LOCUS       sequence-345355-        5693 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expressing WT CoV2 nsp1 in mammalian cells. This contains a 3XFLAG 
            tag fusion to nsp1's C-terminus..
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 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: pCDNA4 CoV-2 nsp1 3xFLAG
FEATURES             Location/Qualifiers
     source          1..5693
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(33..52)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        224..603
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        604..807
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     758..778
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    809..827
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    830..848
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     858..882
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1612..1677
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(1703..1720)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1709..1933
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1979..2407
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2066..2085)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2276..2297
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2416..2436)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2421..2750
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2601..2736
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2663..2682
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2851..3225
                     /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    3355..3476
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3392..3411)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3446..3465
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3525..3541)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3525..3541)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3538..3560)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3549..3565
                     /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(3573..3579,3580..3597,3598..3603))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3618..3639
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3756..3773)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3927..4515)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4007..4026)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4686..5477,5478..5546))
                     /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     5309..5328
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5547..5651)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
       61 agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt
      121 taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct tagggttagg
      181 cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gttgacattg attattgact
      241 agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc
      301 gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg
      361 acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa
      421 tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca
      481 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac
      541 atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc
      601 atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga
      661 tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg
      721 gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta
      781 cggtgggagg tctatataag cagagctctc cctatcagtg atagagatct ccctatcagt
      841 gatagagatc gtcgacgagc tcgtttagtg aaccgtcaga tcgcctggag acgccatcca
      901 cgctgttttg acctccatag aagacaccgg gaccgatcca gcctccggac tctagcgttt
      961 aaacttaagc ttggtaccga gctcggatcc actagtccag tgtggtggaa ttctgcagat
     1021 atccagcaca gtggcggccg catggagagc cttgtccctg gtttcaacga gaaaacacac
     1081 gtccaactca gtttgcctgt tttacaggtt cgcgacgtgc tcgtacgtgg ctttggagac
     1141 tccgtggagg aggtcttatc agaggcacgt caacatctta aagatggcac ttgtggctta
     1201 gtagaagttg aaaaaggcgt tttgcctcaa cttgaacagc cctatgtgtt catcaaacgt
     1261 tcggatgctc gaactgcacc tcatggtcat gttatggttg agctggtagc agaactcgaa
     1321 ggcattcagt acggtcgtag tggtgagaca cttggtgtcc ttgtccctca tgtgggcgaa
     1381 ataccagtgg cttaccgcaa ggttcttctt cgtaagaacg gtaataaagg agctggtggc
     1441 catagttacg gcgccgatct aaagtcattt gacttaggcg acgagcttgg cactgatcct
     1501 tatgaagatt ttcaagaaaa ctggaacact aaacatagca gtggtgttac ccgtgaactc
     1561 atgcgtgagc ttaacggagg gtggcggccg ctcgagggag gcggtggagc cgactacaag
     1621 gaccacgacg gcgactacaa ggaccacgac atcgactaca aggacgacga cgacaagggg
     1681 cccgtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt
     1741 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     1801 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
     1861 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg
     1921 gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac
     1981 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     2041 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     2101 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     2161 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     2221 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     2281 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     2341 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     2401 gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag
     2461 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc
     2521 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag
     2581 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     2641 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc
     2701 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg
     2761 gagcttgtat atccattttc ggatctgatc agcacgtgtt gacaattaat catcggcata
     2821 gtataatacg acaaggtgag gaactaaacc atggccaagt tgaccagtgc cgttccggtg
     2881 ctcaccgcgc gcgacgtcgc cggagcggtc gagttctgga ccgaccggct cgggttctcc
     2941 cgggacttcg tggaggacga cttcgccggt gtggtccggg acgacgtgac cctgttcatc
     3001 agcgcggtcc aggaccaggt ggtgccggac aacaccctgg cctgggtgtg ggtgcgcggc
     3061 ctggacgagc tgtacgccga gtggtcggag gtcgtgtcca cgaacttccg ggacgcctcc
     3121 gggccggcca tgaccgagat cggcgagcag ccgtgggggc gggagttcgc cctgcgcgac
     3181 ccggccggca actgcgtgca cttcgtggcc gaggagcagg actgacacgt gctacgagat
     3241 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
     3301 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg
     3361 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     3421 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     3481 gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct
     3541 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     3601 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     3661 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     3721 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     3781 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     3841 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     3901 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     3961 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     4021 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     4081 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     4141 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     4201 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     4261 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     4321 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     4381 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     4441 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     4501 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     4561 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     4621 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     4681 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     4741 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     4801 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     4861 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     4921 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     4981 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     5041 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     5101 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     5161 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     5221 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     5281 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     5341 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     5401 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     5461 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     5521 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     5581 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     5641 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc ggg
//
