LOCUS       sequence-422485-        7361 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral plasmid for the measurement of RfxCas13d (CasRx) nuclease
            activity. As a marker, the plasmid also encodes the tRFP657 
            fluorescence protein..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7361)
  AUTHORS   Montero JJ, Trozzo R, Sugden M, Ollinger R, Belka A, Zhigalova E, 
            Waetzig P, Engleitner T, Schmidt-Supprian M, Saur D, Rad R
  TITLE     Genome-scale pan-cancer interrogation of lncRNA dependencies using 
            CasRx.
  JOURNAL   Nat Methods. 2024 Feb 26. doi: 10.1038/s41592-024-02190-0.
  PUBMED    38409225
REFERENCE   2  (bases 1 to 7361)
  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: "Nat 
            Methods. 2024 Feb 26. doi: 10.1038/s41592-024-02190-0."
COMMENT     Sequence Label: pLKO5.U6.DR130(CasRX)_Non-targeting
            sgRNA.EFS....
FEATURES             Location/Qualifiers
     source          1..7361
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        5..231
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             232..412
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    459..584
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1077..1310
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1495..1539
                     /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        1717..1965
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1717..1737
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1888..1907
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1968..1996
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d 
                     gRNA (Konermann et al., 2018)"
     misc_feature    2072..2189
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2258..2469
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2495..3196
                     /codon_start=1
                     /product="far-red monomeric fluorescent protein with an 
                     emission peak at 657 nm (Morozova et al., 2010)"
                     /label=TagRFP657
                     /note="derived from mKate"
                     /translation="MSELITENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTQRIKV
                     VEGGPLPFAFDILATSFMYGSHTFINHTQGIPDFWKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEAHTEMLYPADGGLEGRTALALK
                     LVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDYRLERIKEADKETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     misc_feature    3218..3806
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3271..3291)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3689..3700)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3878..4111
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4189..4310
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4226..4245)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4280..4299
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4350..4485
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4505..4524)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4506..4524)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4534..4551)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4534..4550)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4543..4565)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4692..5147
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4779..4798)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4989..5010
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5173..5277
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5278..5346,5347..6138)
                     /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(5496..5515)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6309..6897
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6798..6817
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7051..7068
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7185..7206
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7221..7226,7227..7244,7245..7251)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7259..7275
                     /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     7264..7286
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7283..7299
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7283..7299
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7318..7338
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7320..7338
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 ttaatgtagt cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa
       61 catgccttac aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac
      121 gatcgtgcct tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa
      181 ttgccgcatt gcagagatat tgtatttaag tgcctagctc gatacataaa cgggtctctc
      241 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
      301 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
      361 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc
      421 cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc
      481 ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt
      541 tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga
      601 gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat
      661 taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt
      721 tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag
      781 gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa
      841 ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa
      901 gtaagaccac cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg
      961 gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta
     1021 gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga
     1081 gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg
     1141 ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg
     1201 agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc
     1261 caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg
     1321 ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat
     1381 aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac
     1441 aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat
     1501 gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca
     1561 aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga
     1621 atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg
     1681 tttcagaccc acctcccaac cccgagggga cccagagagg gcctatttcc catgattcct
     1741 tcatatttgc atatacgata caaggctgtt agagagataa ttagaattaa tttgactgta
     1801 aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt
     1861 gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat
     1921 ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccgtacc cctaccaact
     1981 ggtcggggtt tgaaacggta atgcctggct tgtcgacgca tagtcttttt taattctaga
     2041 tcttgagaca aatggcagta ttcatccaca attttaaaag aaaagggggg attggggggt
     2101 acagtgcagg ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac
     2161 aaaaacaaat tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccac
     2221 tttggcgccg gctcgagtgg ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca
     2281 gtccccgaga agttgggggg aggggtcggc aattgaaccg gtgcctagag aaggtggcgc
     2341 ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga gggtggggga
     2401 gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc
     2461 agaacacagg tgtcgtgacg cgggatctgc caccatgagc gagctgatca ccgagaacat
     2521 gcacatgaag ctgtacatgg agggcaccgt gaacaaccac cacttcaagt gcacatccga
     2581 gggcgaaggc aagccctacg agggcaccca gacccagaga atcaaggtgg tcgagggcgg
     2641 ccctctcccc ttcgccttcg acatcctggc taccagcttc atgtacggca gtcacacctt
     2701 catcaaccac acccagggca tccccgactt ctggaagcag tccttccctg agggcttcac
     2761 atgggagaga gtcaccacat acgaagacgg gggcgtgctg accgctaccc aggacaccag
     2821 cctccaggac ggctgcctca tctacaacgt caagatcaga ggggtgaact tcccatccaa
     2881 cggccctgtg atgcagaaga aaacactcgg ctgggaggcc cacaccgaga tgctgtaccc
     2941 cgctgacggc ggcctggaag gcagaaccgc gctggccctg aagctcgtgg gcgggggcca
     3001 cctgatctgc aacttcaaga ccacatacag atccaagaaa cccgctaaga acctcaagat
     3061 gcccggcgtc tactatgtgg actacagact ggaaagaatc aaggaggccg acaaagagac
     3121 ctacgtcgag cagcacgagg tggctgtggc cagatactgc gacctcccta gcaaactggg
     3181 gcacaagctt aattaaggta ccacgcgtta agtcgacaat caacctctgg attacaaaat
     3241 ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc
     3301 tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt
     3361 gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg
     3421 cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg
     3481 tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc
     3541 cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt
     3601 gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca cctggattct
     3661 gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg
     3721 cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg
     3781 gatctccctt tgggccgcct ccccgcgtcg actttaagac caatgactta caaggcagct
     3841 gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat tcactcccaa
     3901 cgaagacaag atctgctttt tgcttgtact gggtctctct ggttagacca gatctgagcc
     3961 tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga
     4021 gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga
     4081 cccttttagt cagtgtggaa aatctctagc agtacgtata gtagttcatg tcatcttatt
     4141 attcagtatt tataacttgc aaagaaatga atatcagaga gtgagaggaa cttgtttatt
     4201 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     4261 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg
     4321 ctctagctat cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     4381 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg
     4441 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggg acgtacccaa
     4501 ttcgccctat agtgagtcgt attacgcgcg ctcactggcc gtcgttttac aacgtcgtga
     4561 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
     4621 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
     4681 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     4741 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     4801 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     4861 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     4921 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     4981 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     5041 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     5101 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
     5161 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     5221 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     5281 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     5341 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     5401 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     5461 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     5521 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     5581 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     5641 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     5701 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     5761 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     5821 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     5881 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     5941 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     6001 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     6061 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     6121 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     6181 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     6241 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     6301 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     6361 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     6421 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     6481 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     6541 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     6601 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     6661 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     6721 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     6781 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     6841 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     6901 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     6961 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     7021 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     7081 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
     7141 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc
     7201 actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt
     7261 gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc caagcgcgca
     7321 attaaccctc actaaaggga acaaaagctg gagctgcaag c
//
