LOCUS       sequence-338555-        5924 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of 6xHis tagged protein for crystallization, includes TEV
            protease site for tag removal.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5924)
  AUTHORS   Schuller M, Correy GJ, Gahbauer S, Fearon D, Wu T, Diaz RE, Young 
            ID, Carvalho Martins L, Smith DH, Schulze-Gahmen U, Owens TW, 
            Deshpande I, Merz GE, Thwin AC, Biel JT, Peters JK, Moritz M, 
            Herrera N, Kratochvil HT, Aimon A, Bennett JM, Brandao Neto J, Cohen
            AE, Dias A, Douangamath A, Dunnett L, Fedorov O, Ferla MP, Fuchs MR,
            Gorrie-Stone TJ, Holton JM, Johnson MG, Krojer T, Meigs G, Powell 
            AJ, Rack JGM, Rangel VL, Russi S, Skyner RE, Smith CA, Soares AS, 
            Wierman JL, Zhu K, O'Brien P, Jura N, Ashworth A, Irwin JJ, Thompson
            MC, Gestwicki JE, von Delft F, Shoichet BK, Fraser JS, Ahel I
  TITLE     Fragment binding to the Nsp3 macrodomain of SARS-CoV-2 identified 
            through crystallographic screening and computational docking.
  JOURNAL   Sci Adv. 2021 Apr 14;7(16). pii: 7/16/eabf8711. doi: 
            10.1126/sciadv.abf8711. Print 2021 Apr.
  PUBMED    33853786
REFERENCE   2  (bases 1 to 5924)
  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: "Sci Adv."; 
            date: "2021-04-14"; volume: "7(16). pii: 7/16/eabf8711. doi"; pages:
            " 10.1126/sciadv.abf8711. Print 2021 Apr"
COMMENT     Sequence Label: Nsp3 Mac1 - C2 (UCSF)
FEATURES             Location/Qualifiers
     source          1..5924
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             7..24
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             25..45
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             571..588
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(641..659)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      655..702
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      739..1194
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(826..845)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1036..1057
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1220..1324
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1325..1393,1394..2185)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1543..1562)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2356..2944
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2845..2864
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3098..3115
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3130..3269
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3195..3214)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3355..3377
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3371..3562)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4371..5453)
                     /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     5415..5434
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5454..5531)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5737..5756
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5840..5859
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5840..5858
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5859..5883
                     /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             5898..5920
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 catatgcacc atcaccatca ccatgaaaac ctgtattttc agtcgaatgc cggcgaagta
       61 aattctttca gtggttatct gaagttgacg gataacgtct atattaaaaa cgctgacatt
      121 gtggaagagg cgaaaaaggt aaagcctaca gttgtggtta atgcggcaaa cgtctattta
      181 aaacatggtg gaggtgtggc gggggctctc aacaaagcga ctaacaatgc aatgcaggtg
      241 gaaagcgatg attacattgc gaccaacggc ccactgaaag ttggcggttc gtgcgtgctc
      301 tcaggtcaca atctggctaa acattgtctg cacgtcgtgg ggcccaacgt taataaaggc
      361 gaggatatcc aactattaaa aagcgcatac gaaaacttta accagcatga agttctgctg
      421 gcgccgttgc ttagcgccgg aatattcggc gccgacccga tccactccct gcgcgtctgc
      481 gttgatacgg tacgtaccaa tgtgtatctg gccgtctttg acaaaaatct ttacgataaa
      541 ctggtgagtt cctttctcga atgactcgag caccaccacc accaccactg agatccggct
      601 gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca
      661 taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata
      721 tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
      781 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
      841 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
      901 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
      961 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     1021 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     1081 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     1141 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg
     1201 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     1261 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     1321 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     1381 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     1441 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     1501 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     1561 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     1621 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     1681 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     1741 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     1801 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     1861 gatgcctgca gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     1921 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     1981 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     2041 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     2101 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     2161 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     2221 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     2281 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     2341 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     2401 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     2461 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
     2521 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     2581 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     2641 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     2701 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     2761 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     2821 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     2881 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     2941 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     3001 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     3061 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     3121 ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa
     3181 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc
     3241 tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc
     3301 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     3361 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa
     3421 gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct
     3481 cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg
     3541 cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg
     3601 taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg
     3661 cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga
     3721 gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg
     3781 gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc
     3841 gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg
     3901 cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct
     3961 aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc
     4021 gcacccgtgg ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg
     4081 cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca
     4141 ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg
     4201 ccggcacctg tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag
     4261 tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc
     4321 gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg
     4381 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     4441 gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac
     4501 agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt
     4561 tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg
     4621 tcttcggtat cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg
     4681 gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga
     4741 acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg
     4801 ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc
     4861 agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga
     4921 cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata
     4981 ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca
     5041 gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg
     5101 cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc
     5161 atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt
     5221 tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg
     5281 cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc
     5341 actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc
     5401 tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc
     5461 accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat
     5521 tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc
     5581 cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat
     5641 ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct
     5701 catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc
     5761 agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga
     5821 gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat
     5881 tcccctctag aaataatttt gtttaacttt aagaaggaga tata
//
