LOCUS       sequence-394460-        6008 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of codon optimized SARS-CoV-2 nsp8 tagged with 
            an N-terminus His-tag followed by a TEV protease cleavage site. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6008)
  AUTHORS   Ghelichkhani F, Gonzalez FA, Kapitonova MA, Rozovsky S
  TITLE     Selenoprotein S Interacts with the Replication and Transcription 
            Complex of SARS-CoV-2 by Binding nsp7.
  JOURNAL   J Mol Biol. 2023 Apr 15;435(8):168008. doi: 
            10.1016/j.jmb.2023.168008. Epub 2023 Feb 10.
  PUBMED    36773692
REFERENCE   2  (bases 1 to 6008)
  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: "J Mol 
            Biol."; date: "2023-04-15"; volume: "435(8):168008. doi"; pages: " 
            10.1016/j.jmb.2023.168008. Epub 2023 Feb 10"
COMMENT     Sequence Label: pETDuet-1-6xHis-TEV-Nsp8
FEATURES             Location/Qualifiers
     source          1..6008
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /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)."
     RBS             42..64
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             83..100
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             110..130
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     promoter        805..823
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    824..848
                     /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             957..1001
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1039..1057)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1053..1100
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1137..1592
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1224..1243)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1434..1455
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(1707..2498,2499..2567))
                     /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     2330..2349
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2568..2660)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2741..3329
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3230..3249
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3483..3500
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3515..3654
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3580..3599)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3740..3762
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3756..3947)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4519..5601)
                     /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     5563..5582
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5602..5679)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5885..5904
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(5992..6008,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
       61 gagatatacc atgggcagca gccatcacca tcatcaccac agcagcggcg aaaacctgta
      121 ttttcaggga tccatggcca ttgcgagcga attttcaagt cttccatcct atgctgcatt
      181 cgctactgca caagaggctt acgaacaggc ggtggccaac ggcgactcgg aagtcgttct
      241 gaagaaactg aaaaaatctt taaatgttgc gaaaagcgaa tttgatcgtg acgctgcgat
      301 gcagcgcaag ttggagaaaa tggctgatca ggcaatgacg caaatgtaca aacaggctcg
      361 ttccgaagac aaaagggcca aagtaacctc tgctatgcag accatgctgt tcacaatgct
      421 gcgcaagctg gataacgacg cgctcaacaa catcatcaac aatgcacgtg atggttgcgt
      481 acctctcaac attatcccgc ttactactgc tgcaaaactg atggtggtta taccggacta
      541 caacacctat aagaacacct gtgatgggac gactttcacc tacgccagcg cactgtggga
      601 aatccagcag gttgtcgatg ctgacagtaa aatcgttcag ctgagcgaga tttctatgga
      661 caattctccg aacctggcgt ggccactgat cgtaactgct ctgcgtgcga atagcgcggt
      721 caaattacaa taaaagcttg cggccgcata atgcttaagt cgaacagaaa gtaatcgtat
      781 tgtacacggc cgcataatcg aaattaatac gactcactat aggggaattg tgagcggata
      841 acaattcccc atcttagtat attagttaag tataagaagg agatatacat atggcagatc
      901 tcaattggat atcggccggc cacgcgatcg ctgacgtcgg taccctcgag tctggtaaag
      961 aaaccgctgc tgcgaaattt gaacgccagc acatggactc gtctactagc gcagcttaat
     1021 taacctaggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac
     1081 gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc
     1141 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     1201 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     1261 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     1321 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     1381 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     1441 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     1501 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     1561 gaattttaac aaaatattaa cgtttacaat ttctggcggc acgatggcat gagattatca
     1621 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     1681 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     1741 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     1801 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     1861 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     1921 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     1981 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     2041 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     2101 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     2161 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     2221 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     2281 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     2341 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     2401 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     2461 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     2521 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     2581 caatcatgat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     2641 tatttagaaa aataaacaaa taggtcatga ccaaaatccc ttaacgtgag ttttcgttcc
     2701 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     2761 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     2821 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     2881 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     2941 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     3001 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     3061 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     3121 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     3181 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     3241 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     3301 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     3361 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     3421 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     3481 gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc
     3541 atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc
     3601 atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga
     3661 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     3721 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     3781 aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg
     3841 tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt
     3901 ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg
     3961 taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg
     4021 atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg
     4081 gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt
     4141 aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac
     4201 ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag
     4261 accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc
     4321 tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc
     4381 ctcaacgaca ggagcacgat catgctagtc atgccccgcg cccaccggaa ggagctgact
     4441 gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt
     4501 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     4561 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt
     4621 ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga
     4681 gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt
     4741 ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat
     4801 gtccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg
     4861 atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg
     4921 ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt
     4981 gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg
     5041 gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg
     5101 cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag
     5161 aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag
     5221 cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt
     5281 acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc
     5341 ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt
     5401 ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat
     5461 gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct
     5521 ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc
     5581 gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca
     5641 tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct
     5701 tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg
     5761 ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg
     5821 ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc
     5881 catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg
     5941 ccacgatgcg tccggcgtag aggatcgaga tcgatctcga tcccgcgaaa ttaatacgac
     6001 tcactata
//
