LOCUS       sequence-338554-        5936 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 5936)
  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 5936)
  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 - P43 (Oxford)
FEATURES             Location/Qualifiers
     source          1..5936
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             7..24
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             49..69
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             583..600
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(653..671)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      667..714
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      751..1206
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(838..857)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1048..1069
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1232..1336
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1337..1405,1406..2197)
                     /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(1555..1574)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2368..2956
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2857..2876
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3110..3127
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3142..3281
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3207..3226)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3367..3389
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3383..3574)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4383..5465)
                     /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     5427..5446
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5466..5543)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5749..5768
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5852..5871
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5852..5870
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5871..5895
                     /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             5910..5932
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 catatgcacc atcaccatca ccattcttct ggtgtagatc tgggtaccga aaacctgtat
       61 tttcagtcga tggtaaattc tttcagtggt tatctgaagt tgacggataa cgtctatatt
      121 aaaaacgctg acattgtgga agaggcgaaa aaggtaaagc ctacagttgt ggttaatgcg
      181 gcaaacgtct atttaaaaca tggtggaggt gtggcggggg ctctcaacaa agcgactaac
      241 aatgcaatgc aggtggaaag cgatgattac attgcgacca acggcccact gaaagttggc
      301 ggttcgtgcg tgctctcagg tcacaatctg gctaaacatt gtctgcacgt cgtggggccc
      361 aacgttaata aaggcgagga tatccaacta ttaaaaagcg catacgaaaa ctttaaccag
      421 catgaagttc tgctggcgcc gttgcttagc gccggaatat tcggcgccga cccgatccac
      481 tccctgcgcg tctgcgttga tacggtacgt accaatgtgt atctggccgt ctttgacaaa
      541 aatctttacg ataaactggt gagttccttt ctctgactcg agcaccacca ccaccaccac
      601 tgagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag
      661 caataactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa
      721 ggaggaacta tatccggatt ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg
      781 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
      841 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
      901 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
      961 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     1021 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     1081 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     1141 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta
     1201 caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     1261 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     1321 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     1381 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     1441 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     1501 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     1561 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     1621 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     1681 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     1741 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     1801 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     1861 gcgtgacacc acgatgcctg cagcaatggc aacaacgttg cgcaaactat taactggcga
     1921 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     1981 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     2041 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     2101 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     2161 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     2221 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     2281 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     2341 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     2401 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     2461 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
     2521 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     2581 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     2641 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     2701 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     2761 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     2821 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     2881 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     2941 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     3001 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     3061 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     3121 gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     3181 ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     3241 tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac
     3301 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     3361 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc
     3421 agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat
     3481 ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg
     3541 ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc
     3601 tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg
     3661 atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc
     3721 ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag
     3781 gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg
     3841 gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg
     3901 ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg
     3961 attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga
     4021 gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga
     4081 aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata
     4141 ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga
     4201 cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg
     4261 cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca
     4321 agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc
     4381 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     4441 aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt
     4501 gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg
     4561 tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata
     4621 taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc
     4681 agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc
     4741 atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg
     4801 gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta
     4861 tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg
     4921 atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg
     4981 gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca
     5041 ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc
     5101 agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg
     5161 cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc
     5221 gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc
     5281 aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc
     5341 atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg
     5401 cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt
     5461 ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag
     5521 gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat
     5581 taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc
     5641 atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc
     5701 gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc
     5761 gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc
     5821 gtagaggatc gagatctcga tcccgcgaaa ttaatacgac tcactatagg ggaattgtga
     5881 gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga gatata
//
