LOCUS       sequence-403791-        5926 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GST-tagged UFBP1 F196A V198A in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5926)
  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 5926)
  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-UFBP1 F196A V198A
FEATURES             Location/Qualifiers
     source          1..5926
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(184..189,190..205,206..212)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    220..236
                     /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             259..912
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     870..892
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             919..942
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     regulatory      950..959
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(1977..1999)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2144..2162)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2230..2334
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2335..2403,2404..3195)
                     /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(2553..2572)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3366..3954
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3855..3874
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4108..4125
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4198..4275
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4276..5358
                     /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(4295..4314)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5407..5412,5413..5430,5431..5437)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5445..5461
                     /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     5469..5485
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5469..5485
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5481..5926,1..82)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5501..5518)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5501..5517)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5510..5532)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 tagcttatcg actgcacggt gcaccaatgc ttctggcgtc aggcagccat cggaagctgt
       61 ggtatggctg tgcaggtcgt aaatcactgc ataattcgtg tcgctcaagg cgcactcccg
      121 ttctggataa tgttttttgc gccgacatca taacggttct ggcaaatatt ctgaaatgag
      181 ctgttgacaa ttaatcatcg gctcgtataa tgtgtggaat tgtgagcgga taacaatttc
      241 acacaggaaa cagtattcat gtcccctata ctaggttatt ggaaaattaa gggccttgtg
      301 caacccactc gacttctttt ggaatatctt gaagaaaaat atgaagagca tttgtatgag
      361 cgcgatgaag gtgataaatg gcgaaacaaa aagtttgaat tgggtttgga gtttcccaat
      421 cttccttatt atattgatgg tgatgttaaa ttaacacagt ctatggccat catacgttat
      481 atagctgaca agcacaacat gttgggtggt tgtccaaaag agcgtgcaga gatttcaatg
      541 cttgaaggag cggttttgga tattagatac ggtgtttcga gaattgcata tagtaaagac
      601 tttgaaactc tcaaagttga ttttcttagc aagctacctg aaatgctgaa aatgttcgaa
      661 gatcgtttat gtcataaaac atatttaaat ggtgatcatg taacccatcc tgacttcatg
      721 ttgtatgacg ctcttgatgt tgttttatac atggacccaa tgtgcctgga tgcgttccca
      781 aaattagttt gttttaaaaa acgtattgaa gctatcccac aaattgataa gtacttgaaa
      841 tccagcaagt atatagcatg gcctttgcag ggctggcaag ccacgtttgg tggtggcgac
      901 catcctccaa aatcggatct ggaagttctg ttccaggggc ccctgggatc catggtggcg
      961 cctgtgtggt acttggtagc ggcggctctg ctagtcggct ttatcctctt cctgactcgc
     1021 agccggggcc gggcggcatc agccggccaa gagccactgc acaatgagga gctggcagga
     1081 gcaggccggg tggcccagcc tgggcccctg gagcctgagg agccgagagc tggaggcagg
     1141 cctcggcgcc ggagggacct gggcagccgc ctacaggccc agcgtcgagc ccagcgggtg
     1201 gcctgggcag aagcagatga gaacgaggag gaagctgtca tcctagccca ggaggaggaa
     1261 ggtgtcgaga agccagcgga aactcacctg tcggggaaaa ttggagctaa gaaactgcgg
     1321 aagctggagg agaaacaagc gcgaaaggcc cagcgtgagg cagaggaggc tgaacgtgag
     1381 gagcggaaac gactcgagtc ccagcgcgaa gctgagtgga agaaggagga ggagcggctt
     1441 cgcctggagg aggagcagaa ggaggaggag gagaggaagg cccgcgagga gcaggcccag
     1501 cgggagcatg aggagtacct gaaactgaag gaggccgctg tggcggagga ggaaggcgta
     1561 ggagagacca tgactgagga acagtcccag agcttcctga cagagttcat caactacatc
     1621 aagcagtcca aggttgtgct cttggaagac ctggcttccc aggtgggcct acgcactcag
     1681 gacaccataa atcgcatcca ggacctgctg gctgagggga ctataacagg tgtgattgac
     1741 gaccggggca agttcatcta cataacccca gaggaactgg ccgccgtggc caacttcatc
     1801 cgacagcggg gccgggtgtc catcgccgag cttgcccaag ccagcaactc cctcatcgcc
     1861 tggggccggg agtcccctgc ccaagcccca gcctgagaat tcccgggtcg actcgagcgg
     1921 ccgcatcgtg actgactgac gatctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc
     1981 tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga
     2041 caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag
     2101 tcacgtagcg atagcggagt gtataattct tgaagacgaa agggcctcgt gatacgccta
     2161 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     2221 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     2281 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     2341 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     2401 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     2461 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     2521 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt
     2581 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     2641 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     2701 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     2761 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     2821 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgca
     2881 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     2941 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     3001 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     3061 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     3121 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     3181 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     3241 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     3301 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     3361 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     3421 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     3481 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     3541 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     3601 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     3661 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     3721 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     3781 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     3841 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     3901 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     3961 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     4021 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     4081 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     4141 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat aaattccgac
     4201 accatcgaat ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga agagagtcaa
     4261 ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg tcgcagagta tgccggtgtc
     4321 tcttatcaga ccgtttcccg cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg
     4381 gaaaaagtgg aagcggcgat ggcggagctg aattacattc ccaaccgcgt ggcacaacaa
     4441 ctggcgggca aacagtcgtt gctgattggc gttgccacct ccagtctggc cctgcacgcg
     4501 ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc cagcgtggtg
     4561 gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc
     4621 gcgcaacgcg tcagtgggct gatcattaac tatccgctgg atgaccagga tgccattgct
     4681 gtggaagctg cctgcactaa tgttccggcg ttatttcttg atgtctctga ccagacaccc
     4741 atcaacagta ttattttctc ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc
     4801 gcattgggtc accagcaaat cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt
     4861 ctgcgtctgg ctggctggca taaatatctc actcgcaatc aaattcagcc gatagcggaa
     4921 cgggaaggcg actggagtgc catgtccggt tttcaacaaa ccatgcaaat gctgaatgag
     4981 ggcatcgttc ccactgcgat gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc
     5041 gccattaccg agtccgggct gcgcgttggt gcggatatct cggtagtggg atacgacgat
     5101 accgaagaca gctcatgtta tatcccgccg ttaaccacca tcaaacagga ttttcgcctg
     5161 ctggggcaaa ccagcgtgga ccgcttgctg caactctctc agggccaggc ggtgaagggc
     5221 aatcagctgt tgcccgtctc actggtgaaa agaaaaacca cgctagcgcc caatacgcaa
     5281 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga
     5341 ctggaaagcg ggcagtgagc gcaacgcaat taatgtaagt tagctcactc attaggcacc
     5401 ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca
     5461 atttcacaca ggaaacagct atgaccatga ttacggattc actggccgtc gttttacaac
     5521 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
     5581 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
     5641 gcctgaatgg cgaatggcgc tttgcctggt ttccggcacc agaagcggtg ccggaaagct
     5701 ggctggagtg cgatcttcct gaggccgata ctgtcgtcgt cccctcaaac tggcagatgc
     5761 acggttacga tgcgcccatc tacaccaacg tgacctatcc cattacggtc aatccgccgt
     5821 ttgttcccac ggagaatccg acgggttgtt actcgctcac atttaatgtt gatgaaagct
     5881 ggctacagga aggccagacg cgaattattt ttgatggcgt tggaat
//
