LOCUS Exported 8313 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION sgRNA1-2XPP7. ACCESSION . VERSION . KEYWORDS pLH-sgRNA1-2XPP7 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8313) AUTHORS Ma H, Tu LC, Naseri A, Huisman M, Zhang S, Grunwald D, Pederson T TITLE Multiplexed labeling of genomic loci with dCas9 and engineered sgRNAs using CRISPRainbow. JOURNAL Nat Biotechnol. 2016 Apr 18. doi: 10.1038/nbt.3526. PUBMED 27088723 REFERENCE 2 (bases 1 to 8313) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8313 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 230..470 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 230..250 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 401..420 /label=LKO.1 5' /note="Human U6 promoter, forward primer" CDS 819..1124 /codon_start=1 /gene="ccdB" /product="CcdB, a bacterial toxin that poisons DNA gyrase" /label=ccdB /note="Plasmids containing the ccdB gene cannot be propagated in standard E. coli strains." /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI" primer_bind 1003..1022 /label=ccdB-fwd /note="ccdB gene, forward primer" misc_feature 1315..1432 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 1481..1991 /label=hPGK promoter /note="human phosphoglycerate kinase 1 promoter" primer_bind 1910..1928 /label=hPGK-F /note="Human PGK promoter, forward primer" CDS 2081..3106 /codon_start=1 /gene="aph(4)-Ia" /product="aminoglycoside phosphotransferase from E. coli" /label=HygR /note="confers resistance to hygromycin" /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK E" primer_bind complement(3130..3146) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3131..3147) /label=pBluescriptKS /note="For pBluescript vector" LTR 3229..3462 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 3534..3655 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3571..3590) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 3625..3644 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 3695..3830 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3757..3776 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(3850..3869) /label=T7 /note="T7 promoter, forward primer" promoter complement(3851..3869) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(3879..3896) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(3879..3895) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3888..3910) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 4037..4492 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4124..4143) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4334..4355 /label=F1ori-F /note="F1 origin, forward primer" promoter 4518..4622 /gene="bla" /label=AmpR promoter CDS 4623..5483 /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(4841..4860) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5654..6242 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6143..6162 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 6396..6413 /label=L4440 /note="L4440 vector, forward primer" protein_bind 6530..6551 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 6566..6596 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6604..6620 /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 6609..6631 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 6628..6644 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 6628..6644 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 6663..6683 /label=T3 /note="T3 promoter, forward primer" promoter 6665..6683 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 6711..6937 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" LTR 6938..7118 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 7165..7290 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 7783..8016 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 8201..8245 /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 ggtaggttta agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata 61 ttcaccatta tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg 121 aatagaagaa gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc 181 tcgacggtat cgatcacgag actagcctcg agcggccgcc cccttcaccg agggcctatt 241 tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga taattggaat 301 taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt 361 tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt 421 aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccgg 481 tgtcttcgag gcttactaaa agccagataa cagtatgcgt atttgcgcgc tgatttttgc 541 ggtataagaa tatatactga tatgtatacc cgaagtatgt caaaaagagg tgtgctatga 601 agcagcgtat tacagtgaca gttgacagcg acagctatca gttgctcaag gcatatatga 661 tgtcaatatc tccggtctgg taagcacaac catgcagaat gaagcccgtc gtctgcgtgc 721 cgaacgctgg aaagcggaaa atcaggaagg gatggctgag gtcgcccggt ttattgaaat 781 gaacggctct tttgctgacg agaacaggga ctggtgaaat gcagtttaag gtttacacct 841 ataaaagaga gagccgttat cgtctgtttg tggatgtaca gagtgatatt attgacacgc 901 ccgggcgacg gatggtgatc cccctggcca gtgcacgtct gctgtcagat aaagtctccc 961 gtgaacttta cccggtggtg catatcgggg atgaaagctg gcgcatgatg accaccgata 1021 tggccagtgt gccggtctcc gttatcgggg aagaagtggc tgatctcagc caccgcgaaa 1081 atgacatcaa aaacgccatt aacctgatgt tctggggaat ataaaccctg caggacgaag 1141 acccgtttga gagctaccgg agcagacgat atggcgtcgc tccggtagca agttcaaata 1201 aggctagtcc gttatcaact tggagcagac gatatggcgt cgctccaagt ggcaccgagt 1261 cggtgctttt tttgaattct cgacctcgag acaaatggca gtattcatcc acaattttaa 1321 aagaaaaggg gggattgggg ggtacagtgc aggggaaaga atagtagaca taatagcaac 1381 agacatacaa actaaagaat tacaaaaaca aattacaaaa attcaaaatt ttcgggttta 1441 ttacagggac agcagagatc cactttggcc gcggctcgag ggggttgggg ttgcgccttt 1501 tccaaggcag ccctgggttt gcgcagggac gcggctgctc tgggcgtggt tccgggaaac 1561 gcagcggcgc cgaccctggg tctcgcacat tcttcacgtc cgttcgcagc gtcacccgga 1621 tcttcgccgc tacccttgtg ggccccccgg cgacgcttcc tgctccgccc ctaagtcggg 1681 aaggttcctt gcggttcgcg gcgtgccgga cgtgacaaac ggaagccgca cgtctcacta 1741 gtaccctcgc agacggacag cgccagggag caatggcagc gcgccgaccg cgatgggctg 1801 tggccaatag cggctgctca gcagggcgcg ccgagagcag cggccgggaa ggggcggtgc 1861 gggaggcggg gtgtggggcg gtagtgtggg ccctgttcct gcccgcgcgg tgttccgcat 1921 tctgcaagcc tccggagcgc acgtcggcag tcggctccct cgttgaccga atcaccgacc 1981 tctctcccca gggggatcca ccggatcaag cttgccagct ggggcgccct ctggtaaggt 2041 tgggaagccc tgcagatctg atcccggggg gcaatgagat atgaaaaagc ctgaactcac 2101 cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac agcgtctccg acctgatgca 2161 gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt 2221 cctgcgggta aatagctgcg ccgatggttt ctacaaagat