LOCUS       sequence-461550-        5859 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of the SARS-CoV-2 nsp3 macrodomain for 
            crystallization of SARS-CoV-2 nsp3 macrodomain in P 4 3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5859)
  TITLE     ASAP Discovery Plasmids 2024
REFERENCE   2  (bases 1 to 5859)
  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"
COMMENT     Sequence Label: SARS-CoV-2 isolate:Wuhan-Hu-1 nsp3 macrodomain
FEATURES             Location/Qualifiers
     source          1..5859
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(215..235)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             complement(260..277)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             288..310
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    325..349
                     /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)."
     primer_bind     complement(349..368)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(350..368)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(452..471)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        677..754
                     /gene="lacI"
                     /label=lacI promoter
     CDS             755..1837
                     /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     complement(774..793)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2646..2837
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(2831..2853)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    2939..3078
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     2994..3013
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3093..3110)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3264..3852)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3344..3363)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(3963..3982)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3974..4789
                     /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             3974..4789
                     /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     complement(4047..4066)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(4881..5336)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5018..5039)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5230..5249
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(5373..5420)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     5416..5434
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(5487..5504)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
ORIGIN
        1 cattggttgc aatataatcg tcgctctcaa cctgcattgc attattggtt gctttattca
       61 gtgcaccggc aacaccacca ccatgtttca ggtaaacatt ggctgcatta acaacaacgg
      121 tcggtttaac ttttttggcc tcttccacaa tatcggcgtt tttgatatac acgttatcgg
      181 tcagtttcag ataaccgcta aagctattaa ccatggattg gaagtacagg ttctcggtac
      241 ccagatctac accagaagaa tgatgatgat gatggtgcat atgtatatct ccttcttaaa
      301 gttaaacaaa attatttcta gaggggaatt gttatccgct cacaattccc ctatagtgag
      361 tcgtattaat ttcgcgggat cgagatctcg atcctctacg ccggacgcat cgtggccggc
      421 atcaccggcg ccacaggtgc ggttgctggc gcctatatcg ccgacatcac cgatggggaa
      481 gatcgggctc gccacttcgg gctcatgagc gcttgtttcg gcgtgggtat ggtggcaggc
      541 cccgtggccg ggggactgtt gggcgccatc tccttgcatg caccattcct tgcggcggcg
      601 gtgctcaacg gcctcaacct actactgggc tgcttcctaa tgcaggagtc gcataaggga
      661 gagcgtcgag atcccggaca ccatcgaatg gcgcaaaacc tttcgcggta tggcatgata
      721 gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt
      781 cgcagagtat gccggtgtct cttatcagac cgtttcccgc gtggtgaacc aggccagcca
      841 cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg gcggagctga attacattcc
      901 caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg ctgattggcg ttgccacctc
      961 cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca
     1021 actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc
     1081 ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg atcattaact atccgctgga
     1141 tgaccaggat gccattgctg tggaagctgc ctgcactaat gttccggcgt tatttcttga
     1201 tgtctctgac cagacaccca tcaacagtat tattttctcc catgaagacg gtacgcgact
     1261 gggcgtggag catctggtcg cattgggtca ccagcaaatc gcgctgttag cgggcccatt
     1321 aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat aaatatctca ctcgcaatca
     1381 aattcagccg atagcggaac gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac
     1441 catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca acgatcagat
     1501 ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg cggatatctc
     1561 ggtagtggga tacgacgata ccgaagacag ctcatgttat atcccgccgt taaccaccat
     1621 caaacaggat tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc aactctctca
     1681 gggccaggcg gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac
