LOCUS Exported 8844 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_FLAG.VSVg.C_NGFR SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8844) 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 8844) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8844 /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..2049 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" CDS 2068..2100 /codon_start=1 /product="epitope tag from vesicular stomatitis virus G protein" /label=VSV-G tag /translation="YTDIEMNRLGK" misc_feature 2917..3505 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(2970..2990) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3388..3399) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 3580..3813 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 3885..4006 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3922..3941) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 3976..3995 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 4046..4181 /label=SV40 ori /note="SV40 origin of replication" primer_bind 4108..4127 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(4201..4220) /label=T7 /note="T7 promoter, forward primer" promoter complement(4202..4220) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(4230..4247) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(4230..4246) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4239..4261) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 4388..4843 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4475..4494) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4685..4706 /label=F1ori-F /note="F1 origin, forward primer" promoter 4869..4973 /gene="bla" /label=AmpR promoter CDS 4974..5834 /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(5192..5211) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6005..6593 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6494..6513 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 6747..6764 /label=L4440 /note="L4440 vector, forward primer" protein_bind 6881..6902 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 6917..6947 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6955..6971 /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 6960..6982 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 6979..6995 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 6979..6995 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 7014..7034 /label=T3 /note="T3 promoter, forward primer" promoter 7016..7034 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 7062..7288 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" LTR 7289..7469 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 7516..7641 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 8134..8367 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 8552..8596 /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 catgcgacta caaagacgat 2041 gacgacaagg gaggaggcgg gggagggtat acagatatcg aaatgaatag actcggaaag 2101 ggaggaggcg ggggagggga ggaccaggtg gaccccaggc tgatcgacgg caagcccaca 2161 ggcctgtaca cacacagcgg tgagtgctgc aaagcctgca acctgggcga gggtgtggcc 2221 cagccttgtg gagccaacca gaccgtgtgt gagccctgcc tggacagcgt gacgttctcc 2281 gacgtggtga gcgcgaccga gccgtgcaag ccgtgcaccg agtgcgtggg gctccagagc 2341 atgtcggcgc cgtgcgtgga ggccgacgac gccgtgtgcc gctgcgccta cggctactac 2401 caggatgaga cgactgggcg ctgcgaggcg tgccgcgtgt gcgaggcggg ctcgggcctc 2461 gtgttctcct gccaggacaa gcagaacacc gtgtgcgagg agtgccccga cggcacgtat 2521 tccgacgagg ccaaccacgt ggacccgtgc ctgccctgca ccgtgtgcga ggacaccgag 2581 cgccagctcc gcgagtgcac acgctgggcc gacgccgagt gcgaggagat ccctggccgt 2641 tggattacac ggtccacacc cccagagggc tcggacagca cagcccccag cacccaggag 2701 cctgaggcac ctccagaaca agacctcata gccagcacgg tggcaggtgt ggtgaccaca 2761 gtgatgggca gctcccagcc cgtggtgacc cgaggcacca ccgacaacct catccctgtc 2821 tattgctcca tcctggctgc tgtggttgtg ggccttgtgg cctacatagc cttcaagagg 2881 tggaacaggg gtatcctcta gagcggccgc gtcgacaatc aacctctgga ttacaaaatt 2941 tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct 3001 gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg 3061 tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc 3121 gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt 3181 cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc 3241 gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg 3301 ttgtcgggga agctgacgtc ctttccatgg ctgctcgcct gtgttgccac ctggattctg 3361 cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc 3421 ggcctgctgc cggctctgcg gcctcttccg catcttcgcc ttcgccctca gacgagtcgg 3481 atctcccttt gggccgcctc cccgcctggg tacctttaag accaatgact tacaaggcag 3541 ctgtagatct tagccacttt ttaaaagaaa aggggggact ggaagggcta attcactccc 3601 aacgaagata agatctgctt tttgcttgta ctgggtctct ctggttagac cagatctgag 3661 cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt 3721 gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca 3781 gaccctttta gtcagtgtgg aaaatctcta gcagtagtag ttcatgtcat cttattattc 3841 agtatttata acttgcaaag aaatgaatat cagagagtga gaggaacttg tttattgcag 3901 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt 3961 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggctct 4021 agctatcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc 4081 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc 4141 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctagggacgt acccaattcg 4201 ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg 4261 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg 4321 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc 4381 gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc 4441 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt 4501 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc 4561 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt 4621 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt 4681 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt 4741 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa 4801 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg 4861 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc 4921 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta 4981 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg 5041 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg 5101 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac 5161 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg 5221 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt 5281 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg 5341 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac 5401 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt 5461 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag 5521 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc 5581 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc 5641 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta 5701 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg 5761 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga 5821 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac 5881 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa 5941 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat 6001 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc 6061 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg 6121 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc 6181 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg 6241 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg 6301 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa 6361 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg 6421 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga 6481 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct 6541 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca 6601 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc 6661 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg 6721 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc 6781 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca 6841 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc 6901 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga 6961 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag cgcgcaatta 7021 accctcacta aagggaacaa aagctggagc tgcaagctta atgtagtctt atgcaatact 7081 cttgtagtct tgcaacatgg taacgatgag ttagcaacat gccttacaag gagagaaaaa 7141 gcaccgtgca tggcgattgg tggaagtaag gtggtacgat cgtgccttat taggaaggca 7201 acagacgggt ctgacatgga ttggacgaac cactgaattg ccgcattgca gagatattgt 7261 atttaagtgc ctagctcgat acataaacgg gtctctctgg ttagaccaga tctgagcctg 7321 ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt 7381 gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc 7441 cttttagtca gtgtggaaaa tctctagcag tggcgcccga acagggactt gaaagcgaaa 7501 gggaaaccag aggagctctc tcgacgcagg actcggcttg ctgaagcgcg cacggcaaga 7561 ggcgaggggc ggcgactggt gagtacgcca aaaattttga ctagcggagg ctagaaggag 7621 agagatgggt gcgagagcgt cagtattaag cgggggagaa ttagatcgcg atgggaaaaa 7681 attcggttaa ggccaggggg aaagaaaaaa tataaattaa aacatatagt atgggcaagc 7741 agggagctag aacgattcgc agttaatcct ggcctgttag aaacatcaga aggctgtaga 7801 caaatactgg gacagctaca accatccctt cagacaggat cagaagaact tagatcatta 7861 tataatacag tagcaaccct ctattgtgtg catcaaagga tagagataaa agacaccaag 7921 gaagctttag acaagataga ggaagagcaa aacaaaagta agaccaccgc acagcaagcg 7981 gccgctgatc ttcagacctg gaggaggaga tatgagggac aattggagaa gtgaattata 8041 taaatataaa gtagtaaaaa ttgaaccatt aggagtagca cccaccaagg caaagagaag 8101 agtggtgcag agagaaaaaa gagcagtggg aataggagct ttgttccttg ggttcttggg 8161 agcagcagga agcactatgg gcgcagcgtc aatgacgctg acggtacagg ccagacaatt 8221 attgtctggt atagtgcagc agcagaacaa tttgctgagg gctattgagg cgcaacagca 8281 tctgttgcaa ctcacagtct ggggcatcaa gcagctccag gcaagaatcc tggctgtgga 8341 aagataccta aaggatcaac agctcctggg gatttggggt tgctctggaa aactcatttg 8401 caccactgct gtgccttgga atgctagttg gagtaataaa tctctggaac agatttggaa 8461 tcacacgacc tggatggagt gggacagaga aattaacaat tacacaagct taatacactc 8521 cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa caagaattat tggaattaga 8581 taaatgggca agtttgtgga attggtttaa cataacaaat tggctgtggt atataaaatt 8641 attcataatg atagtaggag gcttggtagg tttaagaata gtttttgctg tactttctat 8701 agtgaataga gttaggcagg gatattcacc attatcgttt cagacccacc tcccaacccc 8761 gaggggaccc gacaggcccg aaggaataga agaagaaggt ggagagagag acagagacag 8821 atccattcga ttagtgaacg gatc //