LOCUS       sequence-366421-        7279 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Low-level transient expression of IF2 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: pPW3761 : CMVd3-HsIRE1(WLLI-GSGS)[359-362]-Hal...
FEATURES             Location/Qualifiers
     source          1..7279
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        99..143
                     /label=CMVd3 promoter
                     /note="truncated human cytomegalovirus (CMV) immediate 
                     early promoter that yields strongly reduced expression 
                     relative to the full-length promoter"
     CDS             3155..4045
                     /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    4177..4298
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4214..4233)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4268..4287
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4305..4760)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4442..4463)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4654..4673
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4787..4891
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4916..4936)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4960..5170
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        4982..5178
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5029..5164
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5213..6007
                     /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             5213..6007
                     /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(5267..5286)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5877..5896
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6195..6214)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6239..6286
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6615..7203
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7104..7123
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cccctgattc tgtggataac cgtattaccg ccatgcatta gttattaata gtaatcaatt
       61 acggggtcat tagttcatag cccatatatg gagttccgat gggcggtagg cgtgtacggt
      121 gggaggtcta tataagcaga gctggtttag tgaaccgtca gatccgctag cgctaccggt
      181 cgccaccatg ccggcccggc ggctgctgct gctgctgacg ctgctgctgc ccggcctcgg
      241 gatttttgga agtaccagca cagtgacgct tcctgaaacc ttgttgtttg tgtcaacgct
      301 ggatggaagt ttgcatgctg tcagcaagag gacaggctca atcaaatgga ctttaaaaga
      361 agatccagtc ctgcaggtcc caacacatgt ggaagagcct gcctttctcc cagatcctaa
      421 tgatggcagc ctgtatacgc ttggaagcaa gaataatgaa ggcctgacga aacttccttt
      481 taccatccca gaattggtgc aggcatcccc atgccgaagt tcagatggaa tcctctacat
      541 gggtaaaaag caggacatct ggtatgttat tgacctcctg accggagaga agcagcagac
      601 tttgtcatcg gcctttgcag atagtctctg cccatcaacc tctcttctgt atcttgggcg
      661 aacagaatac accatcacca tgtacgacac caaaacccga gagctccggt ggaatgccac
      721 ctactttgac tatgcggcct cactgcctga ggacgacgtg gactacaaga tgtcccactt
      781 tgtgtccaat ggtgatgggc tggtggtgac tgtggacagt gaatctgggg acgtcctgtg
      841 gatccaaaac tacgcctccc ctgtggtggc cttttatgtc tggcagcggg agggtctgag
      901 gaaggtgatg cacatcaatg tcgctgtgga gaccctgcgc tatctgacct tcatgtctgg
      961 ggaggtgggg cgcatcacaa agtggaagta cccgttcccc aaggagacag aggccaagag
     1021 caagctgacg cccactctgt atgttgggaa atactctacc agcctctatg cctctccctc
     1081 aatggtacac gagggggttg ctgtcgtgcc ccgcggcagc acacttcctt tgctggaagg
     1141 gccccagact gatggcgtca ccatcgggga caagggggag tgtgtgatca cgcccagcac
     1201 ggacgtcaag tttgatcccg gactcaaaag caagaacaag ctcaactact tgaggaatta
     1261 cggctctggc tctggacacc atgaaacccc actgtctgcg tctaccaaga tgctggagag
     1321 atttcccaac aatctaccca aacatcggga aaatgtgatt cctgctgatt cagagaaaaa
     1381 gagctttgag gaagttatca acctggttga ccagacttca gaaaacgcac ctaccaccgt
     1441 gtctcgggat gtggaggaga agcccgccca tgcccctgcc cggcccgagg cccccgtgga
     1501 ctccatgctt aaggacatgg ctaccatcat cctgagcacc ttcctgctga ttggctgggt
     1561 ggccttcatc atcacctatc ccctgagcat gcatcagcag cagcagctcc agcaccagca
     1621 gttccagaag gaactggaga agatccagct cctgcagcag cagcagcagc agctgccctt
     1681 ccacccacct ggagacacgg ctcaggacgg cgagctcctg gacacgtctg gcccgtactc
     1741 agagagctcg ggcaccagca gccccagcac gtcccccagg gcctccaacc actcgctctg
     1801 ctccggcagc tctgcctcca aggctggcag cagcccctcc ctggaacaag acgatggaga
     1861 tgaggaaacc agcgtggtga tagttgggaa aatttccttc tgtcccaagg atgtcctggg
     1921 ccatggagct gagggcacaa ttgtgtaccg gggcatgttt gacaaccgcg acgtggccgt
     1981 gaagaggatc ctccccgagt gttttagctt cgcagaccgt gaggtccagc tgttgcgaga
     2041 atcggatgag cacccgaacg tgatccgcta cttctgcacg gagaaggacc ggcaattcca
     2101 gtacattgcc atcgagctgt gtgcagccac cctgcaagag tatgtggagc agaaggactt
     2161 tgcgcatctc ggcctggagc ccatcacctt gctgcagcag accacctcgg gcctggccca
     2221 cctccactcc ctcaacatcg ttcacagaga cctaaagcca cacaacatcc tcatatccat
     2281 gcccaatgca cacggcaaga tcaaggccat gatctccgac tttggcctct gcaagaagct
     2341 ggcagtgggc agacacagtt tcagccgccg atctggggtg cctggcacag aaggctggat
     2401 cgctccagag atgctgagcg aagactgtaa ggagaaccct acctacacgg tggacatctt
     2461 ttctgcaggc tgcgtctttt actacgtaat ctctgagggc agccaccctt ttggcaagtc
     2521 cctgcagcgg caggccaaca tcctcctggg tgcctgcagc cttgactgct tgcacccaga
     2581 gaagcacgaa gacgtcattg cacgtgaatt gatagagaag atgattgcga tggatcctca
     2641 gaaacgcccc tcagcgaagc acgtgctcaa acacccgttc ttctggagcc tagagaagca
