LOCUS       sequence-366415-        6151 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Low-level transient expression of two tandem HaloTag proteins 
            targeted to the ER membrane, fused with a GST for constitutive 
            dimerization (making a HaloTag tetramer). .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6151)
  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 6151)
  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: pPW3783 : CMVd3-ERmembrane-GST-2xHaloTag-KKMP
FEATURES             Location/Qualifiers
     source          1..6151
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        51..95
                     /label=CMVd3 promoter
                     /note="truncated human cytomegalovirus (CMV) immediate 
                     early promoter that yields strongly reduced expression 
                     relative to the full-length promoter"
     CDS             326..979
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     937..959
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             1007..1897
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     CDS             1967..2857
                     /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    3001..3122
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3038..3057)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3092..3111
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3129..3584)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3266..3287)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3478..3497
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3611..3715
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3740..3760)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3784..3994
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        3806..4002
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3853..3988
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4037..4831
                     /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             4037..4831
                     /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(4091..4110)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4701..4720
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5019..5038)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5063..5110
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5439..6027
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5928..5947
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg atgggcggta
       61 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct
      121 agcgctaccg gtcgccacca tgccggcccg gcggctgctg ctgctgctga cgctgctgct
      181 gcccggcctc gggatttttg gaagtatgct taaggacatg gctaccatca tcctgagcac
      241 cttcctgctg attggctggg tggccttcat catcacctat cccctgagca tgcatggagg
      301 atctggaggc ggcgggtccg gcggaatgtc ccctatacta ggttattgga aaattaaggg
      361 ccttgtgcaa cccactcgac ttcttttgga atatcttgaa gaaaaatatg aagagcattt
      421 gtatgagcgc gatgaaggtg ataaatggcg aaacaaaaag tttgaattgg gtttggagtt
      481 tcccaatctt ccttattata ttgatggtga tgttaaatta acacagtcta tggccatcat
      541 acgttatata gctgacaagc acaacatgtt gggtggttgt ccaaaagagc gtgcagagat
      601 ttcaatgctt gaaggagcgg ttttggatat tagatacggt gtttcgagaa ttgcatatag
      661 taaagacttt gaaactctca aagttgattt tcttagcaag ctacctgaaa tgctgaaaat
      721 gttcgaagat cgtttatgtc ataaaacata tttaaatggt gatcatgtaa cccatcctga
      781 cttcatgttg tatgacgctc ttgatgttgt tttatacatg gacccaatgt gcctggatgc
      841 gttcccaaaa ttagtttgtt ttaaaaaacg tattgaagct atcccacaaa ttgataagta
      901 cttgaaatcc agcaagtata tagcatggcc tttgcagggc tggcaagcca cgtttggtgg
      961 tggcgaccat cctccaaaag gatctggagg cggcgggtcc ggcggaatgg cagaaatcgg
     1021 tactggcttt ccattcgacc cccattatgt ggaagtcctg ggcgagcgca tgcactacgt
     1081 cgatgttggt ccgcgcgatg gcacccctgt gctgttcctg cacggtaacc cgacctcctc
     1141 ctacgtgtgg cgcaacatca tcccgcatgt tgcaccgacc catcgctgca ttgctccaga
     1201 cctgatcggt atgggcaaat ccgacaaacc agacctgggt tatttcttcg acgaccacgt
     1261 ccgcttcatg gatgccttca tcgaagccct gggtctggaa gaggtcgtcc tggtcattca
     1321 cgactggggc tccgctctgg gtttccactg ggccaagcgc aatccagagc gcgtcaaagg
     1381 tattgcattt atggagttca tccgccctat cccgacctgg gacgaatggc cagaatttgc
     1441 ccgcgagacc ttccaggcct tccgcaccac cgacgtcggc cgcaagctga tcatcgatca
     1501 gaacgttttt atcgagggta cgctgccgat gggtgtcgtc cgcccgctga ctgaagtcga
     1561 gatggaccat taccgcgagc cgttcctgaa tcctgttgac cgcgagccac tgtggcgctt
     1621 cccaaacgag ctgccaatcg ccggtgagcc agcgaacatc gtcgcgctgg tcgaagaata
     1681 catggactgg ctgcaccagt cccctgtccc gaagctgctg ttctggggca ccccaggcgt
     1741 tctgatccca ccggccgaag ccgctcgcct ggccaaaagc ctgcctaact gcaaggctgt
     1801 ggacatcggc ccgggtctga atctgctgca agaagacaac ccggacctga tcggcagcga
     1861 gatcgcgcgc tggctgtcga cgctcgagat ttccggcatg gacgagctgt acaagtccgg
