LOCUS       sequence-332848-        7198 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 7198)
  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 7198)
  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-hDDI2-D252N_pET11a
FEATURES             Location/Qualifiers
     source          1..7198
                     /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"
     primer_bind     complement(1699..1717)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1713..1760
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1935..1940,1941..1957,1958..1963))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1990..2008)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2076..2180
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2181..2249,2250..3041)
                     /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(2399..2418)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3212..3800
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3701..3720
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3954..3971
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3986..4125
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4051..4070)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4211..4233
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4227..4418)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5730..6812)
                     /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     6774..6793
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6813..6890)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7096..7115
                     /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 cagcggtctg gttccgcgtg gcagcgcgag catgagcgac agcgaagtta atcaggaagc
      181 gaagccggaa gtgaaaccgg aagttaagcc ggaaacccac attaacctga aagtgagcga
      241 cggcagcagc gagatcttct ttaagattaa gaaaaccacc ccgctgcgtc gtctgatgga
      301 ggcgttcgcg aaacgtcagg gcaaggaaat ggacagcctg cgttttctgt acgatggtat
      361 ccgtattcaa gcggaccaga ccccggaaga cctggatatg gaggacaacg atatcattga
      421 agcgcaccgt gagcaaatcg gtggcggtat gctgctgacc gtgtattgcg ttcgtcgtga
      481 tctgagcgag gtgaccttca gcctgcaggt tgacgcggat ttcgaactgc acaactttcg
      541 tgcgctgtgc gagctggaaa gcggtatccc ggcggcggaa agccaaattg tttatgcgga
      601 gcgtccgctg accgacaacc accgtagcct ggcgagctat ggcctgaaag acggtgatgt
      661 ggttattctg cgtcaaaagg agaacgcgga cccgcgtccg ccggtgcagt tcccgaacct
      721 gccgcgtatc gactttagca gcatcgcggt tccgggtacc agcagcccgc gtcaacgtca
      781 gccgccgggt acccagcaaa gccacagcag cccgggtgaa atcaccagca gcccgcaggg
      841 tctggataac ccggcgctgc tgcgtgacat gctgctggcg aacccgcacg aactgagcct
      901 gctgaaagaa cgtaacccgc cgctggcgga agcgctgctg agcggtgatc tggagaagtt
      961 tagccgtgtg ctggttgaac agcaacagga ccgtgcgcgt cgtgagcaag aacgtattcg
     1021 tctgttcagc gcggacccgt ttgatctgga ggcgcaggcg aaaatcgagg aagatattcg
     1081 tcaacagaac atcgaggaaa acatgaccat tgcgatggag gaagcgccgg aaagcttcgg
     1141 ccaagtggtt atgctgtaca tcaactgcaa agtgaacggc cacccggtga aggcgtttgt
     1201 taacagcggt gcgcaaatga ccatcatgag ccaggcgtgc gcggagcgtt gcaacattat
     1261 gcgtctggtg gaccgtcgtt gggcgggtat cgcgaaaggc gttggtaccc agaagatcat
     1321 tggccgtgtg cacctggcgc aagttcagat tgagggtgat ttcctgccgt gcagctttag
     1381 catcctggag gaacaaccga tggacatgct gctgggcctg gatatgctga agcgtcacca
     1441 gtgcagcatc gacctgaaga aaaacgtgct ggttattggc accaccggta gccagaccac
     1501 cttcctgccg gaaggtgaac tgccggagtg cgcgcgtctg gcgtatggcg cgggtcgtga
     1561 agacgttcgt ccggaggaaa tcgcggacca agaactggcg gaggcgctgc aaaagagcgc
     1621 ggaagatgcg gaacgtcaga aaccgtaggg atccggctgc taacaaagcc cgaaaggaag
     1681 ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac
     1741 gggtcttgag gggttttttg ctgaaaggag gaactatatc cggatatccc gcaagaggcc
     1801 cggcagtacc ggcataacca agcctatgcc tacagcatcc agggtgacgg tgccgaggat
     1861 gacgatgagc gcattgttag atttcataca cggtgcctga ctgcgttagc aatttaactg
     1921 tgataaacta ccgcattaaa gcttatcgat gataagctgt caaacatgag aattcttgaa
     1981 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     2041 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     2101 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     2161 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     2221 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     2281 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     2341 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     2401 tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac
     2461 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     2521 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     2581 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     2641 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     2701 acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg
     2761 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     2821 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     2881 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     2941 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     3001 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     3061 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     3121 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     3181 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     3241 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     3301 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     3361 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     3421 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     3481 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     3541 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     3601 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     3661 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     3721 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     3781 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     3841 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     3901 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     3961 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
     4021 gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     4081 ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca
     4141 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     4201 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     4261 aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt
     4321 tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag
     4381 cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga
     4441 tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt
     4501 actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg
     4561 atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat
     4621 gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg
     4681 cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat
     4741 gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc
     4801 ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac
     4861 aggagcacga tcatgcgcac ccgtggccag gacccaacgc tgcccgagat gcgccgcgtg
     4921 cggctgctgg agatggcgga cgcgatggat atgttctgcc aagggttggt ttgcgcattc
     4981 acagttctcc gcaagaattg attggctcca attcttggag tggtgaatcc gttagcgagg
     5041 tgccgccggc ttccattcag gtcgaggtgg cccggctcca tgcaccgcga cgcaacgcgg
     5101 ggaggcagac aaggtatagg gcggcgccta caatccatgc caacccgttc catgtgctcg
     5161 ccgaggcggc ataaatcgcc gtgacgatca gcggtccaat gatcgaagtt aggctggtaa
     5221 gagccgcgag cgatccttga agctgtccct gatggtcgtc atctacctgc ctggacagca
     5281 tggcctgcaa cgcgggcatc ccgatgccgc cggaagcgag aagaatcata atggggaagg
     5341 ccatccagcc tcgcgtcgcg aacgccagca agacgtagcc cagcgcgtcg gccgccatgc
     5401 cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt
     5461 gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc
     5521 agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt
     5581 gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga
     5641 aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag
     5701 tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     5761 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     5821 gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc
     5881 tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc
     5941 tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc
     6001 actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc
     6061 agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt
     6121 tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc
     6181 tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca
     6241 gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc
     6301 acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca
     6361 gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc
     6421 tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc
     6481 accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca
     6541 cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc
     6601 agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg
     6661 cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca
     6721 gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac
     6781 tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc
     6841 gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg
     6901 acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt
     6961 gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc
     7021 cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc
     7081 ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc
     7141 ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctcgatcc cgcgaaat
//
