LOCUS       sequence-245579-        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"
     protein_bind    87..108
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(123..128,129..146,147..153)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    161..177
                     /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     166..188
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     185..201
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     185..201
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     220..240
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        222..240
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        268..494
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             495..675
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    722..847
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1340..1573
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1758..1802
                     /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        2100..2348
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2100..2120
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2271..2290
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2445..2562
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2611..3121
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3040..3058
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3143..3742
                     /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(3143..3162)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3639..3659
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             3870..4103
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4175..4296
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4212..4231)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4266..4285
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4336..4471
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4491..4510)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4492..4510)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4520..4537)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4520..4536)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4529..4551)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4678..5133
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4765..4784)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4975..4996
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5159..5263
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5264..5332,5333..6124)
                     /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(5482..5501)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6295..6883
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6784..6803
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7037..7054
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
       61 aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag
      121 gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt
      181 cacacaggaa acagctatga ccatgattac gccaagcgcg caattaaccc tcactaaagg
      241 gaacaaaagc tggagctgca agcttaatgt agtcttatgc aatactcttg tagtcttgca
      301 acatggtaac gatgagttag caacatgcct tacaaggaga gaaaaagcac cgtgcatgcc
      361 gattggtgga agtaaggtgg tacgatcgtg ccttattagg aaggcaacag acgggtctga
      421 catggattgg acgaaccact gaattgccgc attgcagaga tattgtattt aagtgcctag
      481 ctcgatacat aaacgggtct ctctggttag accagatctg agcctgggag ctctctggct
      541 aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt
      601 gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt
      661 ggaaaatctc tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga aaccagagga
      721 gctctctcga cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg
      781 actggtgagt acgccaaaaa ttttgactag cggaggctag aaggagagag atgggtgcga
      841 gagcgtcagt attaagcggg ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc
      901 agggggaaag aaaaaatata aattaaaaca tatagtatgg gcaagcaggg agctagaacg
      961 attcgcagtt aatcctggcc tgttagaaac atcagaaggc tgtagacaaa tactgggaca
     1021 gctacaacca tcccttcaga caggatcaga agaacttaga tcattatata atacagtagc
     1081 aaccctctat tgtgtgcatc aaaggataga gataaaagac accaaggaag ctttagacaa
     1141 gatagaggaa gagcaaaaca aaagtaagac caccgcacag caagcggccg ctgatcttca
     1201 gacctggagg aggagatatg agggacaatt ggagaagtga attatataaa tataaagtag
     1261 taaaaattga accattagga gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag
     1321 aaaaaagagc agtgggaata ggagctttgt tccttgggtt cttgggagca gcaggaagca
     1381 ctatgggcgc agcgtcaatg acgctgacgg tacaggccag acaattattg tctggtatag
     1441 tgcagcagca gaacaatttg ctgagggcta ttgaggcgca acagcatctg ttgcaactca
     1501 cagtctgggg catcaagcag ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg
     1561 atcaacagct cctggggatt tggggttgct ctggaaaact catttgcacc actgctgtgc
     1621 cttggaatgc tagttggagt aataaatctc tggaacagat ttggaatcac acgacctgga
     1681 tggagtggga cagagaaatt aacaattaca caagcttaat acactcctta attgaagaat
     1741 cgcaaaacca gcaagaaaag aatgaacaag aattattgga attagataaa tgggcaagtt
     1801 tgtggaattg gtttaacata acaaattggc tgtggtatat aaaattattc ataatgatag
     1861 taggaggctt ggtaggttta agaatagttt ttgctgtact ttctatagtg aatagagtta
     1921 ggcagggata ttcaccatta tcgtttcaga cccacctccc aaccccgagg ggacccgaca
     1981 ggcccgaagg aatagaagaa gaaggtggag agagagacag agacagatcc attcgattag
     2041 tgaacggatc tcgacggtat cgatcacgag actagcctcg agcggccgcc cccttcaccg
     2101 agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga
     2161 taattggaat taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa
     2221 agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata
     2281 tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac
     2341 gaaacaccgg cagcactgcc aatggttcca actcgagttg gaaccattgg cagtgctgtt
     2401 tttgaattct cgacctcgag acaaatggca gtattcatcc acaattttaa aagaaaaggg
     2461 gggattgggg ggtacagtgc aggggaaaga atagtagaca taatagcaac agacatacaa
     2521 actaaagaat tacaaaaaca aattacaaaa attcaaaatt ttcgggttta ttacagggac
     2581 agcagagatc cactttggcc gcggctcgag ggggttgggg ttgcgccttt tccaaggcag
     2641 ccctgggttt gcgcagggac gcggctgctc tgggcgtggt tccgggaaac gcagcggcgc
     2701 cgaccctggg tctcgcacat tcttcacgtc cgttcgcagc gtcacccgga tcttcgccgc
     2761 tacccttgtg ggccccccgg cgacgcttcc tgctccgccc ctaagtcggg aaggttcctt
     2821 gcggttcgcg gcgtgccgga cgtgacaaac ggaagccgca cgtctcacta gtaccctcgc
     2881 agacggacag cgccagggag caatggcagc gcgccgaccg cgatgggctg tggccaatag
     2941 cggctgctca gcagggcgcg ccgagagcag cggccgggaa ggggcggtgc gggaggcggg
     3001 gtgtggggcg gtagtgtggg ccctgttcct gcccgcgcgg tgttccgcat tctgcaagcc
     3061 tccggagcgc acgtcggcag tcggctccct cgttgaccga atcaccgacc tctctcccca
     3121 gggggatcca ccggagctta ccatgaccga gtacaagccc acggtgcgcc tcgccacccg
     3181 cgacgacgtc cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac
     3241 gcgccacacc gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt
     3301 cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt
     3361 ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc
     3421 gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct
     3481 ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggcg tctcgcccga
     3541 ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg
     3601 cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct tctacgagcg
     3661 gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat
     3721 gacccgcaag cccggtgcct gacgcccgcc ccacgacccg cagcgcccga ccgaaaggag
     3781 cgcacgaccc catgcatcgg tacctttaag accaatgact tacaaggcag ctgtagatct
     3841 tagccacttt ttaaaagaaa aggggggact ggaagggcta attcactccc aacgaagaca
     3901 agatctgctt tttgcttgta ctgggtctct ctggttagac cagatctgag cctgggagct
     3961 ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca
     4021 agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta
     4081 gtcagtgtgg aaaatctcta gcagtagtag ttcatgtcat cttattattc agtatttata
     4141 acttgcaaag aaatgaatat cagagagtga gaggaacttg tttattgcag cttataatgg
     4201 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     4261 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggctct agctatcccg
     4321 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     4381 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc
     4441 cagaagtagt gaggaggctt ttttggaggc ctagggacgt acccaattcg ccctatagtg
     4501 agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg
     4561 gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg
     4621 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatgggacg
     4681 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     4741 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     4801 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     4861 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     4921 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     4981 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     5041 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     5101 cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg
     5161 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     5221 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     5281 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     5341 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     5401 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     5461 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     5521 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     5581 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     5641 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     5701 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     5761 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     5821 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     5881 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     5941 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     6001 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     6061 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     6121 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     6181 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     6241 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     6301 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     6361 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     6421 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     6481 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     6541 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     6601 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     6661 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     6721 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     6781 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     6841 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     6901 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     6961 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     7021 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa
     7081 accg
//
