LOCUS Exported 8318 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION sgRNA1-MS2-PP7. ACCESSION . VERSION . KEYWORDS pLH-sgRNA1-MS2-PP7 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8318) 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 8318) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8318 /organism="synthetic DNA construct" /mol_type="other DNA" misc_feature 83..200 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 249..759 /label=hPGK promoter /note="human phosphoglycerate kinase 1 promoter" primer_bind 678..696 /label=hPGK-F /note="Human PGK promoter, forward primer" CDS 849..1874 /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(1898..1914) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(1899..1915) /label=pBluescriptKS /note="For pBluescript vector" LTR 1997..2230 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 2302..2423 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2339..2358) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 2393..2412 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 2463..2598 /label=SV40 ori /note="SV40 origin of replication" primer_bind 2525..2544 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(2618..2637) /label=T7 /note="T7 promoter, forward primer" promoter complement(2619..2637) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(2647..2664) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(2647..2663) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(2656..2678) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 2805..3260 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2892..2911) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3102..3123 /label=F1ori-F /note="F1 origin, forward primer" promoter 3286..3390 /gene="bla" /label=AmpR promoter CDS 3391..4251 /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(3609..3628) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 4422..5010 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4911..4930 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 5164..5181 /label=L4440 /note="L4440 vector, forward primer" protein_bind 5298..5319 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 5334..5364 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5372..5388 /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 5377..5399 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 5396..5412 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 5396..5412 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 5431..5451 /label=T3 /note="T3 promoter, forward primer" promoter 5433..5451 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 5479..5705 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" LTR 5706..5886 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 5933..6058 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 6551..6784 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 6969..7013 /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" promoter 7311..7551 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 7311..7331 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 7482..7501 /label=LKO.1 5' /note="Human U6 promoter, forward primer" CDS 7900..8205 /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 8084..8103 /label=ccdB-fwd /note="ccdB gene, forward primer" misc_RNA 8243..8261 /label=MS2 stem loop /note="stem loop that binds the bacteriophage MS2 coat protein" ORIGIN 1 tatggcgtcg ctccaagtgg caccgagtcg gtgctttttt tgaattctcg acctcgagac 61 aaatggcagt attcatccac aattttaaaa gaaaaggggg gattgggggg tacagtgcag 121 gggaaagaat agtagacata atagcaacag acatacaaac taaagaatta caaaaacaaa 181 ttacaaaaat tcaaaatttt cgggtttatt acagggacag cagagatcca ctttggccgc 241 ggctcgaggg ggttggggtt gcgccttttc caaggcagcc ctgggtttgc gcagggacgc 301 ggctgctctg ggcgtggttc cgggaaacgc agcggcgccg accctgggtc tcgcacattc 361 ttcacgtccg ttcgcagcgt cacccggatc ttcgccgcta cccttgtggg ccccccggcg 421 acgcttcctg ctccgcccct aagtcgggaa ggttccttgc ggttcgcggc gtgccggacg 481 tgacaaacgg aagccgcacg tctcactagt accctcgcag acggacagcg ccagggagca 541 atggcagcgc gccgaccgcg atgggctgtg gccaatagcg gctgctcagc agggcgcgcc 601 gagagcagcg gccgggaagg ggcggtgcgg gaggcggggt gtggggcggt agtgtgggcc 661 ctgttcctgc ccgcgcggtg ttccgcattc tgcaagcctc cggagcgcac gtcggcagtc 721 ggctccctcg ttgaccgaat caccgacctc tctccccagg gggatccacc ggatcaagct 781 tgccagctgg ggcgccctct ggtaaggttg ggaagccctg cagatctgat cccggggggc 841 aatgagatat gaaaaagcct gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa 901 agttcgacag cgtctccgac ctgatgcagc tctcggaggg cgaagaatct cgtgctttca 961 gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct 1021 acaaagatcg ttatgtttat cggcactttg catcggccgc gctcccgatt ccggaagtgc 1081 ttgacattgg ggaatttagc gagagcctga cctattgcat ctcccgccgt gcacagggtg 1141 tcacgttgca agatctgcct gaaaccgaac tgcccgctgt tctgcagccg gtcgcggagg 1201 ccatggatgc gatcgctgcg gccgatctta gccagacgag cgggttcggc ccattcggac 1261 cgcaaggaat cggtcaatac actacatggc gtgatttcat atgcgcgatt gctgatcccc 1321 atgtgtatca ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc 1381 tcgatgagct gatgctttgg gccgaggact gccccgaagt ccggcacctc gtgcacgcgg 1441 atttcggctc caacaatgtc ctgacggaca atggccgcat aacagcggtc attgactgga 1501 gcgaggcgat gttcggggat tcccaatacg aggtcgccaa catcttcttc tggaggccgt 1561 ggttggcttg tatggagcag cagacgcgct acttcgagcg gaggcatccg gagcttgcag 1621 gatcgccgcg gctccgggcg tatatgctcc gcattggtct tgaccaactc tatcagagct 1681 tggttgacgg caatttcgat gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc 1741 gatccggagc cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga 1801 ccgatggctg tgtagaagta ctcgccgata gtggaaaccg acgccccagc actcgtccga 1861 gggcaaagga atagagtaga tgccgaccgg gatctatcga taccgtcgac ctcgaggggg 1921 ggcccggtac ctttaagacc aatgacttac aaggcagctg tagatcttag ccacttttta 1981 aaagaaaagg ggggactgga agggctaatt cactcccaac gaagaaaaga tctgcttttt 2041 gcttgtactg ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta 2101 gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc 2161 cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa 2221 atctctagca gtagtagttc atgtcatctt attattcagt atttataact tgcaaagaaa 2281 tgaatatcag agagtgagag gaacttgttt attgcagctt ataatggtta caaataaagc 2341 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg 2401 tccaaactca tcaatgtatc ttatcatgtc tggctctagc tatcccgccc ctaactccgc 2461 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt 