LOCUS       sequence-403739-        6320 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses mCherry-EGFP-tagged CYB5R3 K214R in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6320)
  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 6320)
  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: pmCherry-EGFP-N3-CYB5R3 K214R
FEATURES             Location/Qualifiers
     source          1..6320
                     /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"
     CDS             1543..2250
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1688..1706)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1956..1975
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     regulatory      2260..2269
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2266..2268,2269..2271,2272..2985)
                     /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(2266..2268,2269..2271,2272..2982)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2311..2332)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2572..2591)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2919..2940
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3108..3229
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3145..3164)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3199..3218
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3236..3691)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3373..3394)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3585..3604
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3718..3822
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3824..4181
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3824..4015
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3847..3867)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4032..4167
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4216..5010
                     /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             4216..5010
                     /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(4270..4289)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4880..4899
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5198..5217)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5242..5289
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5618..6206
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6107..6126
                     /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 accgagaggg 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 ccatggtgag caagggcgag
     1561 gaggataaca tggccatcat caaggagttc atgcgcttca aggtgcacat ggagggctcc
     1621 gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc
     1681 cagaccgcca agctgaaggt gaccaagggt ggccccctgc ccttcgcctg ggacatcctg
     1741 tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc accccgccga catccccgac
     1801 tacttgaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac
     1861 ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg acggcgagtt catctacaag
     1921 gtgaagctgc gcggcaccaa cttcccctcc gacggccccg taatgcagaa gaagaccatg
     1981 ggctgggagg cctcctccga gcggatgtac cccgaggacg gcgccctgaa gggcgagatc
     2041 aagcagaggc tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa gaccacctac
     2101 aaggccaaga agcccgtgca gctgcccggc gcctacaacg tcaacatcaa gttggacatc
     2161 acctcccaca acgaggacta caccatcgtg gaacagtacg aacgcgccga gggccgccac
     2221 tccaccggcg gcatggacga gctgtacaag agatccatcg ccaccatggt gagcaagggc
     2281 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
     2341 cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg
     2401 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg
     2461 acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc
     2521 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc
     2581 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     2641 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     2701 tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac
     2761 ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag
     2821 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag
     2881 tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg
     2941 accgccgccg ggatcactct cggcatggac gagctgtaca agtaaagcgg ccgcgactct
     3001 agatcataat cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac
     3061 acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg
     3121 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     3181 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa ggcgtaaatt
     3241 gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     3301 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
     3361 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     3421 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
     3481 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     3541 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     3601 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     3661 gccgcgctta atgcgccgct acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc
     3721 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     3781 taaccctgat aaatgcttca ataatattga aaaaggaaga gtcctgaggc ggaaagaacc
     3841 agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa
     3901 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc
     3961 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc
     4021 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     4081 gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga
     4141 agtagtgagg aggctttttt ggaggcctag gcttttgcaa agatcgatca agagacagga
     4201 tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg
     4261 gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc
     4321 gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt
     4381 gccctgaatg aactgcaaga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt
     4441 ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc
     4501 gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc
     4561 atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac
     4621 caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag
     4681 gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag
     4741 gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat
     4801 atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg
     4861 gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa
     4921 tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc
     4981 ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc
     5041 aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt
     5101 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca
     5161 tgctggagtt cttcgcccac cctaggggga ggctaactga aacacggaag gagacaatac
     5221 cggaaggaac ccgcgctatg acggcaataa aaagacagaa taaaacgcac ggtgttgggt
     5281 cgtttgttca taaacgcggg gttcggtccc agggctggca ctctgtcgat accccaccga
     5341 gaccccattg gggccaatac gcccgcgttt cttccttttc cccaccccac cccccaagtt
     5401 cgggtgaagg cccagggctc gcagccaacg tcggggcggc aggccctgcc atagcctcag
     5461 gttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     5521 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     5581 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     5641 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     5701 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     5761 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     5821 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     5881 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     5941 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     6001 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     6061 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     6121 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     6181 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     6241 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     6301 accgtattac cgccatgcat
//
