LOCUS       sequence-248726-        7146 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  shRNA to knock down RE1-silencing transcription factor (REST).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7146)
  AUTHORS   Drouin-Ouellet J, Lau S, Brattas PL, Rylander Ottosson D, Pircs K, 
            Grassi DA, Collins LM, Vuono R, Andersson Sjoland A, 
            Westergren-Thorsson G, Graff C, Minthon L, Toresson H, Barker RA, 
            Jakobsson J, Parmar M
  TITLE     REST suppression mediates neural conversion of adult human 
            fibroblasts via microRNA-dependent and -independent pathways.
  JOURNAL   EMBO Mol Med. 2017 Aug;9(8):1117-1131. doi: 10.15252/emmm.201607471.
  PUBMED    28646119
REFERENCE   2  (bases 1 to 7146)
  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"; doi: "10.15252/emmm"; 
            journalName: "EMBO Mol Med"; date: "2017-08"; volume: "9"; issue: 
            "8"; pages: "1117-1131"
COMMENT     Sequence Label: pLKO.1REST_sh_seq2_WPRE
FEATURES             Location/Qualifiers
     source          1..7146
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..22
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(37..42,43..60,61..67)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    75..91
                     /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     80..102
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     99..115
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     99..115
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     134..154
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        136..154
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        182..408
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             409..589
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    636..761
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1254..1487
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1672..1716
                     /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        2014..2262
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2014..2034
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2185..2204
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2362..2479
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2528..3038
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2957..2975
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     primer_bind     complement(3268..3288)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3686..3697)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3846..4079
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4151..4272
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4188..4207)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4242..4261
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4312..4447
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4467..4486)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4468..4486)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4496..4513)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4496..4512)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4505..4527)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4654..5109
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4741..4760)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4951..4972
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5135..5239
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5240..5308,5309..6100)
                     /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(5458..5477)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6271..6859
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6760..6779
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7013..7030
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
       61 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
      121 ttacgccaag cgcgcaatta accctcacta aagggaacaa aagctggagc tgcaagctta
      181 atgtagtctt atgcaatact cttgtagtct tgcaacatgg taacgatgag ttagcaacat
      241 gccttacaag gagagaaaaa gcaccgtgca tgccgattgg tggaagtaag gtggtacgat
      301 cgtgccttat taggaaggca acagacgggt ctgacatgga ttggacgaac cactgaattg
      361 ccgcattgca gagatattgt atttaagtgc ctagctcgat acataaacgg gtctctctgg
      421 ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct
      481 caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt
      541 aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag tggcgcccga
      601 acagggactt gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg actcggcttg
      661 ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt gagtacgcca aaaattttga
      721 ctagcggagg ctagaaggag agagatgggt gcgagagcgt cagtattaag cgggggagaa
      781 ttagatcgcg atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa tataaattaa
      841 aacatatagt atgggcaagc agggagctag aacgattcgc agttaatcct ggcctgttag
      901 aaacatcaga aggctgtaga caaatactgg gacagctaca accatccctt cagacaggat
      961 cagaagaact tagatcatta tataatacag tagcaaccct ctattgtgtg catcaaagga
     1021 tagagataaa agacaccaag gaagctttag acaagataga ggaagagcaa aacaaaagta
     1081 agaccaccgc acagcaagcg gccgctgatc ttcagacctg gaggaggaga tatgagggac
     1141 aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt aggagtagca
     1201 cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg aataggagct
     1261 ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc aatgacgctg
     1321 acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa tttgctgagg
     1381 gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa gcagctccag
     1441 gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg gatttggggt
     1501 tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg gagtaataaa
     1561 tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga aattaacaat
     1621 tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa
     1681 caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa cataacaaat
     1741 tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg tttaagaata
     1801 gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc attatcgttt
     1861 cagacccacc tcccaacccc gaggggaccc gacaggcccg aaggaataga agaagaaggt
     1921 ggagagagag acagagacag atccattcga ttagtgaacg gatctcgacg gtatcgatca
     1981 cgagactagc ctcgagcggc cgcccccttc accgagggcc tatttcccat gattccttca
     2041 tatttgcata tacgatacaa ggctgttaga gagataattg gaattaattt gactgtaaac
     2101 acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca
     2161 gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc
     2221 gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccggtagtac acattaacca
     2281 aatggcacac gtgagccatt tggttaatgt gtactttttt gaattctcga cctcgagaca
     2341 aatggcagta ttcatccaca attttaaaag aaaagggggg attggggggt acagtgcagg
     2401 ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac aaaaacaaat
     2461 tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccac tttggccgcg
     2521 gctcgagggg gttggggttg cgccttttcc aaggcagccc tgggtttgcg cagggacgcg
     2581 gctgctctgg gcgtggttcc gggaaacgca gcggcgccga ccctgggtct cgcacattct
     2641 tcacgtccgt tcgcagcgtc acccggatct tcgccgctac ccttgtgggc cccccggcga
     2701 cgcttcctgc tccgccccta agtcgggaag gttccttgcg gttcgcggcg tgccggacgt
     2761 gacaaacgga agccgcacgt ctcactagta ccctcgcaga cggacagcgc cagggagcaa
     2821 tggcagcgcg ccgaccgcga tgggctgtgg ccaatagcgg ctgctcagca gggcgcgccg
     2881 agagcagcgg ccgggaaggg gcggtgcggg aggcggggtg tggggcggta gtgtgggccc
     2941 tgttcctgcc cgcgcggtgt tccgcattct gcaagcctcc ggagcgcacg tcggcagtcg
     3001 gctccctcgt tgaccgaatc accgacctct ctccccaggg ggatccaatc aatgaacaaa
     3061 attttgatgc ttacgtgact actttgacgg aaatgtatac aaatcaagat cgttatcaga
     3121 gtccagaaaa taaagcccta ctggaaaata taaagcaggc tgtgagagga attcaggtct
     3181 gacaattcga tatcaagctt atcgataagt cgacaatcaa cctctggatt acaaaatttg
     3241 tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc
     3301 tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta
     3361 taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt
     3421 ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca
     3481 gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc
     3541 ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt
     3601 gtcggggaag ctgacgtcct ttccatggct gctcgcctgt gttgccacct ggattctgcg
     3661 cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg
     3721 cctgctgccg gctctagagc ctcttccgcg tcttcgcctt cccgggtcga gctcggtacc
     3781 tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg
     3841 gggactggaa gggctaattc actcccaacg aagacaagat ctgctttttg cttgtactgg
     3901 gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact
     3961 gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg
     4021 tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag
     4081 tagtagttca tgtcatctta ttattcagta tttataactt gcaaagaaat gaatatcaga
     4141 gagtgagagg aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     4201 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     4261 caatgtatct tatcatgtct ggctctagct atcccgcccc taactccgcc catcccgccc
     4321 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     4381 gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt
     4441 ggaggcctag ggacgtaccc aattcgccct atagtgagtc gtattacgcg cgctcactgg
     4501 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg
     4561 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt
     4621 cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg
     4681 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     4741 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     4801 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     4861 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     4921 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     4981 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     5041 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc
     5101 ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     5161 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     5221 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     5281 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     5341 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     5401 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     5461 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     5521 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     5581 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     5641 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     5701 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     5761 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     5821 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     5881 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     5941 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     6001 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     6061 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     6121 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     6181 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     6241 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     6301 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     6361 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     6421 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     6481 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     6541 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     6601 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     6661 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     6721 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     6781 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     6841 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     6901 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     6961 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     7021 agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc
     7081 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa
     7141 cgcaat
//
