LOCUS       sequence-454335-        8288 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  hU6-driven expression of RfxCas13d gRNAs and arrays compatible. 
            Contains AarI sites for guide cloning flanked by 5' DR36.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8288)
  AUTHORS   El Kassem G, Hillmer J, Boettcher M
  TITLE     Evaluation of Cas13d as a tool for genetic interaction mapping.
  JOURNAL   Nat Commun. 2025 Feb 14;16(1):1631. doi: 10.1038/s41467-025-56747-4.
  PUBMED    39952934
REFERENCE   2  (bases 1 to 8288)
  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 
            Commun."; date: "2025-02-14"; volume: "16(1):1631. doi"; pages: " 
            10.1038/s41467-025-56747-4"
COMMENT     Sequence Label: 783-Rx-hU6: RfxCas13d gRNA and array cloning
            b...
FEATURES             Location/Qualifiers
     source          1..8288
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    226..459
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    956..1081
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             1128..1308
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(1326..1524)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(1351..1371)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        1526..1905
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     2077..2096
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        2171..2275
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2276..2344,2345..3136)
                     /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(2494..2513)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3307..3895
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3796..3815
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     LTR             3957..4137
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     4646..4662
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     4647..4663
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    4666..5254
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             4772..4783
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     5181..5201
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5334..5354)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(5364..6071)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(5639..5658)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     5908..5926
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      6068..6077
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(6084..6137)
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             complement(6147..6743)
                     /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(6227..6247)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     6724..6743
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(6756..6967)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     misc_RNA        complement(7526..7561)
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     promoter        complement(7563..7811)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(7621..7640)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(7791..7811)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    7870..7987
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     CDS             complement(join(8285..8288,1..41))
                     /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"
ORIGIN
        1 cttgcccatt tatctaattc caataattct tgttcattct tttcttgctg gttttgcgat
       61 tcttcaatta aggagtgtat taagcttgtg taattgttaa tttctctgtc ccactccatc
      121 caggtcgtgt gattccaaat ctgttccaga gatttattac tccaactagc attccaaggc
      181 acagcagtgg tgcaaatgag ttttccagag caaccccaaa tccccaggag ctgttgatcc
      241 tttaggtatc tttccacagc caggattctt gcctggagct gcttgatgcc ccagactgtg
      301 agttgcaaca gatgctgttg cgcctcaata gccctcagca aattgttctg ctgctgcact
      361 ataccagaca ataattgtct ggcctgtacc gtcagcgtca ttgacgctgc gcccatagtg
      421 cttcctgctg ctcccaagaa cccaaggaac aaagctccta ttcccactgc tcttttttct
      481 ctctgcacca ctcttctctt tgccttggtg ggtgctactc ctaatggttc aatttttact
      541 actttatatt tatataattc acttctccaa ttgtccctca tatctcctcc tccaggtctg
      601 aagatcagcg gccggccgct tgctgtgcgg tggtcttact tttgttttgc tcttcctcta
      661 tcttgtctaa agcttccttg gtgtctttta tctctatcct ttgatgcaca caatagaggg
      721 ttgctactgt attatataat gatctaagtt cttctgatcc tgtctgaagg gatggttgta
      781 gctgtcccag tatttgtcta cagccttctg atgtttctaa caggccagga ttaactgcga
