LOCUS Exported 8862 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_V5.VSVg.C_NGFR SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8862) 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 8862) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8862 /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..2067 /codon_start=1 /product="epitope tag from simian virus 5" /label=V5 tag /translation="GKPIPNPLLGLDST" CDS 2086..2118 /codon_start=1 /product="epitope tag from vesicular stomatitis virus G protein" /label=VSV-G tag /translation="YTDIEMNRLGK" misc_feature 2935..3523 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(2988..3008) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3406..3417) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 3598..3831 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 3903..4024 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3940..3959) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 3994..4013 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 4064..4199 /label=SV40 ori /note="SV40 origin of replication" primer_bind 4126..4145 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(4219..4238) /label=T7 /note="T7 promoter, forward primer" promoter complement(4220..4238) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(4248..4265) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(4248..4264) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4257..4279) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 4406..4861 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4493..4512) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4703..4724 /label=F1ori-F /note="F1 origin, forward primer" promoter 4887..4991 /gene="bla" /label=AmpR promoter CDS 4992..5852 /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(5210..5229) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6023..6611 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6512..6531 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 6765..6782 /label=L4440 /note="L4440 vector, forward primer" protein_bind 6899..6920 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 6935..6965 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6973..6989 /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 6978..7000 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 6997..7013 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 6997..7013 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 7032..7052 /label=T3 /note="T3 promoter, forward primer" promoter 7034..7052 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 7080..7306 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" LTR 7307..7487 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 7534..7659 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 8152..8385 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 8570..8614 /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 catgcggcaa accaattcca 2041 aacccactgc tgggactgga ttccacagga ggaggcgggg gagggtatac agatatcgaa 2101 atgaatagac tcggaaaggg aggaggcggg ggaggggagg accaggtgga ccccaggctg 2161 atcgacggca agcccacagg cctgtacaca cacagcggtg agtgctgcaa agcctgcaac 2221 ctgggcgagg gtgtggccca gccttgtgga gccaaccaga ccgtgtgtga gccctgcctg 2281 gacagcgtga cgttctccga cgtggtgagc gcgaccgagc cgtgcaagcc gtgcaccgag 2341 tgcgtggggc tccagagcat gtcggcgccg tgcgtggagg ccgacgacgc cgtgtgccgc 2401 tgcgcctacg gctactacca ggatgagacg actgggcgct gcgaggcgtg ccgcgtgtgc 2461 gaggcgggct cgggcctcgt gttctcctgc caggacaagc agaacaccgt gtgcgaggag 2521 tgccccgacg gcacgtattc cgacgaggcc aaccacgtgg acccgtgcct gccctgcacc 2581 gtgtgcgagg acaccgagcg ccagctccgc gagtgcacac gctgggccga cgccgagtgc 2641 gaggagatcc ctggccgttg gattacacgg tccacacccc cagagggctc ggacagcaca 2701 gcccccagca cccaggagcc tgaggcacct ccagaacaag acctcatagc cagcacggtg 2761 gcaggtgtgg tgaccacagt gatgggcagc tcccagcccg tggtgacccg aggcaccacc 2821 gacaacctca tccctgtcta ttgctccatc ctggctgctg tggttgtggg ccttgtggcc 2881 tacatagcct tcaagaggtg gaacaggggt atcctctaga gcggccgcgt cgacaatcaa 2941 cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt 3001 acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct 3061 ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc 3121 gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg 3181 ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc 3241 acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc 3301 actgacaatt ccgtggtgtt gtcggggaag ctgacgtcct ttccatggct gctcgcctgt 3361 gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca 3421 gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgca tcttcgcctt 3481 cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcctgggta cctttaagac 3541 caatgactta caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactgg 3601 aagggctaat tcactcccaa cgaagataag atctgctttt tgcttgtact gggtctctct 3661 ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc 3721 ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg 3781 gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc agtagtagtt 3841 catgtcatct tattattcag tatttataac ttgcaaagaa atgaatatca gagagtgaga 3901 ggaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca 3961 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat 4021 cttatcatgt ctggctctag ctatcccgcc cctaactccg cccatcccgc ccctaactcc 4081 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc 4141 cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct 4201 agggacgtac ccaattcgcc ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt 4261 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat 4321 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag 4381 ttgcgcagcc tgaatggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt 4441 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc 4501 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg 4561 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat 4621 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg 4681 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct 4741 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa 4801 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt 4861 taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac 4921 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa 4981 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat 5041 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc 5101 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga 5161 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg 5221 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc 5281 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag 5341 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc 5401 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg 5461 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg 5521 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac 5581 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac 5641 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg 5701 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg 5761 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg 5821 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac 5881 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg 5941 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg 6001 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc 6061 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc 6121 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt 6181 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc 6241 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact 6301 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac 6361 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag 6421 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg 6481 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg 6541 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga 6601 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt 6661 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct 6721 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg 6781 aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt 6841 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta 6901 atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta 6961 tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt 7021 acgccaagcg cgcaattaac cctcactaaa gggaacaaaa gctggagctg caagcttaat 7081 gtagtcttat gcaatactct tgtagtcttg caacatggta acgatgagtt agcaacatgc 7141 cttacaagga gagaaaaagc accgtgcatg gcgattggtg gaagtaaggt ggtacgatcg 7201 tgccttatta ggaaggcaac agacgggtct gacatggatt ggacgaacca ctgaattgcc 7261 gcattgcaga gatattgtat ttaagtgcct agctcgatac ataaacgggt ctctctggtt 7321 agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca 7381 ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa 7441 ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac 7501 agggacttga aagcgaaagg gaaaccagag gagctctctc gacgcaggac tcggcttgct 7561 gaagcgcgca cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact 7621 agcggaggct agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt 7681 agatcgcgat gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa 7741 catatagtat gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa 7801 acatcagaag gctgtagaca aatactggga cagctacaac catcccttca gacaggatca 7861 gaagaactta gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata 7921 gagataaaag acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag 7981 accaccgcac agcaagcggc cgctgatctt cagacctgga ggaggagata tgagggacaa 8041 ttggagaagt gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc 8101 caccaaggca aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt 8161 gttccttggg ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac 8221 ggtacaggcc agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc 8281 tattgaggcg caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc 8341 aagaatcctg gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg 8401 ctctggaaaa ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc 8461 tctggaacag atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta 8521 cacaagctta atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca 8581 agaattattg gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg 8641 gctgtggtat ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt 8701 ttttgctgta ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca 8761 gacccacctc ccaaccccga ggggacccga caggcccgaa ggaatagaag aagaaggtgg 8821 agagagagac agagacagat ccattcgatt agtgaacgga tc //