LOCUS       sequence-366414-        5170 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Low-level transient expression of a HaloTag protein targeted to the 
            ER membrane, fused with a GST for constitutive dimerization. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5170)
  AUTHORS   Belyy V, Zuazo-Gaztelu I, Alamban A, Ashkenazi A, Walter P
  TITLE     Endoplasmic reticulum stress activates human IRE1alpha through 
            reversible assembly of inactive dimers into small oligomers.
  JOURNAL   Elife. 2022 Jun 22;11:e74342. doi: 10.7554/eLife.74342.
  PUBMED    35730415
REFERENCE   2  (bases 1 to 5170)
  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: "Elife."; 
            date: "2022-06-22"; volume: "11:e74342. doi"; pages: " 
            10.7554/eLife.74342"
COMMENT     Sequence Label: pPW3781 : CMVd3-ERmembrane-GST-HaloTag-KKMP
FEATURES             Location/Qualifiers
     source          1..5170
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        98..142
                     /label=CMVd3 promoter
                     /note="truncated human cytomegalovirus (CMV) immediate 
                     early promoter that yields strongly reduced expression 
                     relative to the full-length promoter"
     CDS             373..1026
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     984..1006
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             1033..1923
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     polyA_signal    2067..2188
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2104..2123)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2158..2177
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2195..2650)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2332..2353)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2544..2563
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2677..2781
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2806..2826)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2850..3060
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        2872..3068
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      2919..3054
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3103..3897
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             3103..3897
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3157..3176)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3767..3786
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4085..4104)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4129..4176
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4505..5093
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4994..5013
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ccctgattct gtggataacc gtattaccgc catgcattag ttattaatag taatcaatta
       61 cggggtcatt agttcatagc ccatatatgg agttccgatg ggcggtaggc gtgtacggtg
      121 ggaggtctat ataagcagag ctggtttagt gaaccgtcag atccgctagc gctaccggtc
      181 gccaccatgc cggcccggcg gctgctgctg ctgctgacgc tgctgctgcc cggcctcggg
      241 atttttggaa gtatgcttaa ggacatggct accatcatcc tgagcacctt cctgctgatt
      301 ggctgggtgg ccttcatcat cacctatccc ctgagcatgc atggaggatc tggaggcggc
      361 gggtccggcg gaatgtcccc tatactaggt tattggaaaa ttaagggcct tgtgcaaccc
      421 actcgacttc ttttggaata tcttgaagaa aaatatgaag agcatttgta tgagcgcgat
      481 gaaggtgata aatggcgaaa caaaaagttt gaattgggtt tggagtttcc caatcttcct
      541 tattatattg atggtgatgt taaattaaca cagtctatgg ccatcatacg ttatatagct
      601 gacaagcaca acatgttggg tggttgtcca aaagagcgtg cagagatttc aatgcttgaa
      661 ggagcggttt tggatattag atacggtgtt tcgagaattg catatagtaa agactttgaa
      721 actctcaaag ttgattttct tagcaagcta cctgaaatgc tgaaaatgtt cgaagatcgt
      781 ttatgtcata aaacatattt aaatggtgat catgtaaccc atcctgactt catgttgtat
      841 gacgctcttg atgttgtttt atacatggac ccaatgtgcc tggatgcgtt cccaaaatta
      901 gtttgtttta aaaaacgtat tgaagctatc ccacaaattg ataagtactt gaaatccagc
      961 aagtatatag catggccttt gcagggctgg caagccacgt ttggtggtgg cgaccatcct
     1021 ccaaaaggat ccatggcaga aatcggtact ggctttccat tcgaccccca ttatgtggaa
     1081 gtcctgggcg agcgcatgca ctacgtcgat gttggtccgc gcgatggcac ccctgtgctg
     1141 ttcctgcacg gtaacccgac ctcctcctac gtgtggcgca acatcatccc gcatgttgca
     1201 ccgacccatc gctgcattgc tccagacctg atcggtatgg gcaaatccga caaaccagac
     1261 ctgggttatt tcttcgacga ccacgtccgc ttcatggatg ccttcatcga agccctgggt
     1321 ctggaagagg tcgtcctggt cattcacgac tggggctccg ctctgggttt ccactgggcc
     1381 aagcgcaatc cagagcgcgt caaaggtatt gcatttatgg agttcatccg ccctatcccg
     1441 acctgggacg aatggccaga atttgcccgc gagaccttcc aggccttccg caccaccgac
     1501 gtcggccgca agctgatcat cgatcagaac gtttttatcg agggtacgct gccgatgggt
     1561 gtcgtccgcc cgctgactga agtcgagatg gaccattacc gcgagccgtt cctgaatcct
     1621 gttgaccgcg agccactgtg gcgcttccca aacgagctgc caatcgccgg tgagccagcg
     1681 aacatcgtcg cgctggtcga agaatacatg gactggctgc accagtcccc tgtcccgaag
     1741 ctgctgttct ggggcacccc aggcgttctg atcccaccgg ccgaagccgc tcgcctggcc
     1801 aaaagcctgc ctaactgcaa ggctgtggac atcggcccgg gtctgaatct gctgcaagaa
     1861 gacaacccgg acctgatcgg cagcgagatc gcgcgctggc tgtcgacgct cgagatttcc
     1921 ggcaaaaaga tgccataaga tccaccggat ctagataact gatcataatc agccatacca
     1981 catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac
     2041 ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat
     2101 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     2161 gtttgtccaa actcatcaat gtatcttaac gcgtaaattg taagcgttaa tattttgtta
     2221 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc
     2281 aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg
     2341 aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat
     2401 cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc
     2461 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag
     2521 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg
     2581 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta
     2641 cagggcgcgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     2701 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     2761 taatattgaa aaaggaagag tcctgaggcg gaaagaacca gctgtggaat gtgtgtcagt
     2821 tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     2881 attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca
     2941 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg
     3001 ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct
     3061 tttgcaaaga tcgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga
     3121 ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa
     3181 cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt
     3241 ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg
     3301 ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa
     3361 gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac
     3421 cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt
     3481 gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact
     3541 cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg
     3601 ccagccgaac tgttcgccag gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg
     3661 acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc
     3721 atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt
     3781 gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc
     3841 gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg
     3901 ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg
     3961 attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct
     4021 ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccaccct agggggaggc
     4081 taactgaaac acggaaggag acaataccgg aaggaacccg cgctatgacg gcaataaaaa
     4141 gacagaataa aacgcacggt gttgggtcgt ttgttcataa acgcggggtt cggtcccagg
     4201 gctggcactc tgtcgatacc ccaccgagac cccattgggg ccaatacgcc cgcgtttctt
     4261 ccttttcccc accccacccc ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg
     4321 gggcggcagg ccctgccata gcctcaggtt actcatatat actttagatt gatttaaaac
     4381 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     4441 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     4501 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     4561 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     4621 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     4681 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     4741 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     4801 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     4861 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     4921 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     4981 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     5041 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     5101 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     5161 ctgcgttatc
//
