LOCUS       sequence-59329-e        6569 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6569)
  AUTHORS   Lamothe B, Besse A, Campos AD, Webster WK, Wu H, Darnay BG
  TITLE     Site-specific Lys-63-linked tumor necrosis factor 
            receptor-associated factor 6 auto-ubiquitination is a critical 
            determinant of I kappa B kinase activation.
  JOURNAL   J Biol Chem. 2007 Feb 9;282(6):4102-12. Epub 2006 Nov 29.
  PUBMED    17135271
REFERENCE   2  (bases 1 to 6569)
  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: "J Biol 
            Chem."; date: "2007-02-9"; volume: "282(6)"; pages: "4102-12. Epub 
            2006 Nov 29"
COMMENT     Sequence Label: pGEX4T1-TRAF6-C70A
FEATURES             Location/Qualifiers
     source          1..6569
                     /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..935
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(2619..2641)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2786..2804)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2872..2976
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2977..3045,3046..3837)
                     /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(3195..3214)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4008..4596
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4497..4516
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4750..4767
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4840..4917
                     /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             4918..6000
                     /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     complement(4937..4956)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    6013..6034
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6049..6054,6055..6072,6073..6079)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6087..6103
                     /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     6111..6127
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6111..6127
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(6123..6569,1..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(6143..6160)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6143..6159)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6152..6174)
                     /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 gttccgcgtg gatccccgga attcatgagt ctcttaaact
      961 gtgagaacag ctgcgggtcc agccagtcgt ccagtgactg ctgcgctgcc atggccgcct
     1021 cctgcagcgc tgcagtgaaa gatgacagcg tgagtggctc tgccagcacc gggaacctct
     1081 ccagctcctt catggaggag atccagggct acgatgtgga gtttgaccca cctctggaga
     1141 gcaagtatga ggctcccatc tgcttgatgg ctttacggga agcagtgcaa acaccatgtg
     1201 gccacaggtt ctgcaaagcc tgcatcatca aatccataag ggatgcaggg cacaagtgcc
     1261 cagttgacaa tgaaatactg ctggaaaatc aactgtttcc cgacaatttt gcaaagcgag
     1321 agattctttc cctgacggta aagtgcccaa ataaaggctg tttgcaaaag atggaactga
     1381 gacatctcga ggatcatcaa gtacattgtg aatttgctct agtgaattgt ccccagtgcc
     1441 aacgtccttt ccagaagtgc caggttaata cacacattat tgaggattgt cccaggaggc
     1501 aggtttcttg tgtaaactgt gctgtgtcca tggcatatga agagaaagag atccatgatc
     1561 aaagctgtcc tctggcaaat atcatctgtg aatactgtgg tacaatcctc atcagagaac
     1621 agatgcctaa tcattatgat ctggactgcc caacagctcc aatcccttgc acattcagtg
     1681 tttttggctg tcatgaaaag atgcagagga atcacttggc acgacacttg caagagaata
     1741 cccagttgca catgagactg ttggcccagg ctgttcataa tgttaacctt gctttgcgtc
     1801 cgtgcgatgc cgcctctcca tcccggggat gtcgtccaga ggacccaaat tatgaggaaa
     1861 ctatcaaaca gttggagagt cgcctagtaa gacaggacca tcagatccgg gagctgactg
     1921 ccaaaatgga aactcagagt atgtacgtgg gcgagctcaa acggaccatt cggaccctgg
     1981 aggacaaggt tgccgaaatg gaagcacagc agtgtaacgg gatctacatt tggaagattg
     2041 gcaactttgg gatgcacttg aaatcccaag aagaggaaag acctgttgtc atccatagcc
     2101 ctggattcta cacaggcaga cctgggtaca agctgtgcat gcgcctgcat cttcagttac
     2161 cgacagctca gcgctgtgca aactatatat ccctttttgt ccacacaatg caaggagaat
     2221 atgacagcca cctcccctgg cccttccagg gtacaatacg ccttacaatt ctcgaccagt
     2281 ctgaagcact tataaggcaa aaccacgaag aggtcatgga cgccaaacca gaactgcttg
     2341 cctttcagcg acccacaatc ccacggaacc ccaaaggttt tggctatgta acatttatgc
     2401 acctggaagc cttaagacag ggaaccttca ttaaggatga tacattacta gtgcgctgtg
     2461 aagtctctac ccgctttgac atgggtggcc ttcggaagga gggtttccag ccacgaagta
     2521 ctgatgcggg ggtgtaggtc gagcatgcat ctagtcgagc ggccgcatcg tgactgactg
     2581 acgatctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg
