LOCUS       sequence-240814-        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-shWRN1
FEATURES             Location/Qualifiers
     source          1..7084
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     441..461
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             672..905
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    977..1098
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1014..1033)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1068..1087
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      1138..1273
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(1293..1312)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1294..1312)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1322..1339)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1322..1338)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1331..1353)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1480..1935
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1567..1586)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1777..1798
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1961..2065
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2066..2134,2135..2926)
                     /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(2284..2303)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3097..3685
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3586..3605
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3839..3856
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3973..3994
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4009..4014,4015..4032,4033..4039)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4047..4063
                     /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     4052..4074
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4071..4087
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4071..4087
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     4106..4126
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        4108..4126
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        4154..4380
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             4381..4561
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    4608..4733
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    5226..5459
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             5644..5688
                     /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        5986..6234
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     5986..6006
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     6157..6176
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    6331..6448
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        6497..7007
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     6926..6944
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             join(7029..7084,1..544)
                     /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(7029..7048)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
ORIGIN
        1 cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc
       61 ggaccgccac atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct
      121 cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc
      181 ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag
      241 cggttcccgg ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa
      301 ggagcccgcg tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct
      361 gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt
      421 cctggagacc tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac
      481 cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc
      541 ctgacgcccg ccccacgacc cgcagcgccc gaccgaaagg agcgcacgac cccatgcatc
      601 ggtaccttta agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga
      661 aaagggggga ctggaagggc taattcactc ccaacgaaga caagatctgc tttttgcttg
      721 tactgggtct ctctggttag accagatctg agcctgggag ctctctggct aactagggaa
      781 cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct
      841 gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc
      901 tagcagtagt agttcatgtc atcttattat tcagtattta taacttgcaa agaaatgaat
      961 atcagagagt gagaggaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     1021 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     1081 actcatcaat gtatcttatc atgtctggct ctagctatcc cgcccctaac tccgcccatc
     1141 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     1201 atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc
     1261 ttttttggag gcctagggac gtacccaatt cgccctatag tgagtcgtat tacgcgcgct
     1321 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc
     1381 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc
     1441 gcccttccca acagttgcgc agcctgaatg gcgaatggga cgcgccctgt agcggcgcat
     1501 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     1561 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     1621 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     1681 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     1741 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     1801 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     1861 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     1921 taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     1981 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     2041 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     2101 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     2161 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     2221 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     2281 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     2341 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     2401 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     2461 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     2521 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     2581 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     2641 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     2701 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     2761 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     2821 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     2881 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     2941 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     3001 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     3061 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     3121 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     3181 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     3241 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     3301 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     3361 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     3421 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     3481 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     3541 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     3601 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     3661 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     3721 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     3781 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     3841 cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg
     3901 ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga
     3961 gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat
     4021 gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag
     4081 ctatgaccat gattacgcca agcgcgcaat taaccctcac taaagggaac aaaagctgga
     4141 gctgcaagct taatgtagtc ttatgcaata ctcttgtagt cttgcaacat ggtaacgatg
     4201 agttagcaac atgccttaca aggagagaaa aagcaccgtg catgccgatt ggtggaagta
     4261 aggtggtacg atcgtgcctt attaggaagg caacagacgg gtctgacatg gattggacga
     4321 accactgaat tgccgcattg cagagatatt gtatttaagt gcctagctcg atacataaac
     4381 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca
     4441 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg
     4501 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc
     4561 agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagctc tctcgacgca
     4621 ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc
     4681 caaaaatttt gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta
     4741 agcgggggag aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa
     4801 aatataaatt aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc
     4861 ctggcctgtt agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc
     4921 ttcagacagg atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg
     4981 tgcatcaaag gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc
     5041 aaaacaaaag taagaccacc gcacagcaag cggccgctga tcttcagacc tggaggagga
     5101 gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca
     5161 ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg
     5221 ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg
     5281 tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac
     5341 aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc
     5401 aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg
     5461 gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt
     5521 tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga
     5581 gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa
     5641 gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt
     5701 aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta
     5761 ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca
     5821 ccattatcgt ttcagaccca cctcccaacc ccgaggggac ccgacaggcc cgaaggaata
     5881 gaagaagaag gtggagagag agacagagac agatccattc gattagtgaa cggatctcga
     5941 cggtatcgat cacgagacta gcctcgagcg gccgccccct tcaccgaggg cctatttccc
     6001 atgattcctt catatttgca tatacgatac aaggctgtta gagagataat tggaattaat
     6061 ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt
     6121 gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact
     6181 tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccggcagc
     6241 actgccaatg gttccaactc gagttggaac cattggcagt gctgtttttg aattctcgac
     6301 ctcgagacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga ttggggggta
     6361 cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca
     6421 aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca gagatccact
     6481 ttggccgcgg ctcgaggggg cccgggttgc gccttttcca aggcagccct gggtttgcgc
     6541 agggacgcgg ctgctctggg cgtggttccg ggaaacgcag cggcgccgac cctgggtctc
     6601 gcacattctt cacgtccgtt cgcagcgtca cccggatctt cgccgctacc cttgtgggcc
     6661 ccccggcgac gcttcctgct ccgcccctaa gtcgggaagg ttccttgcgg ttcgcggcgt
     6721 gccggacgtg acaaacggaa gccgcacgtc tcactagtac cctcgcagac ggacagcgcc
     6781 agggagcaat ggcagcgcgc cgaccgcgat gggctgtggc caatagcggc tgctcagcag
     6841 ggcgcgccga gagcagcggc cgggaagggg cggtgcggga ggcggggtgt ggggcggtag
     6901 tgtgggccct gttcctgccc gcgcggtgtt ccgcattctg caagcctccg gagcgcacgt
     6961 cggcagtcgg ctccctcgtt gaccgaatca ccgacctctc tccccagggg gatccaccgg
     7021 agcttaccat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca
     7081 gggc
//
