LOCUS       sequence-420311-        7441 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Knockdown of DNAJC5.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7441)
  AUTHORS   Wu S, Hernandez Villegas NC, Sirkis DW, Thomas-Wright I, 
            Wade-Martins R, Schekman R
  TITLE     Unconventional secretion of alpha-synuclein mediated by 
            palmitoylated DNAJC5 oligomers.
  JOURNAL   Elife. 2023 Jan 10;12:e85837. doi: 10.7554/eLife.85837.
  PUBMED    36626307
REFERENCE   2  (bases 1 to 7441)
  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: "Elife."; 
            date: "2023-01-10"; volume: "12:e85837. doi"; pages: " 
            10.7554/eLife.85837"
COMMENT     Sequence Label: plKO.1-DNAJC5-ShRNA
FEATURES             Location/Qualifiers
     source          1..7441
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     375..394
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     628..645
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    762..783
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(798..803,804..821,822..828)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    836..852
                     /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     841..863
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     860..876
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     860..876
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     895..915
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        897..915
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        943..1169
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             1170..1350
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1397..1522
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    2015..2248
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2433..2477
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     promoter        2775..3023
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2775..2795
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2946..2965
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    3120..3237
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        3286..3796
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3715..3733
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3805..4830
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             3805..4830
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     LTR             4902..5135
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5207..5328
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5244..5263)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5298..5317
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5368..5503
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5523..5542)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5524..5542)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5552..5569)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5552..5568)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5561..5583)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5710..6165
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5797..5816)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6007..6028
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6191..6295
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6296..6364,6365..7156)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6514..6533)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(7327..7441,1..474)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
       61 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
      121 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
      181 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
      241 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
      301 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
      361 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
      421 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
      481 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
      541 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
      601 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
      661 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
      721 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact
      781 cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg
      841 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa gcgcgcaatt
      901 aaccctcact aaagggaaca aaagctggag ctgcaagctt aatgtagtct tatgcaatac
      961 tcttgtagtc ttgcaacatg gtaacgatga gttagcaaca tgccttacaa ggagagaaaa
     1021 agcaccgtgc atgccgattg gtggaagtaa ggtggtacga tcgtgcctta ttaggaaggc
     1081 aacagacggg tctgacatgg attggacgaa ccactgaatt gccgcattgc agagatattg
     1141 tatttaagtg cctagctcga tacataaacg ggtctctctg gttagaccag atctgagcct
     1201 gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag
     1261 tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac
     1321 ccttttagtc agtgtggaaa atctctagca gtggcgcccg aacagggact tgaaagcgaa
     1381 agggaaacca gaggagctct ctcgacgcag gactcggctt gctgaagcgc gcacggcaag
     1441 aggcgagggg cggcgactgg tgagtacgcc aaaaattttg actagcggag gctagaagga
     1501 gagagatggg tgcgagagcg tcagtattaa gcgggggaga attagatcgc gatgggaaaa
     1561 aattcggtta aggccagggg gaaagaaaaa atataaatta aaacatatag tatgggcaag
     1621 cagggagcta gaacgattcg cagttaatcc tggcctgtta gaaacatcag aaggctgtag
     1681 acaaatactg ggacagctac aaccatccct tcagacagga tcagaagaac ttagatcatt
     1741 atataataca gtagcaaccc tctattgtgt gcatcaaagg atagagataa aagacaccaa
     1801 ggaagcttta gacaagatag aggaagagca aaacaaaagt aagaccaccg cacagcaagc
     1861 ggccgctgat cttcagacct ggaggaggag atatgaggga caattggaga agtgaattat
     1921 ataaatataa agtagtaaaa attgaaccat taggagtagc acccaccaag gcaaagagaa
     1981 gagtggtgca gagagaaaaa agagcagtgg gaataggagc tttgttcctt gggttcttgg
     2041 gagcagcagg aagcactatg ggcgcagcgt caatgacgct gacggtacag gccagacaat
     2101 tattgtctgg tatagtgcag cagcagaaca atttgctgag ggctattgag gcgcaacagc
     2161 atctgttgca actcacagtc tggggcatca agcagctcca ggcaagaatc ctggctgtgg
