LOCUS Exported 9548 bp ds-DNA circular SYN 19-OCT-2022 DEFINITION Lentiviral sgRNA vector with mU6 sgRNA promoter, CR1 constant region, and UCOE EF1alpha driving PURO-GFP marker expression. ACCESSION . VERSION . KEYWORDS pJR104 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9548) AUTHORS Replogle JM, Bonnar JL, Pogson AN, Liem CR, Maier NK, Ding Y, Russell BJ, Wang X, Leng K, Guna A, Norman TM, Pak RA, Ramos DM, Ward ME, Gilbert LA, Kampmann M, Weissman JS, Jost M TITLE Maximizing CRISPRi efficacy and accessibility with dual-sgRNA libraries and optimal effectors JOURNAL bioRxiv 2022.07.13.499814 REFERENCE 2 (bases 1 to 9548) AUTHORS . TITLE Direct Submission JOURNAL Exported Oct 19, 2022 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9548 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(77..96) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 268..647 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 649..847 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 802..822 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" LTR 865..1045 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 1092..1217 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1714..1947 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 2132..2176 /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 2474..2591 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" protein_bind 2844..2877 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind 2881..2901 /label=mU6-F /note="Mouse U6 promoter, forward primer" promoter 3796..4007 /label=EF-1-alpha core promoter /note="core promoter for human elongation factor EF-1-alpha" intron 4008..4946 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 4903..4923 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" primer_bind complement(4967..4986) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" CDS 5573..5626 /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 6378..6411 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." misc_feature 6467..7055 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(6520..6540) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(6938..6949) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind complement(7058..7074) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(7059..7075) /label=pBluescriptKS /note="For pBluescript vector" LTR 7584..7764 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" rep_origin complement(7826..8414) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(7906..7925) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(8585..9445) /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 9208..9227 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(9446..2) /gene="bla" /label=AmpR promoter ORIGIN 1 cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatct cccgatcccc 61 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc 121 ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc 181 aaggcttgac cgacaattgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc 241 gcgatgtacg ggccagatat acgcgttgac attgattatt gactagttat taatagtaat 301 caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg 361 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt 421 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac 481 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg 541 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact 601 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt 661 ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc 721 ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc 781 gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata 841 taagcagcgc gttttgcctg tactgggtct ctctggttag accagatctg agcctgggag 901 ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt 961 caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt 1021 tagtcagtgt ggaaaatctc tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga 1081 aaccagagga gctctctcga cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg 1141 aggggcggcg actggtgagt acgccaaaaa ttttgactag cggaggctag aaggagagag 1201 atgggtgcga gagcgtcagt attaagcggg ggagaattag atcgcgatgg gaaaaaattc 1261 ggttaaggcc agggggaaag aaaaaatata aattaaaaca tatagtatgg gcaagcaggg 1321 agctagaacg attcgcagtt aatcctggcc tgttagaaac atcagaaggc tgtagacaaa 1381 tactgggaca gctacaacca tcccttcaga caggatcaga agaacttaga tcattatata 1441 atacagtagc aaccctctat tgtgtgcatc aaaggataga gataaaagac accaaggaag 1501 ctttagacaa gatagaggaa gagcaaaaca aaagtaagac caccgcacag caagcggccg 1561 gccgctgatc ttcagacctg gaggaggaga tatgagggac aattggagaa gtgaattata 1621 taaatataaa gtagtaaaaa ttgaaccatt aggagtagca cccaccaagg caaagagaag 1681 agtggtgcag agagaaaaaa gagcagtggg aataggagct ttgttccttg ggttcttggg 1741 agcagcagga agcactatgg gcgcagcgtc aatgacgctg acggtacagg ccagacaatt 1801 attgtctggt atagtgcagc agcagaacaa tttgctgagg gctattgagg cgcaacagca 1861 tctgttgcaa ctcacagtct ggggcatcaa gcagctccag gcaagaatcc tggctgtgga 1921 aagataccta aaggatcaac agctcctggg gatttggggt tgctctggaa aactcatttg 1981 caccactgct gtgccttgga atgctagttg gagtaataaa tctctggaac agatttggaa 2041 tcacacgacc tggatggagt gggacagaga aattaacaat tacacaagct taatacactc 2101 cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa caagaattat tggaattaga 2161 taaatgggca agtttgtgga attggtttaa cataacaaat tggctgtggt atataaaatt 2221 attcataatg atagtaggag gcttggtagg tttaagaata gtttttgctg tactttctat 2281 agtgaataga gttaggcagg gatattcacc attatcgttt cagacccacc tcccaacccc 2341 gaggggaccc gacaggcccg aaggaataga agaagaaggt ggagagagag acagagacag 2401 atccattcga ttagtgaacg gatcggcact gcgtgcgcca attctgcaga caaatggcag 2461 tattcatcca caattttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa 2521 tagtagacat aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa 2581 ttcaaaattt tcgggtttat tacagggaca gcagagatcc agtttggtta gtaccgggcc 2641 cgctctagag atccgacgcg ccatctctag gcccgcgccg gccccctcgc acggacttgt 2701 gggagaagct cggctactcc cctgccccgg ttaatttgca tataatattt cctagtaact 2761 atagaggctt aatgtgcgat aaaagacaga taatctgttc tttttaatac tagctacatt 2821 ttacatgata ggcttggatt tctataactt cgtatagcat acattatacg aagttataaa 2881 cagcacaaaa ggaaactcac cctaactgta aagtaattgt gtgttttgag actataagta 2941 tcccttggag aaccaccttg ttggaccagg atgggcacca cccgtttaag agctaagctg 3001 gaaacagcat agcaagttta aataaggcta gtccgttatc aacttgaaaa agtggcaccg 3061 agtcggtgct ttttttctcg agtactagga tccattaggc ggccgccggg aggtggtccc 3121 tgcagttacg ccaatgataa cccccgccag aaaaatctta gtagccttcc ctttttgttt 3181 tccgtgcccc aactcggcgg attgactcgg ccccttccgg aaacacccga atcaacttct 3241 agtcaaatta ttgttcacgc cgcaatgacc cacccctggc ccgcgtctgt ggaactgacc 3301 cctggtgtac aggagagttc gctgctgaaa gtggtcccaa aggggtacta gtttttaagc 3361 tcccaactcc ccctccccca gcgtctggag gattccacac cctcgcaccg caggggcgag 3421 gaagtgggcg gagtccggtt ttggcgccag ccgctgaggc tgccaagcag aaaagccacc 3481 gctgaggaga ctccggtcac tgtcctcgcc ccgcctcccc cttccctccc cttggggacc 3541 accgggcgcc acgccgcgaa cggtaagtgc cgcggtcgtc ggcgcctccg ccctccccct 3601 agggccccaa ttcccagcgg gcgcggcgcg cggcccctcc ccccgccggg cgcgcgcccg 3661 ctgccccgcc cttcgtggcc gcccggcgtg ggcggtgcca cccctccccc cggcggcccc 3721 gcgcgcagct cccggctccc tcccccttcg gatgtggctt gagctgtagg cgcggagatg 3781 catgtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag ttggggggag 3841 gggtcggcaa ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg 3901 tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata agtgcagtag 3961 tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacaggta agtgccgtgt 4021 gtggttcccg cgggcctggc ctctttacgg gttatggccc ttgcgtgcct tgaattactt 4081 ccacctggct gcagtacgtg attcttgatc ccgagcttcg ggttggaagt gggtgggaga 4141 gttcgaggcc ttgcgcttaa ggagcccctt cgcctcgtgc ttgagttgag gcctggcctg 4201 ggcgctgggg ccgccgcgtg cgaatctggt ggcaccttcg cgcctgtctc gctgctttcg 4261 ataagtctct agccatttaa aatttttgat gacctgctgc gacgcttttt ttctggcaag 4321 atagtcttgt aaatgcgggc caagatctgc acactggtat ttcggttttt ggggccgcgg 4381 gcggcgacgg ggcccgtgcg tcccagcgca catgttcggc gaggcggggc ctgcgagcgc 4441 ggccaccgag aatcggacgg gggtagtctc aagctggccg gcctgctctg gtgcctggcc 4501 tcgcgccgcc gtgtatcgcc ccgccctggg cggcaaggct ggcccggtcg gcaccagttg 4561 cgtgagcgga aagatggccg cttcccggcc ctgctgcagg gagctcaaaa tggaggacgc 4621 ggcgctcggg agagcgggcg ggtgagtcac ccacacaaag gaaaagggcc tttccgtcct 4681 cagccgtcgc ttcatgtgac tccacggagt accgggcgcc gtccaggcac ctcgattagt 4741 tctcgcgctt ttggagtacg tcgtctttag gttgggggga ggggttttat gcgatggagt 4801 ttccccacac tgagtgggtg gagactgaag ttaggccagc ttggcacttg atgtaattct 4861 ccttggaatt tgcccttttt gagtttggat cttggttcat tctcaagcct cagacagtgg 4921 ttcaaagttt ttttcttcca tttcaggtgt cgtgagctag cccaccatga ccgagtacaa 4981 gcccaccgtt cgcctggcca caagagatga cgtgcctcgc gccgtgagaa ctttggctgc 5041 ggcgttcgcc gattatccag caaccaggca cacagtggac cctgatagac acatcgagcg 5101 agtgactgag ctccaggaac tgtttctgac acgggttggt ctggatatcg gaaaagtgtg 5161 ggtcgctgac gatggggccg ccgtggcagt ctggacaacc ccagaatccg tggaggccgg 5221 agcagtattc gccgagatag gcccccgcat ggcggagctt tcggggtcaa ggctggctgc 5281 acagcagcaa atggagggcc tactcgctcc acaccggcct aaagaacccg cctggttcct 5341 agccactgtt ggcgtgtcac cggaccatca gggaaaaggg ctgggctctg ctgtggtgct 5401 gcctggtgtc gaggctgcgg agagggccgg agtgccggct tttcttgaaa ctagcgcacc 5461 taggaactta cctttctacg agcgtctggg atttaccgtc acagccgacg tggaggttcc 5521 agaggggccc cggacttggt gtatgacacg gaaaccgggc gcaggttctg gggagggtag 5581 ggggtcactt ctgacgtgcg gggatgtgga agaaaacccc ggcccaccag tagcaacgat 5641 ggtcagtaag ggcgaggaac tttttacagg ggtcgttccc attctggttg aattggatgg 5701 cgatgtgaac ggacataaat ttagcgtgtc cggcgaagga gaaggtgatg ctacctacgg 5761 aaagcttacg ttgaagttta tttgcacgac cggtaagctg cccgtaccat ggccaacatt 5821 ggtgaccact ctgacctatg gagtgcagtg tttcagccgg taccccgacc acatgaaaca 5881 acacgacttc ttcaagtctg ccatgcctga aggttatgtc caggaacgta ccatcttttt 5941 taaagacgac ggaaattata agacccgagc cgaggtaaag tttgagggcg acactctcgt 6001 caaccgcata gaattgaagg ggattgactt caaagaagat ggcaacattc taggccataa 6061 attagagtat aattacaaca gccacaacgt ttacatcatg gctgacaaac agaagaatgg 6121 catcaaggca aatttcaaga taagacacaa tatcgaggac gggagtgtcc aactggccga 6181 ccattatcag cagaatacac ctattggcga cggacctgtc ctcctccccg acaatcatta 6241 cctgagtact caatccgcac tctccaaaga tcccaacgag aagagagatc acatggtatt 6301 actggagttc gtgacagctg ccgggatcac cctcggcatg gatgaactct acaagtgaga 6361 attcgtcgag ggacctaata acttcgtata gcatacatta tacgaagtta tacatgttta 6421 agggttccgg ttccactagg tacaattcga tatcaagctt atcgataatc aacctctgga 6481 ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg 6541 tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt 6601 ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag 6661 gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc 6721 caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga 6781 actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa 6841 ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac 6901 ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct 6961 tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca 7021 gacgagtcgg atctcccttt gggccgcctc cccgcatcga taccgtcgac ctcgatcgag 7081 acctagaaaa acatggagca atcacaagta gcaatacagc agctaccaat gctgattgtg 7141 cctggctaga agcacaagag gaggaggagg tgggttttcc agtcacacct caggtacctt 7201 taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg 7261 gactggaagg gctaattcac tcccaacgaa gacaagatat ccttgatctg tggatctacc 7321 acacacaagg ctacttccct gattggcaga actacacacc agggccaggg atcagatatc 7381 cactgacctt tggatggtgc tacaagctag taccagttga gcaagagaag gtagaagaag 7441 ccaatgaagg agagaacacc cgcttgttac accctgtgag cctgcatggg atggatgacc 7501 cggagagaga agtattagag tggaggtttg acagccgcct agcatttcat cacatggccc 7561 gagagctgca tccggactgt actgggtctc tctggttaga ccagatctga gcctgggagc 7621 tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc 7681 aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt 7741 agtcagtgtg gaaaatctct agcagcatgt gagcaaaagg ccagcaaaag gccaggaacc 7801 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca 7861 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt 7921 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc 7981 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc 8041 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc 8101 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact 8161 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg 8221 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta 8281 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca 8341 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa 8401 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg 8461 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc 8521 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg 8581 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat 8641 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg 8701 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa 8761 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca 8821 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc 8881 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt 8941 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa 9001 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat 9061 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct 9121 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga 9181 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag 9241 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga 9301 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca 9361 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg 9421 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc 9481 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag 9541 gggttccg //