LOCUS Exported 8792 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Rfx Cas13d guide cloning lentiviral vector with constitutively expressed puromycin resistance 2A-tagged with tagBFP. ACCESSION . VERSION . KEYWORDS pSLQ5465_pHR_hU6-crScaffold_EF1a-PuroR-T2A-BFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8792) AUTHORS Abbott TR, Dhamdhere G, Liu Y, Lin X, Goudy L, Zeng L, Chemparathy A, Chmura S, Heaton NS, Debs R, Pande T, Endy D, La Russa MF, Lewis DB, Qi LS TITLE Development of CRISPR as an Antiviral Strategy to Combat SARS-CoV-2 and Influenza. JOURNAL Cell. 2020 May 14;181(4):865-876.e12. doi: 10.1016/j.cell.2020.04.020. Epub 2020 Apr 29. PUBMED 32353252 REFERENCE 2 (bases 1 to 8792) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8792 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(47..66) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 238..617 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 619..817 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 772..792 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" LTR 835..1015 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 1062..1187 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1684..1917 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 2102..2146 /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" misc_feature 2444..2561 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 2620..2860 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 2620..2640 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 2791..2810 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 2870..2905 /label=RfxCas13d DR36 /note="36-base direct repeat for an RfxCas13d pre-gRNA (Konermann et al., 2018)" misc_RNA 2924..2959 /label=RfxCas13d DR36 /note="36-base direct repeat for an RfxCas13d pre-gRNA (Konermann et al., 2018)" promoter 3014..4192 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 3245..4183 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 4140..4160 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" primer_bind complement(4211..4230) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 4707..4727 /label=Puro-F /note="Puromycin resistance gene, forward primer" CDS 4817..4870 /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" protein_bind 5592..5625 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." misc_feature 5681..6269 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(5734..5754) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(6152..6163) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind complement(6272..6288) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(6273..6289) /label=pBluescriptKS /note="For pBluescript vector" LTR 6798..6978 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" rep_origin complement(7040..7628) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(7120..7139) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(7799..8659) /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 8422..8441 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(8660..8764) /gene="bla" /label=AmpR promoter ORIGIN 1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg 61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt 121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc 181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac 241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat 301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg 361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt 421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag 481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc 541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag 601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt 661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc 721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg 781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct 841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt 901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac 961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc 1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc 1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa 1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg 1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata 1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc 1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga 1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc 1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca 1501 aaagtaagac caccgcacag caagcggccg gccgctgatc ttcagacctg gaggaggaga 1561 tatgagggac aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt 1621 aggagtagca cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg 1681 aataggagct ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc 1741 aatgacgctg acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa 1801 tttgctgagg gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa 1861 gcagctccag gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg 1921 gatttggggt tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg 1981 gagtaataaa tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga 2041 aattaacaat tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga 2101 aaagaatgaa caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa 2161 cataacaaat tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg 2221 tttaagaata gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc 2281 attatcgttt cagacccacc tcccaacccc gaggggaccc gacaggcccg aaggaataga 2341 agaagaaggt ggagagagag acagagacag atccattcga ttagtgaacg gatcggcact 2401 gcgtgcgcca attctgcaga caaatggcag tattcatcca caattttaaa agaaaagggg 2461 ggattggggg gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa 2521 ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca 2581 gcagagatcc agtttggtta gtaccgggcc cgctctagag agggcctatt tcccatgatt 2641 ccttcatatt tgcatatacg atacaaggct gttagagaga taattggaat taatttgact 2701 gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag 2761 tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag 2821 tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccgc aagtaaaccc 2881 ctaccaactg gtcggggttt gaaacgggtc ttcgagaaga cctcaagtaa acccctacca 2941 actggtcggg gtttgaaact ttttttttca cctatgtatc gtatcaagta ctagaactcc 3001 ccagtgggaa ttcggctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc 