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
//
