LOCUS       sequence-366401-        4510 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Low-level transient expression of a single HaloTag protein with a 
            C-terminal KKMP ER retention signal, targeted to the ER membrane..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4510)
  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 4510)
  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: pPW3756 : CMVd3-ERmembrane-HaloTag-KKMP
FEATURES             Location/Qualifiers
     source          1..4510
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        113..157
                     /label=CMVd3 promoter
                     /note="truncated human cytomegalovirus (CMV) immediate 
                     early promoter that yields strongly reduced expression 
                     relative to the full-length promoter"
     CDS             388..1278
                     /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    1422..1543
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1459..1478)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1513..1532
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1550..2005)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1687..1708)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1899..1918
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2032..2136
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2161..2181)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2205..2415
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        2227..2423
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      2274..2409
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2458..3252
                     /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             2458..3252
                     /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(2512..2531)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3122..3141
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3440..3459)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3484..3531
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      3860..4448
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4349..4368
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgccatgc attagttatt
       61 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgatgggcgg
      121 taggcgtgta cggtgggagg tctatataag cagagctggt ttagtgaacc gtcagatccg
      181 ctagcgctac cggtcgccac catgccggcc cggcggctgc tgctgctgct gacgctgctg
      241 ctgcccggcc tcgggatttt tggaagtatg cttaaggaca tggctaccat catcctgagc
      301 accttcctgc tgattggctg ggtggccttc atcatcacct atcccctgag catgcatgga
      361 ggatctggag gcggcgggtc cggcggaatg gcagaaatcg gtactggctt tccattcgac
      421 ccccattatg tggaagtcct gggcgagcgc atgcactacg tcgatgttgg tccgcgcgat
      481 ggcacccctg tgctgttcct gcacggtaac ccgacctcct cctacgtgtg gcgcaacatc
      541 atcccgcatg ttgcaccgac ccatcgctgc attgctccag acctgatcgg tatgggcaaa
      601 tccgacaaac cagacctggg ttatttcttc gacgaccacg tccgcttcat ggatgccttc
      661 atcgaagccc tgggtctgga agaggtcgtc ctggtcattc acgactgggg ctccgctctg
      721 ggtttccact gggccaagcg caatccagag cgcgtcaaag gtattgcatt tatggagttc
      781 atccgcccta tcccgacctg ggacgaatgg ccagaatttg cccgcgagac cttccaggcc
      841 ttccgcacca ccgacgtcgg ccgcaagctg atcatcgatc agaacgtttt tatcgagggt
      901 acgctgccga tgggtgtcgt ccgcccgctg actgaagtcg agatggacca ttaccgcgag
      961 ccgttcctga atcctgttga ccgcgagcca ctgtggcgct tcccaaacga gctgccaatc
     1021 gccggtgagc cagcgaacat cgtcgcgctg gtcgaagaat acatggactg gctgcaccag
     1081 tcccctgtcc cgaagctgct gttctggggc accccaggcg ttctgatccc accggccgaa
     1141 gccgctcgcc tggccaaaag cctgcctaac tgcaaggctg tggacatcgg cccgggtctg
     1201 aatctgctgc aagaagacaa cccggacctg atcggcagcg agatcgcgcg ctggctgtcg
     1261 acgctcgaga tttccggcaa aaagatgcca taagatccac cggatctaga taactgatca
     1321 taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc
     1381 ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt
     1441 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     1501 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttaacgcgta aattgtaagc
     1561 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     1621 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     1681 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     1741 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt
     1801 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     1861 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
     1921 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg
     1981 cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     2041 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     2101 tgataaatgc ttcaataata ttgaaaaagg aagagtcctg aggcggaaag aaccagctgt
     2161 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     2221 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc
     2281 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
     2341 atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt
     2401 ttggaggcct aggcttttgc aaagatcgat caagagacag gatgaggatc gtttcgcatg
     2461 attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc
     2521 tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg
     2581 caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa
     2641 gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc
     2701 gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat
     2761 ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg
     2821 cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc
     2881 gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag
     2941 catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgagcat gcccgacggc
     3001 gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc
     3061 cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata
     3121 gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc
     3181 gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac
     3241 gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc
     3301 catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt
     3361 tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc
     3421 accctagggg gaggctaact gaaacacgga aggagacaat accggaagga acccgcgcta
     3481 tgacggcaat aaaaagacag aataaaacgc acggtgttgg gtcgtttgtt cataaacgcg
     3541 gggttcggtc ccagggctgg cactctgtcg ataccccacc gagaccccat tggggccaat
     3601 acgcccgcgt ttcttccttt tccccacccc accccccaag ttcgggtgaa ggcccagggc
     3661 tcgcagccaa cgtcggggcg gcaggccctg ccatagcctc aggttactca tatatacttt
     3721 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     3781 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     3841 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     3901 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     3961 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     4021 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     4081 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     4141 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     4201 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     4261 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     4321 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     4381 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     4441 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     4501 ctcacatgtt
//