3061 cgagaagttg gggggagggg tcggcaattg aaccggtgcc tagagaaggt ggcgcggggt 3121 aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc 3181 gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac 3241 acaggtaagt gccgtgtgtg gttcccgcgg gcctggcctc tttacgggtt atggcccttg 3301 cgtgccttga attacttcca cctggctgca gtacgtgatt cttgatcccg agcttcgggt 3361 tggaagtggg tgggagagtt cgaggccttg cgcttaagga gccccttcgc ctcgtgcttg 3421 agttgaggcc tggcctgggc gctggggccg ccgcgtgcga atctggtggc accttcgcgc 3481 ctgtctcgct gctttcgata agtctctagc catttaaaat ttttgatgac ctgctgcgac 3541 gctttttttc tggcaagata gtcttgtaaa tgcgggccaa gatctgcaca ctggtatttc 3601 ggtttttggg gccgcgggcg gcgacggggc ccgtgcgtcc cagcgcacat gttcggcgag 3661 gcggggcctg cgagcgcggc caccgagaat cggacggggg tagtctcaag ctggccggcc 3721 tgctctggtg cctggcctcg cgccgccgtg tatcgccccg ccctgggcgg caaggctggc 3781 ccggtcggca ccagttgcgt gagcggaaag atggccgctt cccggccctg ctgcagggag 3841 ctcaaaatga aggacgcggc gctcgggaga gcgggcgggt gagtcaccca cacaaaggaa 3901 aagggccttt ccgtcctcag ccgtcgcttc atgtgactcc acggagtacc gggcgccgtc 3961 caggcacctc gattagttct cgagcttttg gagtacgtcg tctttaggtt ggggggaggg 4021 gttttatgcg atggagtttc cccacactga gtgggtggag actgaagtta ggccagcttg 4081 gcacttgatg taattctcct tggaatttgc cctttttgag tttggatctt ggttcattct 4141 caagcctcag acagtggttc aaagtttttt tcttccattt caggtgtcgt gacgtacggc 4201 caccatgagc atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc 4261 cagggctgta cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt 4321 cgacccggac cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt 4381 cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac 4441 cacgccggag agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga 4501 gttgagcggt tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg 4561 gcccaaggag cccgcgtggt tcctggccac agtgggtgtg tcccctgatc accagggcaa 4621 gggtctgggc agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc 4681 cgccttcctg gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac 4741 cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc 4801 cggtgccgga tccggagagg gcagaggaag tctgctaaca tgcggtgacg tcgaggagaa 4861 tcctggccca atgtccgaac tgattaaaga gaacatgcac atgaaacttt atatggaagg 4921 tacggtcgac aaccaccatt tcaaatgtac ctccgaagga gagggcaaac cctacgaggg 4981 cacccaaacg atgagaatta aagtagtaga aggtgggccg ctgccctttg cctttgacat 5041 tctggcaact tcttttctct acgggagtaa aacttttatc aaccatacgc aaggcattcc 5101 agacttcttt aagcagtcat ttcccgaggg gtttacttgg gagcgggtca caacatacga 5161 ggacggcggc gtgctgaccg ctactcagga caccagcctg caagacgggt gtttgatcta 5221 caatgtgaag attcgaggcg taaactttac ttcaaacggc ccagttatgc agaaaaaaac 5281 cctcggctgg gaggctttta cagagactct ttacccagcc gacggtggtt tggaaggccg 5341 aaacgacatg gcattgaaat tggttggcgg gagccacctt atagcgaata taaagacgac 5401 ctatcggtct aaaaagcccg ccaaaaattt gaagatgcct ggtgtatatt acgtcgacta 5461 taggcttgag agaataaaag aagctaacaa cgaaacctat gtggagcaac atgaggttgc 5521 agttgcacgc tactgcgatc tgccgagcaa attggggcac aagctgaatt gatgtacagt 5581 cgagggacct aataacttcg tatagcatac attatacgaa gttatacatg tttaagggtt 5641 ccggttccac taggtacaat tcgatatcaa gcttatcgat aatcaacctc tggattacaa 5701 aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata 5761 cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc 5821 cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg 5881 tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac 5941 ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat 6001 cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt 6061 ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg ccacctggat 6121 tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc 6181 ccgcggcctg ctgccggctc tgcggcctct tccgcttctt cgccttcgcc ctcagacgag 6241 tcggatctcc ctttgggccg cctccccgca tcgataccgt cgacctcgat cgagacctag 6301 aaaaacatgg agcaatcaca agtagcaata cagcagctac caatgctgat tgtgcctggc 6361 tagaagcaca agaggaggag gaggtgggtt ttccagtcac acctcaggta cctttaagac 6421 caatgactta caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactgg 6481 aagggctaat tcactcccaa ccaagacaag atatccttga tctgtggatc taccacacac 6541 aaggctactt ccctgattgg cagaactaca caccagggcc agggatcaga tatccactga 6601 cctttggatg gtgctacaag ctagtaccag ttgagcaaga gaaggtagaa gaagccaatg 6661 aaggagagaa cacccgcttg ttacaccctg tgagcctgca tgggatggat gacccggaga 6721 gagaagtatt agagtggagg tttgacagcc gcctagcatt tcatcacatg gcccgagagc 6781 tgcatccgga ctgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg 6841 gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag 6901 tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag 6961 tgtggaaaat ctctagcagc atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa 7021 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc 7081 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc 7141 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg 7201 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt 7261 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc 7321 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc 7381 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag 7441 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg 7501 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa 7561 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag 7621 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact 7681 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa 7741 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt 7801 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag 7861 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca 7921 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc 7981 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt 8041 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg 8101 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca 8161 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg 8221 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca 8281 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg 8341 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct 8401 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca 8461 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca 8521 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg 8581 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac 8641 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt 8701 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc 8761 cgcgcacatt tccccgaaaa gtgccacctg ac //