LOCUS       sequence-308623-        7387 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial co-expression vector fo Covid-SARS2 NSP14 and NSP10.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7387)
  AUTHORS   Baddock HT, Brolih S, Yosaatmadja Y, Ratnaweera M, Bielinski M, 
            Swift LP, Cruz-Migoni A, Fan H, Keown JR, Walker AP, Morris GM, 
            Grimes JM, Fodor E, Schofield CJ, Gileadi O, McHugh PJ
  TITLE     Characterization of the SARS-CoV-2 ExoN (nsp14ExoN-nsp10) complex: 
            implications for its role in viral genome stability and inhibitor 
            identification.
  JOURNAL   Nucleic Acids Res. 2022 Jan 17. pii: 6509090. doi: 
            10.1093/nar/gkab1303.
  PUBMED    35037045
REFERENCE   2  (bases 1 to 7387)
  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: "Nucleic 
            Acids Res. 2022 Jan 17. pii: 6509090. doi: 10.1093/nar/gkab1303."
COMMENT     Sequence Label: COVID-SARS2 NSP14/10
FEATURES             Location/Qualifiers
     source          1..7387
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             2094..2111
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2164..2182)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2178..2225
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2262..2717
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2349..2368)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2559..2580
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2810..3625)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(2810..3625)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3533..3552
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3617..3636
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3747..4335
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4236..4255
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4489..4506
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4521..4663
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4589..4608)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4749..4771
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4765..4956)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5765..6847)
                     /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     6809..6828
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6848..6925)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7131..7150
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     7234..7253
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7234..7252
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    7253..7277
                     /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             7292..7314
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             7325..7342
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             7367..7387
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
ORIGIN
        1 atggcggaaa atgtgactgg tctgtttaaa gattgtagta aagtcattac cgggctgcat
       61 ccgacccaag caccaacaca tctgagtgtc gacaccaagt tcaaaacgga agggctgtgc
      121 gtagatattc ccggtattcc aaaagatatg acttatcgcc gtctgattag catgatgggc
      181 ttcaaaatga attaccaagt gaacggttat ccgaatatgt tcattacccg cgaggaagct
      241 attcgccatg tgcgcgcatg gatcggcttc gacgtggagg gatgtcatgc gacccgcgaa
      301 gccgtgggga ctaacttacc gctgcagctg ggcttttcta ccggcgtgaa tttagttgcc
      361 gtaccgaccg gatatgtaga cacgccgaac aacacggatt tttcgcgtgt gtcagccaaa
      421 ccacctccgg gcgatcaatt taagcacctg atcccactga tgtacaaagg cttaccatgg
      481 aatgttgtac gcattaagat cgtgcagatg ttgagcgata ctctgaaaaa tttgtcggat
      541 cgggtagtgt ttgtcctgtg ggcgcacggc ttcgagctta cttcgatgaa atactttgtc
      601 aagatcggac cggaacgtac ctgttgtctg tgcgaccgtc gcgccacctg tttcagtacc
      661 gcgtctgaca cttatgcgtg ttggcaccac tcgattggtt ttgattacgt atacaatccg
      721 tttatgatcg atgtccaaca atggggcttt acggggaatt tgcagtctaa tcacgatctg
      781 tactgtcagg tacacggcaa tgcccacgtc gccagctgtg acgctattat gacccgttgc
      841 ctggctgtcc acgaatgttt cgtgaaacgt gttgattgga ccattgaata ccctattatc
      901 ggtgacgagc tgaaaattaa cgctgcgtgc cgtaaagttc aacacatggt ggtcaaagcg
      961 gccctgttgg ccgataaatt cccagttttg cacgacattg gcaatcctaa agcaatcaaa
     1021 tgtgttcccc aagctgatgt ggaatggaaa ttctacgatg cacagccatg ctccgacaag
     1081 gcttacaaaa ttgaagaact gttctatagc tatgccaccc attctgacaa attcacggat
     1141 ggtgtttgtc tgttttggaa ctgtaacgtt gatcgttatc cggcaaactc cattgtgtgt
     1201 cgctttgata ctcgggtcct ctcaaatctc aatttacccg gctgcgatgg cggttctctg
     1261 tacgtgaata aacacgcctt ccatactccg gcatttgata aatcagcgtt cgtcaattta
     1321 aagcagctgc cgtttttcta ttatagcgat agcccctgcg aaagccatgg gaaacaggtg
     1381 gttagcgata tcgactatgt cccgctgaag tcggccacgt gtatcacccg ttgtaactta
     1441 ggaggcgcag tttgccgtca ccatgcgaac gagtaccggc tgtatttaga cgcatataac
     1501 atgatgattt ctgccggctt cagtctttgg gtttacaaac agttcgatac ctataatctg
     1561 tggaacacgt ttactcgtct ccaataataa taatttaagg agatatacat ggcgggaaac
     1621 gcgaccgaag tgcctgcaaa ttctacagtg ctgtccttct gtgcctttgc tgttgacgcg
     1681 gcgaaggcat acaaagacta tctcgcgtct ggaggccagc cgatcacaaa ttgcgtcaaa
     1741 atgctgtgta cacacaccgg cacgggtcag gcaattaccg taaccccgga agccaacatg
     1801 gatcaggaaa gtttcggcgg agcaagctgt tgcctctatt gtcgttgcca tatcgaccac
     1861 cccaacccga aaggcttttg cgatttaaaa ggcaagtatg tgcagatccc gacgacctgc
     1921 gcgaacgatc cggtgggctt cacgttaaaa aacaccgtgt gcaccgtgtg tggtatgtgg
     1981 aaaggctatg gctgtagttg tgatcagtta cgcgagccaa tgttgcagta acagtaaagg
     2041 tggatacgga tccgaattcg agctccgtcg acaagcttgc ggccgcactc gagcaccacc
     2101 accaccacca ctgagatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg
     2161 ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt
