LOCUS       sequence-365353-        7181 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  HDR template used for endogenous CRISPR editing of ERN1 gene, 
            inserting a C-terminal HaloTag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7181)
  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 7181)
  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: pPW3755 : HDR-HsIRE1a-10xGS-HaloTag
FEATURES             Location/Qualifiers
     source          1..7181
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(20..475)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(157..178)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     369..388
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        502..606
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(607..675,676..1467)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(825..844)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1638..2226
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2127..2146
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2380..2397
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2514..2535
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2550..2555,2556..2573,2574..2580)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2588..2604
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     2593..2615
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2612..2628
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2612..2628
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2649..2667
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             3481..4371
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     protein_bind    4396..4429
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4494..4993
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(4529..4552)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     4926..4945
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        5005..5052
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             5071..5874
                     /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="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5134..5153)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5744..5763
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5906..5923)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5912..6136
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    6193..6226
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(7018..7037)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7019..7037)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(7044..7061)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7044..7060)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7053..7075)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 cagcctgaat ggcgaatgga aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
       61 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
      121 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
      181 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
      241 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
      301 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
      361 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
      421 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt
      481 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
      541 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
      601 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
      661 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
      721 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
      781 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
      841 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
      901 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
      961 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     1021 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     1081 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     1141 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     1201 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     1261 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     1321 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     1381 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     1441 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     1501 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     1561 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     1621 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     1681 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     1741 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     1801 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     1861 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggacccaaga
     1921 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     1981 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     2041 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     2101 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     2161 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     2221 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     2281 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     2341 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     2401 gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
     2461 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg
     2521 agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg
     2581 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc
     2641 aagctcgaaa ttaaccctca ctaaagggaa caaaagctgg taccgggccc agctaggcgg
     2701 cttcagctgg acttggcaaa ctgcaggctg ttgcctctct tacgctgatt ttcccttcag
     2761 ggacgtaggt gcaggtgaag ctccgggaag cagtgctctg cggcccatgt ccctcatggt
     2821 ctctggtcgc tgacttttgt ggggccctct ccctgtctga tgcagccact gtgtgtaaac
     2881 atgtataaac atacctcaaa tgcggggacc ccactgggac ggacactcca atctcaggcg
     2941 actttactcc tcctgaggtg gccaggagtg tctccaggtc ctgactgtca cagaaggcct
     3001 gagcaccccc tggactggct gggccttgct cacttcaagg cctcctcctg agtgagggta
     3061 gcaggcgaga tgacggggct ggattcttgg atacataagc cttttttctt tttctttacc
     3121 aaaaaccaac actcacattc tgtctcttcc ttgcatgatg aaagggtgtt ttgccaacat
     3181 ttcccgagct ttctaacccc tttctaatct cccgagcttt ctaactcttt gcagaagcac
     3241 cactaccggg agctgcctgc agaggtgcgg gagacgctgg ggtccctccc cgacgacttc
     3301 gtgtgctact tcacgtctcg cttcccccac ctcctcgcac acacctaccg ggccatggag
     3361 ctgtgcagcc acgagagact cttccagccc tactacttcc acgagccccc agagccccag
     3421 cccccagtga ctccagacgc cctcggtgga ggcggtagcg gaggaggtgg ctctgtcgac
     3481 atggcagaaa tcggtactgg ctttccattc gacccccatt atgtggaagt cctgggcgag
     3541 cgcatgcact acgtcgatgt tggtccgcgc gatggcaccc ctgtgctgtt cctgcacggt
     3601 aacccgacct cctcctacgt gtggcgcaac atcatcccgc atgttgcacc gacccatcgc
     3661 tgcattgctc cagacctgat cggtatgggc aaatccgaca aaccagacct gggttatttc
     3721 ttcgacgacc acgtccgctt catggatgcc ttcatcgaag ccctgggtct ggaagaggtc
     3781 gtcctggtca ttcacgactg gggctccgct ctgggtttcc actgggccaa gcgcaatcca
     3841 gagcgcgtca aaggtattgc atttatggag ttcatccgcc ctatcccgac ctgggacgaa
     3901 tggccagaat ttgcccgcga gaccttccag gccttccgca ccaccgacgt cggccgcaag
     3961 ctgatcatcg atcagaacgt ttttatcgag ggtacgctgc cgatgggtgt cgtccgcccg
     4021 ctgactgaag tcgagatgga ccattaccgc gagccgttcc tgaatcctgt tgaccgcgag
     4081 ccactgtggc gcttcccaaa cgagctgcca atcgccggtg agccagcgaa catcgtcgcg
     4141 ctggtcgaag aatacatgga ctggctgcac cagtcccctg tcccgaagct gctgttctgg
     4201 ggcaccccag gcgttctgat cccaccggcc gaagccgctc gcctggccaa aagcctgcct
     4261 aactgcaagg ctgtggacat cggcccgggt ctgaatctgc tgcaagaaga caacccggac
     4321 ctgatcggca gcgagatcgc gcgctggctg tcgacgctcg agatttccgg ctaagaattc
     4381 ggcgcgcccc ttaatataac ttcgtataat gtatgctata cgaagttatt aggtctgaag
     4441 aggagtttac gtccagccaa gctagcttgg ctgcaggtcg tcgaaattct accgggtagg
     4501 ggaggcgctt ttcccaaggc agtctggagc atgcgcttta gcagccccgc tgggcacttg
     4561 gcgctacaca agtggcctct ggcctcgcac acattccaca tccaccggta ggcgccaacc
     4621 ggctccgttc tttggtggcc ccttcgcgcc accttctact cctcccctag tcaggaagtt
     4681 cccccccgcc ccgcagctcg cgtcgtgcag gacgtgacaa atggaagtag cacgtctcac
     4741 tagtctcgtg cagatggaca gcaccgctga gcaatggaag cgggtaggcc tttggggcag
     4801 cggccaatag cagctttgct ccttcgcttt ctgggctcag aggctgggaa ggggtgggtc
     4861 cgggggcggg cttaggggcg ggctcagggg cggggcgggc gcccgaaggt cctccggagg
     4921 cccggcattc tgcacgcttc aaaagcgcac gtctgccgcg ctgttctcct cttcctcatc
     4981 tccgggcctt tcgacctgca gcctgttgac aattaatcat cggcatagta tatcggcata
     5041 gtataatacg acaaggtgag gaactaaacc atgggatcgg ccattgaaca agatggattg
     5101 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
     5161 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
     5221 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta
     5281 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
     5341 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
     5401 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
     5461 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
     5521 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
     5581 gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgatgatct cgtcgtgacc
     5641 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
     5701 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
     5761 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
     5821 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgaggggat
     5881 caattctcta gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     5941 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     6001 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     6061 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     6121 gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctcgac tagagcttgc
     6181 ggaaccctta atataacttc gtataatgta tgctatacga agttattagg tccctcgagg
     6241 ggatccgcga gggcggtccc tctgttctgg tggccccagc tgtgactgag ggcctggtca
     6301 ccacaattag agcttgatgc ctcccggctt tgcagggaga ccaggcttcc caaaccaagt
     6361 gccttgagct gcctgctctg cagcccacag aggacagtgc tgaccccagg aagtgggaga
     6421 agtggcccct cgtgacctac agggaactgg gaagatgctg gccccaaaag ccttacggtc
     6481 atgatgtctg caaaggaggg cctcagagac agcgcgagta gcacccccag ccatctactg
     6541 gataaacttg cttcagactt tttaaattcc tgcttaatgt cagtctacag gcctttcagg
     6601 aagggagagg agggaatcgt acattttgct tgcgtgctgg gacagctagg ctgagatgca
     6661 ccaagtacag ccttcactgg agaccggaat tgagaggtgg gggatgctga ggagggggag
     6721 gacggagttc agagggtgtc gtcctgcagt gtgagatttc tcattgatca cagatgtgcc
     6781 cagagtagcc caggtcactg ttaactagtg tttctgcaga ggcagcagga gccatgagca
     6841 tgaggtgtgg cattagggac tggtcagcta tgcatgctgg caggtggggt tgtgtctgca
     6901 ggtctcagaa atgaagaggc tgctctgttc tggaggcagc cgtggcccag tgccagtggc
     6961 cagaacagtg gcctttggtg ggtgtgtccc gggccaccgc ggtggagctc caattcgccc
     7021 tatagtgagt cgtattacaa ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac
     7081 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat
     7141 agcgaagagg cccgcaccga tcgcccttcc caacagttgc g
//