     2221 aaagatacct aaaggatcaa cagctcctgg ggatttgggg ttgctctgga aaactcattt
     2281 gcaccactgc tgtgccttgg aatgctagtt ggagtaataa atctctggaa cagatttgga
     2341 atcacacgac ctggatggag tgggacagag aaattaacaa ttacacaagc ttaatacact
     2401 ccttaattga agaatcgcaa aaccagcaag aaaagaatga acaagaatta ttggaattag
     2461 ataaatgggc aagtttgtgg aattggttta acataacaaa ttggctgtgg tatataaaat
     2521 tattcataat gatagtagga ggcttggtag gtttaagaat agtttttgct gtactttcta
     2581 tagtgaatag agttaggcag ggatattcac cattatcgtt tcagacccac ctcccaaccc
     2641 cgaggggacc cgacaggccc gaaggaatag aagaagaagg tggagagaga gacagagaca
     2701 gatccattcg attagtgaac ggatctcgac ggtatcgatc acgagactag cctcgagcgg
     2761 ccgccccctt caccgagggc ctatttccca tgattccttc atatttgcat atacgataca
     2821 aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa
     2881 atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta
     2941 aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata
     3001 tcttgtggaa aggacgaaac accgggcaac ctcagatgac attaaactcg agtttaatgt
     3061 catctgaggt tgctttttga attctcgacc tcgagacaaa tggcagtatt catccacaat
     3121 tttaaaagaa aaggggggat tggggggtac agtgcagggg aaagaatagt agacataata
     3181 gcaacagaca tacaaactaa agaattacaa aaacaaatta caaaaattca aaattttcgg
     3241 gtttattaca gggacagcag agatccactt tggccgcggc tcgagggggt tggggttgcg
     3301 ccttttccaa ggcagccctg ggtttgcgca gggacgcggc tgctctgggc gtggttccgg
     3361 gaaacgcagc ggcgccgacc ctgggtctcg cacattcttc acgtccgttc gcagcgtcac
     3421 ccggatcttc gccgctaccc ttgtgggccc cccggcgacg cttcctgctc cgcccctaag
     3481 tcgggaaggt tccttgcggt tcgcggcgtg ccggacgtga caaacggaag ccgcacgtct
     3541 cactagtacc ctcgcagacg gacagcgcca gggagcaatg gcagcgcgcc gaccgcgatg
     3601 ggctgtggcc aatagcggct gctcagcagg gcgcgccgag agcagcggcc gggaaggggc
     3661 ggtgcgggag gcggggtgtg gggcggtagt gtgggccctg ttcctgcccg cgcggtgttc
     3721 cgcattctgc aagcctccgg agcgcacgtc ggcagtcggc tccctcgttg accgaatcac
     3781 cgacctctct ccccaggggg atccatgaaa aagcctgaac tcaccgcgac gtctgtcgag
     3841 aagtttctga tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc ggagggcgaa
     3901 gaatctcgtg ctttcagctt cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc
     3961 tgcgccgatg gtttctacaa agatcgttat gtttatcggc actttgcatc ggccgcgctc
     4021 ccgattccgg aagtgcttga cattggggaa tttagcgaga gcctgaccta ttgcatctcc
     4081 cgccgtgcac agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctg
     4141 cagccggtcg cggaggccat ggatgcgatc gctgcggccg atcttagcca gacgagcggg
     4201 ttcggcccat tcggaccgca aggaatcggt caatacacta catggcgtga tttcatatgc
     4261 gcgattgctg atccccatgt gtatcactgg caaactgtga tggacgacac cgtcagtgcg
     4321 tccgtcgcgc aggctctcga tgagctgatg ctttgggccg aggactgccc cgaagtccgg
     4381 cacctcgtgc acgcggattt cggctccaac aatgtcctga cggacaatgg ccgcataaca
     4441 gcggtcattg actggagcga ggcgatgttc ggggattccc aatacgaggt cgccaacatc
     4501 ttcttctgga ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt cgagcggagg
     4561 catccggagc ttgcaggatc gccgcggctc cgggcgtata tgctccgcat tggtcttgac
     4621 caactctatc agagcttggt tgacggcaat ttcgatgatg cagcttgggc gcagggtcga
     4681 tgcgacgcaa tcgtccgatc cggagccggg actgtcgggc gtacacaaat cgcccgcaga
     4741 agcgcggccg tctggaccga tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc
     4801 cccagcactc gtccgagggc aaaggaatag ggtaccttta agaccaatga cttacaaggc
     4861 agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc taattcactc
     4921 ccaacgaaga caagatctgc tttttgcttg tactgggtct ctctggttag accagatctg
     4981 agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc
     5041 ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct
     5101 cagacccttt tagtcagtgt ggaaaatctc tagcagtagt agttcatgtc atcttattat
     5161 tcagtattta taacttgcaa agaaatgaat atcagagagt gagaggaact tgtttattgc
     5221 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     5281 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggct
     5341 ctagctatcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
     5401 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc
     5461 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctagggac gtacccaatt
     5521 cgccctatag tgagtcgtat tacgcgcgct cactggccgt cgttttacaa cgtcgtgact
     5581 gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct
     5641 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg
     5701 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     5761 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     5821 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     5881 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     5941 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     6001 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     6061 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     6121 aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg
     6181 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     6241 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     6301 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     6361 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     6421 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     6481 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     6541 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     6601 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     6661 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     6721 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     6781 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     6841 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     6901 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     6961 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     7021 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     7081 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     7141 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     7201 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     7261 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     7321 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     7381 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     7441 t
//
