LOCUS       sequence-245958-        7084 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  constitutive expression of a short-hairpin RNA targeting RFP.
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-shRFP
FEATURES             Location/Qualifiers
     source          1..7084
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    47..172
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    665..898
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1083..1127
                     /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        1425..1673
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1425..1445
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1596..1615
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    1770..1887
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1936..2446
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2365..2383
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             2468..3067
                     /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(2468..2487)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2964..2984
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             3195..3428
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    3500..3621
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3537..3556)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3591..3610
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      3661..3796
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3816..3835)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3817..3835)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3845..3862)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3845..3861)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3854..3876)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4003..4458
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4090..4109)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4300..4321
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4484..4588
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4589..4657,4658..5449)
                     /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(4807..4826)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5620..6208
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6109..6128
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6362..6379
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6496..6517
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6532..6537,6538..6555,6556..6562)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6570..6586
                     /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     6575..6597
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6594..6610
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6594..6610
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6629..6649
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6631..6649
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        6677..6903
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             6904..7084
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
ORIGIN
        1 gtggcgcccg aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag
       61 gactcggctt gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc
      121 aaaaattttg actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa
      181 gcgggggaga attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa
      241 atataaatta aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc
      301 tggcctgtta gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct
      361 tcagacagga tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt
      421 gcatcaaagg atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca
      481 aaacaaaagt aagaccaccg cacagcaagc ggccgctgat cttcagacct ggaggaggag
      541 atatgaggga caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat
      601 taggagtagc acccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg
      661 gaataggagc tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt
      721 caatgacgct gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca
      781 atttgctgag ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca
      841 agcagctcca ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg
      901 ggatttgggg ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt
      961 ggagtaataa atctctggaa cagatttgga atcacacgac ctggatggag tgggacagag
     1021 aaattaacaa ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag
     1081 aaaagaatga acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta
     1141 acataacaaa ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag
     1201 gtttaagaat agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac
     1261 cattatcgtt tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag
     1321 aagaagaagg tggagagaga gacagagaca gatccattcg attagtgaac ggatctcgac
     1381 ggtatcgatc acgagactag cctcgagcgg ccgccccctt caccgagggc ctatttccca
     1441 tgattccttc atatttgcat atacgataca aggctgttag agagataatt ggaattaatt
     1501 tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg
     1561 ggtagtttgc agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt
     1621 gaaagtattt cgatttcttg gctttatata tcttgtggaa aggacgaaac accggctcag
     1681 ttccagtacg gctccactcg agtggagccg tactggaact gagtttttga attctcgacc
     1741 tcgagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat tggggggtac
     1801 agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa agaattacaa
     1861 aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag agatccactt
     1921 tggccgcggc tcgagggggt tggggttgcg ccttttccaa ggcagccctg ggtttgcgca
     1981 gggacgcggc tgctctgggc gtggttccgg gaaacgcagc ggcgccgacc ctgggtctcg
     2041 cacattcttc acgtccgttc gcagcgtcac ccggatcttc gccgctaccc ttgtgggccc
     2101 cccggcgacg cttcctgctc cgcccctaag tcgggaaggt tccttgcggt tcgcggcgtg
     2161 ccggacgtga caaacggaag ccgcacgtct cactagtacc ctcgcagacg gacagcgcca
     2221 gggagcaatg gcagcgcgcc gaccgcgatg ggctgtggcc aatagcggct gctcagcagg
     2281 gcgcgccgag agcagcggcc gggaaggggc ggtgcgggag gcggggtgtg gggcggtagt
     2341 gtgggccctg ttcctgcccg cgcggtgttc cgcattctgc aagcctccgg agcgcacgtc
     2401 ggcagtcggc tccctcgttg accgaatcac cgacctctct ccccaggggg atccaccgga
     2461 gcttaccatg accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag
     2521 ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga
     2581 tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg
     2641 gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac
     2701 gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt
     2761 gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc
     2821 caaggagccc gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc agggcaaggg
     2881 tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc
     2941 cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt
     3001 caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg
     3061 tgcctgacgc ccgccccacg acccgcagcg cccgaccgaa aggagcgcac gaccccatgc
     3121 atcggtacct ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa
     3181 agaaaagggg ggactggaag ggctaattca ctcccaacga agacaagatc tgctttttgc
     3241 ttgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg
     3301 gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg
     3361 tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat
     3421 ctctagcagt agtagttcat gtcatcttat tattcagtat ttataacttg caaagaaatg
     3481 aatatcagag agtgagagga acttgtttat tgcagcttat aatggttaca aataaagcaa
     3541 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     3601 caaactcatc aatgtatctt atcatgtctg gctctagcta tcccgcccct aactccgccc
     3661 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     3721 tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga
     3781 ggcttttttg gaggcctagg gacgtaccca attcgcccta tagtgagtcg tattacgcgc
     3841 gctcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta
     3901 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg
     3961 atcgcccttc ccaacagttg cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg
     4021 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     4081 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     4141 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     4201 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     4261 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     4321 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     4381 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     4441 tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     4501 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     4561 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     4621 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     4681 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     4741 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     4801 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     4861 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     4921 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     4981 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     5041 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     5101 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     5161 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     5221 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     5281 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     5341 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     5401 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     5461 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     5521 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     5581 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     5641 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     5701 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     5761 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     5821 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     5881 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     5941 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     6001 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     6061 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     6121 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     6181 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     6241 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     6301 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     6361 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc
     6421 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     6481 tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt
     6541 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
     6601 cagctatgac catgattacg ccaagcgcgc aattaaccct cactaaaggg aacaaaagct
     6661 ggagctgcaa gcttaatgta gtcttatgca atactcttgt agtcttgcaa catggtaacg
     6721 atgagttagc aacatgcctt acaaggagag aaaaagcacc gtgcatgccg attggtggaa
     6781 gtaaggtggt acgatcgtgc cttattagga aggcaacaga cgggtctgac atggattgga
     6841 cgaaccactg aattgccgca ttgcagagat attgtattta agtgcctagc tcgatacata
     6901 aacgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac
     6961 ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg
     7021 ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct
     7081 agca
//
