LOCUS       sequence-364098-        7628 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Stable expression of HaloTag7-mGFP-KDEL in mammalian cells to 
            measure ER-phagy activity.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7628)
  AUTHORS   Yim WW, Yamamoto H, Mizushima N
  TITLE     A pulse-chasable reporter processing assay for mammalian autophagic 
            flux with HaloTag.
  JOURNAL   Elife. 2022 Aug 8;11. pii: 78923. doi: 10.7554/eLife.78923.
  PUBMED    35938926
REFERENCE   2  (bases 1 to 7628)
  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: "2022-08-8"; volume: "11. pii: 78923. doi"; pages: " 
            10.7554/eLife.78923"
COMMENT     Sequence Label: pMRX-IB-HaloTag7-mGFP-KDEL
FEATURES             Location/Qualifiers
     source          1..7628
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        7..386
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     misc_feature    745..1102
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             1167..1583
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1513..1535
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1551..1567
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    1593..1967
                     /label=pol region
                     /note="Moloney murine leukemia virus (MMLV) pol region 
                     containing the splice acceptor site"
     primer_bind     1905..1921
                     /label=pBMN 5'
                     /note="MMLV sequence, for inserts in pBMN retroviral 
                     vector"
     sig_peptide     2003..2056
                     /codon_start=1
                     /product="Drosophila BiP signal sequence"
                     /label=BiP signal sequence
                     /translation="MKLCILLAVVAFVGLSLG"
     CDS             2060..2944
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
     CDS             join(2951..2953,2954..3664)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2993..3014)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3254..3273)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3601..3622
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    3716..4282
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    3732..4284
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3876..3893)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4103..4122
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     LTR             4960..5429
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(5559..5576)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5730..6318)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5810..5829)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6482..7273,7274..7342))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7105..7124
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7343..7447)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7515..7533
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 catgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
       61 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
      121 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
      181 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
      241 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
      301 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
      361 acgtattagt catcgctatt accatgctag agaaccatca gatgtttcca gggtgcccca
      421 aggacctgaa atgaccctgt gccttatttg aactaaccaa tcagttcgct tctcgcttct
      481 gttcgcgcgc ttctgctccc cgagctcaat aaaagagccc acaacccctc actcggcgcg
      541 ccagtcctcc gattgactga gtcgcccggg tacccgtgta tccaataaac cctcttgcag
      601 ttgcatccga cttgtggtct cgctgttcct tgggagggtc tcctctgagt gattgactac
      661 ccgtcagcgg gggtctttca tttgggggct cgtccgggat cgggagaccc ctgcccaggg
      721 accaccgacc caccaccggg aggtaagctg gccagcaact tatctgtgtc tgtccgattg
      781 tctagtgtct atgactgatt ttatgcgcct gcgtcggtac tagttagcta actagctctg
      841 tatctggcgg acccgtggtg gaactgacga gttcggaaca cccggccgca accctgggag
      901 acgtcccagg gacttcgggg gccgtttttg tggcccgacc tgagtccaaa aatcccgatc
      961 gttttggact ctttggtgca ccccccttag aggagggata tgtggttctg gtaggagacg
     1021 agaacctaaa acagttcccg cctccgtctg aatttttgct ttcggtttgg gaccgaagcc
     1081 gcgccgcgcg tcttgtctgc tgcagcatcg ttctgtgttg tctctgtctg actgtgtttc
     1141 tgtatttgtc tgaaaatatg ggcccgggcc agactgttac cactccctta agtttgacct
     1201 taggtcactg gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga
     1261 gacgttgggt taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag
     1321 acggcacctt taaccgagac ctcatcaccc aggttaagat caaggtcttt tcacctggcc
     1381 cgcatggaca cccagaccag gtcccctaca tcgtgacctg ggaagccttg gcttttgacc
     1441 cccctccctg ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg
     1501 ccccgtctct cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag
     1561 ccctcactcc ttctctaggc gcccccatat ggccatatga gatcttatat ggggcacccc
     1621 cgccccttgt aaacttccct gaccctgaca tgacaagagt tactaacagc ccctctctcc
     1681 aagctcactt acaggctctc tacttagtcc agcacgaagt ctggagacct ctggcggcag
     1741 cctaccaaga acaactggac cgaccggtgg tacctcaccc ttaccgagtc ggcgacacag
     1801 tgtgggtccg ccgacaccag actaagaacc tagaacctcg ctggaaagga ccttacacag
     1861 tcctgctgac cacccccacc gccctcaaag tagacggcat cgcagcttgg atacacgccg
     1921 cccacgtgaa ggctgccgac cccgggggtg gaccatcctc tagactgccg gatctgccat
     1981 cgatataaag cttgccgcca ccatgaagtt atgcatatta ctggccgtcg tggcctttgt
     2041 tggcctctcg ctcggatccg aaatcggtac tggctttcca ttcgaccccc attatgtgga
     2101 agtcctgggc gagcgcatgc actacgtcga tgttggtccg cgcgatggca cccctgtgct
     2161 gttcctgcac ggtaacccga cctcctccta cgtgtggcgc aacatcatcc cgcatgttgc
     2221 accgacccat cgctgcattg ctccagacct gatcggtatg ggcaaatccg acaaaccaga
     2281 cctgggttat ttcttcgacg accacgtccg cttcatggat gccttcatcg aagccctggg
     2341 tctggaagag gtcgtcctgg tcattcacga ctggggctcc gctctgggtt tccactgggc
     2401 caagcgcaat ccagagcgcg tcaaaggtat tgcatttatg gagttcatcc gccctatccc
