LOCUS       sequence-345226-        5490 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For protein expression of R99A 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 R99A CoV2
FEATURES             Location/Qualifiers
     source          1..5490
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(532..537,538..555,556..562)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    570..586
                     /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     594..610
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     594..610
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             606..1133
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(626..643)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(626..642)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(635..657)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(1235..1240,1241..1256,1257..1263)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1271..1287
                     /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             1310..1963
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     1921..1943
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             1970..1993
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(2592..2614)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2759..2777)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2845..2949
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2950..3018,3019..3810)
                     /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(3168..3187)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3981..4569
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4470..4489
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4723..4740
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4813..4890
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(4891..5490,1..483)
                     /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(4910..4929)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 tggcataaat atctcactcg caatcaaatt cagccgatag cggaacggga aggcgactgg
       61 agtgccatgt ccggttttca acaaaccatg caaatgctga atgagggcat cgttcccact
      121 gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc
      181 gggctgcgcg ttggtgcgga tatctcggta gtgggatacg acgataccga agacagctca
      241 tgttatatcc cgccgttaac caccatcaaa caggattttc gcctgctggg gcaaaccagc
      301 gtggaccgct tgctgcaact ctctcagggc caggcggtga agggcaatca gctgttgccc
      361 gtctcactgg tgaaaagaaa aaccacgcta gcgcccaata cgcaaaccgc ctctccccgc
      421 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
      481 tgagcgcaac gcaattaatg taagttagct cactcattag gcaccccagg ctttacactt
      541 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
      601 cagctatgac catgattacg gattcactgg ccgtcgtttt acaacgtcgt gactgggaaa
      661 accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta
      721 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat
      781 ggcgctttgc ctggtttccg gcaccagaag cggtgccgga aagctggctg gagtgcgatc
      841 ttcctgaggc cgatactgtc gtcgtcccct caaactggca gatgcacggt tacgatgcgc
      901 ccatctacac caacgtgacc tatcccatta cggtcaatcc gccgtttgtt cccacggaga
      961 atccgacggg ttgttactcg ctcacattta atgttgatga aagctggcta caggaaggcc
     1021 agacgcgaat tatttttgat ggcgttggaa ttagcttatc gactgcacgg tgcaccaatg
     1081 cttctggcgt caggcagcca tcggaagctg tggtatggct gtgcaggtcg taaatcactg
     1141 cataattcgt gtcgctcaag gcgcactccc gttctggata atgttttttg cgccgacatc
     1201 ataacggttc tggcaaatat tctgaaatga gctgttgaca attaatcatc ggctcgtata
     1261 atgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagtattca tgtcccctat
     1321 actaggttat tggaaaatta agggccttgt gcaacccact cgacttcttt tggaatatct
     1381 tgaagaaaaa tatgaagagc atttgtatga gcgcgatgaa ggtgataaat ggcgaaacaa
     1441 aaagtttgaa ttgggtttgg agtttcccaa tcttccttat tatattgatg gtgatgttaa
     1501 attaacacag tctatggcca tcatacgtta tatagctgac aagcacaaca tgttgggtgg
     1561 ttgtccaaaa gagcgtgcag agatttcaat gcttgaagga gcggttttgg atattagata
     1621 cggtgtttcg agaattgcat atagtaaaga ctttgaaact ctcaaagttg attttcttag
     1681 caagctacct gaaatgctga aaatgttcga agatcgttta tgtcataaaa catatttaaa
     1741 tggtgatcat gtaacccatc ctgacttcat gttgtatgac gctcttgatg ttgttttata
     1801 catggaccca atgtgcctgg atgcgttccc aaaattagtt tgttttaaaa aacgtattga
     1861 agctatccca caaattgata agtacttgaa atccagcaag tatatagcat ggcctttgca
     1921 gggctggcaa gccacgtttg gtggtggcga ccatcctcca aaatcggatc tggaagttct
     1981 gttccagggg cccgagagcc ttgtccctgg tttcaacgag aaaacacacg tccaactcag
     2041 tttgcctgtt ttacaggttc gcgacgtgct cgtacgtggc tttggagact ccgtggagga
     2101 ggtcttatca gaggcacgtc aacatcttaa agatggcact tgtggcttag tagaagttga
     2161 aaaaggcgtt ttgcctcaac ttgaacagcc ctatgtgttc atcaaacgtt cggatgctcg
     2221 aactgcacct catggtcatg ttatggttga gctggtagca gaactcgaag gcattcagta
     2281 cggtgcaagt ggtgagacac ttggtgtcct tgtccctcat gtgggcgaaa taccagtggc
     2341 ttaccgcaag gttcttcttc gtaagaacgg taataaagga gctggtggcc atagttacgg
     2401 cgccgatcta aagtcatttg acttaggcga cgagcttggc actgatcctt atgaagattt
     2461 tcaagaaaac tggaacacta aacatagcag tggtgttacc cgtgaactca tgcgtgagct
     2521 taacggaggg taaggccgca tcgtgactga ctgacgatct gcctcgcgcg tttcggtgat
     2581 gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg
     2641 gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc
     2701 gcagccatga cccagtcacg tagcgatagc ggagtgtata attcttgaag acgaaagggc
     2761 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     2821 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     2881 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     2941 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     3001 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     3061 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     3121 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     3181 gtattatccc gtgttgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     3241 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     3301 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     3361 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     3421 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     3481 accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     3541 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     3601 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     3661 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     3721 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     3781 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     3841 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     3901 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     3961 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     4021 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     4081 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     4141 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     4201 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     4261 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     4321 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     4381 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     4441 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     4501 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     4561 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     4621 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     4681 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     4741 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     4801 cgcataaatt ccgacaccat cgaatggcgc aaaacctttc gcggtatggc atgatagcgc
     4861 ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca
     4921 gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt
     4981 tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac
     5041 cgcgtggcac aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt
     5101 ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg
     5161 ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg
     5221 gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac
     5281 caggatgcca ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc
     5341 tctgaccaga cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc
     5401 gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt
     5461 tctgtctcgg cgcgtctgcg tctggctggc
//
