LOCUS Exported 8829 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_HA.NWS.FLAG_NGFR SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8829) 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 8829) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8829 /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..2052 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 2074..2097 /codon_start=1 /product="peptide that binds Strep-Tactin(R), an engineered form of streptavidin" /label=Strep-Tag II /translation="WSHPQFEK" CDS 2116..2139 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" misc_feature 2902..3490 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(2955..2975) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3373..3384) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 3565..3798 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 3870..3991 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3907..3926) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 3961..3980 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 4031..4166 /label=SV40 ori /note="SV40 origin of replication" primer_bind 4093..4112 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(4186..4205) /label=T7 /note="T7 promoter, forward primer" promoter complement(4187..4205) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(4215..4232) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(4215..4231) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4224..4246) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 4373..4828 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4460..4479) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4670..4691 /label=F1ori-F /note="F1 origin, forward primer" promoter 4854..4958 /gene="bla" /label=AmpR promoter CDS 4959..5819 /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(5177..5196) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5990..6578 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6479..6498 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 6732..6749 /label=L4440 /note="L4440 vector, forward primer" protein_bind 6866..6887 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 6902..6932 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6940..6956 /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 6945..6967 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 6964..6980 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 6964..6980 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 6999..7019 /label=T3 /note="T3 promoter, forward primer" promoter 7001..7019 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 7047..7273 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" LTR 7274..7454 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 7501..7626 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 8119..8352 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 8537..8581 /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 catgctaccc atacgatgtg 2041 ccagattacg ctggaggagg cgggggaggg aactggtccc acccccaatt cgagaaagga 2101 ggaggcgggg gaggggacta caaagacgat gacgacaagc ccacaggcct gtacacacac 2161 agcggtgagt gctgcaaagc ctgcaacctg ggcgagggtg tggcccagcc ttgtggagcc 2221 aaccagaccg tgtgtgagcc ctgcctggac agcgtgacgt tctccgacgt ggtgagcgcg 2281 accgagccgt gcaagccgtg caccgagtgc gtggggctcc agagcatgtc ggcgccgtgc 2341 gtggaggccg acgacgccgt gtgccgctgc gcctacggct actaccagga tgagacgact 2401 gggcgctgcg aggcgtgccg cgtgtgcgag gcgggctcgg gcctcgtgtt ctcctgccag 2461 gacaagcaga acaccgtgtg cgaggagtgc cccgacggca cgtattccga cgaggccaac 2521 cacgtggacc cgtgcctgcc ctgcaccgtg tgcgaggaca ccgagcgcca gctccgcgag 2581 tgcacacgct gggccgacgc cgagtgcgag gagatccctg gccgttggat tacacggtcc 2641 acacccccag agggctcgga cagcacagcc cccagcaccc aggagcctga ggcacctcca 2701 gaacaagacc tcatagccag cacggtggca ggtgtggtga ccacagtgat gggcagctcc 2761 cagcccgtgg tgacccgagg caccaccgac aacctcatcc ctgtctattg ctccatcctg 2821 gctgctgtgg ttgtgggcct tgtggcctac atagccttca agaggtggaa caggggtatc 2881 ctctagagcg gccgcgtcga caatcaacct ctggattaca aaatttgtga aagattgact 2941 ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg 3001 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg 3061 ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg 3121 tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct cctttccggg 3181 actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc 3241 tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaagctg 3301 acgtcctttc catggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc 3361 tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct 3421 ctgcggcctc ttccgcatct tcgccttcgc cctcagacga gtcggatctc cctttgggcc 3481 gcctccccgc ctgggtacct ttaagaccaa tgacttacaa ggcagctgta gatcttagcc 3541 actttttaaa agaaaagggg ggactggaag ggctaattca ctcccaacga agataagatc 3601 tgctttttgc ttgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg 3661 gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag 3721 tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag 3781 tgtggaaaat ctctagcagt agtagttcat gtcatcttat tattcagtat ttataacttg 3841 caaagaaatg aatatcagag agtgagagga acttgtttat tgcagcttat aatggttaca 3901 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt 3961 gtggtttgtc caaactcatc aatgtatctt