     1921 actcagatct ggtggaggcg gtagcggagg aggtggctct gtcgacatgg cagaaatcgg
     1981 tactggcttt ccattcgacc cccattatgt ggaagtcctg ggcgagcgca tgcactacgt
     2041 cgatgttggt ccgcgcgatg gcacccctgt gctgttcctg cacggtaacc cgacctcctc
     2101 ctacgtgtgg cgcaacatca tcccgcatgt tgcaccgacc catcgctgca ttgctccaga
     2161 cctgatcggt atgggcaaat ccgacaaacc agacctgggt tatttcttcg acgaccacgt
     2221 ccgcttcatg gatgccttca tcgaagccct gggtctggaa gaggtcgtcc tggtcattca
     2281 cgactggggc tccgctctgg gtttccactg ggccaagcgc aatccagagc gcgtcaaagg
     2341 tattgcattt atggagttca tccgccctat cccgacctgg gacgaatggc cagaatttgc
     2401 ccgcgagacc ttccaggcct tccgcaccac cgacgtcggc cgcaagctga tcatcgatca
     2461 gaacgttttt atcgagggta cgctgccgat gggtgtcgtc cgcccgctga ctgaagtcga
     2521 gatggaccat taccgcgagc cgttcctgaa tcctgttgac cgcgagccac tgtggcgctt
     2581 cccaaacgag ctgccaatcg ccggtgagcc agcgaacatc gtcgcgctgg tcgaagaata
     2641 catggactgg ctgcaccagt cccctgtccc gaagctgctg ttctggggca ccccaggcgt
     2701 tctgatccca ccggccgaag ccgctcgcct ggccaaaagc ctgcctaact gcaaggctgt
     2761 ggacatcggc ccgggtctga atctgctgca agaagacaac ccggacctga tcggcagcga
     2821 gatcgcgcgc tggctgtcga cgctcgagat ttccggcaaa aagatgccat aagatccacc
     2881 ggatctagat aactgatcat aatcagccat accacatttg tagaggtttt acttgcttta
     2941 aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt
     3001 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     3061 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     3121 taacgcgtaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat
     3181 cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata
     3241 gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt
     3301 ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc
     3361 atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa
     3421 agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg
     3481 gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt
     3541 aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcaggtg gcacttttcg
     3601 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     3661 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtcctga
     3721 ggcggaaaga accagctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
     3781 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc
     3841 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
     3901 tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag
     3961 aagtagtgag gaggcttttt tggaggccta ggcttttgca aagatcgatc aagagacagg
     4021 atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg
     4081 ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc
     4141 cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg
     4201 tgccctgaat gaactgcaag acgaggcagc gcggctatcg tggctggcca cgacgggcgt
     4261 tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg
     4321 cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat
     4381 catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca
     4441 ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca
     4501 ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa
     4561 ggcgagcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa
     4621 tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc
     4681 ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga
     4741 atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc
     4801 cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac
     4861 caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg
     4921 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     4981 atgctggagt tcttcgccca ccctaggggg aggctaactg aaacacggaa ggagacaata
     5041 ccggaaggaa cccgcgctat gacggcaata aaaagacaga ataaaacgca cggtgttggg
     5101 tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc actctgtcga taccccaccg
     5161 agaccccatt ggggccaata cgcccgcgtt tcttcctttt ccccacccca ccccccaagt
     5221 tcgggtgaag gcccagggct cgcagccaac gtcggggcgg caggccctgc catagcctca
     5281 ggttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     5341 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     5401 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     5461 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     5521 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     5581 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     5641 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     5701 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     5761 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     5821 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     5881 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     5941 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     6001 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     6061 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     6121 aaccgtatta ccgccatgca ttagttatta a
//
