LOCUS       sequence-273232-        5917 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of GST-RNF126 RING mutant (C231A) fusion 
            protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5917)
  AUTHORS   Smith CJ, Berry DM, McGlade CJ
  TITLE     The E3 ubiquitin ligases RNF126 and Rabring7 regulate endosomal 
            sorting of the epidermal growth factor receptor.
  JOURNAL   J Cell Sci. 2013 Mar 15;126(Pt 6):1366-80. doi: 10.1242/jcs.116129. 
            Epub 2013 Feb 15.
  PUBMED    23418353
REFERENCE   2  (bases 1 to 5917)
  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 Cell 
            Sci."; date: "2013-03-15"; volume: "126(Pt 6):1366-80. doi"; pages: 
            " 10.1242/jcs.116129. Epub 2013 Feb 15"
COMMENT     Sequence Label: GST-RNF126 C231A
FEATURES             Location/Qualifiers
     source          1..5917
                     /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(1967..1989)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2134..2152)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2220..2324
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2325..2393,2394..3185)
                     /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(2543..2562)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3356..3944
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3845..3864
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4098..4115
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4188..4265
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4266..5348
                     /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(4285..4304)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5397..5402,5403..5420,5421..5427)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5435..5451
                     /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     5459..5475
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5459..5475
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5471..5917,1..81)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5491..5508)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5491..5507)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5500..5522)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 agcttatcga 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 gatccgaggc gtcgccgcag cccggacggt
      961 acttctgcca ctgctgctcg gtagagatcg tgccgcgcct gccggattac atctgcccaa
     1021 ggtgcgagtc tggcttcatt gaggagcttc cagaagagac caggaacaca gagaatggct
     1081 cagccccctc cacagccccc accgaccaga accggcagcc atttgagaat gtggaccagc
     1141 acctgttcac gctgccacag ggatacagcc agtttgcctt tggcatcttc gacgatagct
     1201 ttgagattcc cacgttccct cctggagccc aggccgatga tggcagggac cctgagagcc
     1261 gacgggagag agagcaccag tctcggcatc ggtacggggc ccggcagccc cgtgcccgcc
     1321 tcactgcccg ccgggccact ggccggcatg aaggtgtccc tacgctggaa gggatcatcc
     1381 agcagctcgt gaatggcatc atctctccgg ctgctgtgcc cagcctgggc cttggtccct
     1441 ggggcgtcct gcactcgaac ccaatggact acgcctgggg ggccaacggc ctggacacca
     1501 tcatcacgca gctcctcaat cagtttgaga acaccggccc cccacctgca gacaaggaga
     1561 agattcaggc tctccccacg gtcccagtca cagaggaaca cgtgggctca gggcttgagg
     1621 ccccagtgtg taaagaagac tatgcactgg gtgagagtgt gcggcagttg ccctgcaacc
     1681 acctgttcca cgacagctgc atcgtgccct ggctggagca gcatgacagc tgcccggtct
     1741 gccgtaagag cctcactgga cagaacacag ccaccaatcc cccaggcttg accggtgtag
     1801 gtttctcctc gtcctcgtcc tcatcctcct ccagctcgcc cagcaatgag aacgccacaa
     1861 gcaactcctg attctgaaga tatccagcac agtggcggcc gctcgagcgg ccgcatcgtg
     1921 actgactgac gatctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc
     1981 agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc
     2041 agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg
     2101 atagcggagt gtataattct tgaagacgaa agggcctcgt gatacgccta tttttatagg
     2161 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
     2221 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     2281 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     2341 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     2401 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     2461 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     2521 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc
     2581 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     2641 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     2701 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     2761 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     2821 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgca gcaatggcaa
     2881 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     2941 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     3001 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     3061 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     3121 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     3181 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     3241 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     3301 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     3361 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     3421 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     3481 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     3541 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     3601 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     3661 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     3721 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     3781 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     3841 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     3901 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     3961 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     4021 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     4081 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     4141 attttctcct tacgcatctg tgcggtattt cacaccgcat aaattccgac accatcgaat
     4201 ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga agagagtcaa ttcagggtgg
     4261 tgaatgtgaa accagtaacg ttatacgatg tcgcagagta tgccggtgtc tcttatcaga
     4321 ccgtttcccg cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg
     4381 aagcggcgat ggcggagctg aattacattc ccaaccgcgt ggcacaacaa ctggcgggca
     4441 aacagtcgtt gctgattggc gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa
     4501 ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg
     4561 tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg
     4621 tcagtgggct gatcattaac tatccgctgg atgaccagga tgccattgct gtggaagctg
     4681 cctgcactaa tgttccggcg ttatttcttg atgtctctga ccagacaccc atcaacagta
     4741 ttattttctc ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc
     4801 accagcaaat cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg
     4861 ctggctggca taaatatctc actcgcaatc aaattcagcc gatagcggaa cgggaaggcg
     4921 actggagtgc catgtccggt tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc
     4981 ccactgcgat gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc gccattaccg
     5041 agtccgggct gcgcgttggt gcggatatct cggtagtggg atacgacgat accgaagaca
     5101 gctcatgtta tatcccgccg ttaaccacca tcaaacagga ttttcgcctg ctggggcaaa
     5161 ccagcgtgga ccgcttgctg caactctctc agggccaggc ggtgaagggc aatcagctgt
     5221 tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc caatacgcaa accgcctctc
     5281 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
     5341 ggcagtgagc gcaacgcaat taatgtaagt tagctcactc attaggcacc ccaggcttta
     5401 cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca
     5461 ggaaacagct atgaccatga ttacggattc actggccgtc gttttacaac gtcgtgactg
     5521 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg
     5581 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg
     5641 cgaatggcgc tttgcctggt ttccggcacc agaagcggtg ccggaaagct ggctggagtg
     5701 cgatcttcct gaggccgata ctgtcgtcgt cccctcaaac tggcagatgc acggttacga
     5761 tgcgcccatc tacaccaacg tgacctatcc cattacggtc aatccgccgt ttgttcccac
     5821 ggagaatccg acgggttgtt actcgctcac atttaatgtt gatgaaagct ggctacagga
     5881 aggccagacg cgaattattt ttgatggcgt tggaatt
//
