LOCUS Construct_1 10777 bp DNA circular UNA 20-APR-2016 DEFINITION U6p_20nt1_WT_sgRNA. ACCESSION urn.local...1461188663751.10 VERSION urn.local...1461188663751.10 KEYWORDS . SOURCE Human ORGANISM Human . FEATURES Location/Qualifiers promoter 239..815 /created_by="User" /modified_by="User" /label="CMV_imm_early promoter" misc_feature 318..605 /created_by="User" /label="CAG enhancer" LTR 835..1015 /created_by="User" /modified_by="User" /label="HIV-1_5_LTR" misc_feature 1126..1170 /created_by="User" /modified_by="User" /label="HIV-1_psi_pack" misc_feature 1680..1913 /created_by="User" /label="RRE" misc_feature 2444..2459 /created_by="User" /label="cPPT" primer_bind 2594..2616 /created_by="primer3" /%GC="69.6" /Tm="64.72" /Hairpin="4.0" /Primer-Dimer="4.0" /Monovalent_Salt_Concentration="50.0" /Divalent_Salt_Concentration="0.0" /DNTP_Concentration="0.0" /Sequence="GGGGTTCCGCGCACATTTCCCCG" /Mismatches=0 /annotation_group="PrimerPlugin Group3c68cbfa-121b-4872-a7ab-08a32877e25e_urn:local:.:1365 712513487.384" /label="JP313" CDS complement(<2594..2598) /gene="AMP" /SECDrawAs="Gene" /SECStyleId=1 /label="AMP CDS" promoter 2630..2940 /created_by="User" /modified_by="User" /label="U6 promoter" misc_feature 2727..2734 /created_by="User" /label="Enhancer" misc_feature 2883..2901 /created_by="User" /label="proximal element" misc_feature 2919..2924 /created_by="User" /label="TATA box" misc_feature 2941 /Original_Bases="A" /label="A" /note="Geneious type: Editing History Replacement" gRNA 2949..2967 /created_by="User" /modified_by="User" /label="20nt1" misc_feature 2968..3044 /created_by="User" /modified_by="User" /label="sgRNA scaffold" terminator 3045..3050 /created_by="User" /label="Termination signal" CDS 3095..3610 /gene="PGKp" /SECDrawAs="Gene" /SECStyleId=1 /label="PGKp CDS" misc_signal order(3621..3629,3621..3629) /created_by="User" /label="KOZAK" CDS 3630..4343 /gene="EBFP2" /SECDrawAs="Gene" /SECStyleId=1 /modified_by="User" /label="EBFP2 CDS" sig_peptide 4344..4409 /created_by="User" /label="p2A" CDS 4410..5441 /created_by="User" /label="hygroR" misc_feature 5471..6058 /created_by="User" /label="WPRE" misc_feature 6245..6266 /created_by="User" /modified_by="User" /label="U3PPt" misc_feature 6245..6260 /created_by="User" /label="cPPT" LTR 6582..6762 /created_by="User" /label="truncHIV-1_3_LTR" terminator 6791..7018 /created_by="User" /label="bGH_PA_terminator" rep_origin 7081..7387 /created_by="User" /label="f1_origin" misc_feature complement(7507..7722) /created_by="User" /label="SV40 enhancer" rep_origin 7686..7763 /created_by="User" /label="SV40 origin" promoter 7881..7948 /created_by="User" /label="EM7 promoter" gene 7949..8320 /created_by="User" /modified_by="User" /label="bleo" terminator 8456..8575 /created_by="User" /label="SV40_PA_terminator" promoter complement(8672..8701) /created_by="User" /modified_by="User" /label="lac_promoter" CDS complement(9784..10644) /created_by="User" /label="AmpR" promoter complement(10686..10713) /created_by="User" /label="AmpR promoter" ORIGIN 1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg 61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt 121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc 181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac 241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat 301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg 361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt 421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag 481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc 541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag 601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt 661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc 721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg 781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct 841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt 901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac 961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc 1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc 1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa 1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg 1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata 1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc 1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga 1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc 1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca 1501 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg 1561 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga 1621 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata 1681 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg 1741 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg 1801 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag 1861 