LOCUS       sequence-245968-        7084 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  constitutive expression of a short-hairpin RNA targeting human WRN.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7084)
  AUTHORS   Chan EM, Shibue T, McFarland JM, Gaeta B, Ghandi M, Dumont N, 
            Gonzalez A, McPartlan JS, Li T, Zhang Y, Bin Liu J, Lazaro JB, Gu P,
            Piett CG, Apffel A, Ali SO, Deasy R, Keskula P, Ng RWS, Roberts EA, 
            Reznichenko E, Leung L, Alimova M, Schenone M, Islam M, Maruvka YE, 
            Liu Y, Roper J, Raghavan S, Giannakis M, Tseng YY, Nagel ZD, 
            D'Andrea A, Root DE, Boehm JS, Getz G, Chang S, Golub TR, Tsherniak 
            A, Vazquez F, Bass AJ
  TITLE     WRN helicase is a synthetic lethal target in microsatellite unstable
            cancers.
  JOURNAL   Nature. 2019 Apr 10. pii: 10.1038/s41586-019-1102-x. doi: 
            10.1038/s41586-019-1102-x.
  PUBMED    30971823
REFERENCE   2  (bases 1 to 7084)
  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: "Nature. 
            2019 Apr 10. pii: 10.1038/s41586-019-1102-x. doi: 
            10.1038/s41586-019-1102-x."
COMMENT     Sequence Label: pLKO.1-puro-shWRN2
FEATURES             Location/Qualifiers
     source          1..7084
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    63..84
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(99..104,105..122,123..129)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    137..153
                     /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     142..164
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     161..177
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     161..177
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     196..216
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        198..216
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        244..470
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             471..651
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    698..823
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1316..1549
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1734..1778
                     /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        2076..2324
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2076..2096
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2247..2266
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2421..2538
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2587..3097
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3016..3034
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3119..3718
                     /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(3119..3138)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3615..3635
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     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 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
       61 attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct
      121 cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat
      181 gattacgcca agcgcgcaat taaccctcac taaagggaac aaaagctgga gctgcaagct
      241 taatgtagtc ttatgcaata ctcttgtagt cttgcaacat ggtaacgatg agttagcaac
      301 atgccttaca aggagagaaa aagcaccgtg catgccgatt ggtggaagta aggtggtacg
      361 atcgtgcctt attaggaagg caacagacgg gtctgacatg gattggacga accactgaat
      421 tgccgcattg cagagatatt gtatttaagt gcctagctcg atacataaac gggtctctct
      481 ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc
      541 ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg
      601 gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc agtggcgccc
      661 gaacagggac ttgaaagcga aagggaaacc agaggagctc tctcgacgca ggactcggct
      721 tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt
      781 gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta agcgggggag
      841 aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa aatataaatt
      901 aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc ctggcctgtt
      961 agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc ttcagacagg
     1021 atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg tgcatcaaag
     1081 gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaaaag
     1141 taagaccacc gcacagcaag cggccgctga tcttcagacc tggaggagga gatatgaggg
     1201 acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag
     1261 cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag
     1321 ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg tcaatgacgc
     1381 tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga
     1441 gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc
     1501 aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg
     1561 gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata
     1621 aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga gaaattaaca
     1681 attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa gaaaagaatg
     1741 aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt aacataacaa
     1801 attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta ggtttaagaa
     1861 tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca ccattatcgt
     1921 ttcagaccca cctcccaacc ccgaggggac ccgacaggcc cgaaggaata gaagaagaag
     1981 gtggagagag agacagagac agatccattc gattagtgaa cggatctcga cggtatcgat
     2041 cacgagacta gcctcgagcg gccgccccct tcaccgaggg cctatttccc atgattcctt
     2101 catatttgca tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa
     2161 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg
     2221 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt
     2281 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgggcct taacagtctg
     2341 gttaaacctc gaggtttaac cagactgtta aggctttttg aattctcgac ctcgagacaa
     2401 atggcagtat tcatccacaa ttttaaaaga aaagggggga ttggggggta cagtgcaggg
     2461 gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca aaaacaaatt
     2521 acaaaaattc aaaattttcg ggtttattac agggacagca gagatccact ttggccgcgg
     2581 ctcgaggggg ttggggttgc gccttttcca aggcagccct gggtttgcgc agggacgcgg
     2641 ctgctctggg cgtggttccg ggaaacgcag cggcgccgac cctgggtctc gcacattctt
     2701 cacgtccgtt cgcagcgtca cccggatctt cgccgctacc cttgtgggcc ccccggcgac
     2761 gcttcctgct ccgcccctaa gtcgggaagg ttccttgcgg ttcgcggcgt gccggacgtg
     2821 acaaacggaa gccgcacgtc tcactagtac cctcgcagac ggacagcgcc agggagcaat
     2881 ggcagcgcgc cgaccgcgat gggctgtggc caatagcggc tgctcagcag ggcgcgccga
     2941 gagcagcggc cgggaagggg cggtgcggga ggcggggtgt ggggcggtag tgtgggccct
     3001 gttcctgccc gcgcggtgtt ccgcattctg caagcctccg gagcgcacgt cggcagtcgg
     3061 ctccctcgtt gaccgaatca ccgacctctc tccccagggg gatccaccgg agcttaccat
     3121 gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg
     3181 caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg
     3241 ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat
     3301 cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag
     3361 cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc
     3421 ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc
     3481 cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag
     3541 cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga
     3601 gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga
     3661 cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgacg
     3721 cccgccccac gacccgcagc gcccgaccga aaggagcgca cgaccccatg catcggtacc
     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 gatt
//
