LOCUS Exported 9419 bp ds-DNA circular SYN 18-MAY-2018 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pMK1334 - Kampmann lab SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9419) AUTHORS . TITLE Direct Submission JOURNAL Exported Jan 6, 2019 from SnapGene Viewer 3.0.3 http://www.snapgene.com COMMENT [DEFINITION]: Makes CRISPR gRNAs detectable by single-cell RNA-seq. The gRNA is [LOCUS]: Exported [ACCESSION]: . [SOURCE]: 1..10214 [CDS]: 3655..4515 FEATURES Location/Qualifiers source 1..9419 /organism="synthetic DNA construct" /mol_type="other DNA" misc_feature 48..361 /note="mU6 modified" misc_feature 382..474 /note="modified guide constant region" misc_feature 521..701 /note="5'UTR truncated" promoter 1786..1890 /note="AmpR promoter" /note="gene=""bla"" - label=AmpR promoter - 3550..3654" CDS 1891..2751 /note="confers resistance to ampicillin, carbenicillin..." /note="codon_start=1 - gene=""bla"" - product=""beta-lactamase"" - label=AmpR - note=""confers resistance to ampicillin, carbenicillin, and - related antibiotics"" - translation=""MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI - ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQ" primer_bind complement(2109..2128) /note="Ampicillin resistance gene, reverse primer_Amp-R" /note="label=Amp-R - note=""Ampicillin resistance gene, reverse primer"" - complement(3873..3892)" rep_origin 2922..3510 /direction=RIGHT /note="high-copy-number ColE1/pMB1/pBR322/pUC origin o..." /note="direction=RIGHT - label=ori - note=""high-copy-number ColE1/pMB1/pBR322/pUC origin of - replication"" - 4686..5274" primer_bind 3411..3430 /note="pBR322 origin, forward primer_pBR322ori-F" /note="label=pBR322ori-F - note=""pBR322 origin, forward primer"" - 5175..5194" primer_bind 3664..3681 /note="L4440 vector, forward primer_L4440" /note="label=L4440 - note=""L4440 vector, forward primer"" - 5428..5445" protein_bind 3798..3819 /note="CAP binding activates transcription in the pres..." /note="label=CAP binding site - bound_moiety=""E. coli catabolite activator protein"" - note=""CAP binding activates transcription in the presence - of cAMP."" - 5562..5583" promoter 3834..3864 /note="promoter for the E. coli lac operon_lac promoter" /note="label=lac promoter - note=""promoter for the E. coli lac operon"" - 5598..5628" protein_bind 3872..3888 /note="The lac repressor binds to the lac operator to ..." /note="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" primer_bind 3877..3899 /note="In lacZ gene_M13/pUC Reverse" /note="label=M13/pUC Reverse - note=""In lacZ gene"" - 5641..5663" primer_bind 3896..3912 /note="In lacZ gene. Also called M13-rev_M13 Reverse" /note="label=M13 Reverse - note=""In lacZ gene. Also called M13-rev"" - 5660..5676" primer_bind 3896..3912 /note="common sequencing primer, one of multiple simil..." /note="label=M13 rev - note=""common sequencing primer, one of multiple similar - variants"" - 5660..5676" primer_bind 3931..3951 /note="T3 promoter, forward primer_T3" /note="label=T3 - note=""T3 promoter, forward primer"" - 5695..5715" promoter 3933..3951 /note="promoter for bacteriophage T3 RNA polymerase_T3..." /note="label=T3 promoter - note=""promoter for bacteriophage T3 RNA polymerase"" - 5697..5715" promoter 3979..4205 /note="Rous sarcoma virus enhancer/promoter_RSV promoter" /note="label=RSV promoter - note=""Rous sarcoma virus enhancer/promoter"" - 5743..5969" LTR 4206..4386 /note="truncated 5' long terminal repeat (LTR) from HI..." /note="label=5' LTR (truncated) - note=""truncated 5' long terminal repeat (LTR) from HIV-1"" - 5970..6150" misc_feature 4433..4558 /note="packaging signal of human immunodeficiency viru..." /note="label=HIV-1 Psi - note=""packaging signal of human immunodeficiency virus - type 1"" - 6197..6322" misc_feature 5051..5284 /note="The Rev response element (RRE) of HIV-1 allows ..." /note="label=RRE - note=""The Rev response element (RRE) of HIV-1 allows for - Rev-dependent mRNA export from the nucleus to the - cytoplasm."" - 6815..7048" misc_feature 5694..5811 /note="central polypurine tract and central terminatio..." /note="label=cPPT/CTS - note=""central polypurine tract and central termination - sequence of HIV-1"" - 7458..7575" promoter 5950..7128 /note="strong constitutive promoter for human elongati..." /note="label=EF-1-alpha promoter - note=""strong constitutive promoter for human elongation - factor EF-1-alpha"" - 7714..8892" intron 6181..7119 /note="intron upstream of the start codon of human _EF..." /note="label=EF-1-alpha intron A - note=""intron upstream of the start codon of human - EF-1-alpha"" - 7945..8883" CDS 7141..7740 /note="confers resistance to puromycin_PuroR" /note="codon_start=1 - gene=""pac from Streptomyces alboniger"" - product=""puromycin N-acetyltransferase"" - label=PuroR - note=""confers resistance to puromycin"" - translation=""MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER - VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAG" misc_feature 7741..7806 /note="T2A" misc_feature 7807..7833 /note="mycNLS" misc_feature 7849..7875 /note="mycNLS" misc_feature 7903..8616 /note="mTagBFP2" misc_feature 8715..9313 /note="WPRE" ORIGIN 1 ttcaccatta tcgtttcaga cccacctccc aaccccgagg