LOCUS       sequence-345043-        5490 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For protein expression of K164A/H165A 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 K164A/H165A CoV2
FEATURES             Location/Qualifiers
     source          1..5490
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(125..130,131..148,149..155)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    163..179
                     /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     187..203
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     187..203
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             199..726
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(219..236)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(219..235)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(228..250)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(828..833,834..849,850..856)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    864..880
                     /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             903..1556
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     1514..1536
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             1563..1586
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(2185..2207)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2352..2370)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2438..2542
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2543..2611,2612..3403)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGDRGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2761..2780)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3574..4162
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4063..4082
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4316..4333
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4406..4483
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(4484..5490,1..76)
                     /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(4503..4522)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
       61 ggaaagcggg cagtgagcgc aacgcaatta atgtaagtta gctcactcat taggcacccc
      121 aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat
      181 ttcacacagg aaacagctat gaccatgatt acggattcac tggccgtcgt tttacaacgt
      241 cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc
      301 gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc
      361 ctgaatggcg aatggcgctt tgcctggttt ccggcaccag aagcggtgcc ggaaagctgg
      421 ctggagtgcg atcttcctga ggccgatact gtcgtcgtcc cctcaaactg gcagatgcac
      481 ggttacgatg cgcccatcta caccaacgtg acctatccca ttacggtcaa tccgccgttt
      541 gttcccacgg agaatccgac gggttgttac tcgctcacat ttaatgttga tgaaagctgg
      601 ctacaggaag gccagacgcg aattattttt gatggcgttg gaattagctt atcgactgca
      661 cggtgcacca atgcttctgg cgtcaggcag ccatcggaag ctgtggtatg gctgtgcagg
      721 tcgtaaatca ctgcataatt cgtgtcgctc aaggcgcact cccgttctgg ataatgtttt
      781 ttgcgccgac atcataacgg ttctggcaaa tattctgaaa tgagctgttg acaattaatc
      841 atcggctcgt ataatgtgtg gaattgtgag cggataacaa tttcacacag gaaacagtat
      901 tcatgtcccc tatactaggt tattggaaaa ttaagggcct tgtgcaaccc actcgacttc
      961 ttttggaata tcttgaagaa aaatatgaag agcatttgta tgagcgcgat gaaggtgata
     1021 aatggcgaaa caaaaagttt gaattgggtt tggagtttcc caatcttcct tattatattg
     1081 atggtgatgt taaattaaca cagtctatgg ccatcatacg ttatatagct gacaagcaca
     1141 acatgttggg tggttgtcca aaagagcgtg cagagatttc aatgcttgaa ggagcggttt
     1201 tggatattag atacggtgtt tcgagaattg catatagtaa agactttgaa actctcaaag
     1261 ttgattttct tagcaagcta cctgaaatgc tgaaaatgtt cgaagatcgt ttatgtcata
     1321 aaacatattt aaatggtgat catgtaaccc atcctgactt catgttgtat gacgctcttg
     1381 atgttgtttt atacatggac ccaatgtgcc tggatgcgtt cccaaaatta gtttgtttta
     1441 aaaaacgtat tgaagctatc ccacaaattg ataagtactt gaaatccagc aagtatatag
     1501 catggccttt gcagggctgg caagccacgt ttggtggtgg cgaccatcct ccaaaatcgg
     1561 atctggaagt tctgttccag gggcccgaga gccttgtccc tggtttcaac gagaaaacac
     1621 acgtccaact cagtttgcct gttttacagg ttcgcgacgt gctcgtacgt ggctttggag
     1681 actccgtgga ggaggtctta tcagaggcac gtcaacatct taaagatggc acttgtggct
     1741 tagtagaagt tgaaaaaggc gttttgcctc aacttgaaca gccctatgtg ttcatcaaac
     1801 gttcggatgc tcgaactgca cctcatggtc atgttatggt tgagctggta gcagaactcg
     1861 aaggcattca gtacggtcgt agtggtgaga cacttggtgt ccttgtccct catgtgggcg
     1921 aaataccagt ggcttaccgc aaggttcttc ttcgtaagaa cggtaataaa ggagctggtg
     1981 gccatagtta cggcgccgat ctaaagtcat ttgacttagg cgacgagctt ggcactgatc
     2041 cttatgaaga ttttcaagaa aactggaaca ctgcagctag cagtggtgtt acccgtgaac
     2101 tcatgcgtga gcttaacgga gggtaaggcc gcatcgtgac tgactgacga tctgcctcgc
     2161 gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc
     2221 ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg
     2281 cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat agcggagtgt ataattcttg
     2341 aagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
     2401 ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     2461 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     2521 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     2581 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     2641 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     2701 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     2761 gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat
     2821 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     2881 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     2941 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     3001 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     3061 cgacgagcgt gacaccacga tgcctgcagc aatggcaaca acgttgcgca aactattaac
     3121 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     3181 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     3241 tggagccggt gatcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     3301 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     3361 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     3421 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     3481 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     3541 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     3601 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     3661 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     3721 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     3781 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     3841 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     3901 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     3961 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     4021 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     4081 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     4141 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     4201 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     4261 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     4321 gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg
     4381 cggtatttca caccgcataa attccgacac catcgaatgg cgcaaaacct ttcgcggtat
     4441 ggcatgatag cgcccggaag agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt
     4501 atacgatgtc gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca
     4561 ggccagccac gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa
     4621 ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt
     4681 tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg
     4741 cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc
     4801 ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga tcattaacta
     4861 tccgctggat gaccaggatg ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt
     4921 atttcttgat gtctctgacc agacacccat caacagtatt attttctccc atgaagacgg
     4981 tacgcgactg ggcgtggagc atctggtcgc attgggtcac cagcaaatcg cgctgttagc
     5041 gggcccatta agttctgtct cggcgcgtct gcgtctggct ggctggcata aatatctcac
     5101 tcgcaatcaa attcagccga tagcggaacg ggaaggcgac tggagtgcca tgtccggttt
     5161 tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc actgcgatgc tggttgccaa
     5221 cgatcagatg gcgctgggcg caatgcgcgc cattaccgag tccgggctgc gcgttggtgc
     5281 ggatatctcg gtagtgggat acgacgatac cgaagacagc tcatgttata tcccgccgtt
     5341 aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca
     5401 actctctcag ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag
     5461 aaaaaccacg ctagcgccca atacgcaaac
//
