LOCUS Exported 8841 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_AU1.Ollas.NWS_NGFR SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8841) 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 8841) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8841 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 813..833 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" regulatory 914..923 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1052..1093 /codon_start=1 /product="Escherichia coli OmpF Linker and mouse Langerin fusion sequence tag (Park et al., 2008)" /label=OLLAS tag /translation="SGFANELGPRLMGK" CDS 1118..1141 /codon_start=1 /product="peptide that binds Strep-Tactin(R), an engineered form of streptavidin" /label=Strep-Tag II /translation="WSHPQFEK" misc_feature 1904..2492 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(1957..1977) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(2375..2386) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 2567..2800 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 2872..2993 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2909..2928) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 2963..2982 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 3033..3168 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3095..3114 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(3188..3207) /label=T7 /note="T7 promoter, forward primer" promoter complement(3189..3207) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(3217..3234) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(3217..3233) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3226..3248) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 3375..3830 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3462..3481) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3672..3693 /label=F1ori-F /note="F1 origin, forward primer" promoter 3856..3960 /gene="bla" /label=AmpR promoter CDS 3961..4821 /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(4179..4198) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 4992..5580 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5481..5500 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 5734..5751 /label=L4440 /note="L4440 vector, forward primer" protein_bind 5868..5889 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 5904..5934 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5942..5958 /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 5947..5969 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 5966..5982 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 5966..5982 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 6001..6021 /label=T3 /note="T3 promoter, forward primer" promoter 6003..6021 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 6049..6275 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" LTR 6276..6456 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 6503..6628 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 7121..7354 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 7539..7583 /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 7881..8121 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 7881..7901 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 8052..8071 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 8148..8223 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" misc_feature 8271..8388 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 8528..865 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 8759..856 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" ORIGIN 1 gcagtacgtg attcttgatc ccgagcttcg ggttggaagt gggtgggaga gttcgaggcc 61 ttgcgcttaa ggagcccctt cgcctcgtgc ttgagttgag gcctggcctg ggcgctgggg 121 ccgccgcgtg cgaatctggt ggcaccttcg cgcctgtctc gctgctttcg ataagtctct 181 agccatttaa aatttttgat gacctgctgc gacgcttttt ttctggcaag atagtcttgt 241 aaatgcgggc caagatctgc acactggtat ttcggttttt ggggccgcgg gcggcgacgg 301 ggcccgtgcg tcccagcgca catgttcggc gaggcggggc ctgcgagcgc ggccaccgag 361 aatcggacgg gggtagtctc aagctggccg gcctgctctg gtgcctggcc tcgcgccgcc 421 gtgtatcgcc ccgccctggg cggcaaggct ggcccggtcg gcaccagttg cgtgagcgga 481 aagatggccg cttcccggcc ctgctgcagg gagctcaaaa tggaggacgc ggcgctcggg 541 agagcgggcg ggtgagtcac ccacacaaag gaaaagggcc tttccgtcct cagccgtcgc 601 ttcatgtgac tccacggagt accgggcgcc gtccaggcac ctcgattagt tctcgagctt 661 ttggagtacg tcgtctttag gttgggggga ggggttttat gcgatggagt ttccccacac 721 tgagtgggtg gagactgaag ttaggccagc ttggcacttg atgtaattct ccttggaatt 781 tgcccttttt gagtttggat cttggttcat tctcaagcct cagacagtgg ttcaaagttt 841 ttttcttcca tttcaggtgt cgtgaggaat tctgcagtcg atcgacggta ccgcgggccc 901 gggatccacg gtcgccacca tgggggcagg tgccaccggc cgcgccatgg acgggccgcg 961 cctgctgctg ttgctgcttc tgggggtgtc ccttggaggt gccaaggagg catgcgacac 1021 ctatcggtac attggaggag gcgggggagg gagcggcttc gccaacgagc tgggccccag 1081 gctgatgggc aagtgcggag gaggcggggg agggaactgg tcccaccccc aattcgagaa 1141 acccacaggc ctgtacacac acagcggtga gtgctgcaaa gcctgcaacc tgggcgaggg 1201 tgtggcccag ccttgtggag ccaaccagac cgtgtgtgag ccctgcctgg acagcgtgac 1261 gttctccgac gtggtgagcg cgaccgagcc gtgcaagccg tgcaccgagt gcgtggggct 1321 ccagagcatg tcggcgccgt gcgtggaggc cgacgacgcc gtgtgccgct gcgcctacgg 1381 ctactaccag gatgagacga ctgggcgctg cgaggcgtgc cgcgtgtgcg aggcgggctc 1441 gggcctcgtg ttctcctgcc aggacaagca gaacaccgtg tgcgaggagt gccccgacgg 1501 cacgtattcc gacgaggcca accacgtgga cccgtgcctg ccctgcaccg tgtgcgagga 1561 caccgagcgc cagctccgcg agtgcacacg ctgggccgac gccgagtgcg aggagatccc 1621 tggccgttgg attacacggt ccacaccccc agagggctcg gacagcacag cccccagcac 1681 ccaggagcct gaggcacctc cagaacaaga cctcatagcc agcacggtgg caggtgtggt 1741 gaccacagtg atgggcagct cccagcccgt ggtgacccga ggcaccaccg acaacctcat 1801 ccctgtctat tgctccatcc tggctgctgt ggttgtgggc cttgtggcct acatagcctt 1861 caagaggtgg aacaggggta tcctctagag cggccgcgtc gacaatcaac ctctggatta 1921 caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg 1981 atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc 2041 ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca 2101 acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac 2161 cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact 2221 catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc 2281 cgtggtgttg tcggggaagc tgacgtcctt tccatggctg ctcgcctgtg ttgccacctg 2341 gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc 2401 ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcat cttcgccttc gccctcagac 2461 gagtcggatc tccctttggg ccgcctcccc gcctgggtac ctttaagacc aatgacttac 2521 aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt 2581 cactcccaac gaagataaga tctgcttttt gcttgtactg ggtctctctg gttagaccag 2641 atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc 