LOCUS       sequence-323886-        6902 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  shRNA targeting the coding sequences of VPS35.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6902)
  AUTHORS   Sun X, Mahajan D, Chen B, Song Z, Lu L
  TITLE     A quantitative study of the Golgi retention of glycosyltransferases.
  JOURNAL   J Cell Sci. 2021 Oct 15;134(20). pii: 272560. doi: 
            10.1242/jcs.258564. Epub 2021 Oct 21.
  PUBMED    34533190
REFERENCE   2  (bases 1 to 6902)
  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: "J Cell 
            Sci."; date: "2021-10-15"; volume: "134(20). pii: 272560. doi"; 
            pages: " 10.1242/jcs.258564. Epub 2021 Oct 21"
COMMENT     Sequence Label: VPS35 shRNA1
FEATURES             Location/Qualifiers
     source          1..6902
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        42..552
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     471..489
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             574..1173
                     /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(574..593)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1070..1090
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             1301..1534
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    1606..1727
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1643..1662)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1697..1716
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      1767..1902
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(1922..1941)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1923..1941)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1951..1968)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1951..1967)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1960..1982)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2109..2564
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2196..2215)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2406..2427
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2590..2694
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2695..2763,2764..3555)
                     /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(2913..2932)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3726..4314
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4215..4234
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        join(4456..4461,4462..4479,4480..4486)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4494..4510
                     /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     4499..4521
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4518..4534
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4518..4534
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     4553..4573
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        4555..4573
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        4601..4827
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             4828..5008
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5055..5180
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    5673..5906
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6091..6135
                     /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        6433..6681
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     6433..6453
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     6604..6623
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    6778..6895
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
ORIGIN
        1 attacaggga cagcagagat ccactttggc cgcggctcga gggggttggg gttgcgcctt
       61 ttccaaggca gccctgggtt tgcgcaggga cgcggctgct ctgggcgtgg ttccgggaaa
      121 cgcagcggcg ccgaccctgg gtctcgcaca ttcttcacgt ccgttcgcag cgtcacccgg
      181 atcttcgccg ctacccttgt gggccccccg gcgacgcttc ctgctccgcc cctaagtcgg
      241 gaaggttcct tgcggttcgc ggcgtgccgg acgtgacaaa cggaagccgc acgtctcact
      301 agtaccctcg cagacggaca gcgccaggga gcaatggcag cgcgccgacc gcgatgggct
      361 gtggccaata gcggctgctc agcagggcgc gccgagagca gcggccggga aggggcggtg
      421 cgggaggcgg ggtgtggggc ggtagtgtgg gccctgttcc tgcccgcgcg gtgttccgca
      481 ttctgcaagc ctccggagcg cacgtcggca gtcggctccc tcgttgaccg aatcaccgac
      541 ctctctcccc agggggatcc accggagctt accatgaccg agtacaagcc cacggtgcgc
      601 ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac
      661 taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg
      721 caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac
      781 ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc
      841 gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg
      901 gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc
      961 gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag
     1021 gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc
     1081 ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc
     1141 acctggtgca tgacccgcaa gcccggtgcc tgacgcccgc cccacgaccc gcagcgcccg
     1201 accgaaagga gcgcacgacc ccatgcatcg gtacctttaa gaccaatgac ttacaaggca
     1261 gctgtagatc ttagccactt tttaaaagaa aaggggggac tggaagggct aattcactcc
     1321 caacgaagac aagatctgct ttttgcttgt actgggtctc tctggttaga ccagatctga
     1381 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
     1441 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
     1501 agaccctttt agtcagtgtg gaaaatctct agcagtagta gttcatgtca tcttattatt
     1561 cagtatttat aacttgcaaa gaaatgaata tcagagagtg agaggaactt gtttattgca
     1621 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     1681 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggctc
     1741 tagctatccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     1801 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct
     1861 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctagggacg tacccaattc
     1921 gccctatagt gagtcgtatt acgcgcgctc actggccgtc gttttacaac gtcgtgactg
     1981 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg
     2041 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg
     2101 cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     2161 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     2221 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     2281 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     2341 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     2401 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     2461 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     2521 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg
     2581 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     2641 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     2701 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     2761 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     2821 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     2881 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     2941 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     3001 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     3061 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     3121 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     3181 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     3241 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     3301 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     3361 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     3421 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     3481 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     3541 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     3601 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     3661 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     3721 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     3781 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     3841 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     3901 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     3961 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     4021 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     4081 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     4141 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     4201 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     4261 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     4321 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     4381 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctaactca
     4441 ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag
     4501 cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa
     4561 ccctcactaa agggaacaaa agctggagct gcaagcttaa tgtagtctta tgcaatactc
     4621 ttgtagtctt gcaacatggt aacgatgagt tagcaacatg ccttacaagg agagaaaaag
     4681 caccgtgcat gccgattggt ggaagtaagg tggtacgatc gtgccttatt aggaaggcaa
     4741 cagacgggtc tgacatggat tggacgaacc actgaattgc cgcattgcag agatattgta
     4801 tttaagtgcc tagctcgata cataaacggg tctctctggt tagaccagat ctgagcctgg
     4861 gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg
     4921 cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc
     4981 ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa cagggacttg aaagcgaaag
     5041 ggaaaccaga ggagctctct cgacgcagga ctcggcttgc tgaagcgcgc acggcaagag
     5101 gcgaggggcg gcgactggtg agtacgccaa aaattttgac tagcggaggc tagaaggaga
     5161 gagatgggtg cgagagcgtc agtattaagc gggggagaat tagatcgcga tgggaaaaaa
     5221 ttcggttaag gccaggggga aagaaaaaat ataaattaaa acatatagta tgggcaagca
     5281 gggagctaga acgattcgca gttaatcctg gcctgttaga aacatcagaa ggctgtagac
     5341 aaatactggg acagctacaa ccatcccttc agacaggatc agaagaactt agatcattat
     5401 ataatacagt agcaaccctc tattgtgtgc atcaaaggat agagataaaa gacaccaagg
     5461 aagctttaga caagatagag gaagagcaaa acaaaagtaa gaccaccgca cagcaagcgg
     5521 ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag tgaattatat
     5581 aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc aaagagaaga
     5641 gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg gttcttggga
     5701 gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc cagacaatta
     5761 ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc gcaacagcat
     5821 ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct ggctgtggaa
     5881 agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa actcatttgc
     5941 accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca gatttggaat
     6001 cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt aatacactcc
     6061 ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt ggaattagat
     6121 aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta tataaaatta
     6181 ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt actttctata
     6241 gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct cccaaccccg
     6301 aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga cagagacaga
     6361 tccattcgat tagtgaacgg atctcgacgg tatcgatcac gagactagcc tcgagcggcc
     6421 gcccccttca ccgagggcct atttcccatg attccttcat atttgcatat acgatacaag
     6481 gctgttagag agataattgg aattaatttg actgtaaaca caaagatatt agtacaaaat
     6541 acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa
     6601 atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc
     6661 ttgtggaaag gacgaaacac cggaatcagc ggattcgctt cacactcgag tgtgaagcga
     6721 atccgctgat ttttttgaat tctcgacctc gagacaaatg gcagtattca tccacaattt
     6781 taaaagaaaa ggggggattg gggggtacag tgcaggggaa agaatagtag acataatagc
     6841 aacagacata caaactaaag aattacaaaa acaaattaca aaaattcaaa attttcgggt
     6901 tt
//
