LOCUS       sequence-212818-        6343 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of ISG15 C-term..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6343)
  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 6343)
  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: ISG15 C-term. Domain intein/chitin binding dom...
FEATURES             Location/Qualifiers
     source          1..6343
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     28..47
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        28..46
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    47..71
                     /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             86..108
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             353..946
                     /codon_start=1
                     /gene="Mycobacterium xenopi gyrA"
                     /product="modified GyrA intein (Southworth et al., 1999)"
                     /label=Mxe GyrA intein
                     /note="N-terminal cleavage of this intein can be induced by
                     thiols."
                     /translation="CITGDALVALPEGESVRIADIVPGARPNSDNAIDLKVLDRHGNPV
                     LADRLFHSGEHPVYTVRTVEGLRVTGTANHPLLCLVDVAGVPTLLWKLIDEIKPGDYAV
                     IQRSAFSVDCAGFARGKPEFAPTTYTVGVPGLVRFLEAHHRDPDAQAIADELTDGRFYY
                     AKVASVTDAGVQPVYSLRVDTADHAFITNGFVSHA"
     CDS             977..1132
                     /codon_start=1
                     /gene="Bacillus circulans chiA"
                     /product="chitin binding domain from chitinase A1 (Watanabe
                     et al., 1994)"
                     /label=CBD
                     /translation="TTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNV
                     PALWQLQ"
     primer_bind     complement(1197..1215)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1211..1258
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        1318..1422
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1423..1491,1492..2283)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1641..1660)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(2325..2838)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2520..2541)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2732..2751
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      2949..3537
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3438..3457
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3691..3708
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(3730..3749)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3890..3912
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3906..4097)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4654..5736)
                     /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     5698..5717
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5737..5814)
                     /gene="lacI"
                     /label=lacI promoter
     terminator      5967..6053
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      6150..6236
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 aaaccgggga tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg
       61 ataacaattc ccctctagaa ataattttgt ttaactttaa gaaggagata tacatatgga
      121 cgaacctctg agcatcctgg tgaggaataa caagggccgc agcagcacct acgaggtgcg
      181 gctgacgcag accgtggccc acctgaagca gcaagtgagc gggctggagg gtgtgcagga
      241 cgacctgttc tggctgacct tcgaggggaa gcccctggag gaccagctcc cgctggggga
      301 gtacggcctc aagcccctga gcaccgtgtt catgaatctg cgcctgcggg ggtgcatcac
      361 gggagatgca ctagttgccc tacccgaggg cgagtcggta cgcatcgccg acatcgtgcc
      421 gggtgcgcgg cccaacagtg acaacgccat cgacctgaaa gtccttgacc ggcatggcaa
      481 tcccgtgctc gccgaccggc tgttccactc cggcgagcat ccggtgtaca cggtgcgtac
      541 ggtcgaaggt ctgcgtgtga cgggcaccgc gaaccacccg ttgttgtgtt tggtcgacgt
      601 cgccggggtg ccgaccctgc tgtggaagct gatcgacgaa atcaagccgg gcgattacgc
      661 ggtgattcaa cgcagcgcat tcagcgtcga ctgtgcaggt tttgcccgcg ggaaacccga
      721 atttgcgccc acaacctaca cagtcggcgt ccctggactg gtgcgtttct tggaagcaca
      781 ccaccgagac ccggacgccc aagctatcgc cgacgagctg accgacgggc ggttctacta
      841 cgcgaaagtc gccagtgtca ccgacgccgg cgtgcagccg gtgtatagcc ttcgtgtcga
      901 cacggcagac cacgcgttta tcacgaacgg gttcgtcagc cacgctactg gcctcaccgg
      961 tctgaactca ggcctcacga caaatcctgg tgtatccgct tggcaggtca acacagctta
     1021 tactgcggga caattggtca catataacgg caagacgtat aaatgtttgc agccccacac
     1081 ctccttggca ggatgggaac catccaacgt tcctgccttg tggcagcttc aatgactgca
     1141 ggaaggggat ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc
     1201 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     1261 gaaaggagga actatatccg gataactacg tcaggtggca cttttcgggg aaatgtgcgc
     1321 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     1381 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     1441 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     1501 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     1561 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg tttcccaatg
     1621 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtgttga cgccgggcaa
     1681 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     1741 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     1801 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     1861 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     1921 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     1981 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     2041 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     2101 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     2161 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     2221 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     2281 taactgtcag accaagttta ctcatatata ctttagattg atttaccccg gttgataatc
     2341 agaaaagccc caaaaacagg aagattgtat aagcaaatat ttaaattgta aacgttaata
     2401 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg
     2461 aaatcggcaa aatcccttat aaatcaaaag aatagcccga gatagggttg agtgttgttc
     2521 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
     2581 ccgtctatca gggcgatggc ccactacgtg aaccatcacc caaatcaagt tttttggggt
     2641 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
     2701 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
     2761 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
     2821 cgccgctaca gggcgcgtaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     2881 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     2941 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     3001 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     3061 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     3121 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     3181 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     3241 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     3301 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     3361 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     3421 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     3481 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     3541 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     3601 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     3661 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agctatggtg
     3721 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg
     3781 ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga
     3841 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     3901 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca
     3961 tcagcgtggt cgtgcagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg
     4021 agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt
     4081 ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg
     4141 ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg
     4201 ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa
     4261 atcactcagg gtcaatgcca gccgaacgcc agcaagacgt agcccagcgc gtcggccgcc
     4321 atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag
     4381 gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg
     4441 ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg
     4501 agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac
     4561 cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa
     4621 tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     4681 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     4741 gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac
     4801 cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa
     4861 atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta
     4921 tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc
     4981 gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag
     5041 catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat
     5101 cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga
     5161 gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg
     5221 ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg
     5281 gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc
     5341 atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt
     5401 gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct
     5461 ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag
     5521 ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc
     5581 cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt
     5641 cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc
     5701 atactctgcg acatcgtata acgttactgg tttcacattc accaccctga attgactctc
     5761 ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat
     5821 ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc
     5881 cgttgagcac cgccgccgca aggaatggtg catgccggca tgccgccctt tcgtcttcaa
     5941 gaattaattc ccaattcccc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg
     6001 cctttcgttt tatctgttgt ttgtcggtga acgctctcct gagtaggaca aatccgccgg
     6061 gagcggattt gaacgttgcg aagcaacggc ccggagggtg gcgggcagga cgcccgccat
     6121 aaactgccag gaattaattc cccaggcatc aaataaaacg aaaggctcag tcgaaagact
     6181 gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc
     6241 cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca ggacgcccgc
     6301 cataaactgc caggaattgg ggatcggaat taattcccgg ttt
//