     2461 gacctgggac gaatggccag aatttgcccg cgagaccttc caggccttcc gcaccaccga
     2521 cgtcggccgc aagctgatca tcgatcagaa cgtttttatc gagggtacgc tgccgatggg
     2581 tgtcgtccgc ccgctgactg aagtcgagat ggaccattac cgcgagccgt tcctgaatcc
     2641 tgttgaccgc gagccactgt ggcgcttccc aaacgagctg ccaatcgccg gtgagccagc
     2701 gaacatcgtc gcgctggtcg aagaatacat ggactggctg caccagtccc ctgtcccgaa
     2761 gctgctgttc tggggcaccc caggcgttct gatcccaccg gccgaagccg ctcgcctggc
     2821 caaaagcctg cctaactgca aggctgtgga catcggccca ggtctgaatc tgctgcaaga
     2881 agacaacccg gacctgatcg gcagcgagat cgcgcgctgg ctgtctactc tggagatttc
     2941 cggtctcgag gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga
     3001 gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc
     3061 cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg
     3121 gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca
     3181 catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac
     3241 catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga
     3301 caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct
     3361 ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca
     3421 gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca
     3481 gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga
     3541 caaccactac ctgagcaccc agtccaaact gagcaaagac cccaacgaga agcgcgatca
     3601 catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta
     3661 caagaattca aaagatgagc tctgataact cgagtaaagc ggccgctacg taaattccgc
     3721 ccccccccct aacgttactg gccgaagccg cttggaataa ggccggtgtg cgtttgtcta
     3781 tatgttattt tccaccatat tgccgtcttt tggcaatgtg agggcccgga aacctggccc
     3841 tgtcttcttg acgagcattc ctaggggtct ttcccctctc gccaaaggaa tgcaaggtct
     3901 gttgaatgtc gtgaaggaag cagttcctct ggaagtttct tgaagacaaa caacgtctgt
     3961 agcgaccctt tgcaggcagc ggaacccccc acctggcgac aggtgcctct gcggccaaaa
     4021 gccacgtgta taagatacac ctgcaaaggc ggcacaaccc cagtgccacg ttgtgagttg
     4081 gatagttgtg gaaagagtca aatggctctc ctcaagcgta ttcaacaagg ggctgaagga
     4141 tgcccagaag gtaccccatt gtatgggatc tgatctgggg cctcggtgca catgctttac
     4201 atgtgtttag tcgaggttaa aaaaacgtct aggccccccg aaccacgggg acgtggtttt
     4261 cctttgaaaa acacgatgat aatatggcca caaccgtcga catggtcatg aaaacattta
     4321 acatttctca acaagatcta gaattagtag aagtagcgac agagaagatt acaatgcttt
     4381 atgaggataa taaacatcat gtgggagcgg caattcgtac gaaaacagga gaaatcattt
     4441 cggcagtaca tattgaagcg tatataggac gagtaactgt ttgtgcagaa gccattgcga
     4501 ttggtagtgc agtttcgaat ggacaaaagg attttgacac gattgtagct gttagacacc
     4561 cttattctga cgaagtagat agaagtattc gagtggtaag tccttgtggt atgtgtaggg
     4621 agttgatttc agactatgca ccagattgtt ttgtgttaat agaaatgaat ggcaagttag
     4681 tcaaaactac gattgaagaa ctcattccac tcaaatatac ccgaaattaa gtcgaccgcc
     4741 cgccccacga cccgcagcgc ccgaccgaaa ggagcgcacg accccatggc tcgacgataa
     4801 aataaaagat tttatttagt ctccagaaaa aggggggaat gaaagacccc acctgtaggt
     4861 ttggcaagct agcttaagta acgccatttt gcaaggcatg gaaaaataca taactgagaa
     4921 tagagaagtt cagatcaagg tcaggaacag atggaacagc tgaatatggg ccaaacagga
     4981 tatctgtggt aagcagttcc tgccccggct cagggccaag aacagatgga acagctgaat
     5041 atgggccaaa caggatatct gtggtaagca gttcctgccc cggctcaggg ccaagaacag
     5101 atggtcccca gatgcggtcc agccctcagc agtttctaga gaaccatcag atgtttccag
     5161 ggtgccccaa ggacctgaaa tgaccctgtg ccttatttga actaaccaat cagttcgctt
     5221 ctcgcttctg ttcgcgcgct tctgctcccc gagctcaata aaagagccca caacccctca
     5281 ctcggggcgc cagtcctccg attgactgag tcgcccgggt acccgtgtat ccaataaacc
     5341 ctcttgcagt tgcatccgac ttgtggtctc gctgttcctt gggagggtct cctctgagtg
     5401 attgactacc cgtcagcggg ggtctttcac atgcagcatg tatcaaaatt aatttggttt
     5461 tttttcttaa gtatttacat taaatggcca tagttgcatt aatgaatcgg ccaacgcgcg
     5521 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     5581 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     5641 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     5701 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     5761 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     5821 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     5881 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     5941 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     6001 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     6061 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     6121 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     6181 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     6241 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     6301 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     6361 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     6421 atccttttgc ggccggccgc aaatcaatct aaagtatata tgagtaaact tggtctgaca
     6481 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     6541 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     6601 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     6661 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     6721 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     6781 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     6841 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     6901 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     6961 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     7021 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     7081 gctcttgccc ggcgtcaaca cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     7141 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     7201 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     7261 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     7321 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     7381 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     7441 ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga
     7501 cattaaccta taaaaatagg cgtatcacga ggccctttcg tcttcactat aggagcttgg
     7561 cccattgcat acgttgtatc catatcataa tatgtacatt tatattggct catgtccaac
     7621 attaccgc
//
