LOCUS       sequence-366405-        7279 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Low-level transient expression of IF1 mutant HsIRE1 with a 
            C-terminal HaloTag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7279)
  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 7279)
  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: pPW3762 : CMVd3-HsIRE1(K121Y)-HaloTag
FEATURES             Location/Qualifiers
     source          1..7279
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        150..194
                     /label=CMVd3 promoter
                     /note="truncated human cytomegalovirus (CMV) immediate 
                     early promoter that yields strongly reduced expression 
                     relative to the full-length promoter"
     CDS             3206..4096
                     /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    4228..4349
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4265..4284)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4319..4338
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4356..4811)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4493..4514)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4705..4724
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4838..4942
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4967..4987)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5011..5221
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        5033..5229
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5080..5215
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5264..6058
                     /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             5264..6058
                     /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(5318..5337)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5928..5947
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6246..6265)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6290..6337
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6666..7254
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7155..7174
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
       61 ctgtggataa ccgtattacc gccatgcatt agttattaat agtaatcaat tacggggtca
      121 ttagttcata gcccatatat ggagttccga tgggcggtag gcgtgtacgg tgggaggtct
      181 atataagcag agctggttta gtgaaccgtc agatccgcta gcgctaccgg tcgccaccat
      241 gccggcccgg cggctgctgc tgctgctgac gctgctgctg cccggcctcg ggatttttgg
      301 aagtaccagc acagtgacgc ttcctgaaac cttgttgttt gtgtcaacgc tggatggaag
      361 tttgcatgct gtcagcaaga ggacaggctc aatcaaatgg actttaaaag aagatccagt
      421 cctgcaggtc ccaacacatg tggaagagcc tgcctttctc ccagatccta atgatggcag
      481 cctgtatacg cttggaagca agaataatga aggcctgacg aaacttcctt ttaccatccc
      541 agaattggtg caggcatccc catgccgaag ttcagatgga atcctctaca tgggtaaata
      601 ccaggacatc tggtatgtta ttgacctcct gaccggagag aagcagcaga ctttgtcatc
      661 ggcctttgca gatagtctct gcccatcaac ctctcttctg tatcttgggc gaacagaata
      721 caccatcacc atgtacgaca ccaaaacccg agagctccgg tggaatgcca cctactttga
      781 ctatgcggcc tcactgcctg aggacgacgt ggactacaag atgtcccact ttgtgtccaa
      841 tggtgatggg ctggtggtga ctgtggacag tgaatctggg gacgtcctgt ggatccaaaa
      901 ctacgcctcc cctgtggtgg ccttttatgt ctggcagcgg gagggtctga ggaaggtgat
      961 gcacatcaat gtcgctgtgg agaccctgcg ctatctgacc ttcatgtctg gggaggtggg
     1021 gcgcatcaca aagtggaagt acccgttccc caaggagaca gaggccaaga gcaagctgac
     1081 gcccactctg tatgttggga aatactctac cagcctctat gcctctccct caatggtaca
     1141 cgagggggtt gctgtcgtgc cccgcggcag cacacttcct ttgctggaag ggccccagac
     1201 tgatggcgtc accatcgggg acaaggggga gtgtgtgatc acgcccagca cggacgtcaa
     1261 gtttgatccc ggactcaaaa gcaagaacaa gctcaactac ttgaggaatt actggcttct
     1321 gataggacac catgaaaccc cactgtctgc gtctaccaag atgctggaga gatttcccaa
     1381 caatctaccc aaacatcggg aaaatgtgat tcctgctgat tcagagaaaa agagctttga
     1441 ggaagttatc aacctggttg accagacttc agaaaacgca cctaccaccg tgtctcggga
     1501 tgtggaggag aagcccgccc atgcccctgc ccggcccgag gcccccgtgg actccatgct
     1561 taaggacatg gctaccatca tcctgagcac cttcctgctg attggctggg tggccttcat
     1621 catcacctat cccctgagca tgcatcagca gcagcagctc cagcaccagc agttccagaa
     1681 ggaactggag aagatccagc tcctgcagca gcagcagcag cagctgccct tccacccacc
     1741 tggagacacg gctcaggacg gcgagctcct ggacacgtct ggcccgtact cagagagctc
     1801 gggcaccagc agccccagca cgtcccccag ggcctccaac cactcgctct gctccggcag
     1861 ctctgcctcc aaggctggca gcagcccctc cctggaacaa gacgatggag atgaggaaac
     1921 cagcgtggtg atagttggga aaatttcctt ctgtcccaag gatgtcctgg gccatggagc
     1981 tgagggcaca attgtgtacc ggggcatgtt tgacaaccgc gacgtggccg tgaagaggat
     2041 cctccccgag tgttttagct tcgcagaccg tgaggtccag ctgttgcgag aatcggatga
     2101 gcacccgaac gtgatccgct acttctgcac ggagaaggac cggcaattcc agtacattgc
     2161 catcgagctg tgtgcagcca ccctgcaaga gtatgtggag cagaaggact ttgcgcatct
     2221 cggcctggag cccatcacct tgctgcagca gaccacctcg ggcctggccc acctccactc
     2281 cctcaacatc gttcacagag acctaaagcc acacaacatc ctcatatcca tgcccaatgc
     2341 acacggcaag atcaaggcca tgatctccga ctttggcctc tgcaagaagc tggcagtggg
     2401 cagacacagt ttcagccgcc gatctggggt gcctggcaca gaaggctgga tcgctccaga
     2461 gatgctgagc gaagactgta aggagaaccc tacctacacg gtggacatct tttctgcagg
     2521 ctgcgtcttt tactacgtaa tctctgaggg cagccaccct tttggcaagt ccctgcagcg
     2581 gcaggccaac atcctcctgg gtgcctgcag ccttgactgc ttgcacccag agaagcacga