     2221 ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct gtagcggcgc
     2281 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     2341 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     2401 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     2461 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     2521 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     2581 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     2641 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     2701 attaacgttt acaatttcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     2761 tttatttttc taaatacatt caaatatgta tccgctcatg aattaattct tagaaaaact
     2821 catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt
     2881 gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa
     2941 gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc
     3001 cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg
     3061 agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct
     3121 cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga
     3181 gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc
     3241 gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata
     3301 cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac
     3361 ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca
     3421 tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg
     3481 catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag
     3541 cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag
     3601 acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca
     3661 gttttattgt tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     3721 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     3781 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     3841 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     3901 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     3961 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     4021 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     4081 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     4141 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     4201 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     4261 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     4321 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     4381 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     4441 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     4501 agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     4561 tcacaccgca tatatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     4621 agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac
     4681 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     4741 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     4801 gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc
     4861 atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg
     4921 ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt
     4981 tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac
     5041 tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat
     5101 gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt
     5161 aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca
     5221 gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt
     5281 tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg
     5341 tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag
     5401 gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct gcttctcgcc
     5461 gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa
     5521 taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat
     5581 gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag
     5641 tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct
     5701 caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt
     5761 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     5821 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca
     5881 gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc
     5941 ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga
     6001 tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc
     6061 gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca
     6121 gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca
     6181 tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt
     6241 tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg
     6301 cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat
     6361 gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa
     6421 cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga
     6481 tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc
     6541 cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa
     6601 tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca
     6661 gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg
     6721 ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca
     6781 cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg
     6841 gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa
     6901 aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc
     6961 attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt
     7021 gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg
     7081 ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg
     7141 gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg
     7201 gcgtagagga tcgagatctc gatcccgcga aattaatacg actcactata ggggaattgt
     7261 gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag gagatataca
     7321 tatgcaccat catcatcatc attcttctgg tgtagatctg ggtaccgaga acctgtactt
     7381 ccaatcc
//