2521 tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag 2581 gaggcttttt tggaggccta gggacgtacc caattcgccc tatagtgagt cgtattacgc 2641 gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact 2701 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac 2761 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg 2821 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc 2881 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc 2941 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct 3001 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac 3061 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac 3121 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat 3181 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa 3241 aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt 3301 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa 3361 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt 3421 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa 3481 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac 3541 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt 3601 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt 3661 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat 3721 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac 3781 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg 3841 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc 3901 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa 3961 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag 4021 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct 4081 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat 4141 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa 4201 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac 4261 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc 4321 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc 4381 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg 4441 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg 4501 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca 4561 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg 4621 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg 4681 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga 4741 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac 4801 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat 4861 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc 4921 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga 4981 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc 5041 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg 5101 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag 5161 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc 5221 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc 5281 agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac 5341 tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga 5401 aacagctatg accatgatta cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag 5461 ctggagctgc aagcttaatg tagtcttatg caatactctt gtagtcttgc aacatggtaa 5521 cgatgagtta gcaacatgcc ttacaaggag agaaaaagca ccgtgcatgc cgattggtgg 5581 aagtaaggtg gtacgatcgt gccttattag gaaggcaaca gacgggtctg acatggattg 5641 gacgaaccac tgaattgccg cattgcagag atattgtatt taagtgccta gctcgataca 5701 taaacgggtc tctctggtta gaccagatct gagcctggga gctctctggc taactaggga 5761 acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc 5821 tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct 5881 ctagcagtgg cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg agctctctcg 5941 acgcaggact cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc gactggtgag 6001 tacgccaaaa attttgacta gcggaggcta gaaggagaga gatgggtgcg agagcgtcag 6061 tattaagcgg gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa 6121 gaaaaaatat aaattaaaac atatagtatg ggcaagcagg gagctagaac gattcgcagt 6181 taatcctggc ctgttagaaa catcagaagg ctgtagacaa atactgggac agctacaacc 6241 atcccttcag acaggatcag aagaacttag atcattatat aatacagtag caaccctcta 6301 ttgtgtgcat caaaggatag agataaaaga caccaaggaa gctttagaca agatagagga 6361 agagcaaaac aaaagtaaga ccaccgcaca gcaagcggcc gctgatcttc agacctggag 6421 gaggagatat gagggacaat tggagaagtg aattatataa atataaagta gtaaaaattg 6481 aaccattagg agtagcaccc accaaggcaa agagaagagt ggtgcagaga gaaaaaagag 6541 cagtgggaat aggagctttg ttccttgggt tcttgggagc agcaggaagc actatgggcg 6601 cagcgtcaat gacgctgacg gtacaggcca gacaattatt gtctggtata gtgcagcagc 6661 agaacaattt gctgagggct attgaggcgc aacagcatct gttgcaactc acagtctggg 6721 gcatcaagca gctccaggca agaatcctgg ctgtggaaag atacctaaag gatcaacagc 6781 tcctggggat ttggggttgc tctggaaaac tcatttgcac cactgctgtg ccttggaatg 6841 ctagttggag taataaatct ctggaacaga tttggaatca cacgacctgg atggagtggg 6901 acagagaaat taacaattac acaagcttaa tacactcctt aattgaagaa tcgcaaaacc 6961 agcaagaaaa gaatgaacaa gaattattgg aattagataa atgggcaagt ttgtggaatt 7021 ggtttaacat aacaaattgg ctgtggtata taaaattatt cataatgata gtaggaggct 7081 tggtaggttt aagaatagtt tttgctgtac tttctatagt gaatagagtt aggcagggat 7141 attcaccatt atcgtttcag acccacctcc caaccccgag gggacccgac aggcccgaag 7201 gaatagaaga agaaggtgga gagagagaca gagacagatc cattcgatta gtgaacggat 7261 ctcgacggta tcgatcacga gactagcctc gagcggccgc ccccttcacc gagggcctat 7321 ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa 7381 ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat 7441 ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg 7501 taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg 7561 gtgtcttcga ggcttactaa aagccagata acagtatgcg tatttgcgcg ctgatttttg 7621 cggtataaga atatatactg atatgtatac ccgaagtatg tcaaaaagag gtgtgctatg 7681 aagcagcgta ttacagtgac agttgacagc gacagctatc agttgctcaa ggcatatatg 7741 atgtcaatat ctccggtctg gtaagcacaa ccatgcagaa tgaagcccgt cgtctgcgtg 7801 ccgaacgctg gaaagcggaa aatcaggaag ggatggctga ggtcgcccgg tttattgaaa 7861 tgaacggctc ttttgctgac gagaacaggg actggtgaaa tgcagtttaa ggtttacacc 7921 tataaaagag agagccgtta tcgtctgttt gtggatgtac agagtgatat tattgacacg 7981 cccgggcgac ggatggtgat ccccctggcc agtgcacgtc tgctgtcaga taaagtctcc 8041 cgtgaacttt acccggtggt gcatatcggg gatgaaagct ggcgcatgat gaccaccgat 8101 atggccagtg tgccggtctc cgttatcggg gaagaagtgg ctgatctcag ccaccgcgaa 8161 aatgacatca aaaacgccat taacctgatg ttctggggaa tataaaccct gcaggacgaa 8221 gacccgtttg agagctaggc caacatgagg atcacccatg tctgcagggc ctagcaagtt 8281 caaataaggc tagtccgtta tcaacttgga gcagacga //