LOCUS       sequence-493223-        4887 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of the SNAPf-tagged ER reporter SEC61b; to 
            label ER membranes..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4887)
  AUTHORS   Choi H, Liao YC, Yoon YJ, Grimm J, Wang N, Lavis LD, Singer RH, 
            Lippincott-Schwartz J
  TITLE     Secretome translation shaped by lysosomes and lunapark-marked ER 
            junctions.
  JOURNAL   Nature. 2025 Nov 5. doi: 10.1038/s41586-025-09718-0.
  PUBMED    41193816
REFERENCE   2  (bases 1 to 4887)
  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: "Nature. 
            2025 Nov 5. doi: 10.1038/s41586-025-09718-0."
COMMENT     Sequence Label: pCMV-SNAPf-Sec61b
FEATURES             Location/Qualifiers
     source          1..4887
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        78..381
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        382..585
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     536..556
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             623..1168
                     /codon_start=1
                     /product="human O6-alkylguanine-DNA-alkyltransferase 
                     variant that forms covalent bonds with benzylguanine 
                     derivatives"
                     /label=SNAP-tag(R)
                     /note="fast-reacting variant, optimized for mammalian 
                     expression"
                     /translation="MDKDCEMKRTTLDSPLGKLELSGCEQGLHRIIFLGKGTSAADAVE
                     VPAPAAVLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLK
                     VVKFGEVISYSHLAALAGNPAATAAVKTALSGNPVPILIPCHRVVQGDLDVGGYEGGLA
                     VKEWLLAHEGHRLGKPGLG"
     CDS             1244..1531
                     /codon_start=1
                     /gene="SEC61B"
                     /product="human Sec61-beta subunit of the translocon in the
                     endoplasmic reticulum"
                     /label=Sec61-beta
                     /translation="PGPTPSGTNVGSSGRSPSKAVAARAAGSTVRQRKNASCGTRSAGR
                     TTSAGTGGMWRFYTEDSPGLKVGPVPVLVMSLLFIASVFMLHIWGKYTRS"
     polyA_signal    1692..1813
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1729..1748)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1783..1802
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1820..2275)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1957..1978)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2169..2188
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2302..2406
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2408..2765
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2408..2599
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2431..2451)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2616..2751
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2800..3594
                     /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             2800..3594
                     /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(2854..2873)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3464..3483
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3782..3801)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3826..3873
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4202..4790
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4691..4710
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gtattaccgc catgcattag ttattaatag taatcaatta cggggtcatt agttcatagc
       61 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
      121 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg
      181 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
      241 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc
      301 tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta
      361 ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag
      421 cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt
      481 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa
      541 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt
      601 cagatccgct agcgctaccg gtatggacaa agactgcgaa atgaagcgca ccaccctgga
      661 tagccctctg ggcaagctgg aactgtctgg gtgcgaacag ggcctgcacc gtatcatctt
      721 cctgggcaaa ggaacatctg ccgccgacgc cgtggaagtg cctgccccag ccgccgtgct
      781 gggcggacca gagccactga tgcaggccac cgcctggctc aacgcctact ttcaccagcc
      841 tgaggccatc gaggagttcc ctgtgccagc cctgcaccac ccagtgttcc agcaggagag
      901 ctttacccgc caggtgctgt ggaaactgct gaaagtggtg aagttcggag aggtcatcag
      961 ctacagccac ctggccgccc tggccggcaa tcccgccgcc accgccgccg tgaaaaccgc
     1021 cctgagcgga aatcccgtgc ccattctgat cccctgccac cgggtggtgc agggcgacct
     1081 ggacgtgggg ggctacgagg gcgggctcgc cgtgaaagag tggctgctgg cccacgaggg
     1141 ccacagactg ggcaagcctg ggctgggtct gatggacgag ctgtacaagt ccggactcag
     1201 atctggctcc agcgcaggca gcgcatccgg cggaagcgga agccctggtc cgacccccag
     1261 tggcactaac gtgggatcct cagggcgctc tcccagcaaa gcagtggccg cccgggcggc
     1321 gggatccact gtccggcaga ggaaaaatgc cagctgtggg acaaggagtg caggccgcac
     1381 aacctcggca ggcaccgggg ggatgtggcg attctacaca gaagattcac ctgggctcaa
     1441 agttggccct gttccagtat tggttatgag tcttctgttc atcgcttctg tatttatgtt
     1501 gcacatttgg ggcaagtaca ctcgttcgta ggaattctgc agtcgacggt accgcgggcc
     1561 cgggatccac cggatctaga taactgatca taatcagcca taccacattt gtagaggttt
     1621 tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa
     1681 ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     1741 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     1801 tcaatgtatc ttaacgcgta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
     1861 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     1921 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     1981 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     2041 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     2101 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     2161 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     2221 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt
     2281 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     2341 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     2401 aagagtcctg aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt
     2461 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     2521 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     2581 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     2641 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     2701 cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     2761 gcaaagatcg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg
     2821 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
     2881 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
     2941 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aagacgaggc agcgcggcta
     3001 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
     3061 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
     3121 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
     3181 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
     3241 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
     3301 gccgaactgt tcgccaggct caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc
     3361 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
     3421 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
     3481 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
     3541 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga
     3601 ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt
     3661 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     3721 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccctagg gggaggctaa
     3781 ctgaaacacg gaaggagaca ataccggaag gaacccgcgc tatgacggca ataaaaagac
     3841 agaataaaac gcacggtgtt gggtcgtttg ttcataaacg cggggttcgg tcccagggct
     3901 ggcactctgt cgatacccca ccgagacccc attggggcca atacgcccgc gtttcttcct
     3961 tttccccacc ccacccccca agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg
     4021 cggcaggccc tgccatagcc tcaggttact catatatact ttagattgat ttaaaacttc
     4081 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     4141 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     4201 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     4261 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     4321 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     4381 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     4441 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     4501 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     4561 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     4621 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     4681 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     4741 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     4801 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     4861 cgttatcccc tgattctgtg gataacc
//