     2641 gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
     2701 tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga
     2761 gtgtataatt cttgaagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc
     2821 atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     2881 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     2941 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     3001 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     3061 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     3121 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     3181 acttttaaag ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg gcaagagcaa
     3241 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     3301 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     3361 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     3421 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     3481 gaagccatac caaacgacga gcgtgacacc acgatgcctg cagcaatggc aacaacgttg
     3541 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     3601 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     3661 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     3721 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     3781 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     3841 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     3901 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     3961 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     4021 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     4081 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     4141 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     4201 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     4261 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     4321 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     4381 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     4441 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     4501 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     4561 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     4621 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     4681 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     4741 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
     4801 cttacgcatc tgtgcggtat ttcacaccgc ataaattccg acaccatcga atggtgcaaa
     4861 acctttcgcg gtatggcatg atagcgcccg gaagagagtc aattcagggt ggtgaatgtg
     4921 aaaccagtaa cgttatacga tgtcgcagag tatgccggtg tctcttatca gaccgtttcc
     4981 cgcgtggtga accaggccag ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg
     5041 atggcggagc tgaattacat tcccaaccgc gtggcacaac aactggcggg caaacagtcg
     5101 ttgctgattg gcgttgccac ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg
     5161 gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga
     5221 agcggcgtcg aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg
     5281 ctgatcatta actatccgct ggatgaccag gatgccattg ctgtggaagc tgcctgcact
     5341 aatgttccgg cgttatttct tgatgtctct gaccagacac ccatcaacag tattattttc
     5401 tcccatgaag acggtacgcg actgggcgtg gagcatctgg tcgcattggg tcaccagcaa
     5461 atcgcgctgt tagcgggccc attaagttct gtctcggcgc gtctgcgtct ggctggctgg
     5521 cataaatatc tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg cgactggagt
     5581 gccatgtccg gttttcaaca aaccatgcaa atgctgaatg agggcatcgt tcccactgcg
     5641 atgctggttg ccaacgatca gatggcgctg ggcgcaatgc gcgccattac cgagtccggg
     5701 ctgcgcgttg gtgcggatat ctcggtagtg ggatacgacg ataccgaaga cagctcatgt
     5761 tatatcccgc cgttaaccac catcaaacag gattttcgcc tgctggggca aaccagcgtg
     5821 gaccgcttgc tgcaactctc tcagggccag gcggtgaagg gcaatcagct gttgcccgtc
     5881 tcactggtga aaagaaaaac caccctggcg cccaatacgc aaaccgcctc tccccgcgcg
     5941 ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga
     6001 gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat
     6061 gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag
     6121 ctatgaccat gattacggat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc
     6181 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata
     6241 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc
     6301 gctttgcctg gtttccggca ccagaagcgg tgccggaaag ctggctggag tgcgatcttc
     6361 ctgaggccga tactgtcgtc gtcccctcaa actggcagat gcacggttac gatgcgccca
     6421 tctacaccaa cgtaacctat cccattacgg tcaatccgcc gtttgttccc acggagaatc
     6481 cgacgggttg ttactcgctc acatttaatg ttgatgaaag ctggctacag gaaggccaga
     6541 cgcgaattat ttttgatggc gttggaatt
//
