LOCUS Exported 8838 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.FLAG_NGFR SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8838) 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 8838) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8838 /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 2062..2103 /codon_start=1 /product="Escherichia coli OmpF Linker and mouse Langerin fusion sequence tag (Park et al., 2008)" /label=OLLAS tag /translation="SGFANELGPRLMGK" CDS 2125..2148 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" misc_feature 2911..3499 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(2964..2984) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3382..3393) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 3574..3807 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 3879..4000 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3916..3935) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 3970..3989 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 4040..4175 /label=SV40 ori /note="SV40 origin of replication" primer_bind 4102..4121 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(4195..4214) /label=T7 /note="T7 promoter, forward primer" promoter complement(4196..4214) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(4224..4241) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(4224..4240) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4233..4255) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 4382..4837 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4469..4488) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4679..4700 /label=F1ori-F /note="F1 origin, forward primer" promoter 4863..4967 /gene="bla" /label=AmpR promoter CDS 4968..5828 /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(5186..5205) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5999..6587 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6488..6507 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 6741..6758 /label=L4440 /note="L4440 vector, forward primer" protein_bind 6875..6896 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 6911..6941 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6949..6965 /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 6954..6976 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 6973..6989 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 6973..6989 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 7008..7028 /label=T3 /note="T3 promoter, forward primer" promoter 7010..7028 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 7056..7282 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" LTR 7283..7463 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 7510..7635 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 8128..8361 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 8546..8590 /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 catgcgacac ctatcggtac 2041 attggaggag gcgggggagg gagcggcttc gccaacgagc tgggccccag gctgatgggc 2101 aagtgcggag gaggcggggg aggggactac aaagacgatg acgacaagcc cacaggcctg 2161 tacacacaca gcggtgagtg ctgcaaagcc tgcaacctgg gcgagggtgt ggcccagcct 2221 tgtggagcca accagaccgt gtgtgagccc tgcctggaca gcgtgacgtt ctccgacgtg 2281 gtgagcgcga ccgagccgtg caagccgtgc accgagtgcg tggggctcca gagcatgtcg 2341 gcgccgtgcg tggaggccga cgacgccgtg tgccgctgcg cctacggcta ctaccaggat 2401 gagacgactg ggcgctgcga ggcgtgccgc gtgtgcgagg cgggctcggg cctcgtgttc 2461 tcctgccagg acaagcagaa caccgtgtgc gaggagtgcc ccgacggcac gtattccgac 2521 gaggccaacc acgtggaccc gtgcctgccc tgcaccgtgt gcgaggacac cgagcgccag 2581 ctccgcgagt gcacacgctg ggccgacgcc gagtgcgagg agatccctgg ccgttggatt 2641 acacggtcca cacccccaga gggctcggac agcacagccc ccagcaccca ggagcctgag 2701 gcacctccag aacaagacct catagccagc acggtggcag gtgtggtgac cacagtgatg 2761 ggcagctccc agcccgtggt gacccgaggc accaccgaca acctcatccc tgtctattgc 2821 tccatcctgg ctgctgtggt tgtgggcctt gtggcctaca tagccttcaa gaggtggaac 2881 aggggtatcc tctagagcgg ccgcgtcgac aatcaacctc tggattacaa aatttgtgaa 2941 agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta 3001 atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa 3061 tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg 3121 tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc 3181 ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc 3241 cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg 3301 gggaagctga cgtcctttcc atggctgctc gcctgtgttg ccacctggat tctgcgcggg 3361 acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg 3421 ctgccggctc tgcggcctct tccgcatctt cgccttcgcc ctcagacgag tcggatctcc 3481 ctttgggccg cctccccgcc tgggtacctt taagaccaat gacttacaag gcagctgtag 3541 atcttagcca ctttttaaaa gaaaaggggg gactggaagg gctaattcac tcccaacgaa 3601 gataagatct gctttttgct tgtactgggt ctctctggtt agaccagatc tgagcctggg 3661 agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc 3721 ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct 3781 tttagtcagt gtggaaaatc tctagcagta gtagttcatg tcatcttatt attcagtatt 3841 tataacttgc aaagaaatga atatcagaga gtgagaggaa cttgtttatt gcagcttata 3901 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc 3961 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg ctctagctat 4021 