcgacgagc ttggcactga tccttatgaa gattttcaag aaaactggaa
     1561 cactaaacat agcagtggtg ttacccgtga actcatgcgt gagcttaacg gaggggggcc
     1621 gctcgaggga ggcggtggag ccgactacaa ggaccacgac ggcgactaca aggaccacga
     1681 catcgactac aaggacgacg acgacaaggg gcccgtttaa acccgctgat cagcctcgac
     1741 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     1801 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     1861 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     1921 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
     1981 aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc
     2041 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     2101 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     2161 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     2221 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     2281 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     2341 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     2401 aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag
     2461 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     2521 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     2581 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     2641 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     2701 gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt
     2761 ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat
     2821 cagcacgtgt tgacaattaa tcatcggcat agtataatac gacaaggtga ggaactaaac
     2881 catggccaag ttgaccagtg ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt
     2941 cgagttctgg accgaccggc tcgggttctc ccgggacttc gtggaggacg acttcgccgg
     3001 tgtggtccgg gacgacgtga ccctgttcat cagcgcggtc caggaccagg tggtgccgga
     3061 caacaccctg gcctgggtgt gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga
     3121 ggtcgtgtcc acgaacttcc gggacgcctc cgggccggcc atgaccgaga tcggcgagca
     3181 gccgtggggg cgggagttcg ccctgcgcga cccggccggc aactgcgtgc acttcgtggc
     3241 cgaggagcag gactgacacg tgctacgaga tttcgattcc accgccgcct tctatgaaag
     3301 gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct
     3361 catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata
     3421 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     3481 tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag
     3541 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     3601 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     3661 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     3721 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     3781 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     3841 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     3901 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     3961 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     4021 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     4081 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     4141 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     4201 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     4261 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     4321 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     4381 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     4441 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     4501 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     4561 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     4621 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     4681 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     4741 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     4801 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     4861 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     4921 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     4981 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     5041 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     5101 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     5161 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     5221 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     5281 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     5341 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     5401 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     5461 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     5521 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     5581 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     5641 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatt
//
