LOCUS       sequence-322802-        5470 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SARS-CoV-2 nsp7 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 5470)
  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 5470)
  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: pTKA001
FEATURES             Location/Qualifiers
     source          1..5470
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      33..488
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(120..139)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     330..351
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(603..1394,1395..1463))
                     /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     1226..1245
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1464..1556)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1637..2225
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2126..2145
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2379..2396
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2411..2550
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2476..2495)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2636..2658
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2652..2843)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3415..4497)
                     /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     4459..4478
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4498..4575)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4781..4800
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4888..4907
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4888..4906
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4907..4931
                     /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             4946..4968
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4981..4998
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5002..5016
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             5020..5040
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             5323..5367
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(5405..5423)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5419..5466
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc
       61 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
      121 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
      181 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
      241 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
      301 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
      361 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
      421 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt
      481 tacaatttct ggcggcacga tggcatgaga ttatcaaaaa ggatcttcac ctagatcctt
      541 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
      601 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
      661 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
      721 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
      781 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
      841 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
      901 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
      961 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     1021 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     1081 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     1141 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     1201 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     1261 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     1321 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     1381 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     1441 acacggaaat gttgaatact catactcttc ctttttcaat catgattgaa gcatttatca
     1501 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     1561 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     1621 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     1681 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     1741 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     1801 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     1861 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     1921 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     1981 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     2041 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     2101 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     2161 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     2221 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     2281 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     2341 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     2401 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     2461 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg
     2521 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg
     2581 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     2641 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa
     2701 agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc
     2761 tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg
     2821 gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg
     2881 gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat
     2941 gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag
     3001 agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag
     3061 ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc
     3121 cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc
     3181 gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc
     3241 taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg
     3301 ctagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc
     3361 ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg
     3421 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     3481 gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg
     3541 caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct
     3601 ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga
     3661 gctgtcttcg gtatcgtcgt atcccactac cgagatgtcc gcaccaacgc gcagcccgga
     3721 ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt
     3781 gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca
     3841 gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc
     3901 agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg
     3961 gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat
     4021 aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca
     4081 ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact
     4141 gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc
     4201 taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac
     4261 aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg
     4321 tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc
     4381 ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac
     4441 ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt
     4501 caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg
     4561 ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc
     4621 agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg
     4681 agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag
     4741 cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg
     4801 cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga
     4861 tcgagatcga tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg
     4921 ataacaattc ccctctagaa ataattttgt ttaactttaa gaaggagata taccatggca
     4981 catcaccacc accatcacgt ggatgacgac gacaagatgg agaatcttta ttttcagggt
     5041 tccaaaatgt ccgatgtgaa atgtaccagc gtcgtcctgc tgagcgtcct gcaacaactg
     5101 cgtgtcgagt cctcctccaa actgtgggcg cagtgcgtgc agctgcacaa cgacatcctg
     5161 ctggcgaagg ataccaccga ggctttcgaa aaaatggtga gcctgctgag cgttctgctg
     5221 agcatgcagg gtgcggttga catcaacaag ctgtgcgagg aaatgctgga taaccgtgct
     5281 accctgcagt aaccgggctt ctcctcaaat ctcgagtctg gtaaagaaac cgctgctgcg
     5341 aaatttgaac gccagcacat ggactcgtct actagcgcag cttaattaac ctaggctgct
     5401 gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt
     5461 tttttgctga
//
