LOCUS       sequence-496800-        5574 bp    DNA     circular SYN 09-JAN-2026
DEFINITION  N-term GST tag E2 variant selected for HOIL1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5574)
  TITLE     Schulman lab plasmids 2025
REFERENCE   2  (bases 1 to 5574)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 9, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: GST-tag HOIL1 binding E2 variant L3HL-1
FEATURES             Location/Qualifiers
     source          1..5574
                     /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"
     CDS             1147..1191
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     primer_bind     complement(1367..1389)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1534..1552)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1620..1724
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1725..1793,1794..2585)
                     /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(1943..1962)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2756..3344
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3245..3264
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3498..3515
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3588..3665
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3666..4748
                     /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(3685..3704)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(4797..4802,4803..4820,4821..4827)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4835..4851
                     /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     4859..4875
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4859..4875
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             4871..5398
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(4891..4908)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4891..4907)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4900..4922)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(5500..5505,5506..5521,5522..5528)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    5536..5552
                     /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 actttccaca gcagtaccca attccgccac 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 gtggacggtc ttaacgacat cttcgaggct caaaagattg aatggcatga ataagcggcc
     1201 gcatcgggct gacctagctg aagaatactc taaggaccgt aaaaaattcg caaagaatgc
     1261 tgaagagttt acaaagaaat atggggaaaa gcgacctgtg gactaagcgg ccgcatcgtg
     1321 actgactgac gatctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc
     1381 agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc
     1441 agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg
     1501 atagcggagt gtataattct tgaagacgaa agggcctcgt gatacgccta tttttatagg
     1561 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
     1621 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     1681 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     1741 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     1801 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     1861 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     1921 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc
     1981 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     2041 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     2101 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     2161 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     2221 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgca gcaatggcaa
     2281 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     2341 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     2401 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     2461 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     2521 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     2581 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     2641 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     2701 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     2761 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     2821 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     2881 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     2941 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     3001 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     3061 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     3121 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     3181 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     3241 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     3301 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     3361 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     3421 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     3481 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     3541 attttctcct tacgcatctg tgcggtattt cacaccgcat aaattccgac accatcgaat
     3601 ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga agagagtcaa ttcagggtgg
     3661 tgaatgtgaa accagtaacg ttatacgatg tcgcagagta tgccggtgtc tcttatcaga
     3721 ccgtttcccg cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg
     3781 aagcggcgat ggcggagctg aattacattc ccaaccgcgt ggcacaacaa ctggcgggca
     3841 aacagtcgtt gctgattggc gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa
     3901 ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg
     3961 tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg
     4021 tcagtgggct gatcattaac tatccgctgg atgaccagga tgccattgct gtggaagctg
     4081 cctgcactaa tgttccggcg ttatttcttg atgtctctga ccagacaccc atcaacagta
     4141 ttattttctc ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc
     4201 accagcaaat cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg
     4261 ctggctggca taaatatctc actcgcaatc aaattcagcc gatagcggaa cgggaaggcg
     4321 actggagtgc catgtccggt tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc
     4381 ccactgcgat gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc gccattaccg
     4441 agtccgggct gcgcgttggt gcggatatct cggtagtggg atacgacgat accgaagaca
     4501 gctcatgtta tatcccgccg ttaaccacca tcaaacagga ttttcgcctg ctggggcaaa
     4561 ccagcgtgga ccgcttgctg caactctctc agggccaggc ggtgaagggc aatcagctgt
     4621 tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc caatacgcaa accgcctctc
     4681 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
     4741 ggcagtgagc gcaacgcaat taatgtaagt tagctcactc attaggcacc ccaggcttta
     4801 cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttaacaca
     4861 ggaaacagct atgaccatga ttacggattc actggccgtc gttttacaac gtcgtgactg
     4921 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg
     4981 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg
     5041 cgaatggcgc tttgcctggt ttccggcacc agaagcggtg ccggaaagct ggctggagtg
     5101 cgatcttcct gaggccgata ctgtcgtcgt cccctcaaac tggcagatgc acggttacga
     5161 tgcgcccatc tacaccaacg tgacctatcc cattacggtc aatccgccgt ttgttcccac
     5221 ggagaatccg acgggttgtt actcgctcac atttaatgtt gatgaaagct ggctacagga
     5281 aggccagacg cgaattattt ttgatggcgt tggaattagc ttatcgactg cacggtgcac
     5341 caatgcttct ggcgtcaggc agccatcgga agctgtggta tggctgtgca ggtcgtaaat
     5401 cactgcataa ttcgtgtcgc tcaaggcgca ctcccgttct ggataatgtt ttttgcgccg
     5461 acatcataac ggttctggca aatattctga aatgagctgt tgacaattaa tcatcggctc
     5521 gtataatgtg tggaattgtg agcggataac aatttcacac aggaaacagt attc
//