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt 1921 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt 1981 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt 2041 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag 2101 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata 2161 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta 2221 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta 2281 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa 2341 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt 2401 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat 2461 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa 2521 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag 2581 agatccagtt tggggggttc cgcgcacatt tccccgaaaa gtgccacctt gtacaaaaaa 2641 gcaggcttta aaggaaccaa ttcagtcgac tggatccggt accaaggtcg ggcaggaaga 2701 gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat 2761 aattagaatt aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa 2821 gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat 2881 gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg 2941 gaacaccgta agtcggagta ctgtcctgtt ttagagctag aaatagcaag ttaaaataag 3001 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt ctagacccag 3061 ctttcttgta caaagttggc attagacgtc gaggaattct accgggtagg ggaggcgctt 3121 ttcccaaggc agtctggagc atgcgcttta gcagccccgc tgggcacttg gcgctacaca 3181 agtggcctct ggcctcgcac acattccaca tccaccggta ggcgccaacc ggctccgttc 3241 tttggtggcc ccttcgcgcc accttctact cctcccctag tcaggaagtt cccccccgcc 3301 ccgcagctcg cgtcgtgcag gacgtgacaa atggaagtag cacgtctcac tagtctcgtg 3361 cagatggaca gcaccgctga gcaatggaag cgggtaggcc tttggggcag cggccaatag 3421 cagctttgct ccttcgcttt ctgggctcag aggctgggaa ggggtgggtc cgggggcggg 3481 ctcaggggcg ggctcagggg cggggcgggc gcccgaaggt cctccggagg cccggcattc 3541 tgcacgcttc aaaagcgcac gtctgccgcg ctgttctcct cttcctcatc tccgggcctt 3601 tcgacctgca tcccaagctt gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg 3661 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgaggg 3721 gcgagggcga gggcgatgcc accaacggca agctgaccct gaagttcatc tgcaccaccg 3781 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gagccacggc gtgcagtgct 3841 tcgcccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag 3901 gctacgtcca ggagcgcacc atcttcttca aggacgacgg cacctacaag acccgcgccg 3961 aggtgaagtt cgagggcgac accctagtga accgcatcga gctgaagggc gtcgacttca 4021 aggaggacgg caacatcctg gggcacaagc tggagtacaa cttcaacagc cacaacatct 4081 atatcatggc cgtcaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaacg 4141 tggaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg 4201 gccccgtgct gctgcccgac agccactacc tgagcaccca gtccgtgctg agcaaagacc 4261 ccaacgagaa gcgcgatcac atggtcctgc tggagttccg caccgccgcc gggatcactc 4321 tcggcatgga cgagctgtac aagggaagcg gagctactaa cttcagcctg ctgaagcagg 4381 ctggagacgt ggaggagaac cctggaccta aaaagcctga actcaccgcg acgtctgtcg 4441 agaagtttct gatcgaaaag ttcgacagcg tctccgacct gatgcagctc tcggagggcg 4501 aagaatctcg tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata 4561 gctgcgccga tggtttctac aaagatcgtt atgtttatcg gcactttgca tcggccgcgc 4621 tcccgattcc ggaagtgctt gacattgggg aattcagcga gagcctgacc tattgcatct 4681 cccgccgtgc acagggtgtc acgttgcaag acctgcctga aaccgaactg cccgctgttc 4741 tgcagccggt cgcggaggcc atggatgcga tcgctgcggc cgatcttagc cagacgagcg 4801 ggttcggccc attcggaccg caaggaatcg gtcaatacac tacatggcgt gatttcatat 4861 gcgcgattgc tgatccccat gtgtatcact ggcaaactgt gatggacgac accgtcagtg 4921 cgtccgtcgc gcaggctctc gatgagctga tgctttgggc cgaggactgc cccgaagtcc 4981 ggcacctcgt gcacgcggat ttcggctcca acaatgtcct gacggacaat ggccgcataa 5041 cagcggtcat tgactggagc gaggcgatgt tcggggattc ccaatacgag gtcgccaaca 5101 tcttcttctg gaggccgtgg ttggcttgta tggagcagca gacgcgctac ttcgagcgga 5161 ggcatccgga gcttgcagga tcgccgcggc tccgggcgta tatgctccgc attggtcttg 5221 accaactcta tcagagcttg gttgacggca atttcgatga tgcagcttgg gcgcagggtc 5281 gatgcgacgc aatcgtccga tccggagccg ggactgtcgg gcgtacacaa atcgcccgca 5341 gaagcgcggc cgtctggacc gatggctgtg tagaagtact cgccgatagt ggaaaccgac 5401 gccccagcac tcgtccggat cgggagatgg gggaggctaa ctaagaattc gatatcaagc 5461 ttatcgataa tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact 5521 atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg 5581 cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg 5641 aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa 5701 cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc 5761 ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg 5821 ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt 5881 ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt 5941 cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc 6001 cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcatc 6061 gataccgtcg acctcgagac ctagaaaaac atggagcaat cacaagtagc aatacagcag 6121 ctaccaatgc tgattgtgcc tggctagaag