LOCUS       sequence-422499-        7358 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 7358)
  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 7358)
  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)_GFP-targeting
            sgRNA.EFS....
FEATURES             Location/Qualifiers
     source          1..7358
                     /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    2069..2186
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2255..2466
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2492..3193
                     /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    3215..3803
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3268..3288)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3686..3697)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3875..4108
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4186..4307
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4223..4242)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4277..4296
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4347..4482
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4502..4521)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4503..4521)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4531..4548)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4531..4547)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4540..4562)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4689..5144
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4776..4795)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4986..5007
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5170..5274
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5275..5343,5344..6135)
                     /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(5493..5512)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6306..6894
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6795..6814
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7048..7065
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7182..7203
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7218..7223,7224..7241,7242..7248)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7256..7272
                     /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     7261..7283
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7280..7296
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7280..7296
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7315..7335
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7317..7335
                     /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 tgaaaccacc ccggtgaaca gctcctcgcc ctttttttaa ttctagatct
     2041 tgagacaaat ggcagtattc atccacaatt ttaaaagaaa aggggggatt ggggggtaca
     2101 gtgcagggga aagaatagta gacataatag caacagacat acaaactaaa gaattacaaa
     2161 aacaaattac aaaaattcaa aattttcggg tttattacag ggacagcaga gatccacttt
     2221 ggcgccggct cgagtggctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc
     2281 cccgagaagt tggggggagg ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg
     2341 gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa
     2401 ccgtatataa gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga
     2461 acacaggtgt cgtgacgcgg gatctgccac catgagcgag ctgatcaccg agaacatgca
     2521 catgaagctg tacatggagg gcaccgtgaa caaccaccac ttcaagtgca catccgaggg
     2581 cgaaggcaag ccctacgagg gcacccagac ccagagaatc aaggtggtcg agggcggccc
     2641 tctccccttc gccttcgaca tcctggctac cagcttcatg tacggcagtc acaccttcat
     2701 caaccacacc cagggcatcc ccgacttctg gaagcagtcc ttccctgagg gcttcacatg
     2761 ggagagagtc accacatacg aagacggggg cgtgctgacc gctacccagg acaccagcct
     2821 ccaggacggc tgcctcatct acaacgtcaa gatcagaggg gtgaacttcc catccaacgg
     2881 ccctgtgatg cagaagaaaa cactcggctg ggaggcccac accgagatgc tgtaccccgc
     2941 tgacggcggc ctggaaggca gaaccgcgct ggccctgaag ctcgtgggcg ggggccacct
     3001 gatctgcaac ttcaagacca catacagatc caagaaaccc gctaagaacc tcaagatgcc
     3061 cggcgtctac tatgtggact acagactgga aagaatcaag gaggccgaca aagagaccta
     3121 cgtcgagcag cacgaggtgg ctgtggccag atactgcgac ctccctagca aactggggca
     3181 caagcttaat taaggtacca cgcgttaagt cgacaatcaa cctctggatt acaaaatttg
     3241 tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc
     3301 tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta
     3361 taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt
     3421 ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca
     3481 gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc
     3541 ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt
     3601 gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct ggattctgcg
     3661 cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg
     3721 cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat
     3781 ctccctttgg gccgcctccc cgcgtcgact ttaagaccaa tgacttacaa ggcagctgta
     3841 gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca ctcccaacga
     3901 agacaagatc tgctttttgc ttgtactggg tctctctggt tagaccagat ctgagcctgg
     3961 gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg
     4021 cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc
     4081 ttttagtcag tgtggaaaat ctctagcagt acgtatagta gttcatgtca tcttattatt
     4141 cagtatttat aacttgcaaa gaaatgaata tcagagagtg agaggaactt gtttattgca
     4201 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     4261 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggctc
     4321 tagctatccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     4381 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct
     4441 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctagggacg tacccaattc
     4501 gccctatagt gagtcgtatt acgcgcgctc actggccgtc gttttacaac gtcgtgactg
     4561 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg
     4621 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg
     4681 cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     4741 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     4801 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     4861 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     4921 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     4981 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     5041 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     5101 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg
     5161 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     5221 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     5281 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     5341 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     5401 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     5461 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     5521 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     5581 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     5641 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     5701 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     5761 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     5821 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     5881 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     5941 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     6001 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     6061 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     6121 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     6181 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     6241 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     6301 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     6361 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     6421 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     6481 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     6541 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     6601 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     6661 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     6721 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     6781 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     6841 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     6901 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     6961 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     7021 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     7081 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
     7141 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact
     7201 cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg
     7261 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa gcgcgcaatt
     7321 aaccctcact aaagggaaca aaagctggag ctgcaagc
//
