LOCUS       sequence-311056-        7383 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 7383)
  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 7383)
  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..7383
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     969..988
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1008..1085)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1291..1310
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1394..1413
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1394..1412
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1413..1437
                     /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             1452..1474
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1485..1502
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1527..1547
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             3641..3658
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3711..3729)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3725..3772
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3809..4264
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3896..3915)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4106..4127
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4356..5171)
                     /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(4356..5171)
                     /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     5079..5098
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5163..5182
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5293..5881
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5782..5801
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6035..6052
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6067..6206
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6132..6151)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6292..6314
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6308..6499)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(7308..7383,1..1007))
                     /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"
ORIGIN
        1 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga
       61 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc
      121 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg
      181 gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg
      241 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
      301 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc
      361 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc
      421 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
      481 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
      541 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
      601 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
      661 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
      721 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
      781 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
      841 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
      901 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
      961 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
     1021 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg
     1081 gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag
     1141 taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc
     1201 caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag
     1261 cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac
     1321 cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc
     1381 gatcccgcga aattaatacg actcactata ggggaattgt gagcggataa caattcccct
     1441 ctagaaataa ttttgtttaa ctttaagaag gagatataca tatgcaccat catcatcatc
     1501 attcttctgg tgtagatctg ggtaccgaga acctgtactt ccaatccatg gcggaaaatg
     1561 tgactggtct gtttaaagat tgtagtaaag tcattaccgg gctgcatccg acccaagcac
     1621 caacacatct gagtgtcgac accaagttca aaacggaagg gctgtgcgta gatattcccg
     1681 gtattccaaa agatatgact tatcgccgtc tgattagcat gatgggcttc aaaatgaatt
     1741 accaagtgaa cggttatccg aatatgttca ttacccgcga ggaagctatt cgccatgtgc
     1801 gcgcatggat cggcttcgac gtggagggat gtcatgcgac ccgcgaagcc gtggggacta
     1861 acttaccgct gcagctgggc ttttctaccg gcgtgaattt agttgccgta ccgaccggat
     1921 atgtagacac gccgaacaac acggattttt cgcgtgtgtc agccaaacca cctccgggcg
     1981 atcaatttaa gcacctgatc ccactgatgt acaaaggctt accatggaat gttgtacgca
     2041 ttaagatcgt gcagatgttg agcgatactc tgaaaaattt gtcggatcgg gtagtgtttg
     2101 tcctgtgggc gcacggcttc gagcttactt cgatgaaata ctttgtcaag atcggaccgg
     2161 aacgtacctg ttgtctgtgc gaccgtcgcg ccacctgttt cagtaccgcg tctgacactt
     2221 atgcgtgttg gcaccactcg attggttttg attacgtata caatccgttt atgatcgatg
     2281 tccaacaatg gggctttacg gggaatttgc agtctaatca cgatctgtac tgtcaggtac
     2341 acggcaatgc ccacgtcgcc agctgtgacg ctattatgac ccgttgcctg gctgtccacg
     2401 aatgtttcgt gaaacgtgtt gattggacca ttgaataccc tattatcggt gacgagctga
     2461 aaattaacgc tgcgtgccgt aaagttcaac acatggtggt caaagcggcc ctgttggccg
     2521 ataaattccc agttttgcac gacattggca atcctaaagc aatcaaatgt gttccccaag
     2581 ctgatgtgga atggaaattc tacgatgcac agccatgctc cgacaaggct tacaaaattg
     2641 aagaactgtt ctatagctat gccacccatt ctgacaaatt cacggatggt gtttgtctgt
     2701 tttggaactg taacgttgat cgttatccgg caaactccat tgtgtgtcgc tttgatactc
     2761 gggtcctctc aaatctcaat ttacccggct gcgatggcgg ttctctgtac gtgaataaac
     2821 acgccttcca tactccggca tttgataaat cagcgttcgt caatttaaag cagctgccgt
     2881 ttttctatta tagcgatagc ccctgcgaaa gccatgggaa acaggtggtt agcgatatcg
     2941 actatgtccc gctgaagtcg gccacgtgta tcacccgttg taacttagga ggcgcagttt
     3001 gccgtcacca tgcgaacgag taccggctgt atttagacgc atataacatg atgatttctg
     3061 ccggcttcag tctttgggtt tacaaacagt tcgataccta taatctgtgg aacacgttta
     3121 ctcgtctcca ataataataa tttaaggaga tatacatggc gggaaacgcg accgaagtgc
     3181 ctgcaaattc tacagtgctg tccttctgtg cctttgctgt tgacgcggcg aaggcataca
     3241 aagactatct cgcgtctgga ggccagccga tcacaaattg cgtcaaaatg ctgtgtacac
     3301 acaccggcac gggtcaggca attaccgtaa ccccggaagc caacatggat caggaaagtt
     3361 tcggcggagc aagctgttgc ctctattgtc gttgccatat cgaccacccc aacccgaaag
     3421 gcttttgcga tttaaaaggc aagtatgtgc agatcccgac gacctgcgcg aacgatccgg
     3481 tgggcttcac gttaaaaaac accgtgtgca ccgtgtgtgg tatgtggaaa ggctatggct
     3541 gtagttgtga tcagttacgc gagccaatgt tgcagtaaca gtaaaggtgg atacggatcc
     3601 gaattcgagc tccgtcgaca agcttgcggc cgcactcgag caccaccacc accaccactg
     3661 agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca
     3721 ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg
     3781 aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg
     3841 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     3901 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     3961 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     4021 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     4081 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     4141 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     4201 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
     4261 atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     4321 tacattcaaa tatgtatccg ctcatgaatt aattcttaga aaaactcatc gagcatcaaa
     4381 tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc
     4441 tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg
     4501 tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata
     4561 aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt
     4621 ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca
     4681 ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga
     4741 tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc
     4801 agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt
     4861 ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg
     4921 atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca
     4981 tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca
     5041 tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca
     5101 tataaatcag catccatgtt ggaatttaat cgcggcctag agcaagacgt ttcccgttga
     5161 atatggctca taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat
     5221 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     5281 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     5341 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     5401 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     5461 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     5521 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     5581 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     5641 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     5701 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     5761 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     5821 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     5881 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     5941 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     6001 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     6061 agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg
     6121 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat
     6181 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct
     6241 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     6301 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct
     6361 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt
     6421 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg
     6481 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa
     6541 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc
     6601 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa
     6661 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta
     6721 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg
     6781 tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag
     6841 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac
     6901 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca
     6961 cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg
     7021 gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc
     7081 cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg
     7141 gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca
     7201 tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag
     7261 atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt
     7321 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     7381 gcg
//
