LOCUS       sequence-433377-        6757 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli plasmid (T7lac promoter) for full-length, SARS CoV-2 
            Nsp15-V66L (uridine-specific nidoviral endoribonuclease, 
            expression-optimized gene) with N-terminal TEV protease cleavable 
            His6-tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6757)
  AUTHORS   Sonowal M, Ketawala G, Nagaratnam N, Logeswaran D, Basu S, de 
            Sanctis D, Orlans J, Rose SL, Jernigan RJ, Hu H, Aguilar JDM, 
            Ranaweera ME, Zacks MA, Chen JJ, Hansen DT, Schrag LG, Fromme R, 
            Botha S, Fromme P
  TITLE     Functional implications of hexameric dynamics in SARS-CoV-2 Nsp15.
  JOURNAL   Protein Sci. 2025 Jun;34(6):e70115. doi: 10.1002/pro.70115.
  PUBMED    40411374
REFERENCE   2  (bases 1 to 6757)
  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: "Protein 
            Sci."; date: "2025-06"; volume: "34(6):e70115. doi"; pages: " 
            10.1002/pro.70115"
COMMENT     Sequence Label: pHis-TEV-Nsp15-V66L
FEATURES             Location/Qualifiers
     source          1..6757
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             92..109
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             134..154
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(1258..1276)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1272..1319
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1494..1499,1500..1516,1517..1522))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1549..1567)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1635..1739
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1740..1808,1809..2600)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1958..1977)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2771..3359
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3260..3279
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3513..3530
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3545..3684
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3610..3629)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3770..3792
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3786..3977)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5289..6371)
                     /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     6333..6352
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6372..6449)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6655..6674
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gcaccaccac caccaccata gcagcggcgt
      121 tgacctgggc accgagaacc tgtatttcca aagcaatgcg atgagcctgg agaatgttgc
      181 gtttaacgtg gttaacaagg gtcacttcga tggtcagcaa ggcgaggtgc cggttagcat
      241 cattaacaac accgtgtaca ccaaggttga cggcgtggat gttgagctgt ttgaaaacaa
      301 aaccaccctg ccggtgaacg ttgcgttcga gctgtgggcg aagcgtaaca tcaaaccgct
      361 gccggaagtt aagattctga acaacctggg tgtggacatc gcggcgaaca ccgttatttg
      421 ggactataaa cgtgatgcgc cggcgcacat cagcaccatt ggcgtttgca gcatgaccga
      481 catcgcgaag aaaccgaccg aaaccatttg cgcgccgctg accgtgttct ttgacggtcg
      541 tgtggatggc caggttgacc tgtttcgtaa cgcgcgtaac ggtgtgctga tcaccgaggg
      601 tagcgttaaa ggcctgcagc cgagcgtggg tccgaaacaa gcgagcctga acggtgttac
      661 cctgattggc gaagcggtga agacccagtt caactactat aagaaagtgg atggtgtggt
      721 tcagcaactg ccggaaacct actttaccca gagccgtaac ctgcaagagt tcaagccgcg
      781 tagccaaatg gagatcgatt ttctggaact ggcgatggac gagttcattg aacgttacaa
      841 actggagggt tatgcgtttg aacacatcgt ttacggcgac ttcagccaca gccaactggg
      901 tggcctgcac ctgctgattg gtctggcgaa gcgtttcaaa gagagcccgt ttgagctgga
      961 agatttcatc ccgatggaca gcaccgtgaa gaactatttt attaccgatg cgcagaccgg
     1021 cagcagcaaa tgcgtgtgca gcgttatcga cctgctgctg gacgatttcg ttgagatcat
     1081 taaaagccag gacctgagcg tggtgagcaa ggtggttaaa gtgaccatcg actataccga
     1141 aattagcttc atgctgtggt gcaaagatgg tcacgtggaa accttctacc cgaaactgca
     1201 ataataagga tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg
     1261 ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc
     1321 tgaaaggagg aactatatcc ggatatcccg caagaggccc ggcagtaccg gcataaccaa
     1381 gcctatgcct acagcatcca gggtgacggt gccgaggatg acgatgagcg cattgttaga
     1441 tttcatacac ggtgcctgac tgcgttagca atttaactgt gataaactac cgcattaaag
     1501 cttatcgatg ataagctgtc aaacatgaga attcttgaag acgaaagggc ctcgtgatac
     1561 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt
     1621 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     1681 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     1741 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     1801 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     1861 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     1921 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     1981 gtgttgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     2041 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     2101 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     2161 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     2221 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     2281 ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     2341 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     2401 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     2461 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     2521 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     2581 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     2641 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     2701 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     2761 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     2821 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     2881 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     2941 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     3001 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     3061 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     3121 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     3181 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     3241 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     3301 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     3361 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     3421 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     3481 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     3541 agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg
     3601 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg
     3661 ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga
     3721 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     3781 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca
     3841 tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg
     3901 agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt
     3961 ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg
     4021 ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg
     4081 ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa
     4141 atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc
     4201 cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt
     4261 tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac
     4321 gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca
     4381 gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc
     4441 cgtggccagg acccaacgct gcccgagatg cgccgcgtgc ggctgctgga gatggcggac
     4501 gcgatggata tgttctgcca agggttggtt tgcgcattca cagttctccg caagaattga
     4561 ttggctccaa ttcttggagt ggtgaatccg ttagcgaggt gccgccggct tccattcagg
     4621 tcgaggtggc ccggctccat gcaccgcgac gcaacgcggg gaggcagaca aggtataggg
     4681 cggcgcctac aatccatgcc aacccgttcc atgtgctcgc cgaggcggca taaatcgccg
     4741 tgacgatcag cggtccaatg atcgaagtta ggctggtaag agccgcgagc gatccttgaa
     4801 gctgtccctg atggtcgtca tctacctgcc tggacagcat ggcctgcaac gcgggcatcc
     4861 cgatgccgcc ggaagcgaga agaatcataa tggggaaggc catccagcct cgcgtcgcga
     4921 acgccagcaa gacgtagccc agcgcgtcgg ccgccatgcc ggcgataatg gcctgcttct
     4981 cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc
     5041 cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga
     5101 aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca
     5161 taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg
     5221 ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg
     5281 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     5341 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc
     5401 accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc
     5461 aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc
     5521 gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca
     5581 acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca
     5641 accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg
     5701 gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga
     5761 tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac
     5821 agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct
     5881 tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc
     5941 ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta
     6001 atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg
     6061 acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat
     6121 ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca
     6181 atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc
     6241 tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc
     6301 accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt
     6361 actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg
     6421 cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc
     6481 ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa
     6541 tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc
     6601 cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat
     6661 gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg
     6721 tccggcgtag aggatcgaga tctcgatccc gcgaaat
//
