LOCUS       sequence-219779-        5768 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of Met1 diUb.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5768)
  AUTHORS   Swatek KN, Aumayr M, Pruneda JN, Visser LJ, Berryman S, Kueck AF, 
            Geurink PP, Ovaa H, van Kuppeveld FJM, Tuthill TJ, Skern T, Komander
            D
  TITLE     Irreversible inactivation of ISG15 by a viral leader protease 
            enables alternative infection detection strategies.
  JOURNAL   Proc Natl Acad Sci U S A. 2018 Mar 6;115(10):2371-2376. doi: 
            10.1073/pnas.1710617115. Epub 2018 Feb 20.
  PUBMED    29463763
REFERENCE   2  (bases 1 to 5768)
  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"; doi: 
            "10.1073/pnas.1710617115"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2018-03-6- 6"; volume: "115"; issue: "10"; pages: "2371-2376"
COMMENT     Sequence Label: Met1 diUb (1-152, R42W)
FEATURES             Location/Qualifiers
     source          1..5768
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     106..125
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     209..228
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        209..227
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    228..252
                     /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             267..289
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             308..325
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             335..358
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             359..586
                     /codon_start=1
                     /gene="Homo sapiens UBC"
                     /product="human ubiquitin C"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQWLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     CDS             587..814
                     /codon_start=1
                     /product="human ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     CDS             841..858
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(911..929)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      925..972
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1009..1464
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1096..1115)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1306..1327
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1556..2371)
                     /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             complement(1556..2371)
                     /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     2279..2298
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2363..2382
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2493..3081
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2982..3001
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3235..3252
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3267..3406
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3332..3351)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3492..3514
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3508..3699)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4508..5590)
                     /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     5552..5571
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5591..5668)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
       61 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
      121 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
      181 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
      241 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat ataccatggg
      301 cagcagccat catcatcatc atcacagcag cggtctggaa gttctgtttc agggcccgat
      361 gcagatcttc gtgaagaccc tgactggtaa gaccatcact ctcgaagtgg agccgagtga
      421 caccattgag aatgtcaagg caaagatcca agacaaggaa ggcatccctc ctgaccagca
      481 gtggttgatc tttgctggga agcagctgga agatggacgc accctgtctg actacaacat
      541 ccagaaagag tccaccctgc acctggtcct ccgtctcaga ggtgggatgc aaatatttgt
      601 caaaacacta acaggaaaaa caataacact agaggtcgaa ccctctgata caatagaaaa
      661 cgtgaaagcc aaaatacagg ataaagaggg aatacccccc gatcaacaaa ggctaatatt
      721 cgccggaaaa caactagagg acgggaggac actaagtgat tataatatac aaaaggaaag
      781 tacactacat ctagtgctaa ggttaagagg tgggtgataa agctttctag accatttaaa
      841 caccaccacc accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg
      901 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
      961 aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta
     1021 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     1081 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     1141 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     1201 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     1261 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     1321 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     1381 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     1441 acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     1501 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgaatt aattcttaga
     1561 aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat
     1621 atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga
     1681 tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta
     1741 atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat
     1801 ccggtgagaa tggcaaaagt ttatgcattt ctttccagac ttgttcaaca ggccagccat
     1861 tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct
     1921 gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca
     1981 accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt
     2041 ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag
     2101 gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc
     2161 tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact
     2221 ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat
     2281 cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctag
     2341 agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag
     2401 cagacagttt tattgttcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     2461 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     2521 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     2581 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     2641 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     2701 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     2761 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     2821 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     2881 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     2941 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     3001 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     3061 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     3121 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     3181 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     3241 tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc
     3301 ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     3361 gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc
     3421 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     3481 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     3541 gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg
     3601 ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa
     3661 gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg
     3721 atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt
     3781 tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg
     3841 gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga
     3901 tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt
     3961 gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca
     4021 tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat
     4081 cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga
     4141 caggagcacg atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc
     4201 gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc
     4261 gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa
     4321 aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat
     4381 aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc
     4441 tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc
     4501 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     4561 cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca
     4621 ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca
     4681 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg
     4741 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa
     4801 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa
     4861 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg
     4921 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat
     4981 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca
     5041 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt
     5101 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg
     5161 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa
     5221 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga
     5281 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt
     5341 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa
     5401 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct
     5461 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca
     5521 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta
     5581 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc
     5641 gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc
     5701 tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat
     5761 ggtgcatg
//
