LOCUS       Exported                3267 bp ds-DNA     circular SYN 08-AUG-2019
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    LB001_3xHA_bGHpolyA+TRE#2_Sp_Cas9_gRNA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3267)
  AUTHORS   Invitrogen
  TITLE     Direct Submission
  JOURNAL   Exported Thursday, Aug 8, 2019 from SnapGene Viewer 3.3.3
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..3267
                     /organism="synthetic DNA construct"
                     /note="dnas_title=LB001_3xHA_bGHpolyA+TRE#2_Sp_Cas9_gRNA"
     misc_feature    complement(268..295)
                     /note="rrnB T2 transcription termination sequence"
     misc_feature    complement(427..470)
                     /note="rrnB T1 transcription termination sequence"
     primer_bind     517..533
                     /note="M13 Forward (-40) primer binding site"
     primer_bind     537..552
                     /note="M13 Forward (-20) primer binding site"
     misc_recomb     570..667
                     /note="attL3"
                     /note="attL3 recombination site
                     recognized as attL3"
     misc_feature    665..676
                     /note="flexible linker (GGGS)"
     misc_feature    677..760
                     /note="3x HA epitope tag"
     polyA_signal    788..998
                     /note="bGH Poly A"
                     /note="bGH Poly A
                     
                     "
     promoter        1005..1253
                     /note="human U6 promoter"
     misc_feature    1255..1274
                     /note="SpCas9 TRE #2 gRNA"
     misc_RNA        1275..1366
                     /note="SpCas9 optimized gRNA"
                     /note="SpCas9 optimized gRNA
                     Based on PMID: 26671237 and PMID: 27210042"
     misc_recomb     complement(1380..1477)
                     /note="attL2"
                     /note="attL2 recombination site
                     recognized as attL2"
     misc_feature    1389..658
                     /label=vector
                     /note="pDONR221-P3P2"
                     /note="source=pDONR221-P3P2
                     type=Donor Vector"
     primer_bind     complement(1532..1548)
                     /note="M13 Reverse primer binding site"
     gene            1661..2470
                     /note="Kanamycin resistance gene"
     rep_origin      2591..3264
                     /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 tgtataataa
      661 agttggtggc ggatcctacc catacgatgt tccagattac gcttacccat acgatgttcc
      721 agattacgct tacccatacg atgttccaga ttacgcttaa gaattcctag agctcgctga
      781 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
      841 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
      901 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
      961 ggggaggatt gggaagagaa tagcaggcat gctggggagg taccgagggc ctatttccca
     1021 tgattccttc atatttgcat atacgataca aggctgttag agagataatt ggaattaatt
     1081 tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg
     1141 ggtagtttgc agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt
     1201 gaaagtattt cgatttcttg gctttatata tcttgtggaa aggacgaaac accgctatca
     1261 gtgatagaga acgagtttca gagctatgct ggaaacagca tagcaagttg aaataaggct
     1321 agtccgttat caacttgaaa aagtggcacc gagtcggtgc ttttttgcgg ccgcaggaac
     1381 ccagctttct tgtacaaagt tggcattata agaaagcatt gcttatcaat ttgttgcaac
     1441 gaacaggtca ctatcagtca aaataaaatc attatttgcc atccagctgc agggcggccg
     1501 cgatatcccc tatagtgagt cgtattacat ggtcatagct gtttcctggc agctctggcc
     1561 cgtgtctcaa aatctctgat gttacattgc acaagataaa aatatatcat catgaacaat
     1621 aaaactgtct gcttacataa acagtaatac aaggggtgtt atgagccata ttcaacggga
     1681 aacgtcgagg ccgcgattaa attccaacat ggatgctgat ttatatgggt ataaatgggc
     1741 tcgcgataat gtcgggcaat caggtgcgac aatctatcgc ttgtatggga agcccgatgc
     1801 gccagagttg tttctgaaac atggcaaagg tagcgttgcc aatgatgtta cagatgagat
     1861 ggtcagacta aactggctga cggaatttat gcctcttccg accatcaagc attttatccg
     1921 tactcctgat gatgcatggt tactcaccac tgcgatcccc ggaaaaacag cattccaggt
     1981 attagaagaa tatcctgatt caggtgaaaa tattgttgat gcgctggcag tgttcctgcg
     2041 ccggttgcat tcgattcctg tttgtaattg tccttttaac agcgatcgcg tatttcgtct
     2101 cgctcaggcg caatcacgaa tgaataacgg tttggttgat gcgagtgatt ttgatgacga
     2161 gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg cataaacttt tgccattctc
     2221 accggattca gtcgtcactc atggtgattt ctcacttgat aaccttattt ttgacgaggg
     2281 gaaattaata ggttgtattg atgttggacg agtcggaatc gcagaccgat accaggatct
     2341 tgccatccta tggaactgcc tcggtgagtt ttctccttca ttacagaaac ggctttttca
     2401 aaaatatggt attgataatc ctgatatgaa taaattgcag tttcatttga tgctcgatga
     2461 gtttttctaa tcagaattgg ttaattggtt gtaacactgg cagagcatta cgctgacttg
     2521 acgggacggc gcaagctcat gaccaaaatc ccttaacgtg agttacgcgt cgttccactg
     2581 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     2641 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     2701 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     2761 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     2821 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     2881 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     2941 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     3001 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     3061 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     3121 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     3181 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     3241 cttttgctgg ccttttgctc acatgtt
//
