LOCUS       sequence-366403-        7279 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Low-level transient expression of kinase-dead K599A 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: pPW3760 : CMVd3-HsIRE1-K599A_KinaseDead-HaloTag
FEATURES             Location/Qualifiers
     source          1..7279
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        121..165
                     /label=CMVd3 promoter
                     /note="truncated human cytomegalovirus (CMV) immediate 
                     early promoter that yields strongly reduced expression 
                     relative to the full-length promoter"
     CDS             3177..4067
                     /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    4199..4320
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4236..4255)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4290..4309
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4327..4782)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4464..4485)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4676..4695
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4809..4913
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4938..4958)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4982..5192
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        5004..5200
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5051..5186
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5235..6029
                     /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             5235..6029
                     /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(5289..5308)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5899..5918
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6217..6236)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6261..6308
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6637..7225
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7126..7145
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgccatgcat
       61 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
      121 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt
      181 cagatccgct agcgctaccg gtcgccacca tgccggcccg gcggctgctg ctgctgctga
      241 cgctgctgct gcccggcctc gggatttttg gaagtaccag cacagtgacg cttcctgaaa
      301 ccttgttgtt tgtgtcaacg ctggatggaa gtttgcatgc tgtcagcaag aggacaggct
      361 caatcaaatg gactttaaaa gaagatccag tcctgcaggt cccaacacat gtggaagagc
      421 ctgcctttct cccagatcct aatgatggca gcctgtatac gcttggaagc aagaataatg
      481 aaggcctgac gaaacttcct tttaccatcc cagaattggt gcaggcatcc ccatgccgaa
      541 gttcagatgg aatcctctac atgggtaaaa agcaggacat ctggtatgtt attgacctcc
      601 tgaccggaga gaagcagcag actttgtcat cggcctttgc agatagtctc tgcccatcaa
      661 cctctcttct gtatcttggg cgaacagaat acaccatcac catgtacgac accaaaaccc
      721 gagagctccg gtggaatgcc acctactttg actatgcggc ctcactgcct gaggacgacg
      781 tggactacaa gatgtcccac tttgtgtcca atggtgatgg gctggtggtg actgtggaca
      841 gtgaatctgg ggacgtcctg tggatccaaa actacgcctc ccctgtggtg gccttttatg
      901 tctggcagcg ggagggtctg aggaaggtga tgcacatcaa tgtcgctgtg gagaccctgc
      961 gctatctgac cttcatgtct ggggaggtgg ggcgcatcac aaagtggaag tacccgttcc
     1021 ccaaggagac agaggccaag agcaagctga cgcccactct gtatgttggg aaatactcta
     1081 ccagcctcta tgcctctccc tcaatggtac acgagggggt tgctgtcgtg ccccgcggca
     1141 gcacacttcc tttgctggaa gggccccaga ctgatggcgt caccatcggg gacaaggggg
     1201 agtgtgtgat cacgcccagc acggacgtca agtttgatcc cggactcaaa agcaagaaca
     1261 agctcaacta cttgaggaat tactggcttc tgataggaca ccatgaaacc ccactgtctg
     1321 cgtctaccaa gatgctggag agatttccca acaatctacc caaacatcgg gaaaatgtga
     1381 ttcctgctga ttcagagaaa aagagctttg aggaagttat caacctggtt gaccagactt
     1441 cagaaaacgc acctaccacc gtgtctcggg atgtggagga gaagcccgcc catgcccctg
     1501 cccggcccga ggcccccgtg gactccatgc ttaaggacat ggctaccatc atcctgagca
     1561 ccttcctgct gattggctgg gtggccttca tcatcaccta tcccctgagc atgcatcagc
     1621 agcagcagct ccagcaccag cagttccaga aggaactgga gaagatccag ctcctgcagc
     1681 agcagcagca gcagctgccc ttccacccac ctggagacac ggctcaggac ggcgagctcc
     1741 tggacacgtc tggcccgtac tcagagagct cgggcaccag cagccccagc acgtccccca
     1801 gggcctccaa ccactcgctc tgctccggca gctctgcctc caaggctggc agcagcccct
     1861 ccctggaaca agacgatgga gatgaggaaa ccagcgtggt gatagttggg aaaatttcct
     1921 tctgtcccaa ggatgtcctg ggccatggag ctgagggcac aattgtgtac cggggcatgt
     1981 ttgacaaccg cgacgtggcc gtggctagga tcctccccga gtgttttagc ttcgcagacc
     2041 gtgaggtcca gctgttgcga gaatcggatg agcacccgaa cgtgatccgc tacttctgca
     2101 cggagaagga ccggcaattc cagtacattg ccatcgagct gtgtgcagcc accctgcaag
     2161 agtatgtgga gcagaaggac tttgcgcatc tcggcctgga gcccatcacc ttgctgcagc
     2221 agaccacctc gggcctggcc cacctccact ccctcaacat cgttcacaga gacctaaagc
     2281 cacacaacat cctcatatcc atgcccaatg cacacggcaa gatcaaggcc atgatctccg
     2341 actttggcct ctgcaagaag ctggcagtgg gcagacacag tttcagccgc cgatctgggg
     2401 tgcctggcac agaaggctgg atcgctccag agatgctgag cgaagactgt aaggagaacc
     2461 ctacctacac ggtggacatc ttttctgcag gctgcgtctt ttactacgta atctctgagg
     2521 gcagccaccc ttttggcaag tccctgcagc ggcaggccaa catcctcctg ggtgcctgca
     2581 gccttgactg cttgcaccca gagaagcacg aagacgtcat tgcacgtgaa ttgatagaga
     2641 agatgattgc gatggatcct cagaaacgcc cctcagcgaa gcacgtgctc aaacacccgt