      841 atcgttctag ctccctgctt gcccatacta tatgttttaa tttatatttt ttctttcccc
      901 ctggccttaa ccgaattttt tcccatcgcg atctaattct cccccgctta atactgacgc
      961 tctcgcaccc atctctctcc ttctagcctc cgctagtcaa aatttttggc gtactcacca
     1021 gtcgccgccc ctcgcctctt gccgtgcgcg cttcagcaag ccgagtcctg cgtcgagaga
     1081 gctcctctgg tttccctttc gctttcaagt ccctgttcgg gcgccactgc tagagatttt
     1141 ccacactgac taaaagggtc tgagggatct ctagttacca gagtcacaca acagacgggc
     1201 acacactact tgaagcactc aaggcaagct ttattgaggc ttaagcagtg ggttccctag
     1261 ttagccagag agctcccagg ctcagatctg gtctaaccag agagacccag tacaggcaaa
     1321 acgcgctgct tatatagacc tcccaccgta cacgcctacc gcccatttgc gtcaatgggg
     1381 cggagttgtt acgacatttt ggaaagtccc gttgattttg gtgccaaaac aaactcccat
     1441 tgacgtcaat ggggtggaga cttggaaatc cccgtgagtc aaaccgctat ccacgcccat
     1501 tgatgtactg ccaaaaccgc atcaccatgg taatagcgat gactaatacg tagatgtact
     1561 gccaagtagg aaagtcccat aaggtcatgt actgggcata atgccaggcg ggccatttac
     1621 cgtcattgac gtcaataggg ggcgtacttg gcatatgata cacttgatgt actgccaagt
     1681 gggcagttta ccgtaaatac tccacccatt gacgtcaatg gaaagtccct attggcgtta
     1741 ctatgggaac atacgtcatt attgacgtca atgggcgggg gtcgttgggc ggtcagccag
     1801 gcgggccatt taccgtaagt tatgtaacgc ggaactccat atatgggcta tgaactaatg
     1861 accccgtaat tgattactat taataactag tcaataatca atgtcaacgc gtatatctgg
     1921 cccgtacatc gcgaagcagc gcaaaacgcc taaccctaag cagattcttc atgcaattgt
     1981 cggtcaagcc ttgccttgtt gtagcttaaa ttttgctcgc gcactactca gcgacctcca
     2041 acacacaagc agggagcaga tactggctta actatgcggc atcagagcag attgtactga
     2101 gagtgcacca taggggatcg ggagatctcc cgatccgtcg acgtcaggtg gcacttttcg
     2161 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     2221 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     2281 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     2341 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     2401 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     2461 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     2521 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     2581 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     2641 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     2701 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     2761 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     2821 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     2881 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     2941 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     3001 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     3061 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     3121 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     3181 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     3241 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     3301 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     3361 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     3421 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     3481 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     3541 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     3601 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     3661 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     3721 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     3781 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     3841 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     3901 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgctgct
     3961 agagattttc cacactgact aaaagggtct gagggatctc tagttaccag agtcacacaa
     4021 cagacgggca cacactactt gaagcactca aggcaagctt tattgaggct taagcagtgg
     4081 gttccctagt tagccagaga gctcccaggc tcagatctgg tctaaccaga gagacccagt
     4141 acagtccgga tgcagctctc gggccatgtg atgaaatgct aggcggctgt caaacctcca
     4201 ctctaatact tctctctccg ggtcatccat cccatgcagg ctcacagggt gtaacaagcg
     4261 ggtgttctct ccttcattgg cttcttctac cttctcttgc tcaactggta ctagcttgta
     4321 gcaccatcca aaggtcagtg gatatctgat ccctggccct ggtgtgtagt tctgccaatc
     4381 agggaagtag ccttgtgtgt ggtagatcca cagatcaagg atatcttgtc ttcgttggga
     4441 gtgaattagc ccttccagtc cccccttttc ttttaaaaag tggctaagat ctacagctgc
     4501 cttgtaagtc attggtctta aaggtacctg aggtgtgact ggaaaaccca cctcctcctc
     4561 ctcttgtgct tctagccagg cacaatcagc attggtagct gctgtattgc tacttgtgat
     4621 tgctccatgt ttttctaggt ctcgatcgag gtcgacggta tcgatgcggg gaggcggccc
     4681 aaagggagat ccgactcgtc tgagggcgaa ggcgaagacg cggaagaggc cgcagagccg