     1741 cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     1801 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt
     1861 agctcactca ttaggcaccg ggatctcgac cgatgccctt gagagccttc aacccagtca
     1921 gctccttccg gtgggcgcgg ggcatgacta tcgtcgccgc acttatgact gtcttcttta
     1981 tcatgcaact cgtaggacag gtgccggcag cgctctgggt cattttcggc gaggaccgct
     2041 ttcgctggag cgcgacgatg atcggcctgt cgcttgcggt attcggaatc ttgcacgccc
     2101 tcgctcaagc cttcgtcact ggtcccgcca ccaaacgttt cggcgagaag caggccatta
     2161 tcgccggcat ggcggcccca cgggtgcgca tgatcgtgct cctgtcgttg aggacccggc
     2221 taggctggcg gggttgcctt actggttagc agaatgaatc accgatacgc gagcgaacgt
     2281 gaagcgactg ctgctgcaaa acgtctgcga cctgagcaac aacatgaatg gtcttcggtt
     2341 tccgtgtttc gtaaagtctg gaaacgcgga agtcagcgcc ctgcaccatt atgttccgga
     2401 tctgcatcgc aggatgctgc tggctaccct gtggaacacc tacatctgta ttaacgaagc
     2461 gctggcattg accctgagtg atttttctct ggtcccgccg catccatacc gccagttgtt
     2521 taccctcaca acgttccagt aaccgggcat gttcatcatc agtaacccgt atcgtgagca
     2581 tcctctctcg tttcatcggt atcattaccc ccatgaacag aaatccccct tacacggagg
     2641 catcagtgac caaacaggaa aaaaccgccc ttaacatggc ccgctttatc agaagccaga
     2701 cattaacgct tctggagaaa ctcaacgagc tggacgcgga tgaacaggca gacatctgtg
     2761 aatcgcttca cgaccacgct gatgagcttt accgcagctg cctcgcgcgt ttcggtgatg
     2821 acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg
     2881 atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg
     2941 cagccatgac ccagtcacgt agcgatagcg gagtgtatac tggcttaact atgcggcatc
     3001 agagcagatt gtactgagag tgcaccattg cggtgtgaaa taccgcacag atgcgtaagg
     3061 agaaaatacc gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     3121 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     3181 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     3241 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     3301 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     3361 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     3421 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     3481 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     3541 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     3601 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     3661 acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc
     3721 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     3781 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     3841 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     3901 aactcacgtt aagggatttt ggtcatgaac aataaaactg tctgcttaca taaacagtaa
     3961 tacaaggggt gttatgagcc atattcaacg ggaaacgtct tgctctaggc cgcgattaaa
     4021 ttccaacatg gatgctgatt tatatgggta taaatgggct cgcgataatg tcgggcaatc
     4081 aggtgcgaca atctatcgat tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca
     4141 tggcaaaggt agcgttgcca atgatgttac agatgagatg gtcagactaa actggctgac
     4201 ggaatttatg cctcttccga ccatcaagca ttttatccgt actcctgatg atgcatggtt
     4261 actcaccact gcgatccccg ggaaaacagc attccaggta ttagaagaat atcctgattc
     4321 aggtgaaaat attgttgatg cgctggcagt gttcctgcgc cggttgcatt cgattcctgt
     4381 ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat
     4441 gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct ggcctgttga
     4501 acaagtctgg aaagaaatgc ataaactttt gccattctca ccggattcag tcgtcactca
     4561 tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag gttgtattga
     4621 tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat ggaactgcct
     4681 cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta ttgataatcc
     4741 tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaag aattaattca
     4801 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     4861 ttccccgaaa agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa
     4921 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
     4981 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
     5041 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
     5101 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa
     5161 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc
     5221 gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt
     5281 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca
     5341 ttcgccaatc cggatatagt tcctcctttc agcaaaaaac ccctcaagac ccgtttagag
     5401 gccccaaggg gttatgctag ttattgctca gcggtggcag cagccaactc agcttccttt
     5461 cgggctttgt tagcagccgg atctcagtgg tggtggtggt ggtgctcgag tgcggccgca
     5521 agcttgtcga cggagctcga attcggatcc gtatccacct ttactgtcat aagaagctgc
     5581 tcaccagttt gtcatacagg tttttatcaa acactgccag atacacattg gtacgaacgg
     5641 tatcaacaca aacacgcagg ctatgaatcg gatctgcacc aaaaatacct gcgctcagca
     5701 gcggtgccag cagaacttca tgctgattaa agttttcata ggcgcttttc agcagctgaa
     5761 tatcttcacc tttattcaca ttcggaccaa caacatgcag acagtgtttt gccagattat
     5821 gaccgctcag aacacagcta ccaccaactt tcagcggac
//
