LOCUS       sequence-331340-        5603 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of Nsp3 Macrodomain 2 and Macrodomain 3 
            (residues 413-676).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5603)
  TITLE     Savchenko Lab Plasmids
REFERENCE   2  (bases 1 to 5603)
  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: pMCSG53_Nsp3_Unique
FEATURES             Location/Qualifiers
     source          1..5603
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     477..496
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(516..593)
                     /gene="lacI"
                     /label=lacI promoter
     tRNA            733..808
                     /gene="ileX"
                     /product="E. coli tRNA-Ile recognizing AUA"
                     /label=ileX
     primer_bind     957..976
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     tRNA            1078..1154
                     /gene="argU"
                     /product="E. coli tRNA-Arg recognizing AGA and AGG"
                     /label=argU
     primer_bind     1429..1448
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1429..1447
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1448..1472
                     /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             1487..1509
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1520..1537
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1562..1582
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             2446..2463
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2516..2534)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2530..2577
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(2701..2720)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2887..2990
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2991..3059,3060..3851)
                     /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(3209..3228)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4022..4610
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4511..4530
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4764..4781
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(4861..4880)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(join(5036..5603,1..515))
                     /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 tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg
       61 tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca
      121 tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg
      181 tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg
      241 gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg
      301 gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc
      361 acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc
      421 gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca
      481 tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct
      541 tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc
      601 tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc
      661 gttgagcacc gccgccgcaa ggaatggtgc atgcggattg cgacacggag ttactttata
      721 atccgctacc atggcccctt agctcagtgg ttagagcagg cgactcataa tcgcttggtc
      781 gctggttcaa gtccagcagg ggccaccaga tatagcaaag gctgacgaga aatcgtcagc
      841 ctttttgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata
      901 cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg
      961 atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggcgagatc aaaagccatt
     1021 gactcagcaa gggttgaccg tataattcac gcgattacac cgcattgcgg tatcaacgcg
     1081 cccttagctc agttggatag agcaacgacc ttctaagtcg tgggccgcag gttcgaatcc
     1141 tgcagggcgc gccattacaa ttcaatcagt tacgccttct ttatatcctc cataattcca
     1201 gagtgggaca tatttgggac attatcacca aaaatgtcgt ctattttcct cgcatgctct
     1261 gtcaaatgat taggcgcaag gtgagcatac ctacgaacca tttctatgga ctcccatccg
     1321 cccatttcct gaagcactga taatgggacg cctgactgaa tccaccggtg atgccggcca
     1381 cgatgcgtcc ggcgtagagg atcgagatcg atctcgatcc cgcgaaatta atacgactca
     1441 ctatagggga attgtgagcg gataacaatt cccctctaga aataattttg tttaacttta
     1501 agaaggagat atacatatgc accatcatca tcatcattct tctggtgtag atctgggtac
     1561 cgagaacctg tacttccaat ccaatatgaa aataaaggct tgtgttgaag aggtaacaac
     1621 cacccttgag gaaaccaagt tcttgaccga aaacctgttg ctgtatatcg acattaacgg
     1681 caacctgcac ccggacagcg cgacgttggt gagcgatatc gacattacct ttctcaaaaa
     1741 agacgctccg tatatcgttg gcgatgttgt gcaggagggc gttctgaccg cggttgtcat
     1801 cccgaccaaa aaggctggtg gtacgactga gatgctagcg aaggcgctgc gcaaggtgcc
     1861 tacagataat tacatcacca cgtatccggg tcagggtctg aatggctaca ccgtcgagga
     1921 ggccaaaacc gtgttgaaaa agtgcaaatc cgcgttttac atcctgccga gcatcatcag
     1981 caatgaaaaa caggagatcc tgggtacggt gtcttggaat ctgcgtgaaa tgctggcgca
     2041 cgccgaagag actcgcaagc tgatgccggt ttgtgtagaa acgaaagcca ttgtcagcac
     2101 cattcaacgt aagtataaag gtattaaaat tcaagagggt gtggttgatt acggcgcaag
     2161 attctacttc tataccagca aaaccaccgt cgctagcttg attaacacct taaacgatct
     2221 gaacgaaact ctcgtgacca tgccactggg ctacgttacc catggtctga acttggagga
     2281 ggctgcgcgt tatatgcgta gcctgaaagt gccggcaacc gtgagcgttt catctccgga
     2341 cgcagttact gcgtataacg gctacctgac gtcctcgtcc taaattggaa gtggataacg
     2401 gatccgaatt cgagctccgt cgacaagctt gcggccgcac tcgagcacca ccaccaccac
     2461 cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct
     2521 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
     2581 aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg
     2641 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     2701 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     2761 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     2821 aacttgatta gggtgatggt tcacattaac gcttacaatt taggtggcac ttttcgggga
     2881 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     2941 atgagacaat aaccctgata aatgcttcaa taatttgaaa aaggaagagt atgagtattc
     3001 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     3061 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     3121 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     3181 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     3241 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     3301 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     3361 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     3421 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     3481 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgcagcaa
     3541 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     3601 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     3661 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     3721 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     3781 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     3841 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     3901 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     3961 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     4021 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     4081 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     4141 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     4201 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     4261 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     4321 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     4381 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     4441 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     4501 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     4561 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     4621 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     4681 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     4741 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga
     4801 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag
     4861 tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac
     4921 tgtagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat
     4981 cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact
     5041 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     5101 ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg
     5161 gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc
     5221 tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg
     5281 agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg
     5341 actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag
     5401 tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc
     5461 agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc
     5521 cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct
     5581 ggtgacccaa tgcgaccaga tgc
//
