LOCUS       sequence-332851-        7222 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expression of protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7222)
  AUTHORS   Dirac-Svejstrup AB, Walker J, Faull P, Encheva V, Akimov V, Puglia 
            M, Perkins D, Kumper S, Hunjan SS, Blagoev B, Snijders AP, Powell 
            DJ, Svejstrup JQ
  TITLE     DDI2 Is a Ubiquitin-Directed Endoprotease Responsible for Cleavage 
            of Transcription Factor NRF1.
  JOURNAL   Mol Cell. 2020 Jul 16;79(2):332-341.e7. doi: 
            10.1016/j.molcel.2020.05.035. Epub 2020 Jun 9.
  PUBMED    32521225
REFERENCE   2  (bases 1 to 7222)
  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"; doi: 
            "10.1016/j.molcel.2020.05.035"; journalName: "Mol Cell"; date: 
            "2020-07-16- 16"; volume: "79"; issue: "2"; pages: "332-341.e7"
COMMENT     Sequence Label: His-SUMO- flag-hDDI2-D252N_pET11a
FEATURES             Location/Qualifiers
     source          1..7222
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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)."
     RBS             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             101..118
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             128..145
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             449..472
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(1723..1741)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1737..1784
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1959..1964,1965..1981,1982..1987))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2014..2032)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2100..2204
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2205..2273,2274..3065)
                     /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(2423..2442)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3236..3824
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3725..3744
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3978..3995
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4010..4149
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4075..4094)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4235..4257
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4251..4442)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5754..6836)
                     /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     6798..6817
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6837..6914)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7120..7139
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gggcagcagc catcaccatc accatcacgg
      121 tagcggtctg gttccgcgtg gcagcgcgag catgagcgac agcgaagtta atcaagaggc
      181 gaagccggaa gtgaaaccgg aagttaagcc ggaaacccac attaacctga aagtgagcga
      241 tggcagcagc gagatcttct ttaagattaa gaaaaccacc ccgctgcgtc gtctgatgga
      301 ggcgttcgcg aaacgtcaag gcaaggaaat ggacagcctg cgttttctgt acgatggtat
      361 ccgtattcaa gcggaccaga ccccggaaga cctggatatg gaggacaacg atatcattga
      421 agcgcaccgt gagcagatcg gtggcggtga ttacaaagac gatgacgata agatgctgct
      481 gaccgtgtat tgcgttcgtc gtgacctgag cgaggtgacc ttcagcctgc aagttgacgc
      541 ggatttcgaa ctgcacaact ttcgtgcgct gtgcgagctg gaaagcggta tcccggcggc
      601 ggaaagccag attgtttatg cggagcgtcc gctgaccgac aaccaccgta gcctggcgag
      661 ctatggcctg aaagacggtg atgtggttat tctgcgtcaa aaggagaacg cggacccgcg
      721 tccgccggtg cagttcccga acctgccgcg tatcgacttt agcagcatcg cggttccggg
      781 taccagcagc ccgcgtcaac gtcagccgcc gggtacccag caaagccaca gcagcccggg
      841 tgaaatcacc agcagcccgc agggtctgga taacccggcg ctgctgcgtg acatgctgct
      901 ggcgaacccg cacgaactga gcctgctgaa agaacgtaac ccgccgctgg cggaagcgct
      961 gctgagcggt gatctggaga agtttagccg tgtgctggtt gaacagcaac aggaccgtgc
     1021 gcgtcgtgag caagaacgta ttcgtctgtt cagcgcggac ccgtttgatc tggaggcgca
     1081 ggcgaaaatc gaggaagata ttcgtcaaca gaacatcgag gaaaacatga ccattgcgat
     1141 ggaggaagcg ccggaaagct tcggccaagt ggttatgctg tacatcaact gcaaagtgaa
     1201 cggccacccg gtgaaggcgt ttgttaacag cggtgcgcaa atgaccatca tgagccaggc
     1261 gtgcgcggag cgttgcaaca ttatgcgtct ggtggaccgt cgttgggcgg gtatcgcgaa
     1321 aggcgttggt acccagaaga tcattggccg tgtgcacctg gcgcaagttc agattgaggg
     1381 tgatttcctg ccgtgcagct ttagcatcct ggaggaacaa ccgatggaca tgctgctggg
     1441 cctggatatg ctgaagcgtc accagtgcag catcgacctg aagaaaaacg tgctggttat
     1501 tggcaccacc ggtagccaga ccaccttcct gccggaaggt gaactgccgg agtgcgcgcg
     1561 tctggcgtat ggcgcgggtc gtgaagacgt tcgtccggag gaaatcgcgg accaagaact
     1621 ggcggaggcg ctgcaaaaga gcgcggaaga tgcggaacgt cagaaaccgt agggatccgg
     1681 ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag
     1741 cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta
     1801 tatccggata tcccgcaaga ggcccggcag taccggcata accaagccta tgcctacagc
     1861 atccagggtg acggtgccga ggatgacgat gagcgcattg ttagatttca tacacggtgc
     1921 ctgactgcgt tagcaattta actgtgataa actaccgcat taaagcttat cgatgataag
     1981 ctgtcaaaca tgagaattct tgaagacgaa agggcctcgt gatacgccta tttttatagg
     2041 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
     2101 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     2161 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     2221 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     2281 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     2341 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     2401 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc
     2461 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     2521 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     2581 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     2641 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     2701 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgca gcaatggcaa
     2761 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     2821 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     2881 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     2941 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     3001 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     3061 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     3121 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     3181 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     3241 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     3301 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     3361 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     3421 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     3481 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     3541 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     3601 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     3661 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     3721 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     3781 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     3841 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     3901 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     3961 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     4021 attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat
     4081 ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc
     4141 atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     4201 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     4261 tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga
     4321 agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc
     4381 gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc
     4441 actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga
     4501 gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt
     4561 gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa
     4621 tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg
     4681 atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa
     4741 acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag
     4801 tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc
     4861 cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ccaggaccca
     4921 acgctgcccg agatgcgccg cgtgcggctg ctggagatgg cggacgcgat ggatatgttc
     4981 tgccaagggt tggtttgcgc attcacagtt ctccgcaaga attgattggc tccaattctt
     5041 ggagtggtga atccgttagc gaggtgccgc cggcttccat tcaggtcgag gtggcccggc
     5101 tccatgcacc gcgacgcaac gcggggaggc agacaaggta tagggcggcg cctacaatcc
     5161 atgccaaccc gttccatgtg ctcgccgagg cggcataaat cgccgtgacg atcagcggtc
     5221 caatgatcga agttaggctg gtaagagccg cgagcgatcc ttgaagctgt ccctgatggt
     5281 cgtcatctac ctgcctggac agcatggcct gcaacgcggg catcccgatg ccgccggaag
     5341 cgagaagaat cataatgggg aaggccatcc agcctcgcgt cgcgaacgcc agcaagacgt
     5401 agcccagcgc gtcggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg
     5461 tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg
     5521 acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg
     5581 ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga
     5641 tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg
     5701 gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc
     5761 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     5821 ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc
     5881 aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg
     5941 gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag
     6001 ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac
     6061 tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg
     6121 ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag
     6181 tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca
     6241 gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg
     6301 tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata
     6361 atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag
     6421 gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg
     6481 acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct
     6541 accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca
     6601 atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt
     6661 ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct
     6721 tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg
     6781 gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc
     6841 accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc
     6901 cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca
     6961 gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga
     7021 gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc
     7081 gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc
     7141 gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat
     7201 cgagatctcg atcccgcgaa at
//
