LOCUS       sequence-403789-        5347 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GST-Strep-FLAG-tagged UFM1 in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5347)
  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 5347)
  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-OSF-UFM1
FEATURES             Location/Qualifiers
     source          1..5347
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(206..211,212..227,228..234)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    242..258
                     /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             281..934
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     892..914
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             941..964
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             983..1006
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1043..1066
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1073..1096
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(1420..1442)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1587..1605)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1673..1777
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1778..1846,1847..2638)
                     /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(1996..2015)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2809..3397
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3298..3317
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3551..3568
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3641..3718
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3719..4801
                     /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(3738..3757)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(4850..4855,4856..4873,4874..4880)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4888..4904
                     /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     4912..4928
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4912..4928
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(4924..5347,1..104)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(4944..4961)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4944..4960)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4953..4975)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 ttatttttga tggcgttgga attagcttat cgactgcacg gtgcaccaat gcttctggcg
       61 tcaggcagcc atcggaagct gtggtatggc tgtgcaggtc gtaaatcact gcataattcg
      121 tgtcgctcaa ggcgcactcc cgttctggat aatgtttttt gcgccgacat cataacggtt
      181 ctggcaaata ttctgaaatg agctgttgac aattaatcat cggctcgtat aatgtgtgga
      241 attgtgagcg gataacaatt tcacacagga aacagtattc atgtccccta tactaggtta
      301 ttggaaaatt aagggccttg tgcaacccac tcgacttctt ttggaatatc ttgaagaaaa
      361 atatgaagag catttgtatg agcgcgatga aggtgataaa tggcgaaaca aaaagtttga
      421 attgggtttg gagtttccca atcttcctta ttatattgat ggtgatgtta aattaacaca
      481 gtctatggcc atcatacgtt atatagctga caagcacaac atgttgggtg gttgtccaaa
      541 agagcgtgca gagatttcaa tgcttgaagg agcggttttg gatattagat acggtgtttc
      601 gagaattgca tatagtaaag actttgaaac tctcaaagtt gattttctta gcaagctacc
      661 tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa acatatttaa atggtgatca
      721 tgtaacccat cctgacttca tgttgtatga cgctcttgat gttgttttat acatggaccc
      781 aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa aaacgtattg aagctatccc
      841 acaaattgat aagtacttga aatccagcaa gtatatagca tggcctttgc agggctggca
      901 agccacgttt ggtggtggcg accatcctcc aaaatcggat ctggaagttc tgttccaggg
      961 gcccctggga tccatggcta gctggagcca cccgcagttc gagaaaggtg gaggtgcccg
     1021 aggtggatcg ggaggtggat cgtggagcca cccgcagttc gaaaaaggat tcgattacaa
     1081 ggatgacgat gacaaggaat tcatgtcgaa ggtttccttt aagatcacgc tgacgtcgga
     1141 cccacggctg ccgtacaaag tactcagtgt tcctgaaagt acacctttca cagcagtctt
     1201 aaagtttgca gcagaagaat ttaaagttcc tgctgcaaca agtgcaatta ttaccaatga
     1261 tggaatagga ataaatcctg cacagactgc tggaaatgtt tttctaaaac atggttcaga
     1321 actgcggatt attcctagag atcgtgttgg ataactcgag cggccgcatc gtgactgact
     1381 gacgatctgc ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc
     1441 ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc
     1501 gtcagcgggt gttggcgggt gtcggggcgc agccatgacc cagtcacgta gcgatagcgg
     1561 agtgtataat tcttgaagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     1621 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     1681 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     1741 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     1801 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     1861 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     1921 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     1981 cacttttaaa gttctgctat gtggcgcggt attatcccgt gttgacgccg ggcaagagca
     2041 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     2101 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     2161 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     2221 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     2281 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg caacaacgtt
     2341 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     2401 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     2461 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     2521 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     2581 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     2641 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     2701 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     2761 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     2821 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     2881 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     2941 gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     3001 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     3061 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     3121 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     3181 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     3241 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     3301 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     3361 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     3421 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     3481 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     3541 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct
     3601 ccttacgcat ctgtgcggta tttcacaccg cataaattcc gacaccatcg aatggcgcaa
     3661 aacctttcgc ggtatggcat gatagcgccc ggaagagagt caattcaggg tggtgaatgt
     3721 gaaaccagta acgttatacg atgtcgcaga gtatgccggt gtctcttatc agaccgtttc
     3781 ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg cgggaaaaag tggaagcggc
     3841 gatggcggag ctgaattaca ttcccaaccg cgtggcacaa caactggcgg gcaaacagtc
     3901 gttgctgatt ggcgttgcca cctccagtct ggccctgcac gcgccgtcgc aaattgtcgc
     3961 ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg gtggtgtcga tggtagaacg
     4021 aagcggcgtc gaagcctgta aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg
     4081 gctgatcatt aactatccgc tggatgacca ggatgccatt gctgtggaag ctgcctgcac
     4141 taatgttccg gcgttatttc ttgatgtctc tgaccagaca cccatcaaca gtattatttt
     4201 ctcccatgaa gacggtacgc gactgggcgt ggagcatctg gtcgcattgg gtcaccagca
     4261 aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg cgtctgcgtc tggctggctg
     4321 gcataaatat ctcactcgca atcaaattca gccgatagcg gaacgggaag gcgactggag
     4381 tgccatgtcc ggttttcaac aaaccatgca aatgctgaat gagggcatcg ttcccactgc
     4441 gatgctggtt gccaacgatc agatggcgct gggcgcaatg cgcgccatta ccgagtccgg
     4501 gctgcgcgtt ggtgcggata tctcggtagt gggatacgac gataccgaag acagctcatg
     4561 ttatatcccg ccgttaacca ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt
     4621 ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt
     4681 ctcactggtg aaaagaaaaa ccacgctagc gcccaatacg caaaccgcct ctccccgcgc
     4741 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
     4801 agcgcaacgc aattaatgta agttagctca ctcattaggc accccaggct ttacacttta
     4861 tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca
     4921 gctatgacca tgattacgga ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac
     4981 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat
     5041 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
     5101 cgctttgcct ggtttccggc accagaagcg gtgccggaaa gctggctgga gtgcgatctt
     5161 cctgaggccg atactgtcgt cgtcccctca aactggcaga tgcacggtta cgatgcgccc
     5221 atctacacca acgtgaccta tcccattacg gtcaatccgc cgtttgttcc cacggagaat
     5281 ccgacgggtt gttactcgct cacatttaat gttgatgaaa gctggctaca ggaaggccag
     5341 acgcgaa
//