cacaagagga ggaggaggtg ggttttccag 6181 tcacacctca ggtaccttta agaccaatga cttacaaggc agctgtagat cttagccact 6241 ttttaaaaga aaagggggga ctggaagggc taattcactc ccaacgaaga caagatatcc 6301 ttgatctgtg gatctaccac acacaaggct acttccctga ttggcagaac tacacaccag 6361 ggccagggat cagatatcca ctgacctttg gatggtgcta caagctagta ccagttgagc 6421 aagagaaggt agaagaagcc aatgaaggag agaacacccg cttgttacac cctgtgagcc 6481 tgcatgggat ggatgacccg gagagagaag tattagagtg gaggtttgac agccgcctag 6541 catttcatca catggcccga gagctgcatc cggactgtac tgggtctctc tggttagacc 6601 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa 6661 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga 6721 gatccctcag acccttttag tcagtgtgga aaatctctag cagggcccgt ttaaacccgc 6781 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg 6841 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt 6901 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc 6961 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct 7021 tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc 7081 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc 7141 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc 7201 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc 7261 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg 7321 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact 7381 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt 7441 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt 7501 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc 7561 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag 7621 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc 7681 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa 7741 ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt 7801 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca 7861 ttttcggatc tgatcagcac gtgttgacaa ttaatcatcg gcatagtata tcggcatagt 7921 ataatacgac aaggtgagga actaaaccat ggccaagttg accagtgccg ttccggtgct 7981 caccgcgcgc gacgtcgccg gagcggtcga gttctggacc gaccggctcg ggttctcccg 8041 ggacttcgtg gaggacgact tcgccggtgt ggtccgggac gacgtgaccc tgttcatcag 8101 cgcggtccag gaccaggtgg tgccggacaa caccctggcc tgggtgtggg tgcgcggcct 8161 ggacgagctg tacgccgagt ggtcggaggt cgtgtccacg aacttccggg acgcctccgg 8221 gccggccatg accgagatcg gcgagcagcc gtgggggcgg gagttcgccc tgcgcgaccc 8281 ggccggcaac tgcgtgcact tcgtggccga ggagcaggac tgacacgtgc tacgagattt 8341 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg 8401 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt 8461 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc 8521 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt 8581 ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt 8641 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa 8701 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc 8761 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag 8821 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt 8881 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga 8941 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg 9001 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa 9061 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt 9121 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct 9181 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct 9241 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc 9301 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt 9361 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc 9421 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat 9481 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa 9541 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa 9601 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga 9661 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct 9721 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga 9781 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc 9841 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg 9901 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat 9961 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat 10021 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg 10081 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc 10141 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa 10201 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc 10261 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt 10321 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag 10381 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt 10441 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag 10501 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac 10561 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc 10621 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca 10681 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg 10741 ggttccgcgc acatttcccc gaaaagtgcc acctgac //