LOCUS       sequence-345034-        5496 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses C_K for bioconjugation on nsp1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5496)
  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 5496)
  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 C_K nsp1 K164A/H165A CoV2
FEATURES             Location/Qualifiers
     source          1..5496
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     960..979
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(999..1076)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     complement(1149..1166)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1320..1908)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1400..1419)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2079..2870,2871..2939))
                     /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     2702..2721
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2940..3044)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3112..3130
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     3275..3297
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3902..3925)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(3932..4585)
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     complement(3952..3974)
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     protein_bind    4608..4624
                     /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(4632..4638,4639..4654,4655..4660))
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     CDS             complement(4762..5289)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     5238..5260
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5252..5269
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5253..5269
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5285..5301)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5285..5301)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    5309..5325
                     /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(5333..5339,5340..5357,5358..5363))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     CDS             complement(join(5412..5496,1..998))
                     /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"
ORIGIN
        1 ttgggcgcta gcgtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
       61 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
      121 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
      181 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
      241 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
      301 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
      361 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
      421 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
      481 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
      541 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
      601 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
      661 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
      721 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
      781 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
      841 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
      901 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
      961 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     1021 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtcggaa
     1081 tttatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgctct
     1141 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     1201 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     1261 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     1321 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     1381 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     1441 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     1501 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     1561 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     1621 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     1681 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     1741 aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc
     1801 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     1861 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     1921 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     1981 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     2041 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     2101 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     2161 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     2221 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     2281 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     2341 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctgcaggc
     2401 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     2461 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     2521 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     2581 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     2641 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaacacgg
     2701 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     2761 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     2821 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     2881 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     2941 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     3001 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     3061 gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt
     3121 atcacgaggc cctttcgtct tcaagaatta tacactccgc tatcgctacg tgactgggtc
     3181 atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     3241 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     3301 tcaccgtcat caccgaaacg cgcgaggcag atcgtcagtc agtcacgatg cggccttacc
     3361 ctccgttaag ctcacgcatg agttcacggg taacaccact gctagctgca gtgttccagt
     3421 tttcttgaaa atcttcataa ggatcagtgc caagctcgtc gcctaagtca aatgacttta
     3481 gatcggcgcc gtaactatgg ccaccagctc ctttattacc gttcttacga agaagaacct
     3541 tgcggtaagc cactggtatt tcgcccacat gagggacaag gacaccaagt gtctcaccac
     3601 tacgaccgta ctgaatgcct tcgagttctg ctaccagctc aaccataaca tgaccatgag
     3661 gtgcagttcg agcatccgaa cgtttgatga acacataggg ctgttcaagt tgaggcaaaa
     3721 cgcctttttc aacttctact aagccacaag tgccatcttt aagatgttga cgtgcctctg
     3781 ataagacctc ctccacggag tctccaaagc cacgtacgag cacgtcgcga acctgtaaaa
     3841 caggcaaact gagttggacg tgtgttttct cgttgaaacc agggacaagg ctctctttac
     3901 agggcccctg gaacagaact tccagatccg attttggagg atggtcgcca ccaccaaacg
     3961 tggcttgcca gccctgcaaa ggccatgcta tatacttgct ggatttcaag tacttatcaa
     4021 tttgtgggat agcttcaata cgttttttaa aacaaactaa ttttgggaac gcatccaggc
     4081 acattgggtc catgtataaa acaacatcaa gagcgtcata caacatgaag tcaggatggg
     4141 ttacatgatc accatttaaa tatgttttat gacataaacg atcttcgaac attttcagca
     4201 tttcaggtag cttgctaaga aaatcaactt tgagagtttc aaagtcttta ctatatgcaa
     4261 ttctcgaaac accgtatcta atatccaaaa ccgctccttc aagcattgaa atctctgcac
     4321 gctcttttgg acaaccaccc aacatgttgt gcttgtcagc tatataacgt atgatggcca
     4381 tagactgtgt taatttaaca tcaccatcaa tataataagg aagattggga aactccaaac
     4441 ccaattcaaa ctttttgttt cgccatttat caccttcatc gcgctcatac aaatgctctt
     4501 catatttttc ttcaagatat tccaaaagaa gtcgagtggg ttgcacaagg cccttaattt
     4561 tccaataacc tagtataggg gacatgaata ctgtttcctg tgtgaaattg ttatccgctc
     4621 acaattccac acattatacg agccgatgat taattgtcaa cagctcattt cagaatattt
     4681 gccagaaccg ttatgatgtc ggcgcaaaaa acattatcca gaacgggagt gcgccttgag
     4741 cgacacgaat tatgcagtga tttacgacct gcacagccat accacagctt ccgatggctg
     4801 cctgacgcca gaagcattgg tgcaccgtgc agtcgataag ctaattccaa cgccatcaaa
     4861 aataattcgc gtctggcctt cctgtagcca gctttcatca acattaaatg tgagcgagta
     4921 acaacccgtc ggattctccg tgggaacaaa cggcggattg accgtaatgg gataggtcac
     4981 gttggtgtag atgggcgcat cgtaaccgtg catctgccag tttgagggga cgacgacagt
     5041 atcggcctca ggaagatcgc actccagcca gctttccggc accgcttctg gtgccggaaa
     5101 ccaggcaaag cgccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg
     5161 ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg
     5221 ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg aatccgtaat
     5281 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     5341 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa cttacattaa
     5401 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     5461 gaatcggcca acgcgcgggg agaggcggtt tgcgta
//
