LOCUS       sequence-494534-        5465 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  N-term GST tag E2 variant selected for activated ARIH1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5465)
  TITLE     E2 Variants for Probing E3 Ubiquitin Ligase Activities
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5465)
  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: 
            "Unpublished"
COMMENT     Sequence Label: GST-tag ARIH1 binding E2 variant L3A1-1
FEATURES             Location/Qualifiers
     source          1..5465
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..24,25..31)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    39..55
                     /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     63..79
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     63..79
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             75..602
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(95..112)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(95..111)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(104..126)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(704..709,710..725,726..732)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    740..756
                     /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             779..1432
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     1390..1412
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             1439..1459
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1928..1972
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     primer_bind     complement(2036..2058)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2203..2221)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2289..2393
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2394..2462,2463..3254)
                     /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(2612..2631)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3425..4013
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3914..3933
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4167..4184
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4257..4334
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4335..5417
                     /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(4354..4373)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttaa
       61 cacaggaaac agctatgacc atgattacgg attcactggc cgtcgtttta caacgtcgtg
      121 actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca
      181 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga
      241 atggcgaatg gcgctttgcc tggtttccgg caccagaagc ggtgccggaa agctggctgg
      301 agtgcgatct tcctgaggcc gatactgtcg tcgtcccctc aaactggcag atgcacggtt
      361 acgatgcgcc catctacacc aacgtgacct atcccattac ggtcaatccg ccgtttgttc
      421 ccacggagaa tccgacgggt tgttactcgc tcacatttaa tgttgatgaa agctggctac
      481 aggaaggcca gacgcgaatt atttttgatg gcgttggaat tagcttatcg actgcacggt
      541 gcaccaatgc ttctggcgtc aggcagccat cggaagctgt ggtatggctg tgcaggtcgt
      601 aaatcactgc ataattcgtg tcgctcaagg cgcactcccg ttctggataa tgttttttgc
      661 gccgacatca taacggttct ggcaaatatt ctgaaatgag ctgttgacaa ttaatcatcg
      721 gctcgtataa tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagtattcat
      781 gtcccctata ctaggttatt ggaaaattaa gggccttgtg caacccactc gacttctttt
      841 ggaatatctt gaagaaaaat atgaagagca tttgtatgag cgcgatgaag gtgataaatg
      901 gcgaaacaaa aagtttgaat tgggtttgga gtttcccaat cttccttatt atattgatgg
      961 tgatgttaaa ttaacacagt ctatggccat catacgttat atagctgaca agcacaacat
     1021 gttgggtggt tgtccaaaag agcgtgcaga gatttcaatg cttgaaggag cggttttgga
     1081 tattagatac ggtgtttcga gaattgcata tagtaaagac tttgaaactc tcaaagttga
     1141 ttttcttagc aagctacctg aaatgctgaa aatgttcgaa gatcgtttat gtcataaaac
     1201 atatttaaat ggtgatcatg taacccatcc tgacttcatg ttgtatgacg ctcttgatgt
     1261 tgttttatac atggacccaa tgtgcctgga tgcgttccca aaattagttt gttttaaaaa
     1321 acgtattgaa gctatcccac aaattgataa gtacttgaaa tccagcaagt atatagcatg
     1381 gcctttgcag ggctggcaag ccacgtttgg tggtggcgac catcctccaa aatcggatga
     1441 aaacctgtat tttcagggat ccatgcgggc ggccacgagg aggctgaggt gggagcttga
     1501 agaaatccgc aaagcaggga tgaaaaactt ccgtaacatc caggttgatg aagctaattt
     1561 attgacttgg caagggctta ttgttcctga caaccctcca tatgataagg gagccttcag
     1621 aatcgaaatc aactttccag cagagtaccc attcaaacca ccgaagatca catttaaaac
     1681 aaagatctat cacccaaaca tcgacgaaaa ggggcaggtc tgtctgccag taattagtgc
     1741 cgaaaactgg aagccagcaa ccaaaaccga ccaagtaatc cagtccctca tagcactggt
     1801 gaatgacccc cagcctgagc acccgcttcg ggctgaccta gctgaagaat actctaagga
     1861 ccgtaaaaaa ttcgcaaaga atgctgaaga gtttacaaag aaatatgggg aaaagcgacc
     1921 tgtggacggt cttaacgaca tcttcgaggc tcaaaagatt gaatggcatg aataagcggc
     1981 cgcatcgtga ctgactgacg atctgcctcg cgcgtttcgg tgatgacggt gaaaacctct
     2041 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac
     2101 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt
     2161 cacgtagcga tagcggagtg tataattctt gaagacgaaa gggcctcgtg atacgcctat
     2221 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg
     2281 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     2341 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     2401 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     2461 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     2521 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     2581 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg
     2641 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     2701 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     2761 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     2821 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     2881 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgcag
     2941 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     3001 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     3061 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     3121 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     3181 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     3241 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     3301 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     3361 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     3421 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     3481 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     3541 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     3601 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     3661 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     3721 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     3781 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     3841 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     3901 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     3961 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     4021 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     4081 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     4141 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     4201 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata aattccgaca
     4261 ccatcgaatg gcgcaaaacc tttcgcggta tggcatgata gcgcccggaa gagagtcaat
     4321 tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat gccggtgtct
     4381 cttatcagac cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg
     4441 aaaaagtgga agcggcgatg gcggagctga attacattcc caaccgcgtg gcacaacaac
     4501 tggcgggcaa acagtcgttg ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc
     4561 cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca actgggtgcc agcgtggtgg
     4621 tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg
     4681 cgcaacgcgt cagtgggctg atcattaact atccgctgga tgaccaggat gccattgctg
     4741 tggaagctgc ctgcactaat gttccggcgt tatttcttga tgtctctgac cagacaccca
     4801 tcaacagtat tattttctcc catgaagacg gtacgcgact gggcgtggag catctggtcg
     4861 cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc
     4921 tgcgtctggc tggctggcat aaatatctca ctcgcaatca aattcagccg atagcggaac
     4981 gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg
     5041 gcatcgttcc cactgcgatg ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg
     5101 ccattaccga gtccgggctg cgcgttggtg cggatatctc ggtagtggga tacgacgata
     5161 ccgaagacag ctcatgttat atcccgccgt taaccaccat caaacaggat tttcgcctgc
     5221 tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg gtgaagggca
     5281 atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa
     5341 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
     5401 tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt agctcactca ttaggcaccc
     5461 caggc
//
