LOCUS Exported 8284 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Lentiviral expression of AsCas12a gRNA. ACCESSION . VERSION . KEYWORDS pRDA_052 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8284) AUTHORS DeWeirdt PC, Sanson KR, Sangree AK, Hegde M, Hanna RE, Feeley MN, Griffith AL, Teng T, Borys SM, Strand C, Joung JK, Kleinstiver BP, Pan X, Huang A, Doench JG TITLE Optimization of AsCas12a for combinatorial genetic screens in human cells. JOURNAL Nat Biotechnol. 2020 Jul 13. pii: 10.1038/s41587-020-0600-6. doi: 10.1038/s41587-020-0600-6. PUBMED 32661438 REFERENCE 2 (bases 1 to 8284) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8284 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 1..227 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" LTR 228..408 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 455..580 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1073..1306 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 1491..1535 /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 1719..1959 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 1719..1739 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 1890..1909 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_feature 2075..2192 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 2331..3509 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 2562..3500 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 3457..3477 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" CDS 3522..4121 /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(3522..3541) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 4018..4038 /label=Puro-F /note="Puromycin resistance gene, forward primer" misc_feature 4137..4725 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(4190..4210) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(4608..4619) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 4797..5030 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 5108..5229 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(5145..5164) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 5199..5218 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 5269..5404 /label=SV40 ori /note="SV40 origin of replication" primer_bind 5331..5350 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(5424..5443) /label=T7 /note="T7 promoter, forward primer" promoter complement(5425..5443) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(5453..5470) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(5453..5469) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5462..5484) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 5611..6066 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5698..5717) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5908..5929 /label=F1ori-F /note="F1 origin, forward primer" promoter 6092..6196 /gene="bla" /label=AmpR promoter CDS 6197..7057 /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(6415..6434) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7228..7816 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7717..7736 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 7970..7987 /label=L4440 /note="L4440 vector, forward primer" protein_bind 8104..8125 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 8140..8170 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 8178..8194 /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 8183..8205 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 8202..8218 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 8202..8218 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 8237..8257 /label=T3 /note="T3 promoter, forward primer" promoter 8239..8257 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" ORIGIN 1 tgtagtctta tgcaatactc ttgtagtctt gcaacatggt aacgatgagt tagcaacatg 61 ccttacaagg agagaaaaag caccgtgcat gccgattggt ggaagtaagg tggtacgatc 121 gtgccttatt aggaaggcaa cagacgggtc tgacatggat tggacgaacc actgaattgc 181 cgcattgcag agatattgta tttaagtgcc tagctcgata cataaacggg tctctctggt 241 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc 301 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta 361 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa 421 cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga ctcggcttgc 481 tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa aaattttgac 541 tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc gggggagaat 601 tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat ataaattaaa 661 acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg gcctgttaga 721 aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc agacaggatc 781 agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc atcaaaggat 841 agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa acaaaagtaa 901 gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat atgagggaca 961 attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac 1021 ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt 1081 tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga 1141 cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg 1201 ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg 1261 caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt 1321 gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat 1381 ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt 1441 acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac 1501 aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt 1561 ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag 1621 tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc 1681 agacccacct cccaaccccg aggggaccca gaggtaccga gggcctattt cccatgattc 1741 cttcatattt gcatatacga tacaaggctg ttagagagat aattagaatt aatttgactg 1801 taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt 1861 ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt 1921 atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccggt aatttctact 1981 cttgtagatg gagacgcgtt ttttctcgtc tctgaattcg ctagcaagct tggcgtaact 2041 agatcttgag acaaatggca gtattcatcc acaattttaa aagaaaaggg gggattgggg 2101 ggtacagtgc aggggaaaga atagtagaca taatagcaac agacatacaa actaaagaat 2161 tacaaaaaca aattacaaaa attcaaaatt ttcgggttta ttacagggac agcagagatc 2221 cactttggcg ccggctcgag ggggcccggg tgcaaagatg gataaagttt taaacagaga 2281 ggaatctttg cagctaatgg accttctagg tcttgaaagg agtgggaatt ggctccggtg 2341 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg 2401 gcaattgatc cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt 2461 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg 2521 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt 2581 cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct 2641 ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga 2701 ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct 2761 ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt 2821 ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc 2881 ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg 2941 acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac 3001 cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc 3061 cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag 3121 cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct 3181 cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg 3241 tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga 3301 gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc 3361 acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg 3421 aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa 3481 gtttttttct tccatttcag gtgtcgtgac gtacggccac catgaccgag tacaagccca 3541 cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt 3601 tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc gagcgggtca 3661 ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg 3721 cggacgacgg cgccgccgtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg 3781 tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc 3841 aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca 3901 ccgtcggagt ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg 3961 gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca 4021 acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag 4081 gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg aacgcgttaa gtcgacaatc 4141 aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt 4201 ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg 4261 ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc 4321 ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt 4381 ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg 4441 ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg 4501 gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct 4561 gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc 4621 cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc 4681 ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcgtcga ctttaagacc 4741 aatgacttac aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga 4801 agggctaatt cactcccaac gaagacaaga tctgcttttt gcttgtactg ggtctctctg 4861 gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc 4921 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg 4981 taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gtacgtatag 5041 tagttcatgt catcttatta ttcagtattt ataacttgca aagaaatgaa tatcagagag 5101 tgagaggaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa 5161 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa 5221 tgtatcttat catgtctggc tctagctatc ccgcccctaa ctccgcccat cccgccccta 5281 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca 5341 gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga 5401 ggcctaggga cgtacccaat tcgccctata gtgagtcgta ttacgcgcgc tcactggccg 5461 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag 5521 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc 5581 aacagttgcg cagcctgaat ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg 5641 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc 5701 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa 5761 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac 5821 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt 5881 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca 5941 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt 6001 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta 6061 caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta 6121 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata 6181 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc 6241 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga 6301 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct 6361 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg 6421 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta 6481 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat 6541 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt 6601 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga 6661 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga 6721 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga 6781 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc 6841 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc 6901 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg 6961 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat 7021 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata 7081 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct 7141 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga 7201 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg 7261 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc 7321 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct 7381 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc 7441 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt 7501 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg 7561 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct 7621 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag 7681 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag 7741 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg 7801 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg 7861 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac 7921 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt 7981 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat 8041 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc 8101 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc 8161 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca 8221 tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg agctgcaagc 8281 ttaa //