LOCUS       sequence-501606-        5417 bp    DNA     circular SYN 03-MAR-2026
DEFINITION  N-term GST tag E2 variant selected for RNF14.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5417)
  AUTHORS   Du J, Andree GA, Horn-Ghetko D, Stier L, Singh J, Kostrhon S, Kiss 
            L, Mann M, Sidhu SS, Schulman BA
  TITLE     E2 variants for probing E3 ubiquitin ligase activities.
  JOURNAL   Proc Natl Acad Sci U S A. 2026 Jan 6;123(1):e2524899122. doi: 
            10.1073/pnas.2524899122. Epub 2026 Jan 2.
  PUBMED    41481455
REFERENCE   2  (bases 1 to 5417)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Mar 3, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Proc Natl 
            Acad Sci U S A."; date: "2026-01-6"; volume: "123(1):e2524899122. 
            doi"; pages: " 10.1073/pnas.2524899122. Epub 2026 Jan 2"
COMMENT     Sequence Label: GST-tag RNF14 binding E2 variant L3R14-2
FEATURES             Location/Qualifiers
     source          1..5417
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..654
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     612..634
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             661..681
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     regulatory      683..692
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(1210..1232)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1377..1395)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1463..1567
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1568..1636,1637..2428)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSV
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1786..1805)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2599..3187
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3088..3107
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3341..3358
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3431..3508
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3509..4591
                     /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(3528..3547)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(4640..4645,4646..4663,4664..4670)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4678..4694
                     /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     4702..4718
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4702..4718
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             4714..5241
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(4734..4751)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4734..4750)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4743..4765)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(5343..5348,5349..5364,5365..5371)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    5379..5395
                     /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)."
ORIGIN
        1 atgtccccta tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt
       61 ttggaatatc ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa
      121 tggcgaaaca aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat
      181 ggtgatgtta aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac
      241 atgttgggtg gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg
      301 gatattagat acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt
      361 gattttctta gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa
      421 acatatttaa atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat
      481 gttgttttat acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa
      541 aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca
      601 tggcctttgc agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat
      661 gaaaacctgt attttcaggg atccatggcg gccagcagga ggctgatgaa ggagcttgaa
      721 gaaatccgca aagcagggat gaaaaacttc cgtaacatcc aggttgatga agctaattta
      781 ttgacttggc aagggcttat tgttcctgac aaccctccat atgataaggg agccttcaga
      841 atcgaaatca actttccatt tcggtaccca tttgcgccac cgaagatcac atttaaaaca
      901 aagatctatc acccaaacat cgacgaaaag gggcaggtct gtctgccagt aattagtgcc
      961 gaaaactgga agccagcaac caaaaccgac caagtaatcc agtccctcat agcactggtg
     1021 aatgaccccc agcctgagca cccgcttcgg gctgacctag ctgaagaata ctctaaggac
     1081 cgtaaaaaat tcgcaaagaa tgctgaagag tttacaaaga aatatgggga aaagcgacct
     1141 gtggactaag cggccgcatc gtgactgact gacgatctgc ctcgcgcgtt tcggtgatga
     1201 cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga
     1261 tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtctgggcgc
     1321 agccatgacc cagtcacgta gcgatagcgg agtgtataat tcttgaagac gaaagggcct
     1381 cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg
     1441 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     1501 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     1561 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     1621 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     1681 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     1741 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     1801 attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     1861 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     1921 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     1981 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     2041 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     2101 cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     2161 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     2221 tctgcgctcg gtccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     2281 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     2341 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     2401 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     2461 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     2521 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     2581 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     2641 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     2701 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     2761 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     2821 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     2881 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     2941 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     3001 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     3061 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     3121 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     3181 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     3241 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     3301 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     3361 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     3421 cataaattcc gacaccatcg aatggcgcaa aacctttcgc ggtatggcat gatagcgccc
     3481 ggaagagagt caattcaggg tggtgaatgt gaaaccagta acgttatacg atgtcgcaga
     3541 gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc
     3601 tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag ctgaattaca ttcccaaccg
     3661 cgtggcacaa caactggcgg gcaaacagtc gttgctgatt ggcgttgcca cctccagtct
     3721 ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg
     3781 tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta aagcggcggt
     3841 gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca
     3901 ggatgccatt gctgtggaag ctgcctgcac taatgttccg gcgttatttc ttgatgtctc
     3961 tgaccagaca cccatcaaca gtattatttt ctcccatgaa gacggtacgc gactgggcgt
     4021 ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc cattaagttc
     4081 tgtctcggcg cgtctgcgtc tggctggctg gcataaatat ctcactcgca atcaaattca
     4141 gccgatagcg gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca
     4201 aatgctgaat gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct
     4261 gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt
     4321 gggatacgac gataccgaag acagctcatg ttatatcccg ccgttaacca ccatcaaaca
     4381 ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca
     4441 ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc
     4501 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
     4561 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgta agttagctca
     4621 ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg
     4681 tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgga ttcactggcc
     4741 gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca
     4801 gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc
     4861 caacagttgc gcagcctgaa tggcgaatgg cgctttgcct ggtttccggc accagaagcg
     4921 gtgccggaaa gctggctgga gtgcgatctt cctgaggccg atactgtcgt cgtcccctca
     4981 aactggcaga tgcacggtta cgatgcgccc atctacacca acgtgaccta tcccattacg
     5041 gtcaatccgc cgtttgttcc cacggagaat ccgacgggtt gttactcgct cacatttaat
     5101 gttgatgaaa gctggctaca ggaaggccag acgcgaatta tttttgatgg cgttggaatt
     5161 agcttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg
     5221 gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt
     5281 tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc
     5341 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca
     5401 cacaggaaac agtattc
//
