LOCUS       sequence-345356-        5693 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expressing R124A K125A CoV2 nsp1 in mammalian cells. This contains a
            C-terminal 3xFLAG tag fusion to nsp1..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5693)
  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 5693)
  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: "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 R124A K125A CoV-2 nsp1 3xFLAG
FEATURES             Location/Qualifiers
     source          1..5693
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(34..53)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        225..604
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        605..808
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     759..779
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    810..828
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    831..849
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     859..883
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1613..1678
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(1704..1721)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1710..1934
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1980..2408
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2067..2086)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2277..2298
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2417..2437)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2422..2751
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2602..2737
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2664..2683
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2852..3226
                     /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    3356..3477
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3393..3412)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3447..3466
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3526..3542)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3526..3542)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3539..3561)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3550..3566
                     /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(3574..3580,3581..3598,3599..3604))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3619..3640
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3757..3774)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3928..4516)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4008..4027)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4687..5478,5479..5547))
                     /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     5310..5329
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5548..5652)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
       61 aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat
      121 ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag
      181 gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt gattattgac
      241 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
      301 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      361 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      421 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      481 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      541 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      601 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      661 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      721 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      781 acggtgggag gtctatataa gcagagctct ccctatcagt gatagagatc tccctatcag
      841 tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga gacgccatcc
      901 acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga ctctagcgtt
      961 taaacttaag cttggtaccg agctcggatc cactagtcca gtgtggtgga attctgcaga
     1021 tatccagcac agtggcggcc gcatggagag ccttgtccct ggtttcaacg agaaaacaca
     1081 cgtccaactc agtttgcctg ttttacaggt tcgcgacgtg ctcgtacgtg gctttggaga
     1141 ctccgtggag gaggtcttat cagaggcacg tcaacatctt aaagatggca cttgtggctt
     1201 agtagaagtt gaaaaaggcg ttttgcctca acttgaacag ccctatgtgt tcatcaaacg
     1261 ttcggatgct cgaactgcac ctcatggtca tgttatggtt gagctggtag cagaactcga
     1321 aggcattcag tacggtcgta gtggtgagac acttggtgtc cttgtccctc atgtgggcga
     1381 aataccagtg gcttaccgca aggttcttct tgctgcgaac ggtaataaag gagctggtgg
     1441 ccatagttac ggcgccgatc taaagtcatt tgacttaggc gacgagcttg gcactgatcc
     1501 ttatgaagat tttcaagaaa actggaacac taaacatagc agtggtgtta cccgtgaact
     1561 catgcgtgag cttaacggag ggtggcggcc gctcgaggga ggcggtggag ccgactacaa
     1621 ggaccacgac ggcgactaca aggaccacga catcgactac aaggacgacg acgacaaggg
     1681 gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     1741 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     1801 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     1861 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     1921 ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca
     1981 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     2041 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     2101 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     2161 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     2221 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     2281 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     2341 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     2401 cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
     2461 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc
     2521 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
     2581 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     2641 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag
     2701 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg
     2761 ggagcttgta tatccatttt cggatctgat cagcacgtgt tgacaattaa tcatcggcat
     2821 agtataatac gacaaggtga ggaactaaac catggccaag ttgaccagtg ccgttccggt
     2881 gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg accgaccggc tcgggttctc
     2941 ccgggacttc gtggaggacg acttcgccgg tgtggtccgg gacgacgtga ccctgttcat
     3001 cagcgcggtc caggaccagg tggtgccgga caacaccctg gcctgggtgt gggtgcgcgg
     3061 cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc acgaacttcc gggacgcctc
     3121 cgggccggcc atgaccgaga tcggcgagca gccgtggggg cgggagttcg ccctgcgcga
     3181 cccggccggc aactgcgtgc acttcgtggc cgaggagcag gactgacacg tgctacgaga
     3241 tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc
     3301 cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt
     3361 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     3421 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     3481 tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc
     3541 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     3601 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     3661 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     3721 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     3781 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     3841 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     3901 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     3961 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     4021 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     4081 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     4141 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     4201 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     4261 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     4321 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     4381 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     4441 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     4501 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     4561 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     4621 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     4681 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     4741 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     4801 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     4861 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     4921 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     4981 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     5041 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     5101 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     5161 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     5221 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     5281 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     5341 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     5401 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     5461 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     5521 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     5581 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     5641 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgg
//
