LOCUS       sequence-323630-        5815 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SARS-CoV-2 nsp8 from a pET46 backbone for IPTG-inducible 
            bacterial expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5815)
  AUTHORS   Altincekic N, Korn SM, Qureshi NS, Dujardin M, Ninot-Pedrosa M, 
            Abele R, Abi Saad MJ, Alfano C, Almeida FCL, Alshamleh I, de Amorim 
            GC, Anderson TK, Anobom CD, Anorma C, Bains JK, Bax A, Blackledge M,
            Blechar J, Bockmann A, Brigandat L, Bula A, Butikofer M, 
            Camacho-Zarco AR, Carlomagno T, Caruso IP, Ceylan B, Chaikuad A, Chu
            F, Cole L, Crosby MG, de Jesus V, Dhamotharan K, Felli IC, Ferner J,
            Fleischmann Y, Fogeron ML, Fourkiotis NK, Fuks C, Furtig B, Gallo A,
            Gande SL, Gerez JA, Ghosh D, Gomes-Neto F, Gorbatyuk O, Guseva S, 
            Hacker C, Hafner S, Hao B, Hargittay B, Henzler-Wildman K, Hoch JC, 
            Hohmann KF, Hutchison MT, Jaudzems K, Jovic K, Kaderli J, Kalnins G,
            Kanepe I, Kirchdoerfer RN, Kirkpatrick J, Knapp S, Krishnathas R, 
            Kutz F, Zur Lage S, Lambertz R, Lang A, Laurents D, Lecoq L, Linhard
            V, Lohr F, Malki A, Bessa LM, Martin RW, Matzel T, Maurin D, McNutt 
            SW, Mebus-Antunes NC, Meier BH, Meiser N, Mompean M, Monaca E, 
            Montserret R, Marino Perez L, Moser C, Muhle-Goll C, Neves-Martins 
            TC, Ni X, Norton-Baker B, Pierattelli R, Pontoriero L, Pustovalova 
            Y, Ohlenschlager O, Orts J, Da Poian AT, Pyper DJ, Richter C, Riek 
            R, Rienstra CM, Robertson A, Pinheiro AS, Sabbatella R, Salvi N, 
            Saxena K, Schulte L, Schiavina M, Schwalbe H, Silber M, Almeida MDS,
            Sprague-Piercy MA, Spyroulias GA, Sreeramulu S, Tants JN, Tars K, 
            Torres F, Tows S, Trevino MA, Trucks S, Tsika AC, Varga K, Wang Y, 
            Weber ME, Weigand JE, Wiedemann C, Wirmer-Bartoschek J, Wirtz Martin
            MA, Zehnder J, Hengesbach M, Schlundt A
  TITLE     Large-Scale Recombinant Production of the SARS-CoV-2 Proteome for 
            High-Throughput and Structural Biology Applications.
  JOURNAL   Front Mol Biosci. 2021 May 10;8:653148. doi: 
            10.3389/fmolb.2021.653148. eCollection 2021.
  PUBMED    34041264
REFERENCE   2  (bases 1 to 5815)
  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: "Front Mol 
            Biosci."; date: "2021-05-10"; volume: "8:653148. doi"; pages: " 
            10.3389/fmolb.2021.653148. eCollection 2021"
COMMENT     Sequence Label: pTKA002
FEATURES             Location/Qualifiers
     source          1..5815
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(84..103)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     294..315
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(567..1358,1359..1427))
                     /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     1190..1209
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1428..1520)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1601..2189
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2090..2109
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2343..2360
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2375..2514
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2440..2459)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2600..2622
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2616..2807)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3379..4461)
                     /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     4423..4442
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4462..4539)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4745..4764
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4852..4871
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4852..4870
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4871..4895
                     /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             4910..4932
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4945..4962
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4966..4980
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             4984..5004
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             5632..5676
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(5714..5732)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5728..5775
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      join(5812..5815,1..452)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
       61 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
      121 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
      181 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
      241 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
      301 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
      361 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
      421 gaattttaac aaaatattaa cgtttacaat ttctggcggc acgatggcat gagattatca
      481 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
      541 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
      601 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
      661 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
      721 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
      781 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
      841 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
      901 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
      961 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     1021 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     1081 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     1141 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     1201 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     1261 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     1321 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     1381 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     1441 caatcatgat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     1501 tatttagaaa aataaacaaa taggtcatga ccaaaatccc ttaacgtgag ttttcgttcc
     1561 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     1621 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     1681 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     1741 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     1801 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     1861 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     1921 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     1981 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     2041 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     2101 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     2161 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     2221 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     2281 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     2341 gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc
     2401 atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc
     2461 atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga
     2521 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     2581 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     2641 aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg
     2701 tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt
     2761 ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg
     2821 taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg
     2881 atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg
     2941 gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt
     3001 aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac
     3061 ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag
     3121 accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc
     3181 tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc
     3241 ctcaacgaca ggagcacgat catgctagtc atgccccgcg cccaccggaa ggagctgact
     3301 gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt
     3361 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     3421 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt
     3481 ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga
     3541 gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt
     3601 ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat
     3661 gtccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg
     3721 atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg
     3781 ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt
     3841 gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg
     3901 gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg
     3961 cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag
     4021 aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag
     4081 cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt
     4141 acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc
     4201 ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt
     4261 ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat
     4321 gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct
     4381 ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc
     4441 gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca
     4501 tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct
     4561 tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg
     4621 ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg
     4681 ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc
     4741 catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg
     4801 ccacgatgcg tccggcgtag aggatcgaga tcgatctcga tcccgcgaaa ttaatacgac
     4861 tcactatagg ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact
     4921 ttaagaagga gatataccat ggcacatcac caccaccatc acgtggatga cgacgacaag
     4981 atggagaatc tttattttca gggtgcgatc gctagcgagt tcagcagcct gccgagctac
     5041 gctgctttcg ctaccgctca ggaagcgtac gaacaggcgg tggctaacgg cgacagcgaa
     5101 gtggttctga agaaactgaa gaaaagcctg aacgttgcta aaagcgagtt cgaccgtgat
     5161 gcggctatgc agcgtaagct ggaaaaaatg gcggaccagg ctatgaccca gatgtacaag
     5221 caggcgcgta gcgaggataa gcgtgcgaaa gtgaccagcg ctatgcagac catgctgttc
     5281 accatgctgc gtaaactgga caacgatgcg ctgaacaaca tcatcaacaa cgctcgtgac
     5341 ggttgcgttc cgctgaacat catcccgctg accaccgcgg ctaagctgat ggtggttatc
     5401 ccggactaca acacctacaa aaacacctgc gatggcacca ccttcaccta cgcgagcgct
     5461 ctgtgggaga tccagcaggt ggttgacgct gatagcaaga tcgtgcagct gagcgaaatc
     5521 agcatggata acagcccgaa cctggcttgg ccgctgattg tgaccgccct gcgtgctaac
     5581 tccgctgtga aactgcaata accgggcttc tcctcaaatc tcgagtctgg taaagaaacc
     5641 gctgctgcga aatttgaacg ccagcacatg gactcgtcta ctagcgcagc ttaattaacc
     5701 taggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc
     5761 ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgc
//
