LOCUS       sequence-345038-        5490 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For protein expression of R124A/K125A nsp1 CoV2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5490)
  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 5490)
  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: pGEX nsp1 R124A/K125A CoV2
FEATURES             Location/Qualifiers
     source          1..5490
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(540..545,546..563,564..570)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    578..594
                     /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)."
     primer_bind     602..618
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     602..618
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             614..1141
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(634..651)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(634..650)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(643..665)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(1243..1248,1249..1264,1265..1271)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1279..1295
                     /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)."
     CDS             1318..1971
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     1929..1951
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             1978..2001
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(2600..2622)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2767..2785)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2853..2957
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2958..3026,3027..3818)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3176..3195)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3989..4577
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4478..4497
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4731..4748
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4821..4898
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(4899..5490,1..491)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(4918..4937)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 tggctggctg gcataaatat ctcactcgca atcaaattca gccgatagcg gaacgggaag
       61 gcgactggag tgccatgtcc ggttttcaac aaaccatgca aatgctgaat gagggcatcg
      121 ttcccactgc gatgctggtt gccaacgatc agatggcgct gggcgcaatg cgcgccatta
      181 ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt gggatacgac gataccgaag
      241 acagctcatg ttatatcccg ccgttaacca ccatcaaaca ggattttcgc ctgctggggc
      301 aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc
      361 tgttgcccgt ctcactggtg aaaagaaaaa ccacgctagc gcccaatacg caaaccgcct
      421 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
      481 gcgggcagtg agcgcaacgc aattaatgta agttagctca ctcattaggc accccaggct
      541 ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac
      601 acaggaaaca gctatgacca tgattacgga ttcactggcc gtcgttttac aacgtcgtga
      661 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
      721 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
      781 tggcgaatgg cgctttgcct ggtttccggc accagaagcg gtgccggaaa gctggctgga
      841 gtgcgatctt cctgaggccg atactgtcgt cgtcccctca aactggcaga tgcacggtta
      901 cgatgcgccc atctacacca acgtgaccta tcccattacg gtcaatccgc cgtttgttcc
      961 cacggagaat ccgacgggtt gttactcgct cacatttaat gttgatgaaa gctggctaca
     1021 ggaaggccag acgcgaatta tttttgatgg cgttggaatt agcttatcga ctgcacggtg
     1081 caccaatgct tctggcgtca ggcagccatc ggaagctgtg gtatggctgt gcaggtcgta
     1141 aatcactgca taattcgtgt cgctcaaggc gcactcccgt tctggataat gttttttgcg
     1201 ccgacatcat aacggttctg gcaaatattc tgaaatgagc tgttgacaat taatcatcgg
     1261 ctcgtataat gtgtggaatt gtgagcggat aacaatttca cacaggaaac agtattcatg
     1321 tcccctatac taggttattg gaaaattaag ggccttgtgc aacccactcg acttcttttg
     1381 gaatatcttg aagaaaaata tgaagagcat ttgtatgagc gcgatgaagg tgataaatgg
     1441 cgaaacaaaa agtttgaatt gggtttggag tttcccaatc ttccttatta tattgatggt
     1501 gatgttaaat taacacagtc tatggccatc atacgttata tagctgacaa gcacaacatg
     1561 ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc ttgaaggagc ggttttggat
     1621 attagatacg gtgtttcgag aattgcatat agtaaagact ttgaaactct caaagttgat
     1681 tttcttagca agctacctga aatgctgaaa atgttcgaag atcgtttatg tcataaaaca
     1741 tatttaaatg gtgatcatgt aacccatcct gacttcatgt tgtatgacgc tcttgatgtt
     1801 gttttataca tggacccaat gtgcctggat gcgttcccaa aattagtttg ttttaaaaaa
     1861 cgtattgaag ctatcccaca aattgataag tacttgaaat ccagcaagta tatagcatgg
     1921 cctttgcagg gctggcaagc cacgtttggt ggtggcgacc atcctccaaa atcggatctg
     1981 gaagttctgt tccaggggcc cgagagcctt gtccctggtt tcaacgagaa aacacacgtc
     2041 caactcagtt tgcctgtttt acaggttcgc gacgtgctcg tacgtggctt tggagactcc
     2101 gtggaggagg tcttatcaga ggcacgtcaa catcttaaag atggcacttg tggcttagta
     2161 gaagttgaaa aaggcgtttt gcctcaactt gaacagccct atgtgttcat caaacgttcg
     2221 gatgctcgaa ctgcacctca tggtcatgtt atggttgagc tggtagcaga actcgaaggc
     2281 attcagtacg gtcgtagtgg tgagacactt ggtgtccttg tccctcatgt gggcgaaata
     2341 ccagtggctt accgcaaggt tcttcttgct gcgaacggta ataaaggagc tggtggccat
     2401 agttacggcg ccgatctaaa gtcatttgac ttaggcgacg agcttggcac tgatccttat
     2461 gaagattttc aagaaaactg gaacactaaa catagcagtg gtgttacccg tgaactcatg
     2521 cgtgagctta acggagggta aggccgcatc gtgactgact gacgatctgc ctcgcgcgtt
     2581 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc
     2641 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt
     2701 gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtataat tcttgaagac
     2761 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     2821 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     2881 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     2941 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     3001 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     3061 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     3121 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     3181 gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact
     3241 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     3301 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     3361 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     3421 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     3481 agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     3541 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     3601 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     3661 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     3721 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     3781 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     3841 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     3901 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     3961 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     4021 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     4081 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
     4141 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     4201 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     4261 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     4321 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     4381 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     4441 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     4501 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     4561 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     4621 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     4681 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     4741 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     4801 tttcacaccg cataaattcc gacaccatcg aatggcgcaa aacctttcgc ggtatggcat
     4861 gatagcgccc ggaagagagt caattcaggg tggtgaatgt gaaaccagta acgttatacg
     4921 atgtcgcaga gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca
     4981 gccacgtttc tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag ctgaattaca
     5041 ttcccaaccg cgtggcacaa caactggcgg gcaaacagtc gttgctgatt ggcgttgcca
     5101 cctccagtct ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg
     5161 atcaactggg tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta
     5221 aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc
     5281 tggatgacca ggatgccatt gctgtggaag ctgcctgcac taatgttccg gcgttatttc
     5341 ttgatgtctc tgaccagaca cccatcaaca gtattatttt ctcccatgaa gacggtacgc
     5401 gactgggcgt ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc
     5461 cattaagttc tgtctcggcg cgtctgcgtc
//
