LOCUS       sequence-494436-        7084 bp    DNA     circular SYN 02-DEC-2025
DEFINITION  Mammalian and lentiviral expression of shRNA targeting human CHIP 
            (Stub1).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7084)
  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 7084)
  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: sh-hCHIP
FEATURES             Location/Qualifiers
     source          1..7084
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             185..229
                     /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        527..775
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     527..547
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     698..717
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    872..989
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1038..1548
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1467..1485
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1570..2169
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1570..1589)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2066..2086
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             2297..2530
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2602..2723
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2639..2658)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2693..2712
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      2763..2898
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2918..2937)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2919..2937)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2947..2964)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2947..2963)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2956..2978)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3105..3560
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3192..3211)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3402..3423
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3586..3690
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3691..3759,3760..4551)
                     /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(3909..3928)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4722..5310
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5211..5230
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5464..5481
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5598..5619
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5634..5639,5640..5657,5658..5664)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5672..5688
                     /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     5677..5699
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5696..5712
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5696..5712
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5731..5751
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5733..5751
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5779..6005
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             6006..6186
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6233..6358
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6851..7084
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
ORIGIN
        1 ggggatttgg ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag
       61 ttggagtaat aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag
      121 agaaattaac aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca
      181 agaaaagaat gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt
      241 taacataaca aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt
      301 aggtttaaga atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc
      361 accattatcg tttcagaccc acctcccaac cccgagggga cccgacaggc ccgaaggaat
      421 agaagaagaa ggtggagaga gagacagaga cagatccatt cgattagtga acggatctcg
      481 acggtatcga tcacgagact agcctcgagc ggccgccccc ttcaccgagg gcctatttcc
      541 catgattcct tcatatttgc atatacgata caaggctgtt agagagataa ttggaattaa
      601 tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct
      661 tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac
      721 ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccggccc
      781 aagttctgct gttggactct cgagagtcca acagcagaac ttgggttttt gaattctcga
      841 cctcgagaca aatggcagta ttcatccaca attttaaaag aaaagggggg attggggggt
      901 acagtgcagg ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac
      961 aaaaacaaat tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccac
     1021 tttggccgcg gctcgagggg gttggggttg cgccttttcc aaggcagccc tgggtttgcg
     1081 cagggacgcg gctgctctgg gcgtggttcc gggaaacgca gcggcgccga ccctgggtct
     1141 cgcacattct tcacgtccgt tcgcagcgtc acccggatct tcgccgctac ccttgtgggc
     1201 cccccggcga cgcttcctgc tccgccccta agtcgggaag gttccttgcg gttcgcggcg
     1261 tgccggacgt gacaaacgga agccgcacgt ctcactagta ccctcgcaga cggacagcgc
     1321 cagggagcaa tggcagcgcg ccgaccgcga tgggctgtgg ccaatagcgg ctgctcagca
     1381 gggcgcgccg agagcagcgg ccgggaaggg gcggtgcggg aggcggggtg tggggcggta
     1441 gtgtgggccc tgttcctgcc cgcgcggtgt tccgcattct gcaagcctcc ggagcgcacg
     1501 tcggcagtcg gctccctcgt tgaccgaatc accgacctct ctccccaggg ggatccaccg
     1561 gagcttacca tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc
     1621 agggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc
     1681 gatccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc
     1741 gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc
     1801 acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag
     1861 ttgagcggtt cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg
     1921 cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca ccagggcaag
     1981 ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc
     2041 gccttcctgg agacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc
     2101 gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc
     2161 ggtgcctgac gcccgcccca cgacccgcag cgcccgaccg aaaggagcgc acgaccccat
     2221 gcatcggtac ctttaagacc aatgacttac aaggcagctg tagatcttag ccacttttta
     2281 aaagaaaagg ggggactgga agggctaatt cactcccaac gaagacaaga tctgcttttt
     2341 gcttgtactg ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta
     2401 gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc
     2461 cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa
     2521 atctctagca gtagtagttc atgtcatctt attattcagt atttataact tgcaaagaaa
     2581 tgaatatcag agagtgagag gaacttgttt attgcagctt ataatggtta caaataaagc
     2641 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     2701 tccaaactca tcaatgtatc ttatcatgtc tggctctagc tatcccgccc ctaactccgc
     2761 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     2821 tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag
     2881 gaggcttttt tggaggccta gggacgtacc caattcgccc tatagtgagt cgtattacgc
     2941 gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact
     3001 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac
     3061 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg
     3121 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     3181 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     3241 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     3301 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     3361 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     3421 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     3481 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     3541 aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     3601 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     3661 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     3721 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     3781 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     3841 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     3901 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     3961 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     4021 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     4081 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     4141 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     4201 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     4261 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     4321 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     4381 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     4441 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     4501 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     4561 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     4621 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     4681 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     4741 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     4801 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     4861 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     4921 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     4981 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     5041 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     5101 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     5161 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     5221 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     5281 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     5341 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     5401 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     5461 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc
     5521 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     5581 agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac
     5641 tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga
     5701 aacagctatg accatgatta cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag
     5761 ctggagctgc aagcttaatg tagtcttatg caatactctt gtagtcttgc aacatggtaa
     5821 cgatgagtta gcaacatgcc ttacaaggag agaaaaagca ccgtgcatgc cgattggtgg
     5881 aagtaaggtg gtacgatcgt gccttattag gaaggcaaca gacgggtctg acatggattg
     5941 gacgaaccac tgaattgccg cattgcagag atattgtatt taagtgccta gctcgataca
     6001 taaacgggtc tctctggtta gaccagatct gagcctggga gctctctggc taactaggga
     6061 acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc
     6121 tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct
     6181 ctagcagtgg cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg agctctctcg
     6241 acgcaggact cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc gactggtgag
     6301 tacgccaaaa attttgacta gcggaggcta gaaggagaga gatgggtgcg agagcgtcag
     6361 tattaagcgg gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa
     6421 gaaaaaatat aaattaaaac atatagtatg ggcaagcagg gagctagaac gattcgcagt
     6481 taatcctggc ctgttagaaa catcagaagg ctgtagacaa atactgggac agctacaacc
     6541 atcccttcag acaggatcag aagaacttag atcattatat aatacagtag caaccctcta
     6601 ttgtgtgcat caaaggatag agataaaaga caccaaggaa gctttagaca agatagagga
     6661 agagcaaaac aaaagtaaga ccaccgcaca gcaagcggcc gctgatcttc agacctggag
     6721 gaggagatat gagggacaat tggagaagtg aattatataa atataaagta gtaaaaattg
     6781 aaccattagg agtagcaccc accaaggcaa agagaagagt ggtgcagaga gaaaaaagag
     6841 cagtgggaat aggagctttg ttccttgggt tcttgggagc agcaggaagc actatgggcg
     6901 cagcgtcaat gacgctgacg gtacaggcca gacaattatt gtctggtata gtgcagcagc
     6961 agaacaattt gctgagggct attgaggcgc aacagcatct gttgcaactc acagtctggg
     7021 gcatcaagca gctccaggca agaatcctgg ctgtggaaag atacctaaag gatcaacagc
     7081 tcct
//