atcatgtctg gctctagcta tcccgcccct 4021 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg 4081 actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa 4141 gtagtgagga ggcttttttg gaggcctagg gacgtaccca attcgcccta tagtgagtcg 4201 tattacgcgc gctcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt 4261 acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag 4321 gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg ggacgcgccc 4381 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt 4441 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc 4501 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta 4561 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc 4621 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg 4681 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt 4741 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat 4801 tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa 4861 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac 4921 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg 4981 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc 5041 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg 5101 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga 5161 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc 5221 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag 5281 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga 5341 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg 5401 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga 5461 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt 5521 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact 5581 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt 5641 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg 5701 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta 5761 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac 5821 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta 5881 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt 5941 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt 6001 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt 6061 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc 6121 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg 6181 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg 6241 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt 6301 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac 6361 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg 6421 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg 6481 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat 6541 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt 6601 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg 6661 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa 6721 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc 6781 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga 6841 aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg 6901 ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc 6961 acacaggaaa cagctatgac catgattacg ccaagcgcgc aattaaccct cactaaaggg 7021 aacaaaagct ggagctgcaa gcttaatgta gtcttatgca atactcttgt agtcttgcaa 7081 catggtaacg atgagttagc aacatgcctt acaaggagag aaaaagcacc gtgcatggcg 7141 attggtggaa gtaaggtggt acgatcgtgc cttattagga aggcaacaga cgggtctgac 7201 atggattgga cgaaccactg aattgccgca ttgcagagat attgtattta agtgcctagc 7261 tcgatacata aacgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta 7321 actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg 7381 tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg 7441 gaaaatctct agcagtggcg cccgaacagg gacttgaaag cgaaagggaa accagaggag 7501 ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga 7561 ctggtgagta cgccaaaaat tttgactagc ggaggctaga aggagagaga tgggtgcgag 7621 agcgtcagta ttaagcgggg gagaattaga tcgcgatggg aaaaaattcg gttaaggcca 7681 gggggaaaga aaaaatataa attaaaacat atagtatggg caagcaggga gctagaacga 7741 ttcgcagtta atcctggcct gttagaaaca tcagaaggct gtagacaaat actgggacag 7801 ctacaaccat cccttcagac aggatcagaa gaacttagat cattatataa tacagtagca 7861 accctctatt gtgtgcatca aaggatagag ataaaagaca ccaaggaagc tttagacaag 7921 atagaggaag agcaaaacaa aagtaagacc accgcacagc aagcggccgc tgatcttcag 7981 acctggagga ggagatatga gggacaattg gagaagtgaa ttatataaat ataaagtagt 8041 aaaaattgaa ccattaggag tagcacccac caaggcaaag agaagagtgg tgcagagaga 8101 aaaaagagca gtgggaatag gagctttgtt ccttgggttc ttgggagcag caggaagcac 8161 tatgggcgca gcgtcaatga cgctgacggt acaggccaga caattattgt ctggtatagt 8221 gcagcagcag aacaatttgc tgagggctat tgaggcgcaa cagcatctgt tgcaactcac 8281 agtctggggc atcaagcagc tccaggcaag aatcctggct gtggaaagat acctaaagga 8341 tcaacagctc ctggggattt ggggttgctc tggaaaactc atttgcacca ctgctgtgcc 8401 ttggaatgct agttggagta ataaatctct ggaacagatt tggaatcaca cgacctggat 8461 ggagtgggac agagaaatta acaattacac aagcttaata cactccttaa ttgaagaatc 8521 gcaaaaccag caagaaaaga atgaacaaga attattggaa ttagataaat gggcaagttt 8581 gtggaattgg tttaacataa caaattggct gtggtatata aaattattca taatgatagt 8641 aggaggcttg gtaggtttaa gaatagtttt tgctgtactt tctatagtga atagagttag 8701 gcagggatat tcaccattat cgtttcagac ccacctccca accccgaggg gacccgacag 8761 gcccgaagga atagaagaag aaggtggaga gagagacaga gacagatcca ttcgattagt 8821 gaacggatc //