LOCUS       sequence-403793-        5811 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GST-tagged CYB5R3 deltaN26 in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5811)
  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 5811)
  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: pGEX6p-1-CYB5R3 deltaN26
FEATURES             Location/Qualifiers
     source          1..5811
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(205..210,211..226,227..233)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    241..257
                     /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)."
     CDS             280..933
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     891..913
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             940..963
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(1883..1905)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2050..2068)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2136..2240
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2241..2309,2310..3101)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2459..2478)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3272..3860
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3761..3780
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4014..4031
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4104..4181
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4182..5264
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(4201..4220)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5313..5318,5319..5336,5337..5343)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5351..5367
                     /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)."
     primer_bind     5375..5391
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5375..5391
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5387..5811,1..103)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5407..5424)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5407..5423)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5416..5438)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 tatttttgat ggcgttggaa ttagcttatc gactgcacgg tgcaccaatg cttctggcgt
       61 caggcagcca tcggaagctg tggtatggct gtgcaggtcg taaatcactg cataattcgt
      121 gtcgctcaag gcgcactccc gttctggata atgttttttg cgccgacatc ataacggttc
      181 tggcaaatat tctgaaatga gctgttgaca attaatcatc ggctcgtata atgtgtggaa
      241 ttgtgagcgg ataacaattt cacacaggaa acagtattca tgtcccctat actaggttat
      301 tggaaaatta agggccttgt gcaacccact cgacttcttt tggaatatct tgaagaaaaa
      361 tatgaagagc atttgtatga gcgcgatgaa ggtgataaat ggcgaaacaa aaagtttgaa
      421 ttgggtttgg agtttcccaa tcttccttat tatattgatg gtgatgttaa attaacacag
      481 tctatggcca tcatacgtta tatagctgac aagcacaaca tgttgggtgg ttgtccaaaa
      541 gagcgtgcag agatttcaat gcttgaagga gcggttttgg atattagata cggtgtttcg
      601 agaattgcat atagtaaaga ctttgaaact ctcaaagttg attttcttag caagctacct
      661 gaaatgctga aaatgttcga agatcgttta tgtcataaaa catatttaaa tggtgatcat
      721 gtaacccatc ctgacttcat gttgtatgac gctcttgatg ttgttttata catggaccca
      781 atgtgcctgg atgcgttccc aaaattagtt tgttttaaaa aacgtattga agctatccca
      841 caaattgata agtacttgaa atccagcaag tatatagcat ggcctttgca gggctggcaa
      901 gccacgtttg gtggtggcga ccatcctcca aaatcggatc tggaagttct gttccagggg
      961 cccctgggat ccccggaatt catgttccag cgctccacgc cagccatcac cctcgagagc
     1021 ccggacatca agtacccgct gcggctcatc gaccgggaga tcatcagcca tgacacccgg
     1081 cgcttccgct ttgccctgcc gtcaccccag cacatcctgg gcctccctgt cggccagcac
     1141 atctacctct cggctcgaat tgatggaaac ctggtcgtcc ggccctatac acccatctcc
     1201 agcgatgatg acaagggctt cgtggacctg gtcatcaagg tttacttcaa ggacacccat
     1261 cccaagtttc ccgctggagg gaagatgtct cagtacctgg agagcatgca gattggagac
     1321 accattgagt tccggggccc cagtgggctg ctggtctacc agggcaaagg gaagttcgcc
     1381 atccgacctg acaaaaagtc caaccctatc atcaggacag tgaagtctgt gggcatgatc
     1441 gcgggaggga caggcatcac cccgatgctg caggtgatcc gcgccatcat gaaggaccct
     1501 gatgaccaca ctgtgtgcca cctgctcttt gccaaccaga ccgagaagga catcctgctg
     1561 cgacctgagc tggaggaact caggaacaaa cattctgcac gcttcaagct ctggtacacg
     1621 ctggacagag cccctgaagc ctgggactac ggccagggct tcgtgaatga ggagatgatc
     1681 cgggaccacc ttccaccccc agaggaggag ccgctggtgc tgatgtgtgg ccccccaccc
     1741 atgatccagt acgcctgcct tcccaacctg gaccacgtgg gccaccccac ggagcgctgc
     1801 ttcgtcttct gagtcgactc gagcggccgc atcgtgactg actgacgatc tgcctcgcgc
     1861 gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt
     1921 gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg
     1981 ggtgtcgggg cgcagccatg acccagtcac gtagcgatag cggagtgtat aattcttgaa
     2041 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     2101 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     2161 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     2221 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     2281 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     2341 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     2401 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     2461 tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac
     2521 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     2581 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     2641 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     2701 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     2761 acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg
     2821 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     2881 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     2941 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     3001 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     3061 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     3121 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     3181 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     3241 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     3301 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     3361 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     3421 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     3481 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     3541 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     3601 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     3661 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     3721 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     3781 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     3841 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     3901 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     3961 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     4021 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
     4081 gtatttcaca ccgcataaat tccgacacca tcgaatggcg caaaaccttt cgcggtatgg
     4141 catgatagcg cccggaagag agtcaattca gggtggtgaa tgtgaaacca gtaacgttat
     4201 acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg
     4261 ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt
     4321 acattcccaa ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg attggcgttg
     4381 ccacctccag tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg
     4441 ccgatcaact gggtgccagc gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct
     4501 gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc attaactatc
     4561 cgctggatga ccaggatgcc attgctgtgg aagctgcctg cactaatgtt ccggcgttat
     4621 ttcttgatgt ctctgaccag acacccatca acagtattat tttctcccat gaagacggta
     4681 cgcgactggg cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg
     4741 gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa tatctcactc
     4801 gcaatcaaat tcagccgata gcggaacggg aaggcgactg gagtgccatg tccggttttc
     4861 aacaaaccat gcaaatgctg aatgagggca tcgttcccac tgcgatgctg gttgccaacg
     4921 atcagatggc gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg
     4981 atatctcggt agtgggatac gacgataccg aagacagctc atgttatatc ccgccgttaa
     5041 ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac
     5101 tctctcaggg ccaggcggtg aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa
     5161 aaaccacgct agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
     5221 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
     5281 gtaagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg
     5341 ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac
     5401 ggattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact
     5461 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac
     5521 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgctttg cctggtttcc
     5581 ggcaccagaa gcggtgccgg aaagctggct ggagtgcgat cttcctgagg ccgatactgt
     5641 cgtcgtcccc tcaaactggc agatgcacgg ttacgatgcg cccatctaca ccaacgtgac
     5701 ctatcccatt acggtcaatc cgccgtttgt tcccacggag aatccgacgg gttgttactc
     5761 gctcacattt aatgttgatg aaagctggct acaggaaggc cagacgcgaa t
//
