LOCUS Exported 3716 bp ds-DNA circular SYN 17-JUL-2006 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pDONR221-P5P4 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 3716) AUTHORS Invitrogen TITLE Direct Submission JOURNAL Exported Jan 17, 2019 from SnapGene Viewer 4.2.11 http://www.snapgene.com FEATURES Location/Qualifiers source 1..3716 /organism="synthetic DNA construct" /note="/dnas_title=KB701_hU6_SaCas9gRNA(updated)_CMV" misc_feature complement(268..295) /label=rrnB T2 transcription termination sequence /note="rrnB T2 transcription termination sequence" misc_feature complement(427..470) /label=rrnB T1 transcription termination sequence /note="rrnB T1 transcription termination sequence" primer_bind 517..533 /label=M13 Forward (-40) primer binding site /note="M13 Forward (-40) primer binding site" primer_bind 537..552 /label=M13 Forward (-20) primer binding site /note="M13 Forward (-20) primer binding site" misc_recomb 570..665 /label=attL5 /note="attL5 recombination site" /note="recognized as attL5" promoter 674..922 /note="human U6 promoter" misc_feature 919..942 /label=BsaI sense fragment /note="BsaI sense fragment" /note="Replace with sense protospacer motif with sequence 5%92-CACC(G)N(20-22)-3%92" misc_feature complement(923..946) /label=BsaI antisense fragment /note="BsaI antisense fragment" /note="Replace with antisense protospacer motif with sequence 5%92-AAACN(20-22)(C)-3%92" misc_RNA 943..1032 /label=SaCas9 sgRNA scaffold /note="SaCas9 sgRNA scaffold" /note="adopted from PMID: 26721686 and PMID: 27210042" promoter 1059..1817 /note="CMV promoter" misc_recomb complement(1828..1926) /label=attL4 /note="attL4 recombination site" /note="recognized as attL4" misc_feature join(1838..3716,1..658) /label=nonstandard type: vector /note="/source=pDONR221-P5P4" /note="/type=Donor Vector" primer_bind complement(1981..1997) /label=M13 Reverse primer binding site /note="M13 Reverse primer binding site" gene 2110..2919 /label=Kanamycin resistance gene /note="Kanamycin resistance gene" rep_origin 3040..3713 /label=f1 origin /note="f1 origin" ORIGIN 1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc 241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta 301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc 361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa 421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg 481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa 541 aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac 601 ctgttcgttg caacaaattg atgagcaatg cttttttata atgccaactt tgtatacaaa 661 agttgtggta cctgagggcc tatttcccat gattccttca tatttgcata tacgatacaa 721 ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa 781 tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa 841 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat 901 cttgtggaaa ggacgaaaca ccggagacca cggcaggtct cagtttaagt actctgtgct 961 ggaaacagca cagaatctac taaacaaggc aaaatgccgt gtttatctcg tcaacttgtt 1021 ggcgagattt ttgcggccgc tgtacatacg actctagaac gatctataca ttgaatcaat 1081 attggcaatt agccatatta gtcattggtt atatagcata aatcaatatt ggctattggc 1141 cattgcatac gttgtatcta tatcataata tgtacattta tattggctca tgtccaatat 1201 gaccgccatg ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat 1261 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg 1321 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa 1381 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact 1441 tggcagtaca tcaagtgtat catatgccaa gtccgccccc tattgacgtc aatgacggta 1501 aatggcccgc ctggcattat gcccagtaca tgaccttacg ggactttcct acttggcagt 1561 acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag tacaccaatg 1621 ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg 1681 ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaat aaccccgccc 1741 cgttgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctcgtt 1801 tagtgaaccg tcagatccgg gaattccacc caacttttct atacaaagtt ggcattataa 1861 gaaagcattg cttatcaatt tgttgcaacg aacaggtcac tatcagtcaa aataaaatca 1921 ttatttgcca tccagctgca gggcggccgc gatatcccct atagtgagtc gtattacatg 1981 gtcatagctg tttcctggca gctctggccc gtgtctcaaa atctctgatg ttacattgca 2041 caagataaaa atatatcatc atgaacaata aaactgtctg cttacataaa cagtaataca 2101 aggggtgtta tgagccatat tcaacgggaa acgtcgaggc cgcgattaaa ttccaacatg 2161 gatgctgatt tatatgggta taaatgggct cgcgataatg tcgggcaatc aggtgcgaca 2221 atctatcgct tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt 2281 agcgttgcca atgatgttac agatgagatg gtcagactaa actggctgac ggaatttatg 2341 cctcttccga ccatcaagca ttttatccgt actcctgatg atgcatggtt actcaccact 2401 gcgatccccg gaaaaacagc attccaggta ttagaagaat atcctgattc aggtgaaaat 2461 attgttgatg cgctggcagt gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt 2521 ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat gaataacggt 2581 ttggttgatg cgagtgattt tgatgacgag cgtaatggct ggcctgttga acaagtctgg 2641 aaagaaatgc ataaactttt gccattctca ccggattcag tcgtcactca tggtgatttc 2701 tcacttgata accttatttt tgacgagggg aaattaatag gttgtattga tgttggacga 2761 gtcggaatcg cagaccgata ccaggatctt gccatcctat ggaactgcct cggtgagttt 2821 tctccttcat tacagaaacg gctttttcaa aaatatggta ttgataatcc tgatatgaat 2881 aaattgcagt ttcatttgat gctcgatgag tttttctaat cagaattggt taattggttg 2941 taacactggc agagcattac gctgacttga cgggacggcg caagctcatg accaaaatcc 3001 cttaacgtga gttacgcgtc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga 3061 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg 3121 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact 3181 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac 3241 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg 3301 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg 3361 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga 3421 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc 3481 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg 3541 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc 3601 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc 3661 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgtt //