LOCUS Exported 8865 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Protein Barcode (Pro-Code) for vector/cell tracking. Contains U6 trRNA cassette for sgRNA cloning. Enables phenotypic CRISPR screens at a single cell resolution (using cytometry).. ACCESSION . VERSION . KEYWORDS pLKO_HSV.Ollas.C_NGFR SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8865) AUTHORS Wroblewska A, Dhainaut M, Ben-Zvi B, Rose SA, Park ES, Amir ED, Bektesevic A, Baccarini A, Merad M, Rahman AH, Brown BD TITLE Protein Barcodes Enable High-Dimensional Single-Cell CRISPR Screens. JOURNAL Cell. 2018 Nov 1;175(4):1141-1155.e16. doi: 10.1016/j.cell.2018.09.022. Epub 2018 Oct 18. PUBMED 30343902 REFERENCE 2 (bases 1 to 8865) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8865 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 50..290 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 50..70 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 221..240 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 317..392 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" misc_feature 440..557 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 697..1875 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 928..1866 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 1823..1843 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" regulatory 1924..1933 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 2026..2058 /codon_start=1 /product="HSV (herpes simplex virus) epitope tag" /label=HSV tag /translation="QPELAPEDPED" CDS 2077..2118 /codon_start=1 /product="Escherichia coli OmpF Linker and mouse Langerin fusion sequence tag (Park et al., 2008)" /label=OLLAS tag /translation="SGFANELGPRLMGK" misc_feature 2938..3526 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(2991..3011) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3409..3420) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 3601..3834 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 3906..4027 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3943..3962) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 3997..4016 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 4067..4202 /label=SV40 ori /note="SV40 origin of replication" primer_bind 4129..4148 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(4222..4241) /label=T7 /note="T7 promoter, forward primer" promoter complement(4223..4241) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(4251..4268) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(4251..4267) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4260..4282) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 4409..4864 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4496..4515) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4706..4727 /label=F1ori-F /note="F1 origin, forward primer" promoter 4890..4994 /gene="bla" /label=AmpR promoter CDS 4995..5855 /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(5213..5232) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6026..6614 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6515..6534 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 6768..6785 /label=L4440 /note="L4440 vector, forward primer" protein_bind 6902..6923 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 6938..6968 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6976..6992 /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 6981..7003 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 7000..7016 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 7000..7016 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 7035..7055 /label=T3 /note="T3 promoter, forward primer" promoter 7037..7055 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 7083..7309 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" LTR 7310..7490 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 7537..7662 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 8155..8388 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 8573..8617 /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 tcgacggtat cgatcacgag actagtatcg agcggccgcc cccttcaccg agggcctatt 61 tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga taattggaat 121 taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt 181 tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt 241 aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccgg 301 gtcttcgaga agacctgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt 361 atcaacttga aaaagtggca ccgagtcggt gcttttttta attctcgacc tcgagacaaa 421 tggcagtatt catccacaat tttaaaagaa aaggggggat tggggggtac agtgcagggg 481 aaagaatagt agacataata gcaacagaca tacaaactaa agaattacaa aaacaaatta 541 caaaaattca aaattttcgg gtttattaca gggacagcag agatccactt tggccgcggc 601 tcgaggcgat aagctttgca aagatggata aagttttaaa cagagaggaa tctttgcagc 661 taatggacct tctaggtctt gaaaggagtg ggaattggct ccggtgcccg tcagtgggca 721 gagcgcacat cgcccacagt ccccgagaag ttggggggag gggtcggcaa ttgaaccggt 781 gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt 841 tttcccgagg gtgggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt 901 cgcaacgggt ttgccgccag aacacaggta agtgccgtgt gtggttcccg cgggcctggc 961 ctctttacgg gttatggccc ttgcgtgcct tgaattactt ccacctggct gcagtacgtg 1021 attcttgatc ccgagcttcg ggttggaagt gggtgggaga gttcgaggcc ttgcgcttaa 1081 ggagcccctt cgcctcgtgc ttgagttgag gcctggcctg ggcgctgggg ccgccgcgtg 1141 cgaatctggt ggcaccttcg cgcctgtctc gctgctttcg ataagtctct agccatttaa 1201 aatttttgat gacctgctgc gacgcttttt ttctggcaag atagtcttgt aaatgcgggc 1261 caagatctgc acactggtat ttcggttttt ggggccgcgg gcggcgacgg ggcccgtgcg 1321 tcccagcgca catgttcggc gaggcggggc ctgcgagcgc ggccaccgag aatcggacgg 1381 gggtagtctc aagctggccg gcctgctctg gtgcctggcc tcgcgccgcc gtgtatcgcc 1441 ccgccctggg cggcaaggct ggcccggtcg gcaccagttg cgtgagcgga aagatggccg 1501 cttcccggcc ctgctgcagg gagctcaaaa tggaggacgc ggcgctcggg agagcgggcg 1561 ggtgagtcac ccacacaaag gaaaagggcc tttccgtcct cagccgtcgc ttcatgtgac 1621 tccacggagt accgggcgcc gtccaggcac ctcgattagt tctcgagctt ttggagtacg 1681 tcgtctttag gttgggggga ggggttttat gcgatggagt ttccccacac tgagtgggtg 1741 gagactgaag ttaggccagc ttggcacttg atgtaattct ccttggaatt tgcccttttt 1801 gagtttggat cttggttcat tctcaagcct cagacagtgg ttcaaagttt ttttcttcca 1861 tttcaggtgt cgtgaggaat tctgcagtcg atcgacggta ccgcgggccc gggatccacg 1921 gtcgccacca tgggggcagg tgccaccggc cgcgccatgg acgggccgcg cctgctgctg 1981 ttgctgcttc tgggggtgtc ccttggaggt gccaaggagg catgccagcc cgagctggcc 2041 cccgaggacc ccgaggacgg aggaggcggg ggagggagcg gcttcgccaa cgagctgggc 2101 cccaggctga tgggcaagtg cggaggaggc gggggagggg aggaccaggt ggaccccagg 2161 ctgatcgacg gcaagcccac aggcctgtac acacacagcg gtgagtgctg caaagcctgc 2221 aacctgggcg agggtgtggc ccagccttgt ggagccaacc agaccgtgtg tgagccctgc 2281 ctggacagcg tgacgttctc cgacgtggtg agcgcgaccg agccgtgcaa gccgtgcacc 2341 gagtgcgtgg ggctccagag catgtcggcg ccgtgcgtgg aggccgacga cgccgtgtgc 2401 cgctgcgcct acggctacta ccaggatgag acgactgggc gctgcgaggc gtgccgcgtg 2461 tgcgaggcgg gctcgggcct cgtgttctcc tgccaggaca agcagaacac cgtgtgcgag 2521 gagtgccccg acggcacgta ttccgacgag gccaaccacg tggacccgtg cctgccctgc 2581 accgtgtgcg aggacaccga gcgccagctc cgcgagtgca cacgctgggc cgacgccgag 2641 tgcgaggaga tccctggccg ttggattaca cggtccacac ccccagaggg ctcggacagc 2701 acagccccca gcacccagga gcctgaggca cctccagaac aagacctcat agccagcacg 2761 gtggcaggtg tggtgaccac agtgatgggc agctcccagc ccgtggtgac ccgaggcacc 2821 accgacaacc tcatccctgt ctattgctcc atcctggctg ctgtggttgt gggccttgtg 2881 gcctacatag ccttcaagag gtggaacagg ggtatcctct agagcggccg cgtcgacaat 2941 caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct 3001 tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg 3061 gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg 3121 cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt 3181 tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt 3241 gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg 3301 ggcactgaca attccgtggt gttgtcgggg aagctgacgt cctttccatg gctgctcgcc 3361 tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat 3421 ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcatcttcgc 3481 cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcctgg gtacctttaa 3541 gaccaatgac ttacaaggca gctgtagatc ttagccactt tttaaaagaa aaggggggac 3601 tggaagggct aattcactcc caacgaagat aagatctgct ttttgcttgt actgggtctc 3661 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta 3721 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact 3781 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtagta 3841 gttcatgtca tcttattatt cagtatttat aacttgcaaa gaaatgaata tcagagagtg 3901 agaggaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt 3961 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg 4021 tatcttatca tgtctggctc tagctatccc gcccctaact ccgcccatcc cgcccctaac 4081 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga 4141 ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg 4201 cctagggacg tacccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc 4261 gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca 4321 catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa 4381 cagttgcgca gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg 4441 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct 4501 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat 4561 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt 4621 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg 4681 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac 4741 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta 4801 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca 4861 atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa 4921 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt 4981 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg 5041 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag 5101 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg 5161 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg 5221 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt 5281 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga 5341 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac 5401 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc 5461 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc 5521 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac 5581 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag 5641 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg 5701 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta 5761 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg 5821 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata 5881 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt 5941 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc 6001 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct 6061 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa 6121 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag 6181 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc 6241 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg 6301 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca 6361 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat 6421 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg 6481 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc 6541 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc 6601 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc 6661 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg 6721 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga 6781 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc 6841 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa 6901 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc 6961 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg 7021 attacgccaa gcgcgcaatt aaccctcact aaagggaaca aaagctggag ctgcaagctt 7081 aatgtagtct tatgcaatac tcttgtagtc ttgcaacatg gtaacgatga gttagcaaca 7141 tgccttacaa ggagagaaaa agcaccgtgc atggcgattg gtggaagtaa ggtggtacga 7201 tcgtgcctta ttaggaaggc aacagacggg tctgacatgg attggacgaa ccactgaatt 7261 gccgcattgc agagatattg tatttaagtg cctagctcga tacataaacg ggtctctctg 7321 gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc 7381 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg 7441 taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg 7501 aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag gactcggctt 7561 gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg 7621 actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga 7681 attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa atataaatta 7741 aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta 7801 gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga 7861 tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt gcatcaaagg 7921 atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt 7981 aagaccaccg cacagcaagc ggccgctgat cttcagacct ggaggaggag atatgaggga 8041 caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat taggagtagc 8101 acccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg gaataggagc 8161 tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct 8221 gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag 8281 ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca 8341 ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg 8401 ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt ggagtaataa 8461 atctctggaa cagatttgga atcacacgac ctggatggag tgggacagag aaattaacaa 8521 ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga 8581 acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa 8641 ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat 8701 agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt 8761 tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg 8821 tggagagaga gacagagaca gatccattcg attagtgaac ggatc //