LOCUS       sequence-100371-        6062 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human A20 (OTU domain) in E. coli..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6062)
  AUTHORS   Komander D, Barford D
  TITLE     Structure of the A20 OTU domain and mechanistic insights into 
            deubiquitination.
  JOURNAL   Biochem J. 2008 Jan 1;409(1):77-85.
  PUBMED    17961127
REFERENCE   2  (bases 1 to 6062)
  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: "Biochem 
            J."; date: "2008-01-1"; volume: "409(1)"; pages: "77-85"
COMMENT     Sequence Label: pGEX6P1-A20 (OTU, aa 1-366)
FEATURES             Location/Qualifiers
     source          1..6062
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(183..188,189..204,205..211)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    219..235
                     /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)."
     CDS             258..911
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     869..891
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             918..941
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(2112..2134)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2279..2297)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2365..2469
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2470..2538,2539..3330)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2688..2707)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3501..4089
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3990..4009
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4243..4260
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4333..4410
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             4411..5493
                     /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
                     YIPPSTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(4430..4449)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    5506..5527
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5542..5547,5548..5565,5566..5572)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5580..5596
                     /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)."
     primer_bind     5604..5620
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5604..5620
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5616..6062,1..81)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGITLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5636..5653)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5636..5652)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5645..5667)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 acgttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg
       61 gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt
      121 tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc
      181 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca
      241 cacaggaaac agtattcatg tcccctatac taggttattg gaaaattaag ggccttgtgc
      301 aacccactcg acttcttttg gaatatcttg aagaaaaata tgaagagcat ttgtatgagc
      361 gcgatgaagg tgataaatgg cgaaacaaaa agtttgaatt gggtttggag tttcccaatc
      421 ttccttatta tattgatggt gatgttaaat taacacagtc tatggccatc atacgttata
      481 tagctgacaa gcacaacatg ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc
      541 ttgaaggagc ggttttggat attagatacg gtgtttcgag aattgcatat agtaaagact
      601 ttgaaactct caaagttgat tttcttagca agctacctga aatgctgaaa atgttcgaag
      661 atcgtttatg tcataaaaca tatttaaatg gtgatcatgt aacccatcct gacttcatgt
      721 tgtatgacgc tcttgatgtt gttttataca tggacccaat gtgcctggat gcgttcccaa
      781 aattagtttg ttttaaaaaa cgtattgaag ctatcccaca aattgataag tacttgaaat
      841 ccagcaagta tatagcatgg cctttgcagg gctggcaagc cacgtttggt ggtggcgacc
      901 atcctccaaa atcggatctg gaagttctgt tccaggggcc cctgggatcc atggctgaac
      961 aagtccttcc tcaggctttg tatttgagca atatgcggaa agctgtgaag atacgggaga
     1021 gaactccaga agacattttt aaacctacta atgggatcat tcatcatttt aaaaccatgc
     1081 accgatacac actggaaatg ttcagaactt gccagttttg tcctcagttt cgggagatca
     1141 tccacaaagc cctcatcgac agaaacatcc aggccaccct ggaaagccag aagaaactca
     1201 actggtgtcg agaagtccgg aagcttgtgg cgctgaaaac gaacggtgac ggcaattgcc
     1261 tcatgcatgc cacttctcag tacatgtggg gcgttcagga cacagacttg gtactgagga
     1321 aggcgctgtt cagcacgctc aaggaaacag acacacgcaa ctttaaattc cgctggcaac
     1381 tggagtctct caaatctcag gaatttgttg aaacggggct ttgctatgat actcggaact
     1441 ggaatgatga atgggacaat cttatcaaaa tggcttccac agacacaccc atggcccgaa
     1501 gtggacttca gtacaactca ctggaagaaa tacacatatt tgtcctttgc aacatcctca
     1561 gaaggccaat cattgtcatt tcagacaaaa tgctaagaag tttggaatca ggttccaatt
     1621 tcgccccttt gaaagtgggt ggaatttact tgcctctcca ctggcctgcc caggaatgct
     1681 acagataccc cattgttctc ggctatgaca gccatcattt tgtacccttg gtgaccctga
     1741 aggacagtgg gcctgaaatc cgagctgttc cacttgttaa cagagaccgg ggaagatttg
     1801 aagacttaaa agttcacttt ttgacagatc ctgaaaatga gatgaaggag aagctcttaa
     1861 aagagtactt aatggtgata gaaatccccg tccaaggctg ggaccatggc acaactcatc
     1921 tcatcaatgc cgcaaagttg gatgaagcta acttaccaaa agaaatcaat ctggtagatg
     1981 attactttga acttgttcag catgagtaca agaaatggca ggaaaacagc gagcagggga
     2041 ggagagagta agcggccgca tcgtgactga ctgacgatct gcctcgcgcg tttcggtgat
     2101 gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg
     2161 gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc
     2221 gcagccatga cccagtcacg tagcgatagc ggagtgtata attcttgaag acgaaagggc
     2281 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     2341 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     2401 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     2461 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     2521 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     2581 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     2641 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     2701 gtattatccc gtgttgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     2761 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     2821 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     2881 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     2941 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     3001 accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     3061 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     3121 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     3181 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     3241 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     3301 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     3361 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     3421 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     3481 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     3541 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     3601 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     3661 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     3721 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     3781 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     3841 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     3901 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     3961 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     4021 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     4081 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     4141 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     4201 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     4261 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     4321 cgcataaatt ccgacaccat cgaatggtgc aaaacctttc gcggtatggc atgatagcgc
     4381 ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca
     4441 gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt
     4501 tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac
     4561 cgcgtggcac aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt
     4621 ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg
     4681 ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg
     4741 gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac
     4801 caggatgcca ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc
     4861 tctgaccaga cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc
     4921 gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt
     4981 tctgtctcgg cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt
     5041 cagccgatag cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg
     5101 caaatgctga atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg
     5161 ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga tatctcggta
     5221 gtgggatacg acgataccga agacagctca tgttatatcc cgccgtcaac caccatcaaa
     5281 caggattttc gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc
     5341 caggcggtga agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg
     5401 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
     5461 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
     5521 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
     5581 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg gattcactgg
     5641 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg
     5701 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt
     5761 cccaacagtt gcgcagcctg aatggcgaat ggcgctttgc ctggtttccg gcaccagaag
     5821 cggtgccgga aagctggctg gagtgcgatc ttcctgaggc cgatactgtc gtcgtcccct
     5881 caaactggca gatgcacggt tacgatgcgc ccatctacac caacgtaacc tatcccatta
     5941 cggtcaatcc gccgtttgtt cccacggaga atccgacggg ttgttactcg ctcacattta
     6001 atgttgatga aagctggcta caggaaggcc agacgcgaat tatttttgat ggcgttggaa
     6061 tt
//
