LOCUS       sequence-212886-        5570 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of mutant proISG15 C-term. domain.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5570)
  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 5570)
  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: mature ISG15 C-term. Domain (79-165, W123A)
FEATURES             Location/Qualifiers
     source          1..5570
                     /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             643..660
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(713..731)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      727..774
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      811..1266
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(898..917)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1108..1129
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1358..2173)
                     /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(1358..2173)
                     /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     2081..2100
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2165..2184
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2295..2883
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2784..2803
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3037..3054
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3069..3208
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3134..3153)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3294..3316
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3310..3501)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4310..5392)
                     /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     5354..5373
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5393..5470)
                     /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 agggcccgga
      361 cgaacctctg agcatcctgg tgaggaataa caagggccgc agcagcacct acgaggtgcg
      421 gctgacgcag accgtggccc acctgaagca gcaagtgagc gggctggagg gtgtgcagga
      481 cgacctgttc gcgctgacct tcgaggggaa gcccctggag gaccagctcc cgctggggga
      541 gtacggcctc aagcccctga gcaccgtgtt catgaatctg cgcctgcggg gaggcggcac
      601 agagcctggc gggcggagct aaagctttct agaccattta aacaccacca ccaccaccac
      661 tgagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag
      721 caataactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa
      781 ggaggaacta tatccggatt ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg
      841 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
      901 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
      961 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     1021 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     1081 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     1141 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     1201 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta
     1261 caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     1321 aatacattca aatatgtatc cgctcatgaa ttaattctta gaaaaactca tcgagcatca
     1381 aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt
     1441 tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc
     1501 ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa
     1561 taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa
     1621 gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat
     1681 cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc
     1741 gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg
     1801 ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg
     1861 ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct
     1921 tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa
     1981 catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc
     2041 catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc
     2101 catataaatc agcatccatg ttggaattta atcgcggcct agagcaagac gtttcccgtt
     2161 gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt tttattgttc
     2221 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     2281 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     2341 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     2401 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     2461 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     2521 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     2581 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     2641 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     2701 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     2761 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     2821 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     2881 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     2941 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     3001 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     3061 gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat
     3121 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct
     3181 atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc
     3241 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     3301 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag
     3361 ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc
     3421 gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc
     3481 ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt
     3541 aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc
     3601 ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag
     3661 aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg
     3721 tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg
     3781 cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc
     3841 agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta
     3901 accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg
     3961 cacccgtggg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc
     4021 gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag
     4081 gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc
     4141 cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt
     4201 catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg
     4261 agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc
     4321 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     4381 aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca
     4441 gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt
     4501 gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt
     4561 cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg
     4621 taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa
     4681 cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc
     4741 cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca
     4801 gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac
     4861 ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac
     4921 tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag
     4981 cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc
     5041 gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca
     5101 tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt
     5161 gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc
     5221 ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca
     5281 ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct
     5341 gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca
     5401 ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt
     5461 cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc
     5521 agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg
//
