LOCUS       sequence-494424-        5558 bp    DNA     circular SYN 02-DEC-2025
DEFINITION  Bacterial expression of human DNAJC5 L115R with GST and a cleavage 
            site for TEV protease on its N-terminus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5558)
  AUTHORS   Lee J, Chin N, Zou J, Mazli WNAB, Jarnik M, Saidi L, Xu Y, Jeong E, 
            Suh J, Replogle J, Ward ME, Bonifacino JS, Zheng W, Hao L, Ye Y
  TITLE     CHIP protects lysosomes from CLN4 mutant-induced membrane damage.
  JOURNAL   Nat Cell Biol. 2025 Aug 25. doi: 10.1038/s41556-025-01738-2.
  PUBMED    40855364
REFERENCE   2  (bases 1 to 5558)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 2, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat Cell 
            Biol. 2025 Aug 25. doi: 10.1038/s41556-025-01738-2."
COMMENT     Sequence Label: pGEX GST-tev-hDNAJC5 L115R
FEATURES             Location/Qualifiers
     source          1..5558
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(575..580,581..598,599..605)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    613..629
                     /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     637..653
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     637..653
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             649..1176
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(669..686)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(669..685)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(678..700)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(1278..1283,1284..1299,1300..1306)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1314..1330
                     /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             1353..2006
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     1964..1986
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2019..2039
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(2703..2725)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2870..2888)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2956..3060
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3061..3129,3130..3921)
                     /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(3279..3298)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4092..4680
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4581..4600
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4834..4851
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4924..5001
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(5002..5558,1..526)
                     /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(5021..5040)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gggcccatta agttctgtct cggcgcgtct gcgtctggct ggctggcata aatatctcac
       61 tcgcaatcaa attcagccga tagcggaacg ggaaggcgac tggagtgcca tgtccggttt
      121 tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc actgcgatgc tggttgccaa
      181 cgatcagatg gcgctgggcg caatgcgcgc cattaccgag tccgggctgc gcgttggtgc
      241 ggatatctcg gtagtgggat acgacgatac cgaagacagc tcatgttata tcccgccgtt
      301 aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca
      361 actctctcag ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag
      421 aaaaaccacc ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
      481 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
      541 atgtaagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta
      601 tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt
      661 acggattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa
      721 cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc
      781 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcgctt tgcctggttt
      841 ccggcaccag aagcggtgcc ggaaagctgg ctggagtgcg atcttcctga ggccgatact
      901 gtcgtcgtcc cctcaaactg gcagatgcac ggttacgatg cgcccatcta caccaacgtg
      961 acctatccca ttacggtcaa tccgccgttt gttcccacgg agaatccgac gggttgttac
     1021 tcgctcacat ttaatgttga tgaaagctgg ctacaggaag gccagacgcg aattattttt
     1081 gatggcgttg gaattagctt atcgactgca cggtgcacca atgcttctgg cgtcaggcag
     1141 ccatcggaag ctgtggtatg gctgtgcagg tcgtaaatca ctgcataatt cgtgtcgctc
     1201 aaggcgcact cccgttctgg ataatgtttt ttgcgccgac atcataacgg ttctggcaaa
     1261 tattctgaaa tgagctgttg acaattaatc atcggctcgt ataatgtgtg gaattgtgag
     1321 cggataacaa tttcacacag gaaacagtat tcatgtcccc tatactaggt tattggaaaa
     1381 ttaagggcct tgtgcaaccc actcgacttc ttttggaata tcttgaagaa aaatatgaag
     1441 agcatttgta tgagcgcgat gaaggtgata aatggcgaaa caaaaagttt gaattgggtt
     1501 tggagtttcc caatcttcct tattatattg atggtgatgt taaattaaca cagtctatgg
     1561 ccatcatacg ttatatagct gacaagcaca acatgttggg tggttgtcca aaagagcgtg
     1621 cagagatttc aatgcttgaa ggagcggttt tggatattag atacggtgtt tcgagaattg
     1681 catatagtaa agactttgaa actctcaaag ttgattttct tagcaagcta cctgaaatgc
     1741 tgaaaatgtt cgaagatcgt ttatgtcata aaacatattt aaatggtgat catgtaaccc
     1801 atcctgactt catgttgtat gacgctcttg atgttgtttt atacatggac ccaatgtgcc
     1861 tggatgcgtt cccaaaatta gtttgtttta aaaaacgtat tgaagctatc ccacaaattg
     1921 ataagtactt gaaatccagc aagtatatag catggccttt gcagggctgg caagccacgt
     1981 ttggtggtgg cgaccatcct ccaaaatcgg atactagtga aaacctgtac ttccaatccg
     2041 gttccgcaga ccagagacag cgctcactct ctacttccgg ggaatcgtta taccatgttc
     2101 ttggactgga caagaatgca acctcagatg acattaaaaa gtcctatcgg aagctggccc
     2161 tgaaatatca ccctgacaag aaccctgata acccagaggc tgcagacaag tttaaggaga
     2221 ttaacaacgc ccacgcaatc ctgacagacg ccacgaaaag aaacatttat gacaagtatg
     2281 gctcgctggg gctctatgtg gctgagcaat ttggggagga gaatgtcaac acctacttcg
     2341 tactctccag ctggtgggcc aaggcgctgt tcgtcttctg tggccgcctc acctgctgct
     2401 actgctgctg ctgtttatgc tgttgcttta actgctgctg tgggaagtgc aagcccaagg
     2461 cacctgaggg tgaagagaca gaattctacg tatcccctga agacttggag gcacagctgc
     2521 agtctgatga gagggaggct acagacacac cgatcgtcat acagccagca tccgccacag
     2581 agaccaccca gctgacagct gactcccacc ccagctatca cactgacggg ttcaactaaa
     2641 agcttaattc atcgtgactg actgacgatc tgcctcgcgc gtttcggtga tgacggtgaa
     2701 aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg
     2761 agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg
     2821 acccagtcac gtagcgatag cggagtgtat aattcttgaa gacgaaaggg cctcgtgata
     2881 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact
     2941 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     3001 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     3061 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     3121 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     3181 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     3241 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     3301 cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     3361 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     3421 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     3481 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     3541 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     3601 cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     3661 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     3721 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     3781 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     3841 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     3901 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     3961 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     4021 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     4081 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     4141 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     4201 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     4261 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     4321 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     4381 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     4441 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     4501 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     4561 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     4621 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     4681 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     4741 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     4801 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     4861 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcataaat
     4921 tccgacacca tcgaatggcg caaaaccttt cgcggtatgg catgatagcg cccggaagag
     4981 agtcaattca gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc
     5041 ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa
     5101 acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt acattcccaa ccgcgtggca
     5161 caacaactgg cgggcaaaca gtcgttgctg attggcgttg ccacctccag tctggccctg
     5221 cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc
     5281 gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat
     5341 cttctcgcgc aacgcgtcag tgggctgatc attaactatc cgctggatga ccaggatgcc
     5401 attgctgtgg aagctgcctg cactaatgtt ccggcgttat ttcttgatgt ctctgaccag
     5461 acacccatca acagtattat tttctcccat gaagacggta cgcgactggg cgtggagcat
     5521 ctggtcgcat tgggtcacca gcaaatcgcg ctgttagc
//