ggaccccgat ccgacgcgcc 61 atctctaggc ccgcgccggc cccctcgcac ggacttgtgg gagaagctcg gctactcccc 121 tgccccggtt aatttgcata taatatttcc tagtaactat agaggcttaa tgtgcgataa 181 aagacagata atctgttctt tttaatacta gctacatttt acatgatagg cttggatttc 241 tataacttcg tatagcatac attatacgaa gttataaaca gcacaaaagg aaactcaccc 301 taactgtaaa gtaattgtgt gttttgagac tataagtatc ccttggagaa ccaccttgtt 361 ggaccaggat gggcaccacc cgtttaagag ctaagctgga aacagcatag caagtttaaa 421 taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt ttttaagctt 481 ggcgtaacta gatcttgaga cactgctttt tgcttgtact gggtctctct ggttagacca 541 gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag 601 cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag 661 atccctcaga cccttttagt cagtgtggaa aatctctagc agtacgtata gtagttcatg 721 tcatcttagt atagtagttc atgtcatctt attattcagt atttataact tgcaaagaaa 781 tgaatatcag agagtgagag gaacttgttt attgcagctt ataatggtta caaataaagc 841 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg 901 tccaaactca tcaatgtatc ttatcatgtc tggctctagc tatcccgccc ctaactccgc 961 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt 1021 tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag 1081 gaggcttttt tggaggccta gggacgtacc caattcgccc tatagtgagt cgtattacgc 1141 gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact 1201 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac 1261 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg 1321 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc 1381 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc 1441 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct 1501 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac 1561 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac 1621 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat 1681 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa 1741 aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt 1801 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa 1861 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt 1921 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa 1981 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac 2041 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt 2101 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt 2161 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat 2221 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac 2281 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg 2341 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc 2401 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa 2461 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag 2521 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct 2581 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat 2641 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa 2701 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac 2761 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc 2821 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc 2881 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg 2941 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg 3001 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca 3061 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg 3121 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg 3181 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga 3241 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac 3301 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat 3361 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc 3421 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga 3481 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc 3541 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg 3601 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag 3661 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc 3721 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc 3781 agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac 3841 tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga 3901 aacagctatg accatgatta cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag 3961 ctggagctgc aagcttaatg tagtcttatg caatactctt gtagtcttgc aacatggtaa 4021 cgatgagtta gcaacatgcc ttacaaggag agaaaaagca ccgtgcatgc cgattggtgg 4081 aagtaaggtg gtacgatcgt gccttattag gaaggcaaca gacgggtctg acatggattg 4141 gacgaaccac tgaattgccg cattgcagag atattgtatt taagtgccta gctcgataca 4201 taaacgggtc tctctggtta gaccagatct gagcctggga gctctctggc taactaggga 4261 acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc 4321 tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct 4381 ctagcagtgg cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg agctctctcg 4441 acgcaggact cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc gactggtgag 4501 tacgccaaaa attttgacta gcggaggcta gaaggagaga gatgggtgcg agagcgtcag 4561 tattaagcgg gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa 4621 gaaaaaatat aaattaaaac atatagtatg ggcaagcagg gagctagaac gattcgcagt 4681 taatcctggc ctgttagaaa catcagaagg ctgtagacaa atactgggac agctacaacc 4741 atcccttcag acaggatcag aagaacttag atcattatat aatacagtag caaccctcta 4801 ttgtgtgcat caaaggatag agataaaaga caccaaggaa gctttagaca agatagagga 4861 agagcaaaac aaaagtaaga