cgttatgttt atcggcactt 2281 tgcatcggcc gcgctcccga ttccggaagt gcttgacatt ggggaattta gcgagagcct 2341 gacctattgc atctcccgcc gtgcacaggg tgtcacgttg caagatctgc ctgaaaccga 2401 actgcccgct gttctgcagc cggtcgcgga ggccatggat gcgatcgctg cggccgatct 2461 tagccagacg agcgggttcg gcccattcgg accgcaagga atcggtcaat acactacatg 2521 gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga 2581 cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga 2641 ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga 2701 caatggccgc ataacagcgg tcattgactg gagcgaggcg atgttcgggg attcccaata 2761 cgaggtcgcc aacatcttct tctggaggcc gtggttggct tgtatggagc agcagacgcg 2821 ctacttcgag cggaggcatc cggagcttgc aggatcgccg cggctccggg cgtatatgct 2881 ccgcattggt cttgaccaac tctatcagag cttggttgac ggcaatttcg atgatgcagc 2941 ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg tcgggcgtac 3001 acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag tactcgccga 3061 tagtggaaac cgacgcccca gcactcgtcc gagggcaaag gaatagagta gatgccgacc 3121 gggatctatc gataccgtcg acctcgaggg ggggcccggt acctttaaga ccaatgactt 3181 acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa 3241 ttcactccca acgaagaaaa gatctgcttt ttgcttgtac tgggtctctc tggttagacc 3301 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa 3361 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga 3421 gatccctcag acccttttag tcagtgtgga aaatctctag cagtagtagt tcatgtcatc 3481 ttattattca gtatttataa cttgcaaaga aatgaatatc agagagtgag aggaacttgt 3541 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag 3601 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg 3661 tctggctcta gctatcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc 3721 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc 3781 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc tagggacgta 3841 cccaattcgc cctatagtga gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt 3901 cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc 3961 gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc 4021 ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt 4081 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc 4141 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct 4201 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat 4261 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc 4321 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc 4381 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg 4441 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca 4501 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata 4561 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga 4621 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc 4681 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg 4741 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc 4801 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat 4861 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact 4921 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat 4981 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga 5041 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc 5101 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga 5161 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag 5221 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc 5281 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt 5341 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct 5401 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg 5461 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg 5521 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca 5581 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga 5641 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa 5701 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga 5761 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt 5821 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt 5881 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat 5941 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct 6001 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca 6061 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag 6121 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc 6181 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga 6241 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca 6301 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag 6361 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg 6421 aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct 6481 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt 6541 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg 6601 gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc 6661 gcgcaattaa ccctcactaa agggaacaaa agctggagct gcaagcttaa tgtagtctta 6721 tgcaatactc ttgtagtctt gcaacatggt aacgatgagt tagcaacatg ccttacaagg 6781 agagaaaaag caccgtgcat gccgattggt ggaagtaagg tggtacgatc gtgccttatt 6841 aggaaggcaa cagacgggtc tgacatggat tggacgaacc actgaattgc cgcattgcag 6901 agatattgta tttaagtgcc tagctcgata cataaacggg tctctctggt tagaccagat 6961 ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt 7021 gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc 7081 cctcagaccc ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa cagggacttg 7141 aaagcgaaag ggaaaccaga ggagctctct cgacgcagga ctcggcttgc tgaagcgcgc 7201 acggcaagag gcgaggggcg gcgactggtg agtacgccaa aaattttgac tagcggaggc 7261 tagaaggaga gagatgggtg cgagagcgtc agtattaagc gggggagaat tagatcgcga 7321 tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat ataaattaaa acatatagta 7381 tgggcaagca gggagctaga acgattcgca gttaatcctg gcctgttaga aacatcagaa 7441 ggctgtagac aaatactggg acagctacaa ccatcccttc agacaggatc agaagaactt 7501 agatcattat ataatacagt agcaaccctc tattgtgtgc atcaaaggat agagataaaa 7561 gacaccaagg aagctttaga caagatagag gaagagcaaa acaaaagtaa gaccaccgca 7621 cagcaagcgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag 7681 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc 7741 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg 7801 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc 7861 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc 7921 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct 7981 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa 8041 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca 8101 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt 8161 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt 8221 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta 8281 tataaaatta ttcataatga tagtaggagg ctt //