LOCUS       sequence-345434-        5698 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 5698)
  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 5698)
  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..5698
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    820..838
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    841..859
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     869..893
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1618..1683
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(1709..1726)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1715..1939
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1985..2413
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2072..2091)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2282..2303
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2422..2442)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2427..2756
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2607..2742
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2669..2688
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        2804..2851
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2870..3244
                     /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    3374..3495
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3411..3430)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3465..3484
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3544..3560)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3544..3560)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3557..3579)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3568..3584
                     /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(3592..3598,3599..3616,3617..3622))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3637..3658
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3775..3792)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3946..4531)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4026..4045)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4702..5493,5494..5562))
                     /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     5325..5344
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5563..5667)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggaacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ccctatcagt gatagagatc
      841 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga
      901 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga
      961 ctctagcgtt taaacttaag cttggtaccg agctcggatc cactagtcca gtgtggtgga
     1021 attctgcaga tatccagcac agtggcggcc atggagagcc ttgtccctgg tttcaacgag
     1081 aaaacacacg tccaactcag tttgcctgtt ttacaggttc gcgacgtgct cgtacgtggc
     1141 tttggagact ccgtggagga ggtcttatca gaggcacgtc aacatcttaa agatggcact
     1201 tgtggcttag tagaagttga aaaaggcgtt ttgcctcaac ttgaacagcc ctatgtgttc
     1261 atcaaacgtt cggatgctcg aactgcacct catggtcatg ttatggttga gctggtagca
     1321 gaactcgaag gcattcagta cggtgcaagt ggtgagacac ttggtgtcct tgtccctcat
     1381 gtgggcgaaa taccagtggc ttaccgcaag gttcttcttc gtaagaacgg taataaagga
     1441 gctggtggcc atagttacgg cgccgatcta aagtcatttg acttaggcga cgagcttggc
     1501 actgatcctt atgaagattt tcaagaaaac tggaacacta aacatagcag tggtgttacc
     1561 cgtgaactca tgcgtgagct taacggaggg gggccgctcg agggaggcgg tggagccgac
     1621 tacaaggacc acgacggcga ctacaaggac cacgacatcg actacaagga cgacgacgac
     1681 aaggggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct
     1741 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     1801 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     1861 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg
     1921 gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat
     1981 ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     2041 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     2101 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     2161 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     2221 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     2281 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     2341 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     2401 tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct
     2461 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa
     2521 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     2581 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     2641 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct
     2701 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc
     2761 tcccgggagc ttgtatatcc attttcggat ctgatcagca cgtgttgaca attaatcatc
     2821 ggcatagtat atcggcatag tataatacga caaggtgagg aactaaacca tggccaagtt
     2881 gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc ggagcggtcg agttctggac
     2941 cgaccggctc gggttctccc gggacttcgt ggaggacgac ttcgccggtg tggtccggga
     3001 cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg gtgccggaca acaccctggc
     3061 ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag tggtcggagg tcgtgtccac
     3121 gaacttccgg gacgcctccg ggccggccat gaccgagatc ggcgagcagc cgtgggggcg
     3181 ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac ttcgtggccg aggagcagga
     3241 ctgacacgtg ctacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg
     3301 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt
     3361 cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     3421 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     3481 catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc
     3541 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     3601 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     3661 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     3721 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     3781 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     3841 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     3901 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     3961 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     4021 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     4081 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     4141 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     4201 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     4261 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     4321 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     4381 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     4441 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtttttttg tttgcaagca
     4501 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     4561 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     4621 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     4681 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     4741 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     4801 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     4861 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     4921 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     4981 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     5041 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     5101 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     5161 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     5221 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     5281 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     5341 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     5401 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     5461 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     5521 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     5581 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     5641 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtc
//
