LOCUS       sequence-394447-        5774 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of codon optimized SARS-CoV-2 nsp8 residues 
            76-198, with an N-terminus His tag followed by a TEV cleavage site..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5774)
  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 5774)
  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: pET-Duet-6xHis-TEV-Nsp8(76-198)
FEATURES             Location/Qualifiers
     source          1..5774
                     /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="ENLYFQS"
     promoter        571..589
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    590..614
                     /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             723..767
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(805..823)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      819..866
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      903..1358
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(990..1009)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1200..1221
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(1473..2264,2265..2333))
                     /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     2096..2115
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2334..2426)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2507..3095
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2996..3015
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3249..3266
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3281..3420
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3346..3365)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3506..3528
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3522..3713)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4285..5367)
                     /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     5329..5348
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5368..5445)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5651..5670
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(5758..5774,1..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 ttttcagtcc gaagacaaaa gggccaaagt aacctctgct atgcagacca tgctgttcac
      181 aatgctgcgc aagctggata acgacgcgct caacaacatc atcaacaatg cacgtgatgg
      241 ttgcgtacct ctcaacatta tcccgcttac tactgctgca aaactgatgg tggttatacc
      301 ggactacaac acctataaga acacctgtga tgggacgact ttcacctacg ccagcgcact
      361 gtgggaaatc cagcaggttg tcgatgctga cagtaaaatc gttcagctga gcgagatttc
      421 tatggacaat tctccgaacc tggcgtggcc actgatcgta actgctctgc gtgcgaatag
      481 cgcggtcaaa ttacaataaa agcttgcggc cgcataatgc ttaagtcgaa cagaaagtaa
      541 tcgtattgta cacggccgca taatcgaaat taatacgact cactataggg gaattgtgag
      601 cggataacaa ttccccatct tagtatatta gttaagtata agaaggagat atacatatgg
      661 cagatctcaa ttggatatcg gccggccacg cgatcgctga cgtcggtacc ctcgagtctg
      721 gtaaagaaac cgctgctgcg aaatttgaac gccagcacat ggactcgtct actagcgcag
      781 cttaattaac ctaggctgct gccaccgctg agcaataact agcataaccc cttggggcct
      841 ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga ttggcgaatg
      901 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
      961 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     1021 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     1081 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     1141 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     1201 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     1261 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     1321 taacgcgaat tttaacaaaa tattaacgtt tacaatttct ggcggcacga tggcatgaga
     1381 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     1441 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     1501 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     1561 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     1621 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     1681 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     1741 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     1801 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     1861 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     1921 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     1981 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     2041 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     2101 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     2161 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     2221 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     2281 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     2341 ctttttcaat catgattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     2401 tgaatgtatt tagaaaaata aacaaatagg tcatgaccaa aatcccttaa cgtgagtttt
     2461 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     2521 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     2581 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     2641 taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     2701 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     2761 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     2821 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     2881 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     2941 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     3001 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     3061 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     3121 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     3181 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     3241 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc
     3301 ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa tctgctctga
     3361 tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg
     3421 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     3481 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     3541 tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca
     3601 cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc
     3661 tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc
     3721 tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg
     3781 ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa
     3841 caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc
     3901 ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc
     3961 cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa
     4021 ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac
     4081 gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc
     4141 cgggtcctca acgacaggag cacgatcatg ctagtcatgc cccgcgccca ccggaaggag
     4201 ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc
     4261 taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     4321 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag
     4381 ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc
     4441 ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt
     4501 gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac
     4561 cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc
     4621 catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat
     4681 ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat
     4741 ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact
     4801 taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc
     4861 cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac
     4921 atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc
     4981 atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc
     5041 cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag
     5101 ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact
     5161 ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt
     5221 gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac
     5281 gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc
     5341 gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg
     5401 ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct
     5461 ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca
     5521 ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg
     5581 ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat
     5641 cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga
     5701 tgccggccac gatgcgtccg gcgtagagga tcgagatcga tctcgatccc gcgaaattaa
     5761 tacgactcac tata
//
