LOCUS       sequence-403729-        7192 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 3xFLAG-tagged CYB5R3 K214R in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7192)
  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 7192)
  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: pHis-3xFLAG-CMV14-CYB5R3 K214R
FEATURES             Location/Qualifiers
     source          1..7192
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     126..148
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     140..157
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     141..157
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        318..697
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        698..901
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     852..872
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             1845..1862
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1875..1940
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    1951..2573
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2150..2169)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(2597..2617)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2615..2912
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             2966..3760
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3020..3039)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3630..3649
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4412..4546
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4449..4468)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4503..4522
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4551..4570)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4552..4570)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4584..4600)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4584..4600)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4597..4619)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4608..4624
                     /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)."
     promoter        complement(join(4632..4638,4639..4656,4657..4662))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4677..4698
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4815..4832)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4986..5574)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5066..5085)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5745..6536,6537..6605))
                     /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     6368..6387
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6606..6710)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6737..7165
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6824..6843)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7034..7055
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
       61 attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg
      121 gttttcccag tcacgacgtt gtaaaacgac ggccagtgcc aagctgatct atacattgaa
      181 tcaatattgg caattagcca tattagtcat tggttatata gcataaatca atattggcta
      241 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc
      301 aatatgaccg ccatgttgac attgattatt gactagttat taatagtaat caattacggg
      361 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      421 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      481 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      541 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga
      601 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg
      661 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
      721 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      781 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc
      841 cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
      901 tcgtttagtg aaccgtcaga attaagctta tgggggccca gctcagcacg ttgggccata
      961 tggtgctctt cccagtctgg ttcctgtaca gtctgctcat gaagctgttc cagcgctcca
     1021 cgccagccat caccctcgag agcccggaca tcaagtaccc gctgcggctc atcgaccggg
     1081 agatcatcag ccatgacacc cggcgcttcc gctttgccct gccgtcaccc cagcacatcc
     1141 tgggcctccc tgtcggccag cacatctacc tctcggctcg aattgatgga aacctggtcg
     1201 tccggcccta tacacccatc tccagcgatg atgacaaggg cttcgtggac ctggtcatca
     1261 aggtttactt caaggacacc catcccaagt ttcccgctgg agggaagatg tctcagtacc
     1321 tggagagcat gcagattgga gacaccattg agttccgggg ccccagtggg ctgctggtct
     1381 accagggcaa agggaagttc gccatccgac ctgacaaaaa gtccaaccct atcatcagga
     1441 cagtgaagtc tgtgggcatg atcgcgggag ggacaggcat caccccgatg ctgcaggtga
     1501 tccgcgccat catgaaggac cctgatgacc acactgtgtg ccacctgctc tttgccaacc
     1561 agaccgagag ggacatcctg ctgcgacctg agctggagga actcaggaac aaacattctg
     1621 cacgcttcaa gctctggtac acgctggaca gagcccctga agcctgggac tacggccagg
     1681 gcttcgtgaa tgaggagatg atccgggacc accttccacc cccagaggag gagccgctgg
     1741 tgctgatgtg tggcccccca cccatgatcc agtacgcctg ccttcccaac ctggaccacg
     1801 tgggccaccc cacggagcgc tgcttcgtct tctctagagg atcccatcat caccatcacc
     1861 acagatcccg ggctgactac aaagaccatg acggtgatta taaagatcat gacatcgact
     1921 acaaggatga cgatgacaag tagtgatccc gggtggcatc cctgtgaccc ctccccagtg
     1981 cctctcctgg ccctggaagt tgccactcca gtgcccacca gccttgtcct aataaaatta
     2041 agttgcatca ttttgtctga ctaggtgtcc ttctataata ttatggggtg gaggggggtg
     2101 gtatggagca aggggcaagt tgggaagaca acctgtaggg cctgcggggt ctattgggaa
     2161 ccaagctgga gtgcagtggc acaatcttgg ctcactgcaa tctccgcctc ctgggttcaa
     2221 gcgattctcc tgcctcagcc tcccgagttg ttgggattcc aggcatgcat gaccaggctc
     2281 agctaatttt tgtttttttg gtagagacgg ggtttcacca tattggccag gctggtctcc
     2341 aactcctaat ctcaggtgat ctacccacct tggcctccca aattgctggg attacaggcg
     2401 tgaaccactg ctcccttccc tgtccttctg attttaaaat aactatacca gcaggaggac
     2461 gtccagacac agcataggct acctggccat gcccaaccgg tgggacattt gagttgcttg
     2521 cttggcactg tcctctcatg cgttgggtcc actcagtaga tgcctgttga attgggtacg
     2581 cggccagctt ggctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
     2641 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc
     2701 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
     2761 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     2821 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag
     2881 ctattccaga agtagtgagg aggctttttt ggaggaattg atcagcttgg gatctgatca
     2941 agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc
     3001 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc
     3061 tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga
     3121 cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac
     3181 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct
     3241 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa
     3301 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc
     3361 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct
     3421 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc
     3481 caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg
     3541 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct
     3601 gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct
     3661 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca
     3721 gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa
     3781 atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc
     3841 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     3901 ggggatctca tgctggagtt cttcgcccac cccgggctcg atcccctcgc gagttggttc
     3961 agctgctgcc tgaggctgga cgacctcgcg gagttctacc ggcagtgcaa atccgtcggc
     4021 atccaggaaa ccagcagcgg ctatccgcgc atccatgccc ccgaactgca ggagtgggga
     4081 ggcacgatgg ccgctttggt cgacccggac gggacgctcc tgcgcctgat acagaacgaa
     4141 ttgcttgcag gcatctcatg agtgtgtctt cccgttttcc gcctgaggtc actgcgtgga
     4201 tggagcgctg gcgcctgctg cgcgacggcg agctgctcac cacccactcg ccaagctgga
     4261 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgccgatca taatcagcca
     4321 taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct
     4381 gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta
     4441 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     4501 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggatcaatt ccctatagtg
     4561 agtcgtatta aattcgtaat catgtcatag ctgtttcctg tgtgaaattg ttatccgctc
     4621 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     4681 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     4741 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     4801 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     4861 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     4921 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     4981 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     5041 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     5101 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     5161 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     5221 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     5281 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     5341 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     5401 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     5461 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     5521 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     5581 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     5641 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     5701 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     5761 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     5821 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     5881 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     5941 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     6001 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     6061 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     6121 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     6181 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     6241 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     6301 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     6361 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     6421 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     6481 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     6541 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     6601 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     6661 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     6721 cgaaaagtgc cacctgacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     6781 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     6841 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     6901 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     6961 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     7021 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     7081 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     7141 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat tt
//