     4741 gcagcaggcc gcgggaagga aggtccgctg gattgagggc cgaagggacg tagcagaagg
     4801 acgtcccgcg cagaatccag gtggcaacac aggcgagcag ccaaggaaag gacgatgatt
     4861 tccccgacaa caccacggaa ttgtcagtgc ccaacagccg agcccctgtc cagcagcggg
     4921 caaggcaggc ggcgatgagt tccgccgtgg caatagggag ggggaaagcg aaagtcccgg
     4981 aaaggagctg acaggtggtg gcaatgcccc aaccagtggg ggttgcgtca gcaaacacag
     5041 tgcacaccac gccacgttgc ctgacaacgg gccacaactc ctcataaaga gacagcaacc
     5101 aggatttata caaggaggag aaaatgaaag ccatacggga agcaatagca tgatacaaag
     5161 gcattaaagc agcgtatcca catagcgtaa aaggagcaac atagttaaga ataccagtca
     5221 atctttcaca aattttgtaa tccagaggtt gattatcgat aagcttgata tcgaattgta
     5281 cctagtggaa ccggaaccct taaacatgtt aggtccctcg acttaattaa ctataccttt
     5341 ctcttctttt ttggatcagc tcccttgtac agctcgtcca tgccgccggt ggagtggcgg
     5401 ccctcggcgc gttcgtactg ttccacgatg gtgtagtcct cgttgtggga ggtgatgtcc
     5461 aacttgatgt tgacgttgta ggcgccgggc agctgcacgg gcttcttggc cttgtaggtg
     5521 gtcttgacct cagcgtcgta gtggccgccg tccttcagct tcagcctctg cttgatctcg
     5581 cccttcaggg cgccgtcctc ggggtacatc cgctcggagg aggcctccca gcccatggtc
     5641 ttcttctgca ttacggggcc gtcggagggg aagttggtgc cgcgcagctt caccttgtag
     5701 atgaactcgc cgtcctgcag ggaggagtcc tgggtcacgg tcaccacgcc gccgtcctcg
     5761 aagttcatca cgcgctccca cttgaagccc tcggggaagg acagcttcaa gtagtcgggg
     5821 atgtcggcgg ggtgcttcac gtaggccttg gagccgtaca tgaactgagg ggacaggatg
     5881 tcccaggcga agggcagggg gccacccttg gtcaccttca gcttggcggt ctgggtgccc
     5941 tcgtaggggc ggccctcgcc ctcgccctcg atctcgaact cgtggccgtt cacggagccc
     6001 tccatgtgca ccttgaagcg catgaactcc ttgatgatgg ccatgttatc ctcctcgccc
     6061 ttgctcacca tggtggcgac cggtgggcca ggattctcct cgacgtcacc gcatgttagc
     6121 agacttcctc tgccctctcc cgatccggca ccgggcttgc gggtcatgca ccaggtgcgc
     6181 ggtccttcgg gcacctcgac gtcggcggtg acggtgaagc cgagccgctc gtagaagggg
     6241 aggttgcggg gcgcggaggt ctccaggaag gcgggcaccc cggcgcgctc ggccgcctcc
     6301 actccgggga gcacgacggc gctgcccaga cccttgccct ggtggtcggg cgagacgccg
     6361 acggtggcca ggaaccacgc gggctccttg ggccggtgcg gcgccaggag gccttccatc
     6421 tgttgctgcg cggccagccg ggaaccgctc aactcggcca tgcgcgggcc gatctcggcg
     6481 aacaccgccc ccgcttcgac gctctccggc gtggtccaga ccgccaccgc ggcgccgtcg
     6541 tccgcgaccc acaccttgcc gatgtcgagc ccgacgcgcg tgaggaagag ttcttgcagc
     6601 tcggtgaccc gctcgatgtg gcggtccggg tcgacggtgt ggcgcgtggc ggggtagtcg
     6661 gcgaacgcgg cggcgagggt gcgtacggcc cgggggacgt cgtcgcgggt ggcgaggcgc
     6721 accgtgggct tgtactcggt catggtgggc tagctcactg ttctggcggc aaacccgttg
     6781 cgaaaaagaa cgttcacggc gactactgca cttatatacg gttctccccc accctcggga
     6841 aaaaggcgga gccagtacac gacatcactt tcccagttta ccccgcgcca ccttctctag
     6901 gcaccggttc aattgccgac ccctcccccc aacttctcgg ggactgtggg cgatgtgcgc
     6961 tctgcccact gacgggcaca tgcatggcgg taatacggtt atccacgcgg ccaattcaaa
     7021 aaaagatcgc aggtgacatg tgccgagcca tctctcttag gcgcttgctg ctgctgctgc
     7081 tgcagctgtc acaactccta gctgtcactc aagggaagac gctggtgctg gggaaggaag
     7141 gggaatcagc agaactgccc tgcgagagtt cccagaagaa gatcacagtc ttcacctgga
     7201 agttctctga ccagaggaag attctggggc agcatggcaa aggtgtatta attagaggag
     7261 gttcgccttc gcagtttgat cgttttgatt ccaaaaaagg ggcatgggag aaaggatcgt
     7321 ttcctctcat catcaataaa cttaagatgg aagactctca gacttatatc tgtgagctgg
     7381 agaacaggaa agaggaggtg gagttgtggg tgttcaaagt gaccttcagt ccgggtacca
     7441 gcctgttgca agggcagagc ctgaccctga ccttggatag caactctaag gtctctaacc
     7501 ccttgacaga gtgccacctg cgtctgtttc aaaccccgac cagttggtag gggtttactt
     7561 gcggtgtttc gtcctttcca caagatatat aaagccaaga aatcgaaata ctttcaagtt
     7621 acggtaagca tatgatagtc cattttaaaa cataatttta aaactgcaaa ctacccaaga
     7681 aattattact ttctacgtca cgtattttgt actaatatct ttgtgtttac agtcaaatta
     7741 attccaatta tctctctaac agccttgtat cgtatatgca aatatgaagg aatcatggga
     7801 aataggccct cgctagagcg ggcccggtac taaccaaact ggatctctgc tgtccctgta
     7861 ataaacccga aaattttgaa tttttgtaat ttgtttttgt aattctttag tttgtatgtc
     7921 tgttgctatt atgtctacta ttctttcccc tgcactgtac cccccaatcc ccccttttct
     7981 tttaaaattg tggatgaata ctgccatttg tctgcagaat tggcgcacgc agtgccgatc
     8041 cgttcactaa tcgaatggat ctgtctctgt ctctctctcc accttcttct tctattcctt
     8101 cgggcctgtc gggtcccctc ggggttggga ggtgggtctg aaacgataat ggtgaatatc
     8161 cctgcctaac tctattcact atagaaagta cagcaaaaac tattcttaaa cctaccaagc
     8221 ctcctactat cattatgaat aattttatat accacagcca atttgttatg ttaaaccaat
     8281 tccacaaa
//
