LOCUS       sequence-382570-        6858 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of C9ORF78 R41A variant in E.coli with N-terminal 
            His6-GST-tag which is cleavable with TEV protease.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6858)
  AUTHORS   Bergfort A, Preussner M, Kuropka B, Ilik IA, Hilal T, Weber G, 
            Freund C, Aktas T, Heyd F, Wahl MC
  TITLE     A multi-factor trafficking site on the spliceosome remodeling enzyme
            BRR2 recruits C9ORF78 to regulate alternative splicing.
  JOURNAL   Nat Commun. 2022 Mar 3;13(1):1132. doi: 10.1038/s41467-022-28754-2.
  PUBMED    35241646
REFERENCE   2  (bases 1 to 6858)
  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: "2022-03-3"; volume: "13(1):1132. doi"; pages: " 
            10.1038/s41467-022-28754-2"
COMMENT     Sequence Label: pETM30-C9ORF78_R41A
FEATURES             Location/Qualifiers
     source          1..6858
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      20..475
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(107..126)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     317..338
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(567..1382)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(567..1382)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1290..1309
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1374..1393
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1504..2092
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1993..2012
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2246..2263
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2278..2417
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2343..2362)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2503..2525
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2519..2710)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3519..4601)
                     /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     4563..4582
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4602..4679)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4885..4904
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4988..5007
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4988..5006
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5007..5031
                     /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             5046..5068
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5081..5098
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5117..5767
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPP"
     CDS             5804..5824
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             6710..6727
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6780..6798)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6794..6841
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 atccggattg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
       61 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
      121 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
      181 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
      241 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
      301 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
      361 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
      421 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg
      481 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
      541 atatgtatcc gctcatgaat taattcttag aaaaactcat cgagcatcaa atgaaactgc
      601 aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa
      661 ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt
      721 ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca
      781 agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag tttatgcatt
      841 tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca
      901 accaaaccgt tattcattcg tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta
      961 aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca
     1021 acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg
     1081 atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga
     1141 agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca
     1201 acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga
     1261 tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca
     1321 gcatccatgt tggaatttaa tcgcggccta gagcaagacg tttcccgttg aatatggctc
     1381 ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca tgaccaaaat
     1441 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     1501 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     1561 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     1621 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca
     1681 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     1741 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     1801 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     1861 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     1921 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     1981 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     2041 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     2101 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     2161 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     2221 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct
     2281 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc
     2341 agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg
     2401 actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     2461 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     2521 agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt
     2581 ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct
     2641 ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct
     2701 gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga
     2761 tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg
     2821 aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc
     2881 agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc
     2941 atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac
     3001 tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc
     3061 agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc
     3121 aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtgggg
     3181 ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga
     3241 cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg
     3301 tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc
     3361 ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg
     3421 cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg
     3481 cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg
     3541 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     3601 gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc
     3661 ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg
     3721 cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg
     3781 tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc
     3841 attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca
     3901 ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc
     3961 gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc
     4021 gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc
     4081 agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt
     4141 gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca
     4201 atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga
     4261 agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc
     4321 acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg
     4381 tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt
     4441 tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc
     4501 gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca
     4561 ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga
     4621 ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc
     4681 gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt
     4741 gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag
     4801 tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa
     4861 gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc
     4921 tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tctcgatccc
     4981 gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctagaa
     5041 ataattttgt ttaactttaa gaaggagata taccatgaaa catcaccatc accatcacaa
     5101 cactagtagc aattccatgt cccctatact aggttattgg aaaattaagg gccttgtgca
     5161 acccactcga cttcttttgg aatatcttga agaaaaatat gaagagcatt tgtatgagcg
     5221 cgatgaaggt gataaatggc gaaacaaaaa gtttgaattg ggtttggagt ttcccaatct
     5281 tccttattat attgatggtg atgttaaatt aacacagtct atggccatca tacgttatat
     5341 agctgacaag cacaacatgt tgggtggttg tccaaaagag cgtgcagaga tttcaatgct
     5401 tgaaggagcg gttttggata ttagatacgg tgtttcgaga attgcatata gtaaagactt
     5461 tgaaactctc aaagttgatt ttcttagcaa gctacctgaa atgctgaaaa tgttcgaaga
     5521 tcgtttatgt cataaaacat atttaaatgg tgatcatgta acccatcctg acttcatgtt
     5581 gtatgacgct cttgatgttg ttttatacat ggacccaatg tgcctggatg cgttcccaaa
     5641 attagtttgt tttaaaaaac gtattgaagc tatcccacaa attgataagt acttgaaatc
     5701 cagcaagtat atagcatggc ctttgcaggg ctggcaagcc acgtttggtg gtggcgacca
     5761 tcctccaact agtggatctg gtggtggtgg cggatggatg agcgagaatc tttattttca
     5821 gggcgccatg gcaatgccgg tcgtccggaa gattttccgt cgccgccggg gcgactcgga
     5881 gtcagaggaa gatgagcagg actcagagga ggttcgatta aaactggaag agaccagaga
     5941 ggtacagaac ttggcgaaga ggcccaacgg ggtgagtgct gtggccttgc tggtgggaga
     6001 gaaggtacaa gaggagacca ctctagtgga tgatcccttt cagatgaaga caggtggtat
     6061 ggtggatatg aagaaactga aggaaagggg caaagataag atcagtgagg aggaggacct
     6121 gcacctgggg acatcgtttt ctgcagaaac caaccgaagg gatgaggatg cagacatgat
     6181 gaagtacatt gagacagagc taaagaagag gaaagggatc gtggaacatg aggaacagaa
     6241 agttaagcca aagaatgcag aggactgtct ttatgaactt ccagaaaaca tccgtgtttc
     6301 ctcagcaaag aagaccgagg agatgctttc caaccagatg ctgagtggca ttcctgaggt
     6361 ggacctgggc atcgatgcta aaataaaaaa tatcatttcc acggaggatg ccaaggcccg
     6421 tctgctggca gagcagcaga acaagaagaa agacagcgag acctccttcg tgcctaccaa
     6481 catggctgtg aattatgtgc agcacaacag attttatcat gaggagctca acgcgcccat
     6541 acggagaaac aaagaagagc ccaaggcccg gcccttgaga gtaggtgaca cggagaagcc
     6601 agagcctgag cggtcccctc ctaaccgcaa gcgtcctgct aacgagaagg caactgatga
     6661 ctatcattat gagaagttca agaaaatgaa taggcggtac tgactcgagc accaccacca
     6721 ccaccactga gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac
     6781 cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt
     6841 gctgaaagga ggaactat
//
