LOCUS Exported 8859 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_HSV.V5.VSVg_NGFR SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8859) 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 8859) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8859 /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..2058 /codon_start=1 /product="HSV (herpes simplex virus) epitope tag" /label=HSV tag /translation="QPELAPEDPED" CDS 2077..2118 /codon_start=1 /product="epitope tag from simian virus 5" /label=V5 tag /translation="GKPIPNPLLGLDST" CDS 2137..2169 /codon_start=1 /product="epitope tag from vesicular stomatitis virus G protein" /label=VSV-G tag /translation="YTDIEMNRLGK" misc_feature 2932..3520 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(2985..3005) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3403..3414) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 3595..3828 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 3900..4021 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3937..3956) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 3991..4010 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 4061..4196 /label=SV40 ori /note="SV40 origin of replication" primer_bind 4123..4142 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(4216..4235) /label=T7 /note="T7 promoter, forward primer" promoter complement(4217..4235) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(4245..4262) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(4245..4261) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4254..4276) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 4403..4858 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4490..4509) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4700..4721 /label=F1ori-F /note="F1 origin, forward primer" promoter 4884..4988 /gene="bla" /label=AmpR promoter CDS 4989..5849 /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(5207..5226) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6020..6608 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6509..6528 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 6762..6779 /label=L4440 /note="L4440 vector, forward primer" protein_bind 6896..6917 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 6932..6962 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6970..6986 /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 6975..6997 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 6994..7010 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 6994..7010 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 7029..7049 /label=T3 /note="T3 promoter, forward primer" promoter 7031..7049 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 7077..7303 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" LTR 7304..7484 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 7531..7656 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 8149..8382 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 8567..8611 /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 catgccagcc cgagctggcc 2041 cccgaggacc ccgaggacgg aggaggcggg ggagggggca aaccaattcc aaacccactg 2101 ctgggactgg attccacagg aggaggcggg ggagggtata cagatatcga aatgaataga 2161 ctcggaaagc ccacaggcct gtacacacac agcggtgagt gctgcaaagc ctgcaacctg 2221 ggcgagggtg tggcccagcc ttgtggagcc aaccagaccg tgtgtgagcc ctgcctggac 2281 agcgtgacgt tctccgacgt ggtgagcgcg accgagccgt gcaagccgtg caccgagtgc 2341 gtggggctcc agagcatgtc ggcgccgtgc gtggaggccg acgacgccgt gtgccgctgc 2401 gcctacggct actaccagga tgagacgact gggcgctgcg aggcgtgccg cgtgtgcgag 2461 gcgggctcgg gcctcgtgtt ctcctgccag gacaagcaga acaccgtgtg cgaggagtgc 2521 cccgacggca cgtattccga cgaggccaac cacgtggacc cgtgcctgcc ctgcaccgtg 2581 tgcgaggaca ccgagcgcca gctccgcgag tgcacacgct gggccgacgc cgagtgcgag 2641 gagatccctg gccgttggat tacacggtcc acacccccag agggctcgga cagcacagcc 2701 cccagcaccc aggagcctga ggcacctcca gaacaagacc tcatagccag cacggtggca 2761 ggtgtggtga ccacagtgat gggcagctcc cagcccgtgg tgacccgagg caccaccgac 2821 aacctcatcc ctgtctattg ctccatcctg gctgctgtgg ttgtgggcct tgtggcctac 2881 atagccttca agaggtggaa caggggtatc ctctagagcg gccgcgtcga caatcaacct 2941 ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg 3001 ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc 3061 attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt 3121 gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc 3181 attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg 3241 gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact 3301 gacaattccg tggtgttgtc ggggaagctg acgtcctttc catggctgct cgcctgtgtt 3361 gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg 3421 gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcatct tcgccttcgc 3481 cctcagacga gtcggatctc cctttgggcc gcctccccgc ctgggtacct ttaagaccaa 3541 tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag 3601 ggctaattca ctcccaacga agataagatc tgctttttgc ttgtactggg tctctctggt 3661 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc 3721 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta 3781 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt agtagttcat 3841 gtcatcttat tattcagtat ttataacttg caaagaaatg aatatcagag agtgagagga 3901 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa 3961 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt 4021 atcatgtctg gctctagcta tcccgcccct aactccgccc atcccgcccc taactccgcc 4081 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga 4141 ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg 4201 gacgtaccca attcgcccta tagtgagtcg tattacgcgc gctcactggc cgtcgtttta 4261 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc 4321 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg 4381 cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg 4441 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct 4501 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg 4561 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag 4621 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg 4681 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc 4741 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat 4801 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag 4861 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 4921 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 4981 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 5041 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 5101 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 5161 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 5221 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 5281 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 5341 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 5401 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 5461 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 5521 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 5581 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 5641 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 5701 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 5761 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 5821 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 5881 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 5941 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 6001 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 6061 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 6121 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc 6181 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 6241 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 6301 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 6361 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 6421 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 6481 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 6541 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 6601 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 6661 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 6721 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 6781 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 6841 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg 6901 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 6961 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 7021 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctgcaa gcttaatgta 7081 gtcttatgca atactcttgt agtcttgcaa catggtaacg atgagttagc aacatgcctt 7141 acaaggagag aaaaagcacc gtgcatggcg attggtggaa gtaaggtggt acgatcgtgc 7201 cttattagga aggcaacaga cgggtctgac atggattgga cgaaccactg aattgccgca 7261 ttgcagagat attgtattta agtgcctagc tcgatacata aacgggtctc tctggttaga 7321 ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata 7381 aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta 7441 gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg 7501 gacttgaaag cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa 7561 gcgcgcacgg caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc 7621 ggaggctaga aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga 7681 tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat 7741 atagtatggg caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca 7801 tcagaaggct gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa 7861 gaacttagat cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag 7921 ataaaagaca ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc 7981 accgcacagc aagcggccgc tgatcttcag acctggagga ggagatatga gggacaattg 8041 gagaagtgaa ttatataaat ataaagtagt aaaaattgaa ccattaggag tagcacccac 8101 caaggcaaag agaagagtgg tgcagagaga aaaaagagca gtgggaatag gagctttgtt 8161 ccttgggttc ttgggagcag caggaagcac tatgggcgca gcgtcaatga cgctgacggt 8221 acaggccaga caattattgt ctggtatagt gcagcagcag aacaatttgc tgagggctat 8281 tgaggcgcaa cagcatctgt tgcaactcac agtctggggc atcaagcagc tccaggcaag 8341 aatcctggct gtggaaagat acctaaagga tcaacagctc ctggggattt ggggttgctc 8401 tggaaaactc atttgcacca ctgctgtgcc ttggaatgct agttggagta ataaatctct 8461 ggaacagatt tggaatcaca cgacctggat ggagtgggac agagaaatta acaattacac 8521 aagcttaata cactccttaa ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga 8581 attattggaa ttagataaat gggcaagttt gtggaattgg tttaacataa caaattggct 8641 gtggtatata aaattattca taatgatagt aggaggcttg gtaggtttaa gaatagtttt 8701 tgctgtactt tctatagtga atagagttag gcagggatat tcaccattat cgtttcagac 8761 ccacctccca accccgaggg gacccgacag gcccgaagga atagaagaag aaggtggaga 8821 gagagacaga gacagatcca ttcgattagt gaacggatc //