LOCUS       sequence-403735-        5606 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses EGFP-tagged CYB5R3 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5606)
  AUTHORS   Ishimura R, El-Gowily AH, Noshiro D, Komatsu-Hirota S, Ono Y, Shindo
            M, Hatta T, Abe M, Uemura T, Lee-Okada HC, Mohamed TM, Yokomizo T, 
            Ueno T, Sakimura K, Natsume T, Sorimachi H, Inada T, Waguri S, Noda 
            NN, Komatsu M
  TITLE     The UFM1 system regulates ER-phagy through the ufmylation of CYB5R3.
  JOURNAL   Nat Commun. 2022 Dec 21;13(1):7857. doi: 10.1038/s41467-022-35501-0.
  PUBMED    36543799
REFERENCE   2  (bases 1 to 5606)
  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: "Nat 
            Commun."; date: "2022-12-21"; volume: "13(1):7857. doi"; pages: " 
            10.1038/s41467-022-35501-0"
COMMENT     Sequence Label: pEGFP-N3-CYB5R3
FEATURES             Location/Qualifiers
     source          1..5606
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1546..1555
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1552..1554,1555..1557,1558..2271)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1552..1554,1555..1557,1558..2268)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1597..1618)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1858..1877)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2205..2226
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2394..2515
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2431..2450)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2485..2504
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2522..2977)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2659..2680)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2871..2890
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3004..3108
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3110..3467
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3110..3301
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3133..3153)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3318..3453
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3502..4296
                     /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             3502..4296
                     /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(3556..3575)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4166..4185
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4484..4503)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4528..4575
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4904..5492
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5393..5412
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggactcag atctcgagct caagcttatg ggggcccagc tcagcacgtt gggccatatg
      661 gtgctcttcc cagtctggtt cctgtacagt ctgctcatga agctgttcca gcgctccacg
      721 ccagccatca ccctcgagag cccggacatc aagtacccgc tgcggctcat cgaccgggag
      781 atcatcagcc atgacacccg gcgcttccgc tttgccctgc cgtcacccca gcacatcctg
      841 ggcctccctg tcggccagca catctacctc tcggctcgaa ttgatggaaa cctggtcgtc
      901 cggccctata cacccatctc cagcgatgat gacaagggct tcgtggacct ggtcatcaag
      961 gtttacttca aggacaccca tcccaagttt cccgctggag ggaagatgtc tcagtacctg
     1021 gagagcatgc agattggaga caccattgag ttccggggcc ccagtgggct gctggtctac
     1081 cagggcaaag ggaagttcgc catccgacct gacaaaaagt ccaaccctat catcaggaca
     1141 gtgaagtctg tgggcatgat cgcgggaggg acaggcatca ccccgatgct gcaggtgatc
     1201 cgcgccatca tgaaggaccc tgatgaccac actgtgtgcc acctgctctt tgccaaccag
     1261 accgagaagg acatcctgct gcgacctgag ctggaggaac tcaggaacaa acattctgca
     1321 cgcttcaagc tctggtacac gctggacaga gcccctgaag cctgggacta cggccagggc
     1381 ttcgtgaatg aggagatgat ccgggaccac cttccacccc cagaggagga gccgctggtg
     1441 ctgatgtgtg gccccccacc catgatccag tacgcctgcc ttcccaacct ggaccacgtg
     1501 ggccacccca cggagcgctg cttcgtcttc tctagaggat ccatcgccac catggtgagc
     1561 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
     1621 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
     1681 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     1741 accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac
     1801 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
     1861 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     1921 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     1981 tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag
     2041 gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac
     2101 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     2161 acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
     2221 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagta aagcggccgc
     2281 gactctagat cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc
     2341 tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt
     2401 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     2461 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttaaggcg
     2521 taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca
     2581 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag
     2641 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
     2701 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc
     2761 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
     2821 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
     2881 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
     2941 acacccgccg cgcttaatgc gccgctacag ggcgcgtcag gtggcacttt tcggggaaat
     3001 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     3061 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtcc tgaggcggaa
     3121 agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
     3181 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
     3241 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
     3301 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     3361 atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat
     3421 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaagat cgatcaagag
     3481 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc
     3541 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     3601 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     3661 tccggtgccc tgaatgaact gcaagacgag gcagcgcggc tatcgtggct ggccacgacg
     3721 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     3781 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
     3841 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     3901 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     3961 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     4021 ctcaaggcga gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     4081 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     4141 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     4201 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     4261 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga
     4321 ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg
     4381 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
     4441 atctcatgct ggagttcttc gcccacccta gggggaggct aactgaaaca cggaaggaga
     4501 caataccgga aggaacccgc gctatgacgg caataaaaag acagaataaa acgcacggtg
     4561 ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg ctggcactct gtcgataccc
     4621 caccgagacc ccattggggc caatacgccc gcgtttcttc cttttcccca ccccaccccc
     4681 caagttcggg tgaaggccca gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag
     4741 cctcaggtta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     4801 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     4861 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     4921 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     4981 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     5041 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     5101 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     5161 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     5221 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     5281 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     5341 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     5401 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     5461 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     5521 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     5581 tggataaccg tattaccgcc atgcat
//