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc 4081 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct 4141 attccagaag tagtgaggag gcttttttgg aggcctaggg acgtacccaa ttcgccctat 4201 agtgagtcgt attacgcgcg ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac 4261 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat 4321 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg 4381 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc 4441 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc 4501 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt 4561 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg 4621 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt 4681 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta 4741 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt 4801 aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg 4861 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga 4921 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac 4981 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc 5041 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca 5101 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc 5161 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg 5221 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac 5281 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca 5341 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg 5401 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac 5461 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg 5521 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat 5581 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg 5641 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg 5701 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc 5761 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc 5821 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt 5881 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt 5941 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt 6001 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag 6061 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca 6121 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca 6181 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg 6241 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg 6301 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct 6361 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga 6421 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc 6481 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg 6541 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg 6601 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt 6661 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc 6721 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac 6781 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc 6841 ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg 6901 caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat 6961 aacaatttca cacaggaaac agctatgacc atgattacgc caagcgcgca attaaccctc 7021 actaaaggga acaaaagctg gagctgcaag cttaatgtag tcttatgcaa tactcttgta 7081 gtcttgcaac atggtaacga tgagttagca acatgcctta caaggagaga aaaagcaccg 7141 tgcatggcga ttggtggaag taaggtggta cgatcgtgcc ttattaggaa ggcaacagac 7201 gggtctgaca tggattggac gaaccactga attgccgcat tgcagagata ttgtatttaa 7261 gtgcctagct cgatacataa acgggtctct ctggttagac cagatctgag cctgggagct 7321 ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca 7381 agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta 7441 gtcagtgtgg aaaatctcta gcagtggcgc ccgaacaggg acttgaaagc gaaagggaaa 7501 ccagaggagc tctctcgacg caggactcgg cttgctgaag cgcgcacggc aagaggcgag 7561 gggcggcgac tggtgagtac gccaaaaatt ttgactagcg gaggctagaa ggagagagat 7621 gggtgcgaga gcgtcagtat taagcggggg agaattagat cgcgatggga aaaaattcgg 7681 ttaaggccag ggggaaagaa aaaatataaa ttaaaacata tagtatgggc aagcagggag 7741 ctagaacgat tcgcagttaa tcctggcctg ttagaaacat cagaaggctg tagacaaata 7801 ctgggacagc tacaaccatc ccttcagaca ggatcagaag aacttagatc attatataat 7861 acagtagcaa ccctctattg tgtgcatcaa aggatagaga taaaagacac caaggaagct 7921 ttagacaaga tagaggaaga gcaaaacaaa agtaagacca ccgcacagca agcggccgct 7981 gatcttcaga cctggaggag gagatatgag ggacaattgg agaagtgaat tatataaata 8041 taaagtagta aaaattgaac cattaggagt agcacccacc aaggcaaaga gaagagtggt 8101 gcagagagaa aaaagagcag tgggaatagg agctttgttc cttgggttct tgggagcagc 8161 aggaagcact atgggcgcag cgtcaatgac gctgacggta caggccagac aattattgtc 8221 tggtatagtg cagcagcaga acaatttgct gagggctatt gaggcgcaac agcatctgtt 8281 gcaactcaca gtctggggca tcaagcagct ccaggcaaga atcctggctg tggaaagata 8341 cctaaaggat caacagctcc tggggatttg gggttgctct ggaaaactca tttgcaccac 8401 tgctgtgcct tggaatgcta gttggagtaa taaatctctg gaacagattt ggaatcacac 8461 gacctggatg gagtgggaca gagaaattaa caattacaca agcttaatac actccttaat 8521 tgaagaatcg caaaaccagc aagaaaagaa tgaacaagaa ttattggaat tagataaatg 8581 ggcaagtttg tggaattggt ttaacataac aaattggctg tggtatataa aattattcat 8641 aatgatagta ggaggcttgg taggtttaag aatagttttt gctgtacttt ctatagtgaa 8701 tagagttagg cagggatatt caccattatc gtttcagacc cacctcccaa ccccgagggg 8761 acccgacagg cccgaaggaa tagaagaaga aggtggagag agagacagag acagatccat 8821 tcgattagtg aacggatc //