2701 ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga 2761 tccctcagac ccttttagtc agtgtggaaa atctctagca gtagtagttc atgtcatctt 2821 attattcagt atttataact tgcaaagaaa tgaatatcag agagtgagag gaacttgttt 2881 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca 2941 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc 3001 tggctctagc tatcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg 3061 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct 3121 cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta gggacgtacc 3181 caattcgccc tatagtgagt cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg 3241 tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc 3301 cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct 3361 gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac 3421 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc 3481 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt 3541 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg 3601 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac 3661 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta 3721 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat 3781 ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact 3841 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg 3901 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt 3961 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct 4021 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca 4081 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc 4141 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc 4201 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg 4261 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta 4321 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc 4381 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt 4441 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg 4501 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct 4561 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc 4621 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct 4681 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac 4741 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc 4801 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat 4861 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg 4921 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc 4981 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa 5041 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag 5101 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta 5161 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta 5221 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag 5281 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg 5341 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg 5401 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag 5461 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc 5521 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa 5581 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg 5641 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct 5701 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa 5761 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg 5821 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag 5881 ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga 5941 attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta cgccaagcgc 6001 gcaattaacc ctcactaaag ggaacaaaag ctggagctgc aagcttaatg tagtcttatg 6061 caatactctt gtagtcttgc aacatggtaa cgatgagtta gcaacatgcc ttacaaggag 6121 agaaaaagca ccgtgcatgg cgattggtgg aagtaaggtg gtacgatcgt gccttattag 6181 gaaggcaaca gacgggtctg acatggattg gacgaaccac tgaattgccg cattgcagag 6241 atattgtatt taagtgccta gctcgataca taaacgggtc tctctggtta gaccagatct 6301 gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc 6361 cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc 6421 tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa 6481 agcgaaaggg aaaccagagg agctctctcg acgcaggact cggcttgctg aagcgcgcac 6541 ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta gcggaggcta 6601 gaaggagaga gatgggtgcg agagcgtcag tattaagcgg gggagaatta gatcgcgatg 6661 ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat aaattaaaac atatagtatg 6721 ggcaagcagg gagctagaac gattcgcagt taatcctggc ctgttagaaa catcagaagg 6781 ctgtagacaa atactgggac agctacaacc atcccttcag acaggatcag aagaacttag 6841 atcattatat aatacagtag caaccctcta ttgtgtgcat caaaggatag agataaaaga 6901 caccaaggaa gctttagaca agatagagga agagcaaaac aaaagtaaga ccaccgcaca 6961 gcaagcggcc gctgatcttc agacctggag gaggagatat gagggacaat tggagaagtg 7021 aattatataa atataaagta gtaaaaattg aaccattagg agtagcaccc accaaggcaa 7081 agagaagagt ggtgcagaga gaaaaaagag cagtgggaat aggagctttg ttccttgggt 7141 tcttgggagc agcaggaagc actatgggcg cagcgtcaat gacgctgacg gtacaggcca 7201 gacaattatt gtctggtata gtgcagcagc agaacaattt gctgagggct attgaggcgc 7261 aacagcatct gttgcaactc acagtctggg gcatcaagca gctccaggca agaatcctgg 7321 ctgtggaaag atacctaaag gatcaacagc tcctggggat ttggggttgc tctggaaaac 7381 tcatttgcac cactgctgtg ccttggaatg ctagttggag taataaatct ctggaacaga 7441 tttggaatca cacgacctgg atggagtggg acagagaaat taacaattac acaagcttaa 7501 tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg 7561 aattagataa atgggcaagt ttgtggaatt ggtttaacat aacaaattgg ctgtggtata 7621 taaaattatt cataatgata gtaggaggct tggtaggttt aagaatagtt tttgctgtac 7681 tttctatagt gaatagagtt aggcagggat attcaccatt atcgtttcag acccacctcc 7741 caaccccgag gggacccgac aggcccgaag gaatagaaga agaaggtgga gagagagaca 7801 gagacagatc cattcgatta gtgaacggat ctcgacggta tcgatcacga gactagtatc 7861 gagcggccgc ccccttcacc gagggcctat ttcccatgat tccttcatat ttgcatatac 7921 gatacaaggc tgttagagag ataattggaa ttaatttgac tgtaaacaca aagatattag 7981 tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat 8041 gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt 8101 tatatatctt gtggaaagga cgaaacaccg ggtcttcgag aagacctgtt ttagagctag 8161 aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg 8221 tgcttttttt aattctcgac ctcgagacaa atggcagtat tcatccacaa ttttaaaaga 8281 aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac 8341 atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaattttcg ggtttattac 8401 agggacagca gagatccact ttggccgcgg ctcgaggcga taagctttgc aaagatggat 8461 aaagttttaa acagagagga atctttgcag ctaatggacc ttctaggtct tgaaaggagt 8521 gggaattggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa 8581 gttgggggga ggggtcggca attgaaccgg tgcctagaga aggtggcgcg gggtaaactg 8641 ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat 8701 aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacaggt 8761 aagtgccgtg tgtggttccc gcgggcctgg cctctttacg ggttatggcc cttgcgtgcc 8821 ttgaattact tccacctggc t //