     2641 agacgtcatt gcacgtgaat tgatagagaa gatgattgcg atggatcctc agaaacgccc
     2701 ctcagcgaag cacgtgctca aacacccgtt cttctggagc ctagagaagc agctccagtt
     2761 cttccaggac gtgagcgaca gaatagaaaa ggaatccctg gatggcccga tcgtgaagca
     2821 gttagagaga ggcgggagag ccgtggtgaa gatggactgg cgggagaaca tcactgtccc
     2881 cctccagaca gacctgcgta aattcaggac ctataaaggt ggttctgtca gagatctcct
     2941 ccgagccatg agaaataaga agcaccacta ccgggagctg cctgcagagg tgcgggagac
     3001 gctggggtcc ctccccgacg acttcgtgtg ctacttcacg tctcgcttcc cccacctcct
     3061 cgcacacacc taccgggcca tggagctgtg cagccacgag agactcttcc agccctacta
     3121 cttccacgag cccccagagc cccagccccc agtgactcca gacgccctcg gtggaggcgg
     3181 tagcggagga ggtggctctg tcgacatggc agaaatcggt actggctttc cattcgaccc
     3241 ccattatgtg gaagtcctgg gcgagcgcat gcactacgtc gatgttggtc cgcgcgatgg
     3301 cacccctgtg ctgttcctgc acggtaaccc gacctcctcc tacgtgtggc gcaacatcat
     3361 cccgcatgtt gcaccgaccc atcgctgcat tgctccagac ctgatcggta tgggcaaatc
     3421 cgacaaacca gacctgggtt atttcttcga cgaccacgtc cgcttcatgg atgccttcat
     3481 cgaagccctg ggtctggaag aggtcgtcct ggtcattcac gactggggct ccgctctggg
     3541 tttccactgg gccaagcgca atccagagcg cgtcaaaggt attgcattta tggagttcat
     3601 ccgccctatc ccgacctggg acgaatggcc agaatttgcc cgcgagacct tccaggcctt
     3661 ccgcaccacc gacgtcggcc gcaagctgat catcgatcag aacgttttta tcgagggtac
     3721 gctgccgatg ggtgtcgtcc gcccgctgac tgaagtcgag atggaccatt accgcgagcc
     3781 gttcctgaat cctgttgacc gcgagccact gtggcgcttc ccaaacgagc tgccaatcgc
     3841 cggtgagcca gcgaacatcg tcgcgctggt cgaagaatac atggactggc tgcaccagtc
     3901 ccctgtcccg aagctgctgt tctggggcac cccaggcgtt ctgatcccac cggccgaagc
     3961 cgctcgcctg gccaaaagcc tgcctaactg caaggctgtg gacatcggcc cgggtctgaa
     4021 tctgctgcaa gaagacaacc cggacctgat cggcagcgag atcgcgcgct ggctgtcgac
     4081 gctcgagatt tccggctaag atccaccgga tctagataac tgatcataat cagccatacc
     4141 acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa
     4201 cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa
     4261 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     4321 ggtttgtcca aactcatcaa tgtatcttaa cgcgtaaatt gtaagcgtta atattttgtt
     4381 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg
     4441 caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg
     4501 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta
     4561 tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg
     4621 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa
     4681 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct
     4741 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct
     4801 acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     4861 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     4921 ataatattga aaaaggaaga gtcctgaggc ggaaagaacc agctgtggaa tgtgtgtcag
     4981 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     5041 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     5101 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     5161 gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     5221 ttttgcaaag atcgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg
     5281 attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca
     5341 acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt
     5401 tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg
     5461 gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga
     5521 agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca
     5581 ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct
     5641 tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac
     5701 tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc
     5761 gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt
     5821 gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt
     5881 catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg
     5941 tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat
     6001 cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc
     6061 gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc
     6121 gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc
     6181 tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc tagggggagg
     6241 ctaactgaaa cacggaagga gacaataccg gaaggaaccc gcgctatgac ggcaataaaa
     6301 agacagaata aaacgcacgg tgttgggtcg tttgttcata aacgcggggt tcggtcccag
     6361 ggctggcact ctgtcgatac cccaccgaga ccccattggg gccaatacgc ccgcgtttct
     6421 tccttttccc caccccaccc cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc
     6481 ggggcggcag gccctgccat agcctcaggt tactcatata tactttagat tgatttaaaa
     6541 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     6601 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     6661 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     6721 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     6781 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     6841 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     6901 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     6961 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     7021 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     7081 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     7141 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     7201 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     7261 agcaacgcgg cctttttac
//