ccaccgcaca gcaagcggcc gctgatcttc agacctggag 4921 gaggagatat gagggacaat tggagaagtg aattatataa atataaagta gtaaaaattg 4981 aaccattagg agtagcaccc accaaggcaa agagaagagt ggtgcagaga gaaaaaagag 5041 cagtgggaat aggagctttg ttccttgggt tcttgggagc agcaggaagc actatgggcg 5101 cagcgtcaat gacgctgacg gtacaggcca gacaattatt gtctggtata gtgcagcagc 5161 agaacaattt gctgagggct attgaggcgc aacagcatct gttgcaactc acagtctggg 5221 gcatcaagca gctccaggca agaatcctgg ctgtggaaag atacctaaag gatcaacagc 5281 tcctggggat ttggggttgc tctggaaaac tcatttgcac cactgctgtg ccttggaatg 5341 ctagttggag taataaatct ctggaacaga tttggaatca cacgacctgg atggagtggg 5401 acagagaaat taacaattac acaagcttaa tacactcctt aattgaagaa tcgcaaaacc 5461 agcaagaaaa gaatgaacaa gaattattgg aattagataa atgggcaagt ttgtggaatt 5521 ggtttaacat aacaaattgg ctgtggtata taaaattatt cataatgata gtaggaggct 5581 tggtaggttt aagaatagtt tttgctgtac tttctatagt gaatagagtt aggcagggat 5641 attcaccatt atcgtttcag aatcttgaga caaatggcag tattcatcca caattttaaa 5701 agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat aatagcaaca 5761 gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat 5821 tacagggaca gcagagatcc actttggcgc cggctcgagg gggcccgggt gcaaagatgg 5881 ataaagtttt aaacagagag gaatctttgc agctaatgga ccttctaggt cttgaaagga 5941 gtgggaattg gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag 6001 aagttggggg gaggggtcgg caattgatcc ggtgcctaga gaaggtggcg cggggtaaac 6061 tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat 6121 ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag 6181 gtaagtgccg tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg 6241 ccttgaatta cttccacctg gctgcagtac gtgattcttg atcccgagct tcgggttgga 6301 agtgggtggg agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt 6361 gaggcctggc ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt 6421 ctcgctgctt tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt 6481 tttttctggc aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt 6541 tttggggccg cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg 6601 ggcctgcgag cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct 6661 ctggtgcctg gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg 6721 tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca 6781 aaatggagga cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg 6841 gcctttccgt cctcagccgt cgcttcatgt gactccacgg agtaccgggc gccgtccagg 6901 cacctcgatt agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt 6961 tatgcgatgg agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac 7021 ttgatgtaat tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag 7081 cctcagacag tggttcaaag tttttttctt ccatttcagg tgtcgtgacg tacggccacc 7141 atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta 7201 cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac 7261 cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac 7321 atcggcaagg tgtgggtcgc ggacgacggc gccgccgtgg cggtctggac cacgccggag 7381 agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt 7441 tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag 7501 cccgcgtggt tcctggccac cgtcggagtc tcgcccgacc accagggcaa gggtctgggc 7561 agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg 7621 gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc 7681 gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgccgga 7741 tcgggagagg gcagaggaag tctgctaaca tgcggtgacg tcgaggagaa tcctggccca 7801 ccggtccctg ccgctaaacg ggtcaaactc gacggcggtt ctggaggtcc tgctgcaaag 7861 cgcgttaagt tggatggagg gtcaggggga gccaccgcca ccatggtgtc taagggcgaa 7921 gagctgatta aggagaacat gcacatgaag ctgtacatgg agggcaccgt ggacaaccat 7981 cacttcaagt gcacatccga gggcgaaggc aagccctacg agggcaccca gaccatgaga 8041 atcaaggtgg tcgagggcgg ccctctcccc ttcgccttcg acatcctggc tactagcttc 8101 ctctacggca gcaagacctt catcaaccac acccagggca tccccgactt cttcaagcag 8161 tccttccctg agggcttcac atgggagaga gtcaccacat acgaagacgg gggcgtgctg 8221 accgctaccc aggacaccag cctccaggac ggctgcctca tctacaacgt caagatcaga 8281 ggggtgaact tcacatccaa cggccctgtg atgcagaaga aaacactcgg ctgggaggcc 8341 ttcaccgaga cgctgtaccc cgctgacggc ggcctggaag gcagaaacga catggccctg 8401 aagctcgtgg gcgggagcca tctgatcgca aacgccaaga ccacatatag atccaagaaa 8461 cccgctaaga acctcaagat gcctggcgtc tactatgtgg actacagact ggaaagaatc 8521 aaggaggcca acaacgagac ctacgtcgag cagcacgagg tggcagtggc cagatactgc 8581 gacctcccta gcaaactggg gcacaagctt aattaagaat tcgtcgaggg acctaataac 8641 ttcgtatagc atacattata cgaagttata catgtttaag ggttccggtt ccactaggta 8701 caattcgata tcaagcttat cgataatcaa cctctggatt acaaaatttg tgaaagattg 8761 actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct 8821 ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg 8881 ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact 8941 gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc 9001 gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc 9061 cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa 9121 tcatcgtcct ttccttggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc 9181 ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg 9241 gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg 9301 gccgcctccc cgcgtcgact ttaagaccaa tgacttacaa ggcagctgta gatcttagcc 9361 actttttaaa agaaaagggg ggactggaag ggctaattca ctcccaacga agacaagat //