     2701 tcttctggag cctagagaag cagctccagt tcttccagga cgtgagcgac agaatagaaa
     2761 aggaatccct ggatggcccg atcgtgaagc agttagagag aggcgggaga gccgtggtga
     2821 agatggactg gcgggagaac atcactgtcc ccctccagac agacctgcgt aaattcagga
     2881 cctataaagg tggttctgtc agagatctcc tccgagccat gagaaataag aagcaccact
     2941 accgggagct gcctgcagag gtgcgggaga cgctggggtc cctccccgac gacttcgtgt
     3001 gctacttcac gtctcgcttc ccccacctcc tcgcacacac ctaccgggcc atggagctgt
     3061 gcagccacga gagactcttc cagccctact acttccacga gcccccagag ccccagcccc
     3121 cagtgactcc agacgccctc ggtggaggcg gtagcggagg aggtggctct gtcgacatgg
     3181 cagaaatcgg tactggcttt ccattcgacc cccattatgt ggaagtcctg ggcgagcgca
     3241 tgcactacgt cgatgttggt ccgcgcgatg gcacccctgt gctgttcctg cacggtaacc
     3301 cgacctcctc ctacgtgtgg cgcaacatca tcccgcatgt tgcaccgacc catcgctgca
     3361 ttgctccaga cctgatcggt atgggcaaat ccgacaaacc agacctgggt tatttcttcg
     3421 acgaccacgt ccgcttcatg gatgccttca tcgaagccct gggtctggaa gaggtcgtcc
     3481 tggtcattca cgactggggc tccgctctgg gtttccactg ggccaagcgc aatccagagc
     3541 gcgtcaaagg tattgcattt atggagttca tccgccctat cccgacctgg gacgaatggc
     3601 cagaatttgc ccgcgagacc ttccaggcct tccgcaccac cgacgtcggc cgcaagctga
     3661 tcatcgatca gaacgttttt atcgagggta cgctgccgat gggtgtcgtc cgcccgctga
     3721 ctgaagtcga gatggaccat taccgcgagc cgttcctgaa tcctgttgac cgcgagccac
     3781 tgtggcgctt cccaaacgag ctgccaatcg ccggtgagcc agcgaacatc gtcgcgctgg
     3841 tcgaagaata catggactgg ctgcaccagt cccctgtccc gaagctgctg ttctggggca
     3901 ccccaggcgt tctgatccca ccggccgaag ccgctcgcct ggccaaaagc ctgcctaact
     3961 gcaaggctgt ggacatcggc ccgggtctga atctgctgca agaagacaac ccggacctga
     4021 tcggcagcga gatcgcgcgc tggctgtcga cgctcgagat ttccggctaa gatccaccgg
     4081 atctagataa ctgatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa
     4141 aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa
     4201 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     4261 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     4321 acgcgtaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     4381 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
     4441 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
     4501 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
     4561 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     4621 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     4681 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     4741 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc acttttcggg
     4801 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     4861 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtcctgagg
     4921 cggaaagaac cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc
     4981 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
     5041 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     5101 actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa
     5161 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa gatcgatcaa gagacaggat
     5221 gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg
     5281 tggagaggct attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg
     5341 tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg
     5401 ccctgaatga actgcaagac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc
     5461 cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg
     5521 aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca
     5581 tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc
     5641 aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg
     5701 atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg
     5761 cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata
     5821 tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg
     5881 accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat
     5941 gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct
     6001 tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca
     6061 agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt
     6121 gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat
     6181 gctggagttc ttcgcccacc ctagggggag gctaactgaa acacggaagg agacaatacc
     6241 ggaaggaacc cgcgctatga cggcaataaa aagacagaat aaaacgcacg gtgttgggtc
     6301 gtttgttcat aaacgcgggg ttcggtccca gggctggcac tctgtcgata ccccaccgag
     6361 accccattgg ggccaatacg cccgcgtttc ttccttttcc ccaccccacc ccccaagttc
     6421 gggtgaaggc ccagggctcg cagccaacgt cggggcggca ggccctgcca tagcctcagg
     6481 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     6541 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     6601 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     6661 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     6721 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     6781 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     6841 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     6901 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     6961 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     7021 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     7081 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     7141 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     7201 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     7261 cttttgctgg ccttttgct
//