     2701 gctccagttc ttccaggacg tgagcgacag aatagaaaag gaatccctgg atggcccgat
     2761 cgtgaagcag ttagagagag gcgggagagc cgtggtgaag atggactggc gggagaacat
     2821 cactgtcccc ctccagacag acctgcgtaa attcaggacc tataaaggtg gttctgtcag
     2881 agatctcctc cgagccatga gaaataagaa gcaccactac cgggagctgc ctgcagaggt
     2941 gcgggagacg ctggggtccc tccccgacga cttcgtgtgc tacttcacgt ctcgcttccc
     3001 ccacctcctc gcacacacct accgggccat ggagctgtgc agccacgaga gactcttcca
     3061 gccctactac ttccacgagc ccccagagcc ccagccccca gtgactccag acgccctcgg
     3121 tggaggcggt agcggaggag gtggctctgt cgacatggca gaaatcggta ctggctttcc
     3181 attcgacccc cattatgtgg aagtcctggg cgagcgcatg cactacgtcg atgttggtcc
     3241 gcgcgatggc acccctgtgc tgttcctgca cggtaacccg acctcctcct acgtgtggcg
     3301 caacatcatc ccgcatgttg caccgaccca tcgctgcatt gctccagacc tgatcggtat
     3361 gggcaaatcc gacaaaccag acctgggtta tttcttcgac gaccacgtcc gcttcatgga
     3421 tgccttcatc gaagccctgg gtctggaaga ggtcgtcctg gtcattcacg actggggctc
     3481 cgctctgggt ttccactggg ccaagcgcaa tccagagcgc gtcaaaggta ttgcatttat
     3541 ggagttcatc cgccctatcc cgacctggga cgaatggcca gaatttgccc gcgagacctt
     3601 ccaggccttc cgcaccaccg acgtcggccg caagctgatc atcgatcaga acgtttttat
     3661 cgagggtacg ctgccgatgg gtgtcgtccg cccgctgact gaagtcgaga tggaccatta
     3721 ccgcgagccg ttcctgaatc ctgttgaccg cgagccactg tggcgcttcc caaacgagct
     3781 gccaatcgcc ggtgagccag cgaacatcgt cgcgctggtc gaagaataca tggactggct
     3841 gcaccagtcc cctgtcccga agctgctgtt ctggggcacc ccaggcgttc tgatcccacc
     3901 ggccgaagcc gctcgcctgg ccaaaagcct gcctaactgc aaggctgtgg acatcggccc
     3961 gggtctgaat ctgctgcaag aagacaaccc ggacctgatc ggcagcgaga tcgcgcgctg
     4021 gctgtcgacg ctcgagattt ccggctaaga tccaccggat ctagataact gatcataatc
     4081 agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg
     4141 aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat
     4201 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     4261 tctagttgtg gtttgtccaa actcatcaat gtatcttaac gcgtaaattg taagcgttaa
     4321 tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc
     4381 cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt
     4441 tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa
     4501 aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg
     4561 gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg
     4621 acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc
     4681 tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa
     4741 tgcgccgcta cagggcgcgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     4801 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     4861 aatgcttcaa taatattgaa aaaggaagag tcctgaggcg gaaagaacca gctgtggaat
     4921 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     4981 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
     5041 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     5101 gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     5161 ggcctaggct tttgcaaaga tcgatcaaga gacaggatga ggatcgtttc gcatgattga
     5221 acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga
     5281 ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg
     5341 gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaagacga
     5401 ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt
     5461 tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct
     5521 gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct
     5581 gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg
     5641 agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca
     5701 ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg agcatgcccg acggcgagga
     5761 tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt
     5821 ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt
     5881 ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct
     5941 ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt
     6001 cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca
     6061 cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg
     6121 gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccaccct
     6181 agggggaggc taactgaaac acggaaggag acaataccgg aaggaacccg cgctatgacg
     6241 gcaataaaaa gacagaataa aacgcacggt gttgggtcgt ttgttcataa acgcggggtt
     6301 cggtcccagg gctggcactc tgtcgatacc ccaccgagac cccattgggg ccaatacgcc
     6361 cgcgtttctt ccttttcccc accccacccc ccaagttcgg gtgaaggccc agggctcgca
     6421 gccaacgtcg gggcggcagg ccctgccata gcctcaggtt actcatatat actttagatt
     6481 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     6541 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     6601 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     6661 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     6721 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     6781 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     6841 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     6901 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     6961 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     7021 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     7081 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     7141 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     7201 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     7261 atgttctttc ctgcgttat
//
