LOCUS       sequence-422482-        7347 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral plasmid for the cloning of mature gRNAs of the RfxCas13d 
            (CasRx) CRISPR system. 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 7347)
  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 7347)
  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)_Empty.EFS.tRFP657
FEATURES             Location/Qualifiers
     source          1..7347
                     /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    2058..2175
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2244..2455
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2481..3182
                     /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    3204..3792
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3257..3277)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3675..3686)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3864..4097
                     /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"
     protein_bind    7171..7192
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7207..7212,7213..7230,7231..7237)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7245..7261
                     /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     7250..7272
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7269..7285
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7269..7285
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7304..7324
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7306..7324
                     /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 tgaaacggag acggacgtct cttttttaat tctagatctt gagacaaatg
     2041 gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa
     2101 agaatagtag acataatagc aacagacata caaactaaag aattacaaaa acaaattaca
     2161 aaaattcaaa attttcgggt ttattacagg gacagcagag atccactttg gcgccggctc
     2221 gagtggctcc ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt
     2281 ggggggaggg gtcggcaatt gaaccggtgc ctagagaagg tggcgcgggg taaactggga
     2341 aagtgatgtc gtgtactggc tccgcctttt tcccgagggt gggggagaac cgtatataag
     2401 tgcagtagtc gccgtgaacg ttctttttcg caacgggttt gccgccagaa cacaggtgtc
     2461 gtgacgcggg atctgccacc atgagcgagc tgatcaccga gaacatgcac atgaagctgt
     2521 acatggaggg caccgtgaac aaccaccact tcaagtgcac atccgagggc gaaggcaagc
     2581 cctacgaggg cacccagacc cagagaatca aggtggtcga gggcggccct ctccccttcg
     2641 ccttcgacat cctggctacc agcttcatgt acggcagtca caccttcatc aaccacaccc
     2701 agggcatccc cgacttctgg aagcagtcct tccctgaggg cttcacatgg gagagagtca
     2761 ccacatacga agacgggggc gtgctgaccg ctacccagga caccagcctc caggacggct
     2821 gcctcatcta caacgtcaag atcagagggg tgaacttccc atccaacggc cctgtgatgc
     2881 agaagaaaac actcggctgg gaggcccaca ccgagatgct gtaccccgct gacggcggcc
     2941 tggaaggcag aaccgcgctg gccctgaagc tcgtgggcgg gggccacctg atctgcaact
     3001 tcaagaccac atacagatcc aagaaacccg ctaagaacct caagatgccc ggcgtctact
     3061 atgtggacta cagactggaa agaatcaagg aggccgacaa agagacctac gtcgagcagc
     3121 acgaggtggc tgtggccaga tactgcgacc tccctagcaa actggggcac aagcttaatt
     3181 aaggtaccac gcgttaagtc gacaatcaac ctctggatta caaaatttgt gaaagattga
     3241 ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt
     3301 tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt
     3361 tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg
     3421 tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg
     3481 ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc
     3541 gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat
     3601 catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct
     3661 tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg
     3721 ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg
     3781 ccgcctcccc gcgtcgactt taagaccaat gacttacaag gcagctgtag atcttagcca
     3841 ctttttaaaa gaaaaggggg gactggaagg gctaattcac tcccaacgaa gacaagatct
     3901 gctttttgct tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg
     3961 ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt
     4021 gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt
     4081 gtggaaaatc tctagcagta cgtatagtag 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 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga
     7141 ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc
     7201 ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca
     7261 atttcacaca ggaaacagct atgaccatga ttacgccaag cgcgcaatta accctcacta
     7321 aagggaacaa aagctggagc tgcaagc
//
