LOCUS       sequence-323887-        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 shRNA2
FEATURES             Location/Qualifiers
     source          1..6902
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     425..443
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             528..1127
                     /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(528..547)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1024..1044
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             1255..1488
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    1560..1681
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1597..1616)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1651..1670
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      1721..1856
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(1876..1895)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1877..1895)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1905..1922)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1905..1921)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1914..1936)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2063..2518
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2150..2169)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2360..2381
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2544..2648
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2649..2717,2718..3509)
                     /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(2867..2886)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3680..4268
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4169..4188
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        join(4410..4415,4416..4433,4434..4440)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4448..4464
                     /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     4453..4475
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4472..4488
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4472..4488
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     4507..4527
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        4509..4527
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        4555..4781
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             4782..4962
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5009..5134
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    5627..5860
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6045..6089
                     /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        6387..6635
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     6387..6407
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     6558..6577
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    6732..6849
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        join(6898..6902,1..506)
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
ORIGIN
        1 tggggttgcg ccttttccaa ggcagccctg ggtttgcgca gggacgcggc tgctctgggc
       61 gtggttccgg gaaacgcagc ggcgccgacc ctgggtctcg cacattcttc acgtccgttc
      121 gcagcgtcac ccggatcttc gccgctaccc ttgtgggccc cccggcgacg cttcctgctc
      181 cgcccctaag tcgggaaggt tccttgcggt tcgcggcgtg ccggacgtga caaacggaag
      241 ccgcacgtct cactagtacc ctcgcagacg gacagcgcca gggagcaatg gcagcgcgcc
      301 gaccgcgatg ggctgtggcc aatagcggct gctcagcagg gcgcgccgag agcagcggcc
      361 gggaaggggc ggtgcgggag gcggggtgtg gggcggtagt gtgggccctg ttcctgcccg
      421 cgcggtgttc cgcattctgc aagcctccgg agcgcacgtc ggcagtcggc tccctcgttg
      481 accgaatcac cgacctctct ccccaggggg atccaccgga gcttaccatg accgagtaca
      541 agcccacggt gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc accctcgccg
      601 ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga tccggaccgc cacatcgagc
      661 gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt
      721 gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc gtcgaagcgg
      781 gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc cggctggccg
      841 cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc
      901 tggccaccgt cggcgtctcg cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc
      961 tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag acctccgcgc
     1021 cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc
     1081 ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcctgacgc ccgccccacg
     1141 acccgcagcg cccgaccgaa aggagcgcac gaccccatgc atcggtacct ttaagaccaa
     1201 tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag
     1261 ggctaattca ctcccaacga agacaagatc tgctttttgc ttgtactggg tctctctggt
     1321 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
     1381 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
     1441 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt agtagttcat
     1501 gtcatcttat tattcagtat ttataacttg caaagaaatg aatatcagag agtgagagga
     1561 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     1621 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     1681 atcatgtctg gctctagcta tcccgcccct aactccgccc atcccgcccc taactccgcc
     1741 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     1801 ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     1861 gacgtaccca attcgcccta tagtgagtcg tattacgcgc gctcactggc cgtcgtttta
     1921 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc
     1981 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg
     2041 cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     2101 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     2161 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     2221 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     2281 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     2341 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     2401 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     2461 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag
     2521 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     2581 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     2641 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     2701 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     2761 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     2821 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     2881 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     2941 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     3001 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     3061 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     3121 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     3181 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     3241 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     3301 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     3361 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     3421 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     3481 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     3541 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     3601 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     3661 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     3721 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     3781 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     3841 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     3901 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     3961 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     4021 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     4081 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     4141 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     4201 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     4261 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     4321 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     4381 agtgagctaa ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg
     4441 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc
     4501 aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg agctgcaagc ttaatgtagt
     4561 cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa catgccttac
     4621 aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac gatcgtgcct
     4681 tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa ttgccgcatt
     4741 gcagagatat tgtatttaag tgcctagctc gatacataaa cgggtctctc tggttagacc
     4801 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa
     4861 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga
     4921 gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc cgaacaggga
     4981 cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc ttgctgaagc
     5041 gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg
     5101 aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga gaattagatc
     5161 gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat taaaacatat
     5221 agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt tagaaacatc
     5281 agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag gatcagaaga
     5341 acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa ggatagagat
     5401 aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa gtaagaccac
     5461 cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg gacaattgga
     5521 gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta gcacccacca
     5581 aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga gctttgttcc
     5641 ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg ctgacggtac
     5701 aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg agggctattg
     5761 aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc caggcaagaa
     5821 tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg ggttgctctg
     5881 gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat aaatctctgg
     5941 aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac aattacacaa
     6001 gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat gaacaagaat
     6061 tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca aattggctgt
     6121 ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga atagtttttg
     6181 ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg tttcagaccc
     6241 acctcccaac cccgagggga cccgacaggc ccgaaggaat agaagaagaa ggtggagaga
     6301 gagacagaga cagatccatt cgattagtga acggatctcg acggtatcga tcacgagact
     6361 agcctcgagc ggccgccccc ttcaccgagg gcctatttcc catgattcct tcatatttgc
     6421 atatacgata caaggctgtt agagagataa ttggaattaa tttgactgta aacacaaaga
     6481 tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa
     6541 aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct
     6601 tggctttata tatcttgtgg aaaggacgaa acaccggaat catgagacag tcgcatatct
     6661 cgagatatgc gactgtctca tgattttttt gaattctcga cctcgagaca aatggcagta
     6721 ttcatccaca attttaaaag aaaagggggg attggggggt acagtgcagg ggaaagaata
     6781 gtagacataa tagcaacaga catacaaact aaagaattac aaaaacaaat tacaaaaatt
     6841 caaaattttc gggtttatta cagggacagc agagatccac tttggccgcg gctcgagggg
     6901 